Aboismaiel et al.

Biological Research (2024) 57:47 Biological Research

https://doi.org/10.1186/s40659-024-00527-9

RESEARCH ARTICLE Open Access

Renoprotective effect of a novel combination

of 6-gingerol and metformin in high-fat diet/
streptozotocin-induced diabetic nephropathy
in rats via targeting miRNA-146a, miRNA-223,
TLR4/TRAF6/NLRP3 inflammasome pathway
and HIF-1α
Merna G. Aboismaiel1* , Mohamed N. Amin1 and Laila A. Eissa1*

Abstract
Background MiRNA-146a and miRNA-223 are key epigenetic regulators of toll-like receptor 4 (TLR4)/tumor
necrosis factor-receptor-associated factor 6 (TRAF6)/NOD-like receptor family pyrin domain-containing 3 (NLRP3)
inflammasome pathway, which is involved in diabetic nephropathy (DN) pathogenesis. The currently available oral
anti-diabetic treatments have been insufficient to halt DN development and progression. Therefore, this work aimed
to assess the renoprotective effect of the natural compound 6-gingerol (GR) either alone or in combination with
metformin (MET) in high-fat diet/streptozotocin-induced DN in rats. The proposed molecular mechanisms were also
investigated.
Methods Oral gavage of 6-gingerol (100 mg/kg) and metformin (300 mg/kg) were administered to rats daily for
eight weeks. MiRNA-146a, miRNA-223, TLR4, TRAF6, nuclear factor-kappa B (NF-κB) (p65), NLRP3, caspase-1, and
hypoxia-inducible factor-1 alpha (HIF-1α) mRNA expressions were measured using real-time PCR. ELISA was used
to measure TLR4, TRAF6, NLRP3, caspase-1, tumor necrosis factor-alpha (TNF-α), and interleukin-1-beta (IL-1β) renal
tissue levels. Renal tissue histopathology and immunohistochemical examination of fibronectin and NF-κB (p65) were
performed.
Results 6-Gingerol treatment significantly reduced kidney tissue damage and fibrosis. 6-Gingerol up-regulated
miRNA-146a and miRNA-223 and reduced TLR4, TRAF6, NF-κB (p65), NLRP3, caspase-1, TNF-α, IL-1β, HIF-1α and
fibronectin renal expressions. 6-Gingerol improved lipid profile and renal functions, attenuated renal hypertrophy,

*Correspondence:
Merna G. Aboismaiel
mernagehad@mans.edu.eg
Laila A. Eissa
lailaeissa2002@mans.edu.eg; lailaeissa2002@yahoo.com
Full list of author information is available at the end of the article

© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included
in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.
Aboismaiel et al. Biological Research (2024) 57:47 Page 2 of 25

increased reduced glutathione, and decreased blood glucose and malondialdehyde levels. 6-Gingerol and metformin
combination showed superior renoprotective effects than either alone.
Conclusion 6-Gingerol demonstrated a key protective role in DN by induction of miRNA-146a and miRNA-223
expression and inhibition of TLR4/TRAF6/NLRP3 inflammasome signaling. 6-Gingerol, a safe, affordable, and abundant
natural compound, holds promise for use as an adjuvant therapy with metformin in diabetic patients to attenuate
renal damage and stop the progression of DN.
Keywords 6-Gingerol, Diabetic nephropathy, MiRNA-146a, MiRNA-223, NLRP3 inflammasome, TLR4

Introduction Toll-like receptor 4 (TLR4)/tumor necrosis factor

Diabetes mellitus patients currently experience high rates receptor-associated factor 6 (TRAF6)/NOD-like recep-
of morbidity and mortality due to diabetic nephropa- tor family pyrin domain-containing 3 (NLRP3) inflam-
thy (DN), a serious chronic microvascular complica- masome signaling pathway is one of the most important
tion of diabetes mellitus [1]. DN is the leading cause of inflammatory pathways, which integrates inflammation
chronic kidney disease (CKD) that can progress to end- and fibrosis in diabetes-induced renal injury pathogen-
stage renal disease and is associated with increased car- esis [12, 13]. TLR4 is activated by pathogen-associated
diovascular mortality [2]. CKD is a broader term that molecular patterns (PAMPs) present on microorgan-
encompasses various conditions resulting in progressive isms or damage-associated molecular patterns (DAMPs)
and irreversible kidney damage over time. Aside from released by damaged or stressed tissues (e.g., ATP) [14].
DN, other common causes of CKD include hypertensive Activated TLR4 recruits interleukin-1 receptor-associ-
nephropathy, glomerulonephritis, and polycystic kidney ated kinase-4, interleukin-1 receptor-associated kinase-1
disease [3]. DN is characterized by persistent albumin- (IRAK-1), and TRAF6 to stimulate nuclear factor-kappa
uria and its pathological features include early podocyte B (NF-κB) translocation inside the nucleus and its acti-
injury, glomerular hyperfiltration, mesangial expansion, vation, which triggers transcriptional up-regulation of
glomerular basement membrane thickening, extracellular NLRP3, pro-interleukin-1 beta (IL-1β), and pro-IL-18.
matrix deposition, glomerulosclerosis, and tubulointer- This is the priming step, which is not sufficient to imme-
stitial fibrosis [4, 5]. diately promote NLRP3 inflammasome complex assem-
The prevalence of DN is increasing in tandem with bly and needs an additional activation step [15, 16].
the rising prevalence of diabetes mellitus particularly This activation signal involves triggering numerous
type 2 diabetes mellitus which accounts for about 90% intracellular events by PAMPs and DAMPs, such as
of patients with diabetes [2]. Approximately 20–50% of K + efflux, mitochondrial damage, and reactive oxygen
individuals with type 2 diabetes mellitus will ultimately species (ROS) generation [17]. Both priming and activa-
develop CKD. The prevalence of CKD varies globally, tion signals trigger NLRP3 inflammasome complex for-
with estimates ranging from 5 to 15% of the population, mation, followed by pro-caspase-1 auto-cleavage, which
depending on the region and the criteria used for diagno- becomes activated and cleaves pro-IL-1β, pro-IL-18, and
sis, with DN being responsible for about 50% of cases [6]. gasdermin D to induce inflammation and pyroptosis [18].
In Egypt, the burden of CKD is significant, affecting In addition, NF-κB activation in diabetic kidneys also
approximately 13% of the adult population [7]. In 2017, increases hypoxia-inducible factor-1 alpha (HIF-1α)
the Global Burden of Disease (GBD) Collaboration esti- expression. HIF-1α is the principal regulator of metabolic
mated that there were 7.1 million individuals with CKD responses under hypoxic conditions, which are among
in Egypt, with an age-standardized prevalence of 106 the earliest incidents of DN development. HIF-1α pro-
patients with CKD per 1000 population [8]. Similar to the motes renal fibrosis, perpetuating a cycle of inflamma-
global trend, the burden of CKD has increased by 36% in tion and fibrosis in the diabetic kidneys [19–21].
Egypt, with CKD ranking fifth in leading causes of death MicroRNAs (miRNAs) are small, non-coding RNA
from 2009 to 2019 [9]. DN is a leading cause of CKD in molecules that can bind to targeted mRNAs 3′ untrans-
Egypt due to the high prevalence of diabetes mellitus in lated regions (3′ UTR) and induce their degradation or
the country, with type 2 diabetes mellitus reporting a translational repression [22]. Recent studies have high-
prevalence of approximately 16% of all adults aged 20–79 lighted the critical roles of miRNAs as epigenetic regu-
years [10]. According to the Egyptian Renal Data System lators of numerous pathways involved in the process of
(ERDS) report conducted in 2020, the most common DN development [23–25]. MiRNA-146a is well-known
cause of CKD was hypertension which represented 41% to regulate TLR4-mediated NF-κB activation through a
of cases followed by diabetes mellitus which represented negative feedback mechanism in which NF-κB up-regu-
13% of patients with CKD [11]. lates miRNA-146a gene expression while miRNA-146a
Aboismaiel et al. Biological Research (2024) 57:47 Page 3 of 25

down-regulates its direct targets, IRAK-1 and TRAF6, diabetes mellitus and its complications, including DN, as
downstream of TLR4 signaling to suppress the activity of it involves both insulin resistance and gradual beta-cell
NF-κB [26, 27] while miRNA-223 exerts a direct inhibi- dysfunction which resembles the etiology of human type
tory effect on NLRP3 inflammasome [28]. 2 diabetes mellitus.
Metformin (MET) is a glucose-lowering agent that is The objective of the present study was to assess 6-gin-
used as a first-line therapy for type 2 diabetes mellitus. gerol’s renoprotective effect and its underlying molecu-
It has been shown to slow the progression of kidney dys- lar mechanisms in HFD/STZ-induced DN in rats. The
function through different mechanisms including reduc- hypothesis of targeting miRNA-146a and miRNA-223
ing inflammation, oxidative stress, and fibrosis [29–31]. and modulation of TLR4/TRAF6/NLRP3 inflammasome
Despite current treatment strategies, patients continue pathway was evaluated. Besides, the prospective benefi-
to develop CKD and end-stage renal disease [32]. Also, cial effects of using 6-gingerol and metformin in combi-
oral anti-hyperglycemic drugs have been linked to multi- nation were investigated.
ple adverse effects including metformin-associated lactic
acidosis and gastrointestinal disturbances [33]. Therefore, Materials and methods
the introduction of new therapeutic modalities to pro- Drugs and chemicals
tect against the development and progression of DN has STZ (CAS no.: 18883-66-4), 6-gingerol (CAS no.: 23513-
become mandatory. 14-6), and metformin (CAS no.: 1115-70-4) were pro-
Recently, much attention has been paid to the utiliza- vided by Sigma Aldrich Co., USA. Citric acid and sodium
tion of compounds from natural sources in the manage- citrate (for preparation of citrate buffer) and carboxy-
ment of different conditions due to their safety, efficacy, methyl cellulose (CMC) were provided by El-Gomhouria
and low cost [34–36]. 6-Gingerol (GR) is the major bioac- Co., Mansoura, Egypt. Phosphate-buffered saline (PBS)
tive component of fresh ginger, the rhizome of Zingiber was provided by Biodiagnostic, Giza, Egypt. All the
officinale, which is one of the most commonly used spices study’s chemicals were of standard analytic grade.
worldwide [37]. 6-Gingerol is a phenolic compound that
possesses interesting antioxidant, anti-inflammatory, Animals
anticancer, anti-hyperglycemic, and lipid-lowering effects This research gained approval from the ethics commit-
[34–36]. 6-Gingerol was shown to attenuate myocardial tee of the Faculty of Pharmacy at Mansoura University in
fibrosis by reducing oxidative stress, inflammation, and Mansoura, Egypt (Ethical approval no. 2023 − 159). “Prin-
apoptosis through inhibition of the toll-like receptor 4/ ciples of Laboratory Animal Care” (National Materials
mitogen-activated protein kinase/nuclear factor-kappa Institute of Health publication No. 85 − 23, revised 1985)
B pathway [38]. Also, 6-gingerol was demonstrated to were followed in all animal experiments. Adult male
alleviate pain, anxio-depression, and neuroinflammation Sprague-Dawley rats (200 ± 20 g) were purchased and
in rats with diabetic neuropathy [39]. 6-Gingerol ame- housed in the animal house of the Faculty of Pharmacy,
liorated weight gain and insulin resistance in metabolic Mansoura University. Before the experiment began, rats
syndrome rats by regulating adipocytokines [40]. 6-Gin- were left for two weeks to acclimatize to standard envi-
gerol has also demonstrated a key protective role in DN ronmental conditions of temperature (22 ± 2ºC) and light-
by regulating oxidative stress and inflammation [41]. In ing (12 h light-dark cycle) with unrestricted access to
addition, 6-gingerol could suppress transforming growth food and water.
factor-β1 signaling, thereby inhibiting the activation of
fibroblasts and reducing the deposition of extracellular Induction of type 2 diabetes mellitus
matrix components, which holds promise in mitigating Rats were given high-fat diet (58% fat, 25% protein, and
the progression of renal fibrosis in DN [42]. However, the 17% carbohydrate) for four weeks before receiving a sin-
exact mechanism of the renoprotective effect of 6-gin- gle low-dose (35 mg/kg) intraperitoneal (i.p.) injection of
gerol has not been completely understood. STZ following a night fasting. Cold citrate buffer (0.1 M,
A rat model of type 2 diabetes mellitus, developed by pH 4.5) was used to freshly prepare STZ [44]. Diabetes
Srinivasan et al. in 2005, was used to induce DN through induction was confirmed three days following STZ injec-
a combination of high-fat diet (HFD) and low-dose tion via glucometer (Accu-Check Go, Roche Diagnos-
(35 mg/kg) streptozotocin (STZ) [43]. The use of a low tics, Mannheim, Germany) to measure blood glucose
dose of STZ selectively induces diabetes in high fat-fed levels from the tail vein. Diabetic rats were defined as
rats with insulin resistance, while it fails to induce diabe- those having blood glucose levels ≥ 250 mg/dl. Diabetic
tes in normal rats. This model exhibits stable and persis- rats were continued on high-fat diet until the experiment
tent hyperglycemia in addition to the lipid abnormalities ended. All treatments were started 3 days following STZ
seen in type 2 diabetes mellitus patients. This model injection and continued for 8 weeks [45].
is suitable for studying the pathophysiology of type 2
Aboismaiel et al. Biological Research (2024) 57:47 Page 4 of 25

Experimental design and weighed. Thiopental anesthesia (40 mg/kg i.p.) was
Forty male Sprague-Dawley rats were assigned into five used to obtain blood samples from the retro-orbital
groups (n = 8). A schematic representation of the experi- plexus, then blood samples were centrifuged at 3,000 rpm
mental design was described in Fig. 1. The control group, for 15 min at 4 °C to obtain serum samples which were
where rats were given a typical rat pellet diet. Then a sin- then kept at -20 °C. Then rats were decapitated and sac-
gle i.p. dose of citrate buffer (0.1 M, pH 4.5) was admin- rificed, after which their kidneys were separated and
istered to rats after 4 weeks. Three days later, they were weighed. Two parts of the left kidney were dissected,
administered 0.5% CMC orally every day for 8 weeks. frozen instantly with liquid nitrogen, and later kept at
The diabetic nephropathy (DN) group, where untreated -80 °C. The first part was used for biochemical analysis in
diabetic rats received 0.5% CMC orally every day for kidney homogenate, and the second part was employed
8 weeks. The 6-gingerol (GR) group, in which diabetic for quantitative, real-time polymerase chain reaction
rats were given an oral dose of 6-gingerol (100 mg/kg) (qRT-PCR). 10% neutral buffered formalin was used to
in 0.5% CMC every day for 8 weeks [42]. The metformin fix the right kidney to be utilized in histopathology and
(MET) group, in which diabetic rats were given an oral immunohistochemistry investigations.
dose of metformin (300 mg/kg) in 0.5% CMC every day
for 8 weeks [46]. The 6-gingerol + metformin combina- Biochemical analysis in serum
tion (GR + MET) group, in which diabetic rats received Serum samples were used for measuring fasting blood
a combination of 6-gingerol (100 mg/kg) and metformin glucose, serum creatinine, blood urea nitrogen (BUN),
(300 mg/kg) in 0.5% CMC orally every day for 8 weeks. triglycerides, total cholesterol, and high-density lipopro-
tein (HDL) cholesterol concentrations by spectropho-
Sample collection tometry using biochemical kits (Biodiagnostic Co., Giza,
At the end of the study, 24-hour urine samples were col- Egypt) guided by the manufacturer’s recommendations.
lected from each rat using metabolic cages (Nalgene,
Rochester, NY, USA). Then the rats were fasted overnight

Fig. 1 A schematic representation of the experimental design

DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin; CMC: carboxymethyl cellulose; HFD: high-fat diet; STZ: streptozotocin
Aboismaiel et al. Biological Research (2024) 57:47 Page 5 of 25

Biochemical analysis in urine interstitial fibrosis as indicated by Yamate, et al. [50].

Centrifugation was performed on the collected 24-hour Images were captured using Nikon digital camera-aided
urine samples for 10 min at 4 °C at 2000 rpm. After- computer software. The percentage of Masson’s-positive
ward, urine protein and creatinine concentrations were area was evaluated quantitatively using Image J analysis
measured in the resulting supernatants by spectropho- software (NIH, USA). The histologist was kept unaware
tometry technique using commercial biochemical kits of the experimental groups and a random examination of
(Biodiagnostic Co., Giza, Egypt) as per the manufac- the slides was carried on.
turer’s guidelines. Also, urinary protein excretion level
(mg/24 hr), creatinine clearance (ml/min), and protein- Immunohistochemical examination of renal tissue
uria/creatininuria ratio were calculated [45]. Renal tissue sections were used for immunohistochemi-
cal evaluation of NF-κB (p65) and fibronectin in kid-
Biochemical analysis in kidney homogenate ney tissue. Slides were first deparaffinized in xylene,
First, kidney tissue was rinsed with ice-cold PBS to then rehydrated in graded ethanol, and immersed for
remove any remaining blood. Then, tissue fragments 10 min at room temperature in a 0.3% H2O2/methanol
were weighed and homogenized in PBS (pH 7.4) on ice solution. The anti-NF-κB (p65) (Catalog No. sc-8008)
using a glass homogenizer to prepare 10% (w/v) homog- and anti-fibronectin (Catalog No. sc-8422) from Santa
enate. This was followed by sonication for 60 s to further Cruz Biotechnology, USA were 1:100-diluted and incu-
homogenate the cells. Five minutes were spent centrifug- bated overnight on the slides at 4 °C. PBS was employed
ing the renal tissue homogenate at 10,000 rpm at 4 °C fol- three times to rinse the slides, followed by incubation
lowed by separation of the supernatant to be employed in for thirty minutes at ambient temperature with anti-rat
biochemical assays [47]. IgG secondary antibody (Abcam, Waltham, MA, USA;
Catalog No. ab150165). The slides were then visualized
Enzyme-linked immunosorbent assay (ELISA) with diaminobenzidine and finally counterstained with
Commercial ELISA assay kits were used to determine Mayer’s hematoxylin. For the preparation of the negative
renal concentrations of TLR4 (LifeSpan Biosciences, WA, control procedure, normal rat serum was used instead of
USA; Catalog No. LS-F4846), TRAF6 (LifeSpan Biosci- the primary antibody. Immunostaining of both NF-κB
ences, WA, USA; Catalog No. LS-F20212), NLRP3 (Aviva (p65) and fibronectin showed distinctive brown reac-
Systems Biology, CA, USA; Catalog No. OKCD04232-48), tions. Image J analysis software (NIH, USA) was utilized
caspase-1 (Biovision, CA, USA; Catalog No. E4594-100), for measuring the area of positive expression to provide a
tumor necrosis factor –alpha (TNF-α) (MyBioSource, quantitative assessment [51, 52].
CA, USA; Catalog No. MBS2507393), and IL-1β (Abcam,
Waltham, MA, USA; Catalog No. ab100768) in kidney Quantitative, real-time polymerase chain reaction (qRT-
homogenate according to the manufacturer’s protocols. PCR)
The Bradford method (1976) was applied to assess the The miRNeasy Mini Kit (Qiagen, Hilden, Germany;
tissue’s protein content [48] using a Bradford Assay Kit Catalog No. 217,004) was used for the extraction of
(Abcam, Waltham, MA, USA; Catalog No. ab102535). total RNA, including miRNA, from renal tissue, follow-
ing the guidelines of the manufacturer. Qiagen’s Quan-
Assay of oxidative stress and lipid peroxidation biomarkers tiTect® Reverse Transcription Kit (Catalog No. 205,311)
Spectrophotometric assay kits from Biodiagnostic Co., and miRCURY® LNA® RT Kit (Catalog No. 339,340) were
Giza, Egypt were utilized to determine renal reduced used for synthesizing cDNA from mRNA and miRNA,
glutathione (GSH) and malondialdehyde (MDA) levels in respectively, guided by the manufacturer’s instructions.
renal homogenate as well as serum and urinary MDA lev- PikoReal™ Real-time PCR System (Thermo Fisher Scien-
els following the manufacturer’s guidelines. tific Inc., Waltham, MA, USA) was used for the detection
of mRNA and miRNA using Qiagen’s QuantiTect® SYBR®
Histopathological examination of renal tissue Green RT-PCR Kit (Catalog No. 204,243) and miRCURY®
The formalin-fixed right kidney was dissected longitu- LNA® SYBR® Green PCR Kit (Catalog No. 339,345),
dinally into two halves before being paraffin-embedded. respectively, guided by the manufacturer’s instructions.
Renal tissue Sect. (5 μm thick) were stained differently in The housekeeping genes for mRNA and miRNA were rat
two slide sets. Hematoxylin and eosin (H&E) staining was glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
applied to the first set to assess renal histopathological and U6, respectively. Based on gene sequences derived
alterations [49], which were assessed semi-quantitatively from the GenBank, specific primers were designed for
and given scores from 0 to 3, where 0 is normal, 1 is mild, TLR4, TRAF6, NF-κB p65, NLRP3, caspase-1, HIF-1α,
2 is moderate, and 3 is severe. Masson’s trichrome stain- and GAPDH genes and then analyzed using NetPrimer
ing was applied to the second set for assessment of renal (PREMIER Biosoft, USA). Table 1 shows a list of the
Aboismaiel et al. Biological Research (2024) 57:47 Page 6 of 25

Table 1 Primer sequences of specific genes Results

Gene of Interest Primer Sequence Reference 6-Gingerol, Metformin, and their combination attenuated
Sequence
renal injury and improved renal functions in diabetic rats
TLR4 Forward 5`- ​C​C​A​G​A​G​C​C​G​T ​T​G​G​T​G​T​A​ NM_019178.2
Serum creatinine and BUN concentrations, urinary
T​C ​T-3`
Reverse 5`- ​A​G​A​A​G​A​T​G​T​G​C​C ​T​C​C​C​C​
protein excretion level, creatinine clearance, and pro-
A​G​A-3` teinuria/creatininuria ratio were measured to assess the
TRAF6 Forward 5`- ​T​C ​T​C​C​C​C ​T​G​C​C ​T ​T​C​A​T ​T​G​ NM_001107754.2 renoprotective effects of 6-gingerol, metformin, and their
T​T − 3` combination. As shown in Fig. 2A, B, and C, diabetic
Reverse 5`- ​A​G​G​C ​T​G​G​C​G​A​T ​T ​T ​T​G​T​G​ rats exhibited a 2.8-fold, 2-fold, and 5.8-fold elevation
T​T ​T − 3` of serum creatinine, BUN, and urinary protein excre-
NF-κB Forward 5`- ​T​G​T​G​T​G​A​A​G​A​A​G​C​G​A​G​A​ NM_199267.2 tion levels, respectively, corresponding to control rats
(p65) C​C ​T​G − 3`
(p < 0.0001). Administration of 6-gingerol, metformin,
Reverse 5`- ​A​A​A​A​T​C​G​G​A​T​G​C​G​A​G​A​G​
and their combination to diabetic rats brought about a
G​A​C − 3`
significantly reduced serum creatinine concentration
NLRP3 Forward 5`- ​G​T​A​G​G​T​G​T​G​G​A​A​G​C​A​G​G​ NM_001191642.1
A​C ​T − 3` by 37.7%, 40.6%, and 60.8%, respectively, and BUN by
Reverse 5`- ​C​C ​T ​T ​T​G​C ​T​C​C​A​G​A​C​C​C ​T​ 38.7%, 42.2%, and 49.8%, respectively, while urinary pro-
A​C​A − 3` tein excretion levels were reduced by 51.4%, 62.6%, and
Caspase Forward 5`- ​C​G​T​C ​T ​T​G​C​C​C ​T​C​A​T ​T​A​T​C​ NM_012762.3 77.1%, respectively. Besides, serum creatinine and BUN
1 T​G​C − 3` concentrations, as well as urinary protein excretion lev-
Reverse 5`- ​A​C​A​G​T​A​T​A​C​C​C​C​A​G​A​T​C​C​ els, were significantly reduced upon treatment of diabetic
T​G​C − 3` rats with 6-gingerol + metformin combination as com-
HIF-1α Forward 5`- ​G​C​A​T​C ​T​C​C​A​C​C ​T ​T​C ​T​A​C​C​ NM_024359.2
pared to those treated with either 6-gingerol (p < 0.001),
C​A − 3`
(p < 0.001), (p < 0.0001) or metformin (p < 0.01), (p < 0.05)
Reverse 5`- ​T​C ​T​G​T​C ​T​G​G​T​G​A​G​G​T ​T​G​T​
C​C − 3` and (p < 0.01), respectively. Compared to the control
GAPDH Forward 5`- ​C​C​A​T​C​A​A​C​G​A​C​C​C​C ​T ​T​C​ NM_017008.4 group, 6-gingerol and metformin groups showed a sig-
A​T ​T − 3` nificant increase in serum creatinine (p < 0.0001) and
Reverse 5`- ​C​A​C​G​A​C​A​T​A​C ​T​C​A​G​C​A​C​C​ (p < 0.001), respectively, and BUN (p < 0.0001) concen-
A​G​C − 3` trations and urinary protein excretion level (p < 0.0001),
TLR4: Toll-like receptor 4, TRAF6: Tumor necrosis factor receptor-associated while no significant difference was found in 6-gin-
Factor 6, NF-κB (p65): Nuclear factor-kappa B (p65), NLRP3: NOD-like receptor
family pyrin domain-containing 3, HIF-1α: Hypoxia-inducible factor-1 alpha, gerol + metformin group with respect to control group.
GAPDH: Glyceraldehyde-3-phosphate dehydrogenase On the other hand, Fig. 2D showed that diabetic rats
exhibited a 2.5-fold reduction in creatinine clearance rel-
primer sequences. Qiagen’s miRCURY LNA miRNA ative to the control group (p < 0.0001). Creatinine clear-
PCR assays were used to analyze the expression of ance was significantly elevated in 6-gingerol (p < 0.01),
miRNA. The following primer sets were used: hsa-miR- metformin (p < 0.001), and 6-gingerol + metformin com-
146a-5p, rno-miR-223-3p, and U6 snRNA. The 2−ΔΔCT bination (p < 0.0001) groups relative to the DN group.
method was applied to determine mRNA and miRNA’s Besides, creatinine clearance manifested a significant
relative expression corresponding to GAPDH and U6, elevation in diabetic rats receiving the 6-gingerol + met-
respectively. formin combination when compared to those treated
with 6-gingerol (p < 0.001) or metformin (p < 0.05) alone.
Statistical analysis Compared to the control group, 6-gingerol and metfor-
The results were statistically analyzed using Graph Pad min groups showed a significant decrease in creatinine
Prism 7 (San Diego, CA, USA). Data normality was clearance (p < 0.0001) and (p < 0.001), respectively, while
tested using Shapiro-Wilk test. One-way analysis of vari- no significant difference was found in 6-gingerol + met-
ance (ANOVA) and Tukey’s post-hoc test were employed formin group with respect to control group.
for the analysis of parametric data, which were presented Furthermore, Fig. 2E demonstrated a 3.9-fold eleva-
as mean ± standard error of the mean (SEM). Since statis- tion in proteinuria/creatininuria ratio in diabetic rats
tical analysis for H&E histopathological lesions appeared with respect to control group (p < 0.0001). Proteinuria/
non-parametric, Kruskal-Wallis and Dunn’s tests were creatininuria ratio was markedly reduced in 6-gingerol,
used for the analysis of data, which were presented as metformin, and 6-gingerol + metformin combination
median and range. Differences between groups were groups relative to the DN group (p < 0.0001). In addition,
deemed statistically significant when the p-value was proteinuria/creatininuria ratio exhibited a significant
below 0.05. reduction in diabetic rats receiving the 6-gingerol + met-
formin combination when compared to those treated
Aboismaiel et al. Biological Research (2024) 57:47 Page 7 of 25

Fig. 2 GR, MET, and their combination attenuated renal injury and improved renal functions in diabetic rats
Renal functions assessment by measuring serum levels of A: serum creatinine, B: blood urea nitrogen (BUN); C: urinary protein excretion level; D: creatinine
clearance; and E: proteinuria/creatininuria ratio. DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin. Data are represented as Mean ± SEM (*p <
0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant)
Aboismaiel et al. Biological Research (2024) 57:47 Page 8 of 25

with 6-gingerol (p < 0.001) or metformin (p < 0.05) alone. In addition, the 6-gingerol + metformin group displayed
Also, proteinuria/creatininuria ratio was significantly ele- significantly lower TLR4, TRAF6, and NF-κB (p65)
vated in 6-gingerol and metformin groups with respect mRNA expressions relative to 6-gingerol (p < 0.0001) or
to control group (p < 0.0001) whereas a non-significant metformin (p < 0.001) group and a significant decrease in
difference was observed in 6-gingerol + metformin group NLRP3 and caspase-1 mRNA expressions as compared to
relative to the control group. Interestingly, renal func- 6-gingerol or metformin group (p < 0.0001). 6-Gingerol
tions were markedly improved in rats treated with the and metformin groups showed a significant increase
6-gingerol + metformin combination, as indicated by the in TLR4, TRAF6, NF-κB (p65), NLRP3, and caspase-1
non-significant variation in serum creatinine and BUN mRNA expressions relative to control group (p < 0.0001)
concentrations, urinary protein excretion level, cre- while no significant variation was seen between 6-gin-
atinine clearance, and proteinuria/creatinuria ratio with gerol + metformin group and control group.
respect to control rats.
The renoprotective effects of the 6-gingerol, metfor- 6-Gingerol, Metformin, and their combination suppressed
min, and 6-gingerol + metformin combination were fur- inflammation and pyroptosis in diabetic kidneys
ther examined in kidney tissue segments stained with In addition to gene expression, TLR4, TRAF6, NF-κB
H&E (Fig. 3A and B). Microscopic pictures from the con- (p65), NLRP3, and caspase-1 protein levels were further
trol group showed normal kidney architecture in both determined in renal tissue. As shown in Fig. 5A, B, C, and
the cortex and medulla, with normal glomerular and D, diabetic rats revealed a 4-fold elevation in TLR4, a 4.5-
tubular structures. Severe histopathological lesions were fold elevation in TRAF6, a 3.4-fold elevation in NLRP3,
observed in the DN group in both cortex and medulla, and a 4.6-fold elevation in caspase-1 levels relative to
including diffuse tubular hydropic degeneration, tubular the control group (p < 0.0001). Administering 6-gingerol,
necrosis, hyaline casts, congested glomeruli, and con- metformin, or 6-gingerol + metformin combination to
gested inter-tubular blood vessels. 6-Gingerol group diabetic rats brought about significantly reduced levels
showed moderately decreased histopathological lesions of TLR4 by 38%, 47.3%, and 70.5%, respectively; TRAF6
as compared to the DN group (p < 0.05) and exhibited by 32.3%, 37.6%, and 58.1%; NLRP3 by 40.4%, 36.6%, and
moderate tubular hydropic degeneration, hyaline casts, 57.9%; and caspase-1 by 45.2%, 50.5%, and 70.5%, respec-
and congested inter-tubular blood vessels. Metformin tively. Furthermore, the 6-gingerol + metformin group
group showed moderately decreased histopathologi- revealed significantly reduced levels of TLR4 (p < 0.001),
cal lesions compared to the DN group (p < 0.05) includ- (p < 0.05); TRAF6 (p < 0.001), (p < 0.01); NLRP3 (p < 0.05),
ing moderate tubular hydropic degeneration, congested (p < 0.01); and caspase-1 (p < 0.0001) as compared to
glomeruli, and mildly congested inter-tubular blood either 6-gingerol or metformin groups, respectively. In
vessels. The 6-gingerol + metformin group showed a sig- comparison with the control group, 6-gingerol and met-
nificantly improved histopathology relative to DN group formin groups showed significantly elevated levels of
(p < 0.001) and displayed mild tubular hydropic degen- TLR4 (p < 0.0001), (p < 0.01); TRAF6 (p < 0.0001); NLRP3
eration and mildly congested inter-tubular blood vessels (p < 0.0001); and caspase-1 (p < 0.0001). A non-significant
which seemed non-significantly different from the con- difference was found between 6-gingerol + metformin
trol group. group and control group regarding TLR4, NLRP3, and
caspase-1 levels whereas TRAF6 level was significantly
6-Gingerol, Metformin, and their combination down- higher in 6-gingerol + metformin group compared to con-
regulated TLR4/TRAF6/NLRP3 inflammasome pathway trol group (p < 0.05).
gene expression in DN
As shown in Fig. 4A, B, C, D, and E, the DN group 6-Gingerol, metformin, and their combination decreased
revealed a significantly increased mRNA expression of renal inflammatory cytokines levels and diminished the
each of TLR4 (3.6-fold), TRAF6 (4.3-fold), NF-κB (p65) inflammatory response in DN
(4.2-fold), NLRP3 (3.4 fold), and caspase-1 (4.8 fold) Since inflammatory cytokines are crucial for the onset
relative to the control group (p < 0.0001). Diabetic rats and development of DN, protein levels of TNF-α and
treated with 6-gingerol, metformin, and 6-gingerol + met- IL-1β were measured in kidney tissues. Figure 5E and F
formin combination showed significantly lower mRNA demonstrated a significant elevation in renal TNF-α and
expressions of TLR4 by 52.5%, 55%, and 68%, respec- IL-1β levels in the DN group relative to the control group
tively; TRAF6 by 39.5%, 51.1%, and 74.2%; NF-κB (p65) (p < 0.0001). A significant reduction of TNF-α level was
by 46.6%, 50.1%, and 70.5%; NLRP3 by 47.2%, 53.2%, observed in 6-gingerol, metformin, and 6-gingerol + met-
and 67.9%; and caspase-1 by 36.1%, 47%, and 71.2%, formin combination groups by 40.5%, 43.4%, and
respectively. 52.6%, respectively, while IL-1β level was significantly
decreased by 37.2%, 44.7%, and 63.1%, respectively. The
Aboismaiel et al. Biological Research (2024) 57:47 Page 9 of 25

Fig. 3 Histopathological examination of H&E-stained renal sections showing renoprotective effects of GR, MET, and their combination
A: Microscopic images of hematoxylin and eosin (H&E)-stained renal sections showing normal cortex and medulla in the control group, severe pathologi-
cal changes in the cortex and medulla in DN group, moderately decreased pathological changes in the cortex and medulla in GR group and MET group,
and markedly improved histological picture with mild pathological changes in the cortex and medulla in GR + MET group. Black arrows: diffuse tubular
hydropic degeneration, dashed arrows: tubular necrosis, black arrowheads: cast formation, circular arrows: congested glomeruli, elbow arrows: congested
inter-tubular blood vessels. X: 400, scale bar = 50 micrometer
B: Renal histopathological changes were assessed semi-quantitatively and given scores from 0 to 3, where 0 is normal, 1 is mild, 2 is moderate, and 3 is
severe. DN: diabetic nephropathy, GR: 6-gingerol, MET: metformin. Data are represented as median and range (*p < 0.05, ***p < 0.001, ****p < 0.0001,
ns: non-significant)
Aboismaiel et al. Biological Research (2024) 57:47 Page 10 of 25

Fig. 4 GR, MET, and their combination down-regulated TLR4/TRAF6/NLRP3 inflammasome pathway gene expression in DN
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure mRNA relative expression of TLR4 (A), TRAF6 (B), NF-κB (p65) (C), NLRP3
(D), and caspase-1 (E) in renal tissue. DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin; TLR4: Toll-like receptor 4; TRAF6: Tumor necrosis factor
receptor-associated Factor 6; NF-κB (p65): Nuclear factor-kappa B (p65); NLRP3: NOD-like receptor family pyrin domain-containing 3. Data are represented
as Mean ± SEM (***p < 0.001, ****p < 0.0001, ns: non-significant)

6-gingerol + metformin combination group exhibited a IL-1β (p < 0.0001) levels, respectively, whereas 6-gin-
significantly reduced TNF-α level with regards to 6-gin- gerol + metformin group showed no significant difference
gerol (p < 0.001) and metformin (p < 0.01) groups. Also, as compared to control group.
IL-1β expressed a markedly reduced level in the 6-gin-
gerol + metformin group in comparison with 6-gingerol 6-Gingerol, Metformin, and their combination suppressed
and metformin groups (p < 0.0001). As compared to con- NF-κB (p65) protein expression in kidney tissue
trol group, 6-gingerol and metformin groups manifested Protein expression of NF-κB (p65) in kidney tissue was
significantly elevated TNF-α (p < 0.0001), (p < 0.001) and examined by immunohistochemistry (Fig. 6A and B).
Aboismaiel et al. Biological Research (2024) 57:47 Page 11 of 25

Fig. 5 GR, MET, and their combination suppressed inflammation and pyroptosis in diabetic kidneys
Renal tissue protein levels of TLR4 (A), TRAF6 (B), NLRP3 (C), caspase-1 (D), TNF-α (E), and IL-1β (F). DN: diabetic nephropathy; GR: 6-gingerol; MET: met-
formin; TLR4: Toll-like receptor 4; TRAF6: Tumor necrosis factor receptor-associated Factor 6; NLRP3: NOD-like receptor family pyrin domain-containing 3;
TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin-1 beta. Data are represented as Mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001,
ns: non-significant)
Aboismaiel et al. Biological Research (2024) 57:47 Page 12 of 25

Fig. 6 Immunohistochemical examination of NF-κB (p65) in renal tissue

A: Microscopic images of immunostained renal sections against NF-κB (p65) showing negative staining in the cortex and medulla in the control group,
excess brown tubular staining in the cortex and medulla in the DN group, decreased brown tubular staining in the cortex and medulla in GR group and
MET group, and much more decreased brown tubular staining in the cortex and medulla in GR + MET group. Black arrows refer to nuclear staining. Im-
munohistochemistry is counterstained with Mayer’s hematoxylin. X: 400, scale bar = 50 micrometer
B: Immunostaining of NF-κB (p65) was assessed quantitatively through the area of positive expression. DN: diabetic nephropathy, GR: 6-gingerol, MET:
metformin, NF-κB (p65): nuclear factor-kappa B (p65). Data are represented as Mean ± SEM (****p < 0.0001, ns: non-significant)
Aboismaiel et al. Biological Research (2024) 57:47 Page 13 of 25

Microscopic images of immunostained renal sections a state of high oxidative stress in diabetic rats. Adminis-
against NF-κB (p65) showed no brown staining in either tration of 6-gingerol, metformin, and 6-gingerol + met-
the cortex or medulla in the control group. DN group formin combination to diabetic rats brought about
showed excess brown tubular staining in both the cor- significantly elevated renal GSH levels by 71.2%, 50.2%,
tex and medulla which implied an elevated expression of and 93%, respectively. Renal GSH level was markedly ele-
NF-κB (p65), as well as excess brown nuclear staining in vated in the 6-gingerol group relative to the metformin
tubules, which revealed the elevated nuclear expression group (p < 0.05). Both 6-gingerol and metformin groups
of activated NF-κB (p65). NF-κB (p65) expression was showed a significant decrease in renal GSH level (p < 0.01)
semi-quantified by measuring NF-κB (p65) positive area and (p < 0.0001), respectively, compared to the control
percentage, which was significantly elevated in the DN group. The 6-gingerol + metformin group revealed sig-
group relative to the control group (p < 0.0001). Adminis- nificantly elevated renal GSH levels when compared with
tration of 6-gingerol, metformin, or 6-gingerol + metfor- 6-gingerol (p < 0.001) and metformin (p < 0.0001) groups
min combination to diabetic rats resulted in a significant and no significant difference relative to the control group.
reduction in NF-κB (p65) positive area percentage with Alternatively, Fig. 7D, E and F demonstrated signifi-
regards to untreated diabetic rats (p < 0.0001). 6-Gingerol cantly elevated MDA levels in renal tissue, serum and
and metformin groups exhibited a significant elevation urine, respectively, in the DN group with respect to the
in NF-κB (p65) positive area percentage in compari- control group (p < 0.0001) reflecting a significant increase
son with control group (p < 0.0001). Moreover, the in lipid peroxidation. Administration of 6-gingerol, met-
6-gingerol + metformin group exhibited a significantly formin, and 6-gingerol + metformin combination to dia-
decreased NF-κB (p65) positive area percentage when betic rats led to significantly reduced renal MDA levels
compared with either 6-gingerol or metformin group by 30.8%, 21.4%, and 45.5%; serum MDA levels by 27.2%,
(p < 0.0001) and a non-significant difference with respect 22.9%, and 44%; and urine MDA levels by 36.4%, 25.9%,
to the control group. and 66.7%, respectively. Renal MDA level was markedly
lower in the 6-gingerol group relative to the metfor-
6-Gingerol, Metformin, and their combination min group (p < 0.05). The MDA levels were significantly
up-regulated miRNA-146a and miRNA-223 gene reduced in renal (p < 0.0001), serum (p < 0.01), and uri-
expression in diabetic kidneys nary (p < 0.0001) samples in 6-gingerol + metformin group
As shown in Fig. 7A and B, a significant down-regulation with respect to 6-gingerol group. The 6-gingerol + met-
of miRNA-146a (4.4-fold) and miRNA-223 (2.8-fold) formin group exhibited significantly lower renal, serum,
gene expression was observed in the DN group in com- and urinary MDA levels with respect to metformin
parison with the control group (p < 0.0001). When com- group (p < 0.0001). Compared with control group, renal
pared to the DN group, miRNA-146a gene expression and urinary MDA levels were significantly higher in
was significantly higher in the 6-gingerol, metformin, both 6-gingerol and metformin groups (p < 0.0001) and
and 6-gingerol + metformin groups (p < 0.0001). Also, serum MDA level was significantly higher in 6-gingerol
miRNA-223 gene expression was significantly higher in (p < 0.01) and metformin (p < 0.0001) groups. Besides, the
6-gingerol (p < 0.01), metformin (p < 0.0001), and 6-gin- 6-gingerol + metformin group displayed non-significantly
gerol + metformin (p < 0.0001) groups with respect to different renal and urinary MDA levels and significantly
the DN group. In addition, the 6-gingerol + metformin higher serum MDA level (p < 0.01) when compared with
group revealed significantly up-regulated miRNA-146a the control group.
and miRNA-223 gene expressions with respect to either
the 6-gingerol or metformin group (p < 0.0001). More- 6-Gingerol, Metformin, and their combination suppressed
over, miRNA-146a and miRNA-223 were significantly renal fibrosis in diabetic kidneys
decreased in 6-gingerol and metformin groups with As demonstrated by Fig. 8A and B, Masson trichrome-
respect to control group (p < 0.0001), while no significant stained renal tissue segments showed no excess collagen
difference was observed in 6-gingerol + metformin group deposition in either the cortex or medulla in the control
with respect to control group. group. DN group showed excess bluish-stained colla-
gen deposition in the renal cortex and medulla, reflect-
6-Gingerol, Metformin, and their combination decreased ing significant fibrosis in the DN group. 6-Gingerol and
oxidative stress in diabetic kidneys metformin groups showed markedly less bluish-stained
Our results revealed a markedly improved oxidative sta- collagen deposition. The collagen deposition was even
tus in diabetic rats that received 6-gingerol, metformin, more significantly reduced in the 6-gingerol + metformin
or their combination. As shown in Fig. 7C, a significant group. The fibrosis percentage was markedly decreased
reduction in renal GSH level was observed in the DN by 75.8% in the 6-gingerol group and by 79.3% in the
group relative to the control group (p < 0.0001) reflecting metformin group relative to the DN group. 6-Gingerol
Aboismaiel et al. Biological Research (2024) 57:47 Page 14 of 25

Fig. 7 GR, MET, and their combination up-regulated miRNA-146a and miRNA-223, decreased oxidative stress and lipid peroxidation
Relative expression of miRNA-146a (A) and miRNA-223 (B) was measured using quantitative real-time polymerase chain reaction (qRT-PCR). Oxidative
stress was assessed by spectrophotometric measurement of the levels of reduced glutathione (GSH) (C) and malondialdehyde (MDA) (D) in renal tissue
and the levels of serum MDA (E) and urinary MDA (F). DN: diabetic nephropathy, GR: 6-gingerol; MET: metformin. Data are represented as Mean ± SEM
(*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant)
Aboismaiel et al. Biological Research (2024) 57:47 Page 15 of 25

Fig. 8 Histopathological examination of Masson’s trichrome-stained renal sections and fibrosis percentage assessment in diabetic rats
A: Microscopic images of Masson’s trichrome-stained renal sections showing no collagen deposition in the cortex or medulla in the control group, excess
bluish-stained collagen deposition in the cortex and medulla in the DN group, decreased collagen deposition in the cortex and medulla in the GR group
and MET group, and much more decreased collagen deposition in the cortex and medulla in GR + MET group. Black arrows refer to collagen deposition.
X: 400, scale bar = 50 micrometer
B: Fibrosis percentage was assessed quantitatively through Masson’s-positive area percentage. DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin.
Data are represented as Mean ± SEM (**p < 0.01, ***p < 0.001, ****p < 0.0001, ns: non-significant)
Aboismaiel et al. Biological Research (2024) 57:47 Page 16 of 25

Fig. 9 (See legend on next page.)

Aboismaiel et al. Biological Research (2024) 57:47 Page 17 of 25

(See figure on previous page.)

Fig. 9 Immunohistochemical examination of fibronectin and gene expression of HIF-1α in renal tissue
A: Microscopic images of immunostained renal sections against fibronectin showing negative staining in the cortex and medulla in the control group,
excess brown tubular staining in the cortex and medulla in DN group, decreased brown tubular staining in the cortex and medulla in GR group and MET
group, and much more decreased positive brown tubular staining in the cortex and medulla in GR + MET group. Black arrows refer to fibronectin deposi-
tion. Immunohistochemistry is counterstained with Mayer’s hematoxylin. X: 400, scale bar = 50 micrometer
B: Immunostaining of fibronectin was assessed quantitatively through the area of positive expression. DN: diabetic nephropathy, GR: 6-gingerol, MET:
metformin. Data are represented as Mean ± SEM (****p < 0.0001)
C: Renal hypoxia was assessed by measuring mRNA relative expression of hypoxia-inducible factor-1 alpha (HIF-1α). DN: diabetic nephropathy, GR: 6-gin-
gerol, MET: metformin. Data are represented as Mean ± SEM (***p < 0.001, ****p < 0.0001, ns: non-significant)

and metformin groups exhibited a significant elevation non-significant variation was found with respect to the
in fibrosis percentage in comparison with control group control group.
(p < 0.001). In addition, the 6-gingerol + metformin group
demonstrated a significantly decreased fibrosis percent- 6-Gingerol, Metformin, and their combination reduced
age relative to either 6-gingerol (p < 0.001) or metformin fasting blood glucose and renal hypertrophy in DN
(p < 0.01) groups and non-significant differences with As shown in Table 2, DN group expressed a 4.2-fold ele-
respect to the control group. vated fasting blood glucose level relative to the control
Fibronectin deposition in kidney tissue was stained group (p < 0.0001). When diabetic rats were administered
immunohistochemically to further evaluate renal fibrosis 6-gingerol, metformin, or 6-gingerol + metformin com-
(Fig. 9A and B). Microscopic images of immunostained bination, fasting blood glucose was markedly lowered by
kidney tissue segments against fibronectin showed no 54.9%, 64.3%, and 74.2%, respectively. Noteworthy, fast-
excess brown staining in the cortex and medulla in the ing blood glucose was significantly lower in the 6-gin-
control group. Meanwhile, excess brown tubular stain- gerol group relative to the metformin group (p < 0.05).
ing was expressed by the DN group in both the cortex 6-Gingerol + metformin combination group revealed a
and medulla, reflecting significantly increased fibronec- significantly decreased fasting blood glucose with regards
tin deposition relative to the control group (p < 0.0001). to 6-gingerol (p < 0.0001) or metformin (p < 0.05) groups.
All treatment groups showed a remarkable reduction in Fasting blood glucose was still significantly elevated in
brown tubular staining in both the cortex and medulla. 6-gingerol (p < 0.0001) and metformin (p < 0.01) groups
6-Gingerol and metformin groups demonstrated sig- relative to control group. Whereas the fasting blood
nificantly decreased fibronectin deposition by 71.1% glucose became normalized in rats receiving the 6-gin-
and 84.8%, respectively, in comparison with the DN gerol + metformin combination.
group. However, 6-gingerol and metformin groups DN group showed a 32% increase in kidney weight/
exhibited a significant elevation in fibronectin deposi- body weight (KW/BW) index relative to the control
tion in comparison with control group (p < 0.0001). The group (p < 0.0001), which reveals marked renal hyper-
6-gingerol + metformin group demonstrated significantly trophy in diabetic rats (Table 2). Administration of
decreased fibronectin deposition when compared with 6-gingerol, metformin, and 6-gingerol + metformin com-
6-gingerol and metformin groups (p < 0.0001) and non- bination to diabetic rats has significantly decreased the
significantly different fibronectin deposition relative to KW/BW index by 22.4%, 22.1%, and 32.4%, respectively.
the control group. The 6-gingerol + metformin combination group exhib-
ited a significantly decreased KW/BW index relative to
6-Gingerol, Metformin, and their combination diminished 6-gingerol (p < 0.01) or metformin (p < 0.05) groups. KW/
renal hypoxia in DN BW index was still significantly elevated in 6-gingerol
As shown in Fig. 8C, the DN group showed a 2.9-fold ele- (p < 0.0001) and metformin (p < 0.001) groups relative to
vation in the mRNA expression of HIF-1α with respect control group whereas it was restored in diabetic rats
to the control group (p < 0.0001). This elevation was sig- treated with the 6-gingerol + metformin combination, i.e.,
nificantly reduced when diabetic rats received 6-gingerol, no significant difference was found when compared to
metformin, or 6-gingerol + metformin combination by normal control rats.
46.4%, 40.4%, and 61%, respectively (p < 0.0001). How-
ever, 6-gingerol and metformin groups still exhibited 6-Gingerol, Metformin, and their combination improved
a significant elevation in HIF-1α mRNA expression in lipid profile in diabetic rats
comparison with control group (p < 0.0001). A marked Our results demonstrated an overall improvement
reduction in HIF-1α mRNA expression was seen in in the lipid profile of diabetic rats receiving 6-gin-
the 6-gingerol + metformin group relative to 6-gin- gerol, metformin, or their combination. As shown in
gerol (p < 0.001) or metformin group (p < 0.0001) and a Table 2, diabetic rats showed 2.6-fold, 2.1-fold, and 5.1-
fold elevation in the levels of serum triglycerides, total
Aboismaiel et al. Biological Research (2024) 57:47 Page 18 of 25

Fig. 10 A summary figure of the mechanisms of action of GR, MET, and their combination in HFD/STZ-induced DN in rats
DN: diabetic nephropathy; GR: 6-gingerol; MET: metformin; HFD: high-fat diet; STZ: streptozotocin; TLR4: Toll-like receptor 4; TRAF6: tumor necrosis factor
receptor-associated Factor 6; NF-κB (p65): nuclear factor-kappa B (p65); NLRP3: NOD-like receptor family pyrin domain-containing 3; TNF-α: tumor ne-
crosis factor-alpha; IL-1β: interleukin-1 beta; ROS: reactive oxygen species; GSH: reduced glutathione; MDA: malondialdehyde; HIF-1α: hypoxia-inducible
factor-1 alpha.

Table 2 6-gingerol (GR), metformin (MET), and GR + MET effects on blood glucose, KW/BW index, and lipid profile
Control DN GR MET GR + MET
Fasting blood glucose (mg/dl) 94.7 ± 4.1 395.4 ± 14.5**** 178.3 ± 8.7****, ####, $$$$, % 141.2 ± 4.2**, ####, $ 102.1 ± 4.6####
Body weight (BW) (g) 261.4 ± 6.2 145.2 ± 8.2**** 189.6 ± 11.2****, #, $ 195.3 ± 12.1***, ##, $ 234.1 ± 7.9####
Kidney weight (KW) (g) 0.64 ± 0.02 0.89 ± 0.02**** 0.72 ± 0.01**,####, $$ 0.71 ± 0.01####, $ 0.66 ± 0.01####
KW/BW index (%) 0.25 ± 0.01 0.63 ± 0.03**** 0.39 ± 0.02****, ####, $$ 0.37 ± 0.02 ***, ####, $ 0.28 ± 0.01####
Triglycerides (mg/dl) 71.2 ± 1.4 184.1 ± 2.9**** 84.6 ± 2.5**, ####, $ 86.7 ± 2.6 ***, ####, $$ 74.5 ± 1.2####
Total cholesterol (mg/dl) 72.6 ± 0.8 155.8 ± 3.2**** 87.1 ± 1.8***, ####, $$ 85.3 ± 1.9***, ####, $ 76.9 ± 1.7####
LDL cholesterol (mg/dl) 18.5 ± 1.8 94.3 ± 2.3**** 37.6 ± 1.9****, ####, $$ 31.3 ± 1.7***, #### 25.9 ± 2.3####
HDL cholesterol (mg/dl) 41.1 ± 1.1 24.4 ± 1.3**** 32.6 ± 2.0**, ## 36.9 ± 1.8#### 36.1 ± 1.2####
High-fat diet/streptozotocin (HFD/STZ)-induced diabetic rats were treated daily for eight weeks with oral gavage of GR (100 mg/kg), MET (300 mg/kg), or their
combination. Values are displayed in the form of means ± SEM (n = 8/group)
** represents a significant difference from the control group at p < 0.01.
*** represents a significant difference from the control group at p < 0.001
**** represents a significant difference from the control group at p < 0.0001
# represents a significant difference from the DN group at p < 0.05
## represents a significant difference from the DN group at p < 0.01
#### represents a significant difference from the DN group at p < 0.0001
$ represents a significant difference from the GR + MET group at p < 0.05
$$ represents a significant difference from the GR + MET group at p < 0.01
$$$$ represents a significant difference from the GR + MET group at p < 0.0001
% represents a significant difference from the MET group at p < 0.05
Aboismaiel et al. Biological Research (2024) 57:47 Page 19 of 25

cholesterol, and LDL cholesterol, respectively, as well previously explored. However, further research is war-
as a 1.7-fold decrease in HDL cholesterol levels in com- ranted to fully understand the underlying mechanisms
parison with control rats. 6-Gingerol, metformin, and and validate these findings in clinical settings.
6-gingerol + metformin combination have significantly The DN model was established experimentally in HFD/
decreased triglycerides, total cholesterol, and LDL cho- STZ-induced diabetic rats. High fat diet and a low dose
lesterol (p < 0.0001) while significantly increased HDL of STZ were used to induce both peripheral insulin resis-
cholesterol levels (p < 0.01), (p < 0.0001), and (p < 0.0001), tance as well as impairment of insulin production and
respectively, with regards to the DN group. In addition, secretion via partial degeneration of β-cells to mimic the
the 6-gingerol + metformin group revealed significantly natural pathophysiology of type 2 diabetes mellitus [43].
decreased triglycerides (p < 0.05), (p < 0.01), and total The success of DN induction in diabetic rats was con-
cholesterol (p < 0.01), (p < 0.05) levels relative to 6-gin- firmed by elevated urinary protein excretion levels in
gerol and metformin groups, respectively. The 6-gin- 24-hr urine samples (30.3 mg ± 1.4) which showed micro-
gerol + metformin group revealed significantly lower LDL albuminuria [55]. Elevation of serum levels of creati-
cholesterol levels with regards to the 6-gingerol group nine and BUN, high proteinuria/creatininuria ratio and
(p < 0.01) and non-significantly lower LDL cholesterol decline of creatinine clearance in diabetic rats as well as
levels with regards to the metformin group. No signifi- histopathological lesions observed in the renal cortex and
cant difference in HDL cholesterol existed in diabetic medulla further confirmed the occurrence of renal injury.
rats treated with the 6-gingerol + metformin combina- These results matched those of an earlier DN model
tion relative to those treated with 6-gingerol or metfor- study conducted in rats by Abou-Hany et al. [56].
min. In comparison with the control group, 6-gingerol When 6-gingerol was administered to diabetic rats, a
and metformin groups showed a significant elevation in considerable improvement in renal function tests and a
serum triglycerides (p < 0.01) and (p < 0.001), total cho- remarkable enhancement in the renal glomerular and
lesterol (p < 0.001), and LDL cholesterol (p < 0.0001) and tubular structure were observed, which further con-
(p < 0.001), respectively. Regarding HDL cholesterol, firms the renoprotective effect of 6-gingerol as previ-
a significant decrease was found in 6-gingerol group ously reported by Almatroodi, et al. [41]. In addition, the
(p < 0.01) and a non-significant decrease was observed in combination of 6-gingerol and metformin attained much
metformin group, in comparison with the control group. improved renal function tests and renal histological pic-
Furthermore, no significant difference was observed ture, which indicates a superior renoprotective effect for
between 6-gingerol + metformin group and control group the combination group. This finding suggests that com-
regarding serum triglycerides, total cholesterol, LDL cho- bining these medications may offer enhanced therapeu-
lesterol and HDL cholesterol. tic benefits for the management of DN. Furthermore, it
raises the possibility of reducing the dosage of metformin
Discussion to minimize potential adverse effects while maintaining
Diabetic nephropathy is a serious complication of dia- or even improving its efficacy.
betes mellitus that involves multiple hemodynamic and Diabetic rats demonstrated marked hyperglycemia and
metabolic changes brought on by hyperglycemia, with a considerable elevation in the KW/BW index and renal
inflammation and consequent fibrosis being the ultimate hypertrophy, which are early signs of DN induction [57,
contributors to renal dysfunction [53]. The currently 58]. Our study revealed that 6-gingerol has a remark-
available oral anti-diabetic treatments have been insuf- able glucose-reducing outcome in diabetic rats, which
ficient to halt DN development and progression to end- appeared to be consistent with the results of Singh, et al.
stage renal disease [54]. This work aimed to investigate [59] in diabetic mice. Moreover, 6-gingerol significantly
the proposed renoprotective effects of the natural com- decreased the KW/BW index. However, fasting blood
pound 6-gingerol in DN and its underlying mechanisms glucose levels and KW/BW index were still significantly
of action. Also, the combination of 6-gingerol and the elevated in the 6-gingerol group relative to the control
standard anti-hyperglycemic drug, metformin, was tested group. Yet, the 6-gingerol and metformin combina-
for potential premium renoprotective effects. To the best tion was capable of restoring fasting blood glucose and
of our knowledge, this is the first study to identify the KW/BW index to normal levels and preventing renal
effect of 6-gingerol on renal expression of miRNA-146a hypertrophy.
and miRNA-223 as well as the downstream inflammatory Dyslipidemia is common in type 2 diabetes mel-
pathway in the context of DN in rats. This study sheds litus, as insulin resistance affects enzymes and path-
light on the potential mechanisms underlying the reno- ways of glucose and lipid metabolism [60]. This study’s
protective effects of 6-gingerol in DN. Additionally, our results showed that in diabetic rats, serum triglyc-
findings reveal the beneficial additive effect of combin- erides, total cholesterol, and LDL cholesterol levels
ing 6-gingerol and metformin in DN, which has not been increased while HDL cholesterol levels decreased. This
Aboismaiel et al. Biological Research (2024) 57:47 Page 20 of 25

was correspondent with the findings of Lecamwasam et induced by NF-κB while miRNA-146a possesses a fine-
al. [61] in diabetics suffering from CKD. Treatment of tuning effect that prevents, via a feedback mechanism,
diabetic rats with 6-gingerol brought on a remarkable the over-activation of NF-κB. This is achieved through
improvement in lipid profile that seemed to agree with the down-regulation of IRAK-1 and TRAF6, the direct
Wang, et al. results in HFD/STZ-induced prediabetic target genes of miRNA-146a, downstream of TLR4 and
mice [62]. Besides, the 6-gingerol + metformin combina- upstream of NF-κB. However, miRNA-146a is down-
tion showed more considerable improvements in lipid regulated in diabetic kidneys, which induces an up-reg-
profile biomarkers. ulation of NF-κB and various inflammatory cytokines
In this context, metformin is known to activate AMP- and subsequent augmented inflammation [70, 71]. Our
activated protein kinase which leads to improved glucose findings revealed the ability of 6-gingerol to up-regulate
uptake, enhanced fatty acid oxidation, and inhibition of miRNA-146a expression in diabetic kidneys. This could
pathways associated with excessive energy consumption, explain its ability to inhibit TRAF6 and, consequently,
such as the mechanistic target of rapamycin (mTOR) sig- NF-κB (p65) expression with subsequent inhibition of the
naling [63, 64]. Studies suggest that 6-gingerol may con- inflammatory cytokines TNF-α and IL-1β.
tribute to glycemic control by reducing blood glucose It is worth mentioning that our findings showed that
level, enhancing insulin sensitivity, increasing glucose 6-gingerol treatment significantly reduced TLR4 expres-
uptake, and improving glucose metabolism. A study by sion, which could be partly explained by miRNA-146a’s
Samad, et al. showed that 6-gingerol potentiated gluca- ability to suppress TLR4 expression. In agreement with
gon-like peptide-1 mediated glucose-stimulated insulin our results, miR-146a was shown by He, et al. [72] and
secretion pathway in pancreatic β-cells and improved Liu, et al. [73] to negatively regulate TLR4 expression in
hyperglycemia in type 2 diabetic rats [65]. By regulating an ovarian dysfunction mouse model and fibroblast-like
glucose homeostasis, 6-gingerol may indirectly mitigate synoviocytes in rheumatoid arthritis patients, respec-
the detrimental effects of hyperglycemia on renal func- tively. Contrarily, Morishita, et al. [74] and Petrkova, et
tion [41, 66]. Thus, the combination of 6-gingerol and al. [75] demonstrated that TLR4 was not suppressed by
metformin may have complementary effects in regulating miRNA-146a expression in unilateral ureteral obstruc-
insulin signaling pathways, leading to enhanced glucose tion-induced renal fibrosis in mice and in patients with
utilization, improved lipid metabolism and better glyce- aortic valve stenosis, respectively. This might partly
mic control. reflect a sort of specific localization and will require fur-
Inflammation is a key contributor to DN develop- ther investigations to find out whether TLR4 could be
ment and progression. Activation of the inflammasome a direct target of miRNA-146a in diabetic kidneys and
complex and subsequent pyroptosis, as well as elevation whether 6-gingerol could inhibit TLR4 expression via
of inflammatory cytokine levels, are hallmarks of the miRNA-146a up-regulation or a different mechanism.
inflammatory process [67]. Our results showed the abil- MiRNA-223 proved to be a direct epigenetic regula-
ity of 6-gingerol to suppress inflammation and pyrop- tor of NLRP3 inflammasome expression in different
tosis in diabetic kidneys and attenuate DN by inhibiting experimental models such as calcium oxalate-induced
TLR4/TRAF6/NLRP3 inflammasome signaling. This was renal inflammation [76] and gouty inflammation [77].
evident by the down-regulation of TLR4, TRAF6, NF-κB Our study revealed a significant down-regulation of
(p65), NLRP3, and caspase-1 mRNA and protein expres- renal miRNA-223 expression in rats with DN. These
sion in addition to the TNF-α and IL-1β level reduction results agreed with those obtained by Yu, et al. [78] in
in the renal tissues of diabetic rats receiving 6-gingerol. HBV-transfected podocytes and by Xu, et al. [79] in dia-
These effects were even more pronounced when diabetic betic cardiomyopathy. In our research, treating diabetic
rats received a combination of 6-gingerol and metfor- rats with 6-gingerol led to a significant induction of
min. Our results agreed with those of earlier studies in miRNA-223 expression, which brought about a remark-
liver injury, myocardial fibrosis, and mastitis experimen- able decline in the levels of NLRP3, caspase-1, and IL-1β.
tal models [38, 68, 69]. In addition, a study by Song, et Noteworthy, the 6-gingerol and metformin combination
al. [42] demonstrated that 6-gingerol improved the con- produced much more considerable up-regulation of both
dition of renal tissue in diabetic rats by alteration of p38 miRNA-146a and miRNA-223 expression.
mitogen activated protein kinase and NF-κB and inhibi- The specific molecular mechanisms underlying the
tion of cyclooxygenase-2, prostaglandin E2 and proin- cooperative effects of 6-gingerol and metformin in miti-
flammatory cytokines. gating DN remain to be fully elucidated. In this con-
Noteworthy, our study declared the involvement of text, both 6-gingerol and metformin have been shown
miRNA-146a in the posttranscriptional regulation of to modulate the activity of transcription factors that
TLR4 signaling in the kidneys of DN-affected rats. Under directly regulate miRNA-146a and miRNA-223 expres-
normal conditions, the expression of miRNA-146a is sion, the epigenetic regulators of the TLR4/TRAF6/
Aboismaiel et al. Biological Research (2024) 57:47 Page 21 of 25

NLRP3 inflammasome pathway. For instance, 6-gingerol have reported further evidence supporting the reno-
has been reported to activate nuclear factor erythroid protective effects of 6-gingerol in different renal injury
2-related factor 2 [37], while metformin can activate models. A study by Tahoun, et al. showed the ability of
peroxisome proliferator-activated receptor gamma [80]. 6-gingerol to inhibit inflammation, oxidative stress and
By independently activating these common transcrip- apoptosis in cisplatin-induced nephrotoxicity via reduc-
tion factors, 6-gingerol and metformin may additively tion of IL-1β, TNF-α, interleukin-6, inducible nitric oxide
enhance the expression of miRNA-146a and miRNA-223 synthase, nitric oxide, MDA and caspase-3 in renal tissue
[81, 82]. Furthermore, 6-gingerol and metformin pos- [96]. Also, 6-gingerol was found to protect against genta-
sess complementary anti-inflammatory properties and micin-induced renal cortex apoptosis and oxidative stress
can modulate inflammatory signaling pathways, which in rats via inhibition of caspase-3 and anti-heat shock
are closely linked to miRNA regulation. Both compounds protein 47 [97].
have been shown to inhibit NF-κB signaling, which plays Renal fibrosis is an important pathological feature of
a key role in regulation of miRNA-146a expression [83, diabetic kidneys and represents the final common path-
84]. Additionally, they can suppress pro-inflammatory way in the progression of DN to end-stage renal disease.
cytokines, such as interleukin-6 [42, 85], which can Hyperglycemia-induced metabolic alterations trigger a
indirectly influence miRNA-223 expression [86]. Thus, state of chronic inflammation, which causes persistent
6-gingerol and metformin may cooperatively induce the injury in diabetic kidneys. This promotes various patho-
expression of miRNA-146a and miRNA-223 and thus logical changes, including epithelial-to-mesenchymal
additively inhibit TLR4/TRAF6/NLRP3 inflammasome transition, endothelial-to-mesenchymal transition, and
signaling and subsequent renal damage. activation of fibroblasts and pericytes. These pathological
Diabetes and its complications are greatly influenced changes cause the extracellular matrix components col-
by oxidative stress. Hyperglycemia induces excessive ROS lagen and fibronectin to be deposited in excess, resulting
production, resulting in an imbalance between free radi- in kidney fibrosis [98, 99].
cals and antioxidant defense mechanisms. Lipid peroxi- Hypoxia is an important driving factor for DN and
dation occurs as a result of the interactions between ROS CKD. Excessive oxygen consumption brought on by
and polyunsaturated fatty acids, which plays an impor- diabetes-related metabolic changes causes renal tissue
tant role in diabetes-associated complications, including hypoxia and increased expression of HIF-1α; the cru-
renal injury [87–90]. Our results revealed a high oxida- cial transcriptional regulator of cellular accommodation
tive status in diabetic rats. This was obvious through a with hypoxia [100]. A major crosstalk was found between
considerable reduction in the antioxidant GSH level in hypoxia and inflammation. Previous studies showed that
renal tissue and a remarkable elevation in the reactive activation of NF-κB increased the expression of HIF-1α.
aldehyde MDA levels in the renal tissue, serum and urine. Furthermore, HIF-1α was required for hypoxia-pro-
Both Abou-Hany, et al. [56] and Mi, et al. [91] reported moted TLR4 expression and downstream NF-κB tran-
comparable outcomes in DN models. 6-Gingerol treat- scriptional activation as well [19, 101–103]. In addition,
ment of diabetic rats increased GSH levels, reduced previous studies have shown that increased expression of
MDA levels, and alleviated oxidative stress, possibly due HIF-1α in diabetic kidneys contributed to renal fibrosis
to its well-recognized antioxidant capacity [38, 92]. and the progression of DN [21, 104, 105].
Additionally, the antioxidant potential of 6-gingerol The results of our study revealed significant renal
was further significantly enhanced when metformin was fibrosis in untreated diabetic rats, evidenced by the sig-
used in combination with 6-gingerol. The combination nificantly increased collagen fibril deposition in Mas-
of 6-gingerol and metformin seemed to have a comple- son-stained kidney tissue segments. This was further
mentary effect in reducing oxidative stress and protect- confirmed by the elevated expression of the fibrosis hall-
ing cells from oxidative damage owing to the antioxidant mark protein, fibronectin, in both cortex and medulla of
properties of both compounds. Metformin can reduce immunostained renal tissue from diabetic rats. Addition-
oxidative stress by inhibiting ROS production through ally, our study revealed a significant elevation of renal
AMP-activated protein kinase activation, mitochondrial HIF-1α expression in diabetic rats. These results were in
complex I inhibition, and increased antioxidant enzyme line with those recorded both in vitro and in vivo by Mei,
activity [63, 93]. Whereas 6-gingerol exhibits antioxidant et al. [105], who discovered that elevated HIF-1α expres-
activity via ROS scavenging, nuclear factor erythroid sion and susceptibility to fibrosis in diabetes are signifi-
2-related factor 2 up-regulation, improving the activities cantly correlated.
of antioxidant enzymes catalase, superoxide dismutase, As indicated by our results, 6-gingerol significantly
glutathione peroxidase, and glutathione S-transferase as ameliorated renal fibrosis in diabetic rats possibly as a
well as GSH level and reducing the level of MDA [37, 41, result of its anti-inflammatory and antioxidant capabili-
94, 95]. In addition to these mechanisms, other studies ties. Also, 6-gingerol was able to reduce renal hypoxia
Aboismaiel et al. Biological Research (2024) 57:47 Page 22 of 25

and HIF-1α expression significantly, which could contrib- 6-gingerol and metformin in treatment guidelines and
ute to its anti-fibrotic effect. Moreover, the anti-fibrotic clinical practice guidelines for DN. While the findings of
as well as anti-hypoxic capacities of 6-gingerol were fur- the study are promising, the translation of these findings
ther significantly enhanced when metformin was used into clinical practice requires careful evaluation of safety,
in combination with 6-gingerol suggesting that 6-gin- efficacy, optimal dosing, long term benefits, potential
gerol and metformin may have complementary effects drug interactions, and individual patient factors.
in mitigating hypoxia and fibrosis in DN. 6-Gingerol has Future studies should focus on exploring different dos-
been reported to suppress the expression of HIF-1α and ages and dosing regimens of 6-gingerol and metformin to
reduce hypoxia in lung cancer [106]. 6-Gingerol has been determine the optimal therapeutic dose that achieves the
shown to inhibit renal fibrosis through various mecha- highest efficacy and safety. Conducting long-term studies
nisms, including the suppression of transforming growth would provide valuable insights into the long-term effects
factor-beta 1 signaling and the inhibition of fibroblast and safety profile of the combination therapy. Further
activation and extracellular matrix deposition [42]. Met- mechanistic studies should be performed to deepen our
formin treatment was reported to relieve the processes understanding of the underlying mechanisms by which
of inflammation and fibrosis in individuals with diabetic 6-gingerol and metformin confer renoprotective effects.
kidney disease by reducing the levels of the Tenascin- Clinical trials involving human subjects could be con-
C, p-NF-κB (p65), connective tissue growth factor, and ducted to assess the impact of the combination therapy
fibronectin proteins [83]. Together, these actions may on renal function, glycemic control, inflammation, oxi-
contribute to the reversal of hypoxia and fibrosis in the dative stress, and overall patient outcomes. Comparative
kidney. studies comparing the combination therapy of 6-gingerol
Overall, the findings of this study suggest that 6-gin- and metformin with other existing standard treatments
gerol is promising for the prevention of DN. Our study or emerging therapies for DN could be performed which
showed that 6-gingerol exerted a significant renoprotec- could help guide treatment decisions and determine the
tive effect through multiple mechanisms including inhi- feasibility of 6-gingerol and metformin combination
bition of inflammation via modulation of miRNA-146a, therapy in the management of DN.
miRNA-223 and TLR4/NF-κB/NLRP3 inflammasome
pathway, reduction of oxidative stress, hypoxia, and fibro- Conclusion
sis as well as glycemic control. Being a natural compound, This study disclosed that 6-gingerol is a promising natu-
the renoprotective effects of 6-gingerol, as demonstrated ral compound for the prevention of DN. Inhibition of
in the study, make it an attractive option for patients inflammation, oxidative stress, hypoxia, and fibrosis are
seeking natural alternative therapies or complementary the key mechanisms in 6-gingerol’s renoprotective effect,
therapies alongside conventional pharmacological treat- which proved to be related to the induction of renal
ments. The combination therapy of 6-gingerol and met- expression of miRNA-146a and miRNA-223 and subse-
formin provides superior renoprotection compared to quent inhibition of the TLR4/TRAF6/NLRP3 inflamma-
monotherapy which demonstrates significant potential some pathway. The renoprotective effect of 6-gingerol
for clinical application. The combination therapy has the appeared to be comparable to that of the standard anti-
advantage of addressing multiple mechanisms involved hyperglycemic drug, metformin, in HFD/STZ-induced
in the development and progression of DN by targeting diabetic rats. Moreover, the 6-gingerol and metformin
both metabolic and renal-related pathways. This multi- combination revealed superior renoprotection compared
mechanistic strategy has the potential to yield improved to the use of each drug alone which suggests the potential
outcomes in the management of DN. Another advantage to lower the dosage of metformin to minimize adverse
of the combination therapy is the potential to reduce the effects while still maintaining or even improving its effec-
dosage of metformin while maintaining or enhancing tiveness (Fig. 10). However, further research is needed to
its effectiveness. By lowering the metformin dosage, the determine the optimal dosage and evaluate the balance
adverse effects associated with it, such as gastrointestinal between efficacy and safety when using reduced doses
symptoms, may be minimized, leading to better tolerabil- of metformin in combination with 6-gingerol. Future
ity and adherence to treatment among DN patients. studies should focus on elucidating further underlying
However, it’s important to note that the study was mechanisms and conducting clinical trials to evaluate
conducted in an animal model of DN. Further research, the safety and effectiveness of this drug combination in
particularly in the form of clinical trials involving human patients with DN.
subjects, is essential to validate these findings and
Acknowledgements
assess their applicability to clinical practice. Replicat- Not applicable.
ing the results in clinical studies would provide stron-
ger evidence for considering the combination therapy of
Aboismaiel et al. Biological Research (2024) 57:47 Page 23 of 25

Authors contributions 12. Liu P, Zhang Z, Li Y. Relevance of the pyroptosis-related Inflammasome Path-
MGA contributed to the conceptualization and methodology of the study, way in the Pathogenesis of Diabetic kidney disease. Frontiers in Immunology;
data collection, analysis and interpretation of data, and writing and editing 2021. p. 12.
the original draft of the manuscript. MNA: contributed to data collection, 13. Tang SCW, Yiu WH. Innate immunity in diabetic kidney disease. Nat Rev
analysis, and interpretation of data, and editing and revising the manuscript. Nephrol. 2020;16(4):206–22.
LAE: contributed to the conceptualization of the study, editing, and revising 14. Amarante-Mendes GP, et al. Pattern Recognition Receptors and the Host Cell
the manuscript, and study supervision. All authors read and approved the final Death Molecular Machinery. Frontiers in Immunology; 2018. p. 9.
version of the manuscript. 15. Su Q, et al. Effects of the TLR4/Myd88/NF-κB signaling pathway on NLRP3
inflammasome in Coronary Microembolization-Induced Myocardial Injury.
Funding Cell Physiol Biochem. 2018;47(4):1497–508.
This research did not receive any specific grant from funding agencies in the 16. De Nardo D, et al. Interleukin-1 receptor-associated kinase 4 (IRAK4) plays
public, commercial, or not-for-profit sectors. a dual role in myddosome formation and toll-like receptor signaling. J Biol
Open access funding provided by The Science, Technology & Innovation Chem. 2018;293(39):15195–207.
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank 17. McKee CM, Coll RC. NLRP3 inflammasome priming: a riddle wrapped in a
(EKB). mystery inside an enigma. J Leukoc Biol. 2020;108(3):937–52.
18. Chen MY, et al. The signaling pathways regulating NLRP3 inflammasome
Data availability activation. Inflammation. 2021;44(4):1229–45.
Data will be made available from the corresponding author upon reasonable 19. D’Ignazio L, Bandarra D, Rocha S. NF-κB and HIF crosstalk in immune
request. responses. FEBS J. 2016;283(3):413–24.
20. Stanigut AM et al. Hypoxia-inducible factors and Diabetic kidney Disease-
How Deep can we go? Int J Mol Sci, 2022. 23(18).
Declarations 21. Nayak BK, et al. HIF-1 mediates renal fibrosis in OVE26 type 1 diabetic mice.
Diabetes. 2016;65:1387–97.
Ethics approval and consent to participate 22. Ratti M, et al. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs)
This research gained approval from the ethics committee of the Faculty of as New Tools for Cancer Therapy: first steps from Bench to Bedside. Target
Pharmacy at Mansoura University in Mansoura, Egypt (Ethical approval no. Oncol. 2020;15(3):261–78.
2023 − 159). “Principles of Laboratory Animal Care” (National Materials Institute 23. Cheng L, et al. MicroRNA-122-5p ameliorates tubular injury in diabetic
of Health publication No. 85 − 23, revised 1985) were followed in all animal nephropathy via FIH-1/HIF-1α pathway. Ren Fail. 2022;44(1):293–303.
experiments. 24. Mao R, Shen J, Hu X. BMSCs-derived exosomal microRNA-let-7a plays a
protective role in diabetic nephropathy via inhibition of USP22 expression.
Consent for publication Life Sci. 2021;268:118937.
Not applicable. 25. Hao Y, et al. Mesenchymal stem cell-derived exosomes carry MicroRNA-125a
to protect against Diabetic Nephropathy by Targeting Histone Deacetylase
Competing interests 1 and downregulating Endothelin-1. Diabetes Metabolic Syndrome Obesity:
The authors declare that they have no competing interests. Targets Therapy. 2021;14:1405–18.
26. Fan W et al. MicroRNA-146a is a wide-reaching Neuroinflammatory Regulator
Author details and potential treatment target in neurological diseases. Front Mol Neurosci,
1
Department of Biochemistry, Faculty of Pharmacy, Mansoura University, 2020. 13.
Mansoura 35516, Egypt 27. Liu G-J, et al. MiR-146a ameliorates Hemoglobin-Induced Microglial Inflam-
matory Response via TLR4/IRAK1/TRAF6 Associated pathways. Front Neuro-
Received: 14 October 2023 / Accepted: 29 June 2024 Sci. 2020;14:311–311.
28. Xu W, et al. MicroRNA-223-3p inhibits oxidized low-density lipoprotein-
mediated NLRP3 inflammasome activation via directly targeting NLRP3 and
FOXO3. Clin Hemorheol Microcirc. 2022;81:241–53.
29. Zhou T, et al. A preclinical overview of metformin for the treatment of type 2
References diabetes. Volume 106. Biomedicine & Pharmacotherapy; 2018. pp. 1227–35.
1. Wei J et al. The Influence of Different Types of Diabetes on Vascular Complica- 30. Neven E, et al. Metformin prevents the development of severe chronic
tions Journal of Diabetes Research, 2022. 2022: p. 3448618. kidney disease and its associated mineral and bone disorder. Kidney Int.
2. Reed J, Bain S, Kanamarlapudi V. A review of current trends with type 2 2018;94(1):102–13.
diabetes epidemiology, Aetiology, Pathogenesis, treatments and future 31. Ren H, et al. Metformin alleviates oxidative stress and enhances autophagy in
perspectives. Diabetes Metab Syndr Obes. 2021;14:3567–602. diabetic kidney disease via AMPK/SIRT1-FoxO1 pathway. Mol Cell Endocrinol.
3. Poloni JAT, Rotta LNJJoL, Medicine P. Diabetic kidney disease: pathophysiologi- 2020;500:110628.
cal changes and urinalysis contribution to diagnosis—a narrative review 2022, 32. Yu T, et al. The treatment effectiveness evaluation for slowing the Progression
2022. 7. of Diabetic Nephropathy during Stage 4 chronic kidney disease. Diabetes
4. Lin Y-C, et al. Update of pathophysiology and management of diabetic Therapy: Res Treat Educ Diabetes Relat Disorders. 2021;12(1):301–12.
kidney disease. J Formos Med Assoc. 2018;117(8):662–75. 33. Spiller HA, Sawyer TS. Toxicology of oral antidiabetic medications. Am J
5. Sierra-Mondragon E, et al. All-trans retinoic acid ameliorates inflammatory Health Syst Pharm. 2006;63(10):929–38.
response mediated by TLR4/NF-κB during initiation of diabetic nephropathy. 34. Yang X et al. Clinical efficacy and safety of Chinese herbal medicine for the
J Nutr Biochem. 2018;60:47–60. treatment of patients with early diabetic nephropathy: a protocol for system-
6. Hoogeveen EK. Epidemiol Diabet Kidney Disease. 2022;2(3):433–42. atic review and meta-analysis. Medicine, 2020. 99(29).
7. Nagib SN, et al. What is the prevalence of chronic kidney disease among 35. Sun C, et al. Anti-diabetic effects of natural antioxidants from fruits. Trends
hypertensive non-diabetic Egyptian patients attending primary healthcare? Food Sci Technol. 2021;117:3–14.
Clin Exp Hypertens. 2023;45(1):2203411. 36. Wu X, et al. Autophagy and cardiac diseases: therapeutic potential of natural
8. Global, regional, and national burden of chronic kidney disease, 1990–2017:a products. Med Res Rev. 2021;41(1):314–41.
systematic analysis for the Global Burden of Disease Study 2017. Lancet. 37. Mao Q-Q, et al. Bioactive compounds and bioactivities of Ginger (Zingiber
2020;395(10225):709–33. officinale Roscoe). Foods (Basel Switzerland). 2019;8(6):185.
9. Farag YMK, El-Sayed E. Global Dialysis Perspective: Egypt. 38. Han X, et al. [6]-Gingerol ameliorates ISO‐Induced Myocardial Fibrosis by
2022;Kidney360(7):1263–8. reducing oxidative stress, inflammation, and apoptosis through inhibition of
10. Hegazi R, et al. Epidemiology of and risk factors for type 2 diabetes in Egypt. TLR4/MAPKs/NF‐κB pathway. Volume 64. Molecular nutrition & food research;
Ann Glob Health. 2015;81(6):814–20. 2020. p. 2000003. 13.
11. Hassaballa M et al. Egyptian renal data system (ERDS) 2020: an annual report of 39. Shen CL, et al. Ginger alleviates mechanical hypersensitivity and anxio-
end-stage kidney disease patients on regular hemodialysis 2022. 22(1): pp. 1–28. depressive behavior in rats with diabetic neuropathy through beneficial
Aboismaiel et al. Biological Research (2024) 57:47 Page 24 of 25

actions on gut microbiome composition, mitochondria, and neuroimmune 65. Samad MB, et al. [6]-Gingerol, from Zingiber officinale, potentiates GLP-1
cells of colon and spinal cord. Nutr Res. 2024;124:73–84. mediated glucose-stimulated insulin secretion pathway in pancreatic β-cells
40. Gunawan S, et al. 6-gingerol ameliorates weight gain and insulin resistance and increases RAB8/RAB10-regulated membrane presentation of GLUT4
in metabolic syndrome rats by regulating adipocytokines. Saudi Pharm J. transporters in skeletal muscle to improve hyperglycemia in Lepr(db/db)
2023;31(3):351–8. type 2 diabetic mice. BMC Complement Altern Med. 2017;17(1):395.
41. Almatroodi SA et al. 6-Gingerol, a bioactive compound of Ginger attenuates 66. Alharbi KS et al. Gingerol, a natural antioxidant, attenuates hyperglycemia
renal damage in Streptozotocin-Induced Diabetic rats by regulating the and downstream complications. Metabolites, 2022. 12(12).
oxidative stress and inflammation. Pharmaceutics, 2021. 13(3). 67. Sahakyan G, Vejux A, Sahakyan N. The role of oxidative stress-mediated
42. Song S, Dang M, Kumar M. Anti-inflammatory and renal protective effect of inflammation in the Development of T2DM-Induced Diabetic Nephropathy:
gingerol in high-fat diet/streptozotocin-induced diabetic rats via inflamma- possible preventive action of tannins and other Oligomeric Polyphenols.
tory mechanism. Inflammopharmacology. 2019;27(6):1243–54. Molecules, 2022. 27(24).
43. Srinivasan K, et al. Combination of high-fat diet-fed and low-dose 68. Zahoor A, et al. 6-Gingerol exerts anti-inflammatory effects and protective
streptozotocin-treated rat: a model for type 2 diabetes and pharmacological properties on LTA-induced mastitis. Phytomedicine. 2020;76:153248.
screening. Pharmacol Res. 2005;52(4):313–20. 69. Hong M-K, et al. 6-Gingerol ameliorates sepsis-induced liver injury through
44. Eraky SM, Abdel-Rahman N, Eissa LA. Modulating effects of omega-3 fatty acids the Nrf2 pathway. Int Immunopharmacol. 2020;80:106196.
and pioglitazone combination on insulin resistance through toll-like receptor 4 in 70. Fan B, et al. Treatment of diabetic peripheral neuropathy with engineered
type 2 diabetes mellitus Prostaglandins, Leukotrienes and Essential Fatty Acids, mesenchymal stromal cell-derived exosomes enriched with microRNA-146a
2018. 136: pp. 123–129. provide amplified therapeutic efficacy. Exp Neurol. 2021;341:113694.
45. Samra YA, et al. Cepharanthine and Piperine ameliorate diabetic nephropathy 71. Chen S, et al. miR-146a regulates glucose induced upregulation of inflam-
in rats: role of NF-κB and NLRP3 inflammasome. Life Sci. 2016;157:187–99. matory cytokines extracellular matrix proteins in the retina and kidney in
46. Zhai L, et al. Metformin ameliorates podocyte damage by restoring renal diabetes. PLoS ONE. 2017;12(3):e0173918–0173918.
tissue podocalyxin expression in type 2 Diabetic rats. J Diabetes Res. 72. He F, et al. MicroRNA-146 attenuates lipopolysaccharide induced ovarian
2015;2015:231825. dysfunction by inhibiting the TLR4/NF- κB signaling pathway. Bioengineered.
47. Patlolla AK, et al. Toxicity evaluation of Graphene Oxide in kidneys of 2022;13(5):11611–23.
Sprague-Dawley rats. Int J Environ Res Public Health. 2016;13(4):380. 73. Liu W, et al. MicroRNA-146a suppresses rheumatoid arthritis fibroblast-like
48. Bradford MM. A rapid and sensitive method for the quantitation of micro- synoviocytes proliferation and inflammatory responses by inhibiting the
gram quantities of protein utilizing the principle of protein-dye binding. Anal TLR4/NF-kB signaling. Oncotarget. 2018;9(35):23944–59.
Biochem. 1976;72(1):248–54. 74. Morishita Y, et al. Delivery of microRNA-146a with polyethylenimine nanopar-
49. Feldman AT, Wolfe D. Tissue processing and hematoxylin and eosin staining. ticles inhibits renal fibrosis in vivo. Int J Nanomed. 2015;10:3475–88.
Methods Mol Biol. 2014;1180:31–43. 75. Petrkova J et al. Increased expression of miR-146a in valvular tissue from
50. Yamate J, et al. Immunohistochemical observations on the kinetics of patients with aortic valve stenosis. Front Cardiovasc Med, 2019. 6.
macrophages and myofibroblasts in rat renal interstitial fibrosis induced by 76. Lv P, et al. XIST Inhibition attenuates Calcium Oxalate Nephrocalcinosis-
cis-diamminedichloroplatinum. J Comp Pathol. 1995;112(1):27–39. Induced renal inflammation and oxidative Injury via the miR-223/NLRP3
51. Mohsin Alvi A et al. Post-treatment of Synthetic Polyphenolic 1,3,4 oxadiazole pathway. Oxidative Med Cell Longev. 2021;2021:p1676152.
compound A3, attenuated Ischemic Stroke-Induced Neuroinflammation and 77. Zhang Q-B et al. MicroRNA-223 suppresses IL-1β and TNF-α production in
Neurodegeneration. Biomolecules, 2020. 10(6). gouty inflammation by targeting the NLRP3 inflammasome. Front Pharmacol,
52. Zakzook F, et al. Thymoquinone attenuates dimethoate induced hepatic 2021. 12.
and testicular genotoxicity in rats %J. Kafrelsheikh Veterinary Med J. 78. Yu Y, et al. MicroRNA-223 downregulation promotes HBx-induced
2020;18(2):25–32. podocyte pyroptosis by targeting the NLRP3 inflammasome. Arch Virol.
53. Wu T, et al. The mechanism of Hyperglycemia-Induced Renal Cell Injury 2022;167(9):1841–54.
in Diabetic Nephropathy Disease: an update. Life. 2023;13. https://doi. 79. Xu D, et al. Inhibition of miR-223 attenuates the NLRP3 inflammasome
org/10.3390/life13020539 activation, fibrosis, and apoptosis in diabetic cardiomyopathy. Life Sci.
54. Aziz S, et al. Can newer anti-diabetic therapies Delay the Development of 2020;256:117980.
Diabetic Nephropathy? J Pharm Bioallied Sci. 2022;13:341. 80. Ljubicic V, Jasmin BJ. Metformin increases peroxisome proliferator-activated
55. Bazzano T, et al. Renal biomarkers of male and female Wistar rats (Rattus receptor γ Co-activator-1α and utrophin a expression in dystrophic skeletal
norvegicus) undergoing renal ischemia and reperfusion. Acta Cir Bras. muscle. Muscle Nerve. 2015;52(1):139–42.
2015;30(4):277–88. 81. Rasoulinejad SA, Akbari A, Nasiri K. Interaction of miR-146a-5p with oxidative
56. Abou-Hany HO, et al. Crocin mediated amelioration of oxidative burden stress and inflammation in complications of type 2 diabetes mellitus in
and inflammatory cascade suppresses diabetic nephropathy progression in male rats: anti-oxidant and anti-inflammatory protection strategies in type 2
diabetic rats. Chemico-Biol Interact. 2018;284:90–100. diabetic retinopathy. Iran J Basic Med Sci. 2021;24(8):1078–86.
57. Guo L, et al. Nephroprotective Effect of Adropinin Against Streptozotocin- 82. Gu J, et al. MiR-223 as a Regulator and Therapeutic Target in Liver diseases.
Induced Diabetic Nephropathy in rats: inflammatory mechanism and YAP/ Front Immunol. 2022;13:860661.
TAZ factor. Drug Des Devel Ther. 2021;15:589–600. 83. Zhou Y, et al. Metformin regulates inflammation and fibrosis in diabetic kid-
58. Li X et al. Involvement of histone lysine methylation in p21 gene expression in rat ney disease through TNC/TLR4/NF-κB/miR-155-5p inflammatory loop. World
kidney in vivo and rat mesangial cells in vitro under diabetic conditions Journal J Diabetes. 2021;12(1):19–46.
of Diabetes Research, 2016. 2016. 84. Saedisomeolia A et al. Mechanisms of action of Ginger in Nuclear factor-
59. Singh A et al. Anti-hyperglycaemic, lipid lowering and anti-oxidant properties kappab signaling pathways in diabetes. J Herb Med, 2018. 16.
of [6]-gingerol in db/db mice. Int J Med Med Sci, 2009. 1. 85. Hyun B, et al. Metformin Down-regulates TNF-α secretion via suppression of
60. Li M, et al. Trends in insulin resistance: insights into mechanisms and thera- Scavenger receptors in Macrophages. Immune Netw. 2013;13(4):123–32.
peutic strategy. Signal Transduct Target Therapy. 2022;7(1):216. 86. Chen Q, et al. Inducible microRNA-223 down-regulation promotes TLR-
61. Lecamwasam A, et al. Blood plasma metabolites in Diabetes-Associated triggered IL-6 and IL-1?? Production in macrophages by targeting STAT3. PLoS
chronic kidney disease: a focus on lipid profiles and Cardiovascular Risk. Front ONE. 2012;7:e42971.
Nutr. 2022;9:821209. 87. Augustine J et al. The role of Lipoxidation in the Pathogenesis of Diabetic
62. Wang K, et al. The positive effect of 6-Gingerol on High-Fat Diet and Retinopathy. Front Endocrinol, 2021. 11.
Streptozotocin-Induced Prediabetic mice: potential pathways and underlying 88. Bhatti JS, et al. Oxidative stress in the pathophysiology of type 2 diabetes and
mechanisms. Nutrients. 2023;15. https://doi.org/10.3390/nu15040824 related complications: current therapeutics strategies and future perspec-
63. Bu Y, et al. Protective effects of metformin in various cardiovascular diseases: tives. Free Radic Biol Med. 2022;184:114–34.
clinical evidence and AMPK-dependent mechanisms. J Cell Mol Med. 89. Mansoor G et al. Increased expression of circulating stress markers, inflamma-
2022;26(19):4886–903. tory cytokines and decreased antioxidant level in Diabetic Nephropathy. Med
64. Agius L, Ford BE, Chachra SS. The Metformin Mechanism on Gluconeogenesis (Kaunas), 2022. 58(11).
and AMPK Activation: The Metabolite Perspective 2020. 21(9): p. 3240. 90. Vodošek Hojs N et al. Oxidative stress markers in chronic kidney disease with
emphasis on Diabetic Nephropathy. Antioxid (Basel), 2020. 9(10).
Aboismaiel et al. Biological Research (2024) 57:47 Page 25 of 25

91. Mi W, et al. Analysis of the Renal Protection and antioxidative stress effects of 99. Zheng W, Guo J, Liu Z-S. Effects of metabolic memory on inflammation and
Panax notoginseng Saponins in Diabetic Nephropathy mice. J Immunol Res. fibrosis associated with diabetic kidney disease: an epigenetic perspective.
2022;2022:1–9. Clin Epigenetics. 2021;13(1):87–87.
92. Hong W, et al. 6-Gingerol attenuates ventilator-induced lung injury via 100. Takiyama Y, Haneda M. Hypoxia in diabetic kidneys BioMed research interna-
anti-inflammation and antioxidative stress by modulating the PPARγ/ tional, 2014. 2014: pp. 837421–837421.
NF-κBsignalling pathway in rats. Int Immunopharmacol. 2021;92:107367. 101. van Uden P, Kenneth NS, Rocha S. Regulation of hypoxia-inducible factor-
93. Vial G, Detaille D, Guigas B. Role of Mitochondria in the mechanism(s) of 1alpha by NF-kappaB. Biochem J. 2008;412(3):477–84.
action of Metformin. Front Endocrinol, 2019. 10. 102. van Uden P, et al. Evolutionary conserved regulation of HIF-1β by NF-κB. PLoS
94. Saiah W et al. Antioxidant and gastroprotective actions of butanol fraction of Genet. 2011;7(1):e1001285–1001285.
Zingiber officinale against diclofenac sodium-induced gastric damage in rats. 103. Zhao B et al. TLR4 Agonist and Hypoxia Synergistically Promote the Formation of
2018. 42(1): p. e12456. TLR4/NF-κB/HIF-1α Loop in Human Epithelial Ovarian Cancer Anal Cell Pathol
95. Abolaji AO, et al. Protective properties of 6-gingerol-rich fraction from (Amst), 2022. 2022: p. 4201262.
Zingiber officinale (ginger) on chlorpyrifos-induced oxidative damage and 104. Bessho R, et al. Hypoxia-inducible factor-1α is the therapeutic target of the
inflammation in the brain, ovary and uterus of rats. Chem Biol Interact. SGLT2 inhibitor for diabetic nephropathy. Sci Rep. 2019;9(1):14754–14754.
2017;270:15–23. 105. Mei S, et al. Susceptibility of renal fibrosis in diabetes: role of hypoxia induc-
96. Tahoun E, Elgedawy G, El-Bahrawy A. Cytoprotective effect of ginger extract ible factor-1. FASEB J. 2022;36(8):e22477.
on cisplatin-induced hepatorenal toxicity in rats via modulation of oxidative 106. Kim MJ et al. Reduced HIF-1α Stability Induced by 6-Gingerol inhibits Lung
stress, inflammation and apoptosis: histopathological, biochemical and Cancer Growth through the induction of cell death. Molecules, 2022. 27(7).
immunohistochemical study. Comp Clin Pathol. 2021;30(4):647–63.
97. Hegazy AMS, et al. 6-gingerol ameliorates gentamicin induced renal
cortex oxidative stress and apoptosis in adult male albino rats. Tissue Cell.
2016;48(3):208–16. Publisher’s Note
98. Zhang Y et al. Signaling pathways involved in Diabetic Renal Fibrosis. Front Springer Nature remains neutral with regard to jurisdictional claims in
Cell Dev Biology, 2021. 9. published maps and institutional affiliations.