https://orcid.org/0000-0001-7703-9307
Abstract
Hyperglycemia-induced SIRT1, DNMT1, SODs, as well as oxidative stress, play a pivotal role in the progression of diabetic nephropathy. Cissus quadrangularis, holds antioxidant and hypoglycemic activity; however, a direct link between its activity and prevention of diabetic nephropathy has not been ascertained yet. Accordingly, we aimed to delineate the protective effect of ethanolic extract of Cissus quadrangularis (EECQ) against high-fat diet/streptozotocin (HFD/STZ) induced diabetic nephropathy rats.
The control group was fed with a normal chow diet. Rats kept on an HFD for 12 weeks with a single low dose of STZ manifested the features of diabetic nephropathy. The treatment was done by the oral administration of EECQ (200 mg/kg) for six weeks (six rats in each group).
Treatment with EECQ demonstrated substantial attenuation of elevated insulin resistance, lipid profile and creatinine level. Additionally, EECQ restored albuminuria, glomerular filtration rate and creatinine clearance in diabetic nephropathy rats. Furthermore, HFD consumption in rats culminated in reduced SIRT1 and enhanced DNMT1 expression, nonetheless, rescued by EECQ. Moreover, EECQ augmented the SOD 1 and 3 levels, thereby safeguarded from oxidative damage and renal inflammation. Besides, treatment protected from renal fibrosis by downregulating TGFβ, Smad2/3 and col1/3 expression in diseased rats.
Thus, based on the above findings, we conclude that EECQ shows a protective effect against diabetic nephropathy.
Introduction
Diabetes mellitus prevalence has attained alarming proportions and continues to rise rapidly throughout the globe.[1] Diabetic nephropathy (DN) influences about 40% of all people with diabetes and the incidence is as high as 382 million since 2013, representing 8–10% of the worldwide population.[2] It is a chronic disease that develops over many years and may culminate in end-stage renal disease and renal replacement therapy.[3] Generally, it is caused by continuous exposure to high levels of glucose, as demonstrated by large-scale prospective studies of diabetic patients.[4, 5]
SIRT1, a histone deacetylase 3 involved in numerous physiological and pathological processes, including energy homeostasis and glucose metabolism.[6] It plays an instrumental role in renal protection by countering cellular stress, tubular and glomerular inflammation, and apoptosis and controlling renal lipid metabolism.[7] Increased formation of advanced glycation end products (AGEs) results from diabetic conditions substantially fosters the development and progression of DN[8] by declining the expression of SIRT1.[9] Additionally, SIRT1 controls DNA methylation by modulating the expression of DNA methyltransferase 1 (DNMT1),[10, 11] an epigenetic modulator that exacerbates ROS generation and oxidative stress through facilitating DNA methylation of the antioxidant superoxide dismutase (SOD) genes, thereby reducing its expression.[12] Subsequently, the elevated ROS and oxidative stress predispose the tissues to fibrosis by overexpressing TGF-β,[13] which encourages the progression of diabetic nephropathy.
In recent years, the dependence of diabetic patients on plant-based medication therapy has gradually increased. Besides, it has been reported that about two-thirds of diabetic patients depend on their treatment on plant-based medicine.[14, 15]Cissus quadrangularis flourish mainly in various countries' tropical regions, including India, Sri Lanka, West Africa, Malaysia and Java, and are extensively used as medicinal plants in India.[16] Phytochemical analysis has revealed the presence of several compounds like flavonoids, phytosterols and stilbenes.[17] This plant contains nutritional supplement like carbohydrates, fat, protein, fibre and ash.[18] The stem of C. quadrangularis has remarkable nutritional as well as pharmacological benefits. It helps the skeletal system healthier.[19] It is also used as nutraceutical food (bakery) items.[20] Several studies have asserted its effectiveness in diverse diseases like bone fracture, gastric ulcer and hepatic toxicity.[21, 22] Furthermore, the ethanolic extract of Cissus quadrangularis (EECQ) possesses hypoglycemic and antioxidant activity.[23] Accordingly, this study was aimed to determine the protective effect of EECQ against renal fibrosis, dysfunction and inflammation in diabetic nephropathy rats by altering the SIRT1/DNMT1 pathway.
Method
Cissus quadrangularis extract
The C. quadrangularis stem bark was purchased from Haridass Aggarwal & Sons, Navi Mumbai, and the Botany Division, CSIR-CDRI, Lucknow, carried out its authentication with vide voucher specimen number 25283. The powdered stem bark extraction was prepared and then characterized and quantified by LC-MS/MS, API-QTRAP 4000 (Applied Biosystems Sciex, Toronto, Canada). The aforementioned analysis has been published.[24]
Animal study
Eighteen male Sprague Dawley rats (180–200 g) were received from the Laboratory Animal Division, CSIR–CDRI, Lucknow. All the protocols involving animal experimentations were approved by the Institutional Animal Ethics Committee, CSIR-CDRI, Lucknow [IAEC/2018/91]. The standard housing conditions (temperature 25 ± 2°C and humidity 60 ± 5%) were maintained in an animal quarantine area with a 12 h light and dark cycle. Food and water were available ad libitum.
Induction of diabetic nephropathy
Foremost, all the rats were randomly categorized into two groups. The first group (control) was fed with a normal chow diet (n = 6), while the second group was kept on a high-fat diet (HFD) group (HFD: 60% calories from fat; D12492, Research Diet, NJ, USA). After four weeks, rats on HFD were injected with a single low dose of Streptozotocin (STZ) (35 mg/kg) intraperitoneally. Following seventy-two hours of injection, rats fasted, and blood glucose level was checked, and the rats were considered type 2 diabetic with a glucose level of more than 250 mg/dL.[25] Subsequently, type 2 diabetic rats were further segregated into two groups. DN group: diabetic rats received an HFD diet for 12 weeks (n = 6); DN + EECQ group: diabetic rats received EECQ orally for 40 days at a dose of 200 mg/kg/day and kept on HFD for 12 weeks (n = 6). The dose of EECQ (200 mg/kg) was selected based on the dose–response study, and this dose was comparable to the quercetin (phytoconstituent present in extract) and standard drug metformin.[24]
Body weight, kidney weight and kidney weight to body weight ratio
Before the termination of the animals, the body weight was measured gravimetrically using a weighing balance. Subsequently, the kidney weight was recorded following the sacrifice of the animals. Also, the kidney weight to body weight ratio was determined to ascertain kidney hypertrophy.
Glucose and insulin tolerance tests
The oral glucose tolerance tests (OGTT) were performed following the treatment period. Foremost, rats were fasted for 6 h, and then fasting blood glucose was taken using test strips and glucometer (Arkray USA, Inc.) from the tail vein. Then, glucose (2 g/kg) was inoculated orally, and the blood glucose was checked at different point intervals (15, 30, 60 and 120 min). Similarly, an insulin tolerance test (IPITT) was performed where insulin (0.4 U/kg) was injected intraperitoneally.
Estimation of fasting insulin level
The level of insulin was estimated in serum using a species-specific ELISA kit (ELK Biotechnology), following the instructions embedded by the manufacturer
Analysis of lipid profile
The plasma level of triglyceride (TG), LDL and HDL were estimated by Triglyceride, Low-density Lipoprotein and High-density Lipoprotein Cholesterol Colorimetric Assay Kit (Elabscience Biotechnology Inc. Texas, USA), as per manufacturer's instruction. Furthermore, VLDL and total cholesterol were measured as per the formula described previously.[26]
Measurement of the advanced glycation end product
The AGEs level was determined in whole kidney homogenate according to the manufacturer's protocol of the AGE ELISA kit (Shanghai Korain Biotech, Shanghai, China).
Determination of oxidative stress markers and reactive oxygen species
The SOD activity and the level of malondialdehyde (MDA) and reduced glutathione (GSH) were determined in whole kidney tissue as per the protocol-defined.[27] ROS was estimated in kidney tissue according to the method described previously.[15]
Analysis of renal dysfunction
The kidney function was assessed by measuring serum levels of blood urea nitrogen (BUN) (Elabscience Biotechnology Inc. Texas, USA), creatinine (Sigma Aldrich Chemical Co., St. Louis, MO, USA) and albumin level by Albumin assay kit (Q-line, New Delhi, India) according to the manufacturer's protocol. Also, creatinine clearance and glomerular filtration rate (GFR) were calculated using the following formula. Creatinine clearance (mL/min) = urine volume × urine creatinine/serum creatinine; GFR (mL/min) = (urine creatinine/plasma creatinine) × urine volume/body weight
Assessment of renal inflammation
Renal inflammation was assessed by measuring the expression of inflammatory cytokines in the whole kidney extract as well as in plasma samples. The level of TNF-α, IL-6 and IL-β was estimated in plasma using Rat TNF-α Immunoassay, Rat IL-6 Immunoassay and Rat IL-1β Immunoassay kit (R&D Systems, Inc., USA) according to the manufacturer's protocol.
Histopathology
The formalin-fixed kidney sections were deparaffinized, hydrated and stained with eosin and hematoxylin. Subsequently, sections were dehydrated, cleared in xylene and mounted with DPx. A similar process was undertaken for collagen staining where picro serius red (PSR) stain was used. Images were captured using a bright field microscope (Leica). Moreover, PSR stained area was quantified using Image J (NIH, USA).
Extraction of RNA and quantitative real-time PCR
Total RNA was isolated from the whole kidney tissue using TRIzol reagent. cDNA was synthesized using a cDNA synthesis kit (Thermo Fisher Scientific, USA). Finally, qPCR was performed using prepared cDNA, specific primers (Table 1) and SYBR green in LightCycler 480 Instrument II (Roche Life Science). Gene expression was analyzed by the delta-delta method using obtained Ct value.
List of primers used for real-time qPCR
| rSIRT1 | F | TACCTTGGAGCAGGTTGCAG |
| R | GCTTCATGATGGCAAGTGGC | |
| rDNMT1 | F | TAGTTCGGTGGCTACGAGGA |
| R | CCTGGCCTTACTCGTTCAGG | |
| rSOD1 | F | TTTTGCTCTCCCAGGTTCCG |
| R | TGTCCTGACACCACAACTGG | |
| rSOD3 | F | GAGAGCTTGTCAGGTGTGGA |
| R | CGCCTCCCGTTGTTTTCCTA | |
| rTGFβ | F | CTGGAAAGGGCTCAACACCT |
| R | AGAAGTTGGCATGGTAGCCC | |
| rSMAD2 | F | ATGAGCTCAAGGCGATCGAG |
| R | CAGAGGCGGCAGTTCTGTTA | |
| rSMAD3 | F | GTTAAAAGCGAAGTTCGGGCG |
| R | TTCACCAAGCTCTTGACCGC | |
| rCol1a1 | F | GTACATCAGCCCAAACCCCA |
| R | GGGACTTCTTGAGGTTGCCA | |
| rCol3a1 | F | TGCAATGTGGGACCTGGTTT |
| R | GGGCAGTCTAGTGGCTCATC | |
| rIL6 | F | TCCGGAGAGGAGACTTCACA |
| R | TCTGACAGTGCATCATCGCT | |
| rIL1β | F | ATGAAAGACGGCACACCCAC |
| R | TGTCCCGACCATTGCTGTTT | |
| rTNFα | F | ATCCGAGATGTGGAACTGGC |
| R | ACTGATGAGAGGGAGCCCAT | |
| rGAPDH | F | GCGAGATCCCGCTAACATCA |
| R | CTCGTGGTTCACACCCATCA |
| rSIRT1 | F | TACCTTGGAGCAGGTTGCAG |
| R | GCTTCATGATGGCAAGTGGC | |
| rDNMT1 | F | TAGTTCGGTGGCTACGAGGA |
| R | CCTGGCCTTACTCGTTCAGG | |
| rSOD1 | F | TTTTGCTCTCCCAGGTTCCG |
| R | TGTCCTGACACCACAACTGG | |
| rSOD3 | F | GAGAGCTTGTCAGGTGTGGA |
| R | CGCCTCCCGTTGTTTTCCTA | |
| rTGFβ | F | CTGGAAAGGGCTCAACACCT |
| R | AGAAGTTGGCATGGTAGCCC | |
| rSMAD2 | F | ATGAGCTCAAGGCGATCGAG |
| R | CAGAGGCGGCAGTTCTGTTA | |
| rSMAD3 | F | GTTAAAAGCGAAGTTCGGGCG |
| R | TTCACCAAGCTCTTGACCGC | |
| rCol1a1 | F | GTACATCAGCCCAAACCCCA |
| R | GGGACTTCTTGAGGTTGCCA | |
| rCol3a1 | F | TGCAATGTGGGACCTGGTTT |
| R | GGGCAGTCTAGTGGCTCATC | |
| rIL6 | F | TCCGGAGAGGAGACTTCACA |
| R | TCTGACAGTGCATCATCGCT | |
| rIL1β | F | ATGAAAGACGGCACACCCAC |
| R | TGTCCCGACCATTGCTGTTT | |
| rTNFα | F | ATCCGAGATGTGGAACTGGC |
| R | ACTGATGAGAGGGAGCCCAT | |
| rGAPDH | F | GCGAGATCCCGCTAACATCA |
| R | CTCGTGGTTCACACCCATCA |
r, Rat; F, forward primer; R, reverse primer.
List of primers used for real-time qPCR
| rSIRT1 | F | TACCTTGGAGCAGGTTGCAG |
| R | GCTTCATGATGGCAAGTGGC | |
| rDNMT1 | F | TAGTTCGGTGGCTACGAGGA |
| R | CCTGGCCTTACTCGTTCAGG | |
| rSOD1 | F | TTTTGCTCTCCCAGGTTCCG |
| R | TGTCCTGACACCACAACTGG | |
| rSOD3 | F | GAGAGCTTGTCAGGTGTGGA |
| R | CGCCTCCCGTTGTTTTCCTA | |
| rTGFβ | F | CTGGAAAGGGCTCAACACCT |
| R | AGAAGTTGGCATGGTAGCCC | |
| rSMAD2 | F | ATGAGCTCAAGGCGATCGAG |
| R | CAGAGGCGGCAGTTCTGTTA | |
| rSMAD3 | F | GTTAAAAGCGAAGTTCGGGCG |
| R | TTCACCAAGCTCTTGACCGC | |
| rCol1a1 | F | GTACATCAGCCCAAACCCCA |
| R | GGGACTTCTTGAGGTTGCCA | |
| rCol3a1 | F | TGCAATGTGGGACCTGGTTT |
| R | GGGCAGTCTAGTGGCTCATC | |
| rIL6 | F | TCCGGAGAGGAGACTTCACA |
| R | TCTGACAGTGCATCATCGCT | |
| rIL1β | F | ATGAAAGACGGCACACCCAC |
| R | TGTCCCGACCATTGCTGTTT | |
| rTNFα | F | ATCCGAGATGTGGAACTGGC |
| R | ACTGATGAGAGGGAGCCCAT | |
| rGAPDH | F | GCGAGATCCCGCTAACATCA |
| R | CTCGTGGTTCACACCCATCA |
| rSIRT1 | F | TACCTTGGAGCAGGTTGCAG |
| R | GCTTCATGATGGCAAGTGGC | |
| rDNMT1 | F | TAGTTCGGTGGCTACGAGGA |
| R | CCTGGCCTTACTCGTTCAGG | |
| rSOD1 | F | TTTTGCTCTCCCAGGTTCCG |
| R | TGTCCTGACACCACAACTGG | |
| rSOD3 | F | GAGAGCTTGTCAGGTGTGGA |
| R | CGCCTCCCGTTGTTTTCCTA | |
| rTGFβ | F | CTGGAAAGGGCTCAACACCT |
| R | AGAAGTTGGCATGGTAGCCC | |
| rSMAD2 | F | ATGAGCTCAAGGCGATCGAG |
| R | CAGAGGCGGCAGTTCTGTTA | |
| rSMAD3 | F | GTTAAAAGCGAAGTTCGGGCG |
| R | TTCACCAAGCTCTTGACCGC | |
| rCol1a1 | F | GTACATCAGCCCAAACCCCA |
| R | GGGACTTCTTGAGGTTGCCA | |
| rCol3a1 | F | TGCAATGTGGGACCTGGTTT |
| R | GGGCAGTCTAGTGGCTCATC | |
| rIL6 | F | TCCGGAGAGGAGACTTCACA |
| R | TCTGACAGTGCATCATCGCT | |
| rIL1β | F | ATGAAAGACGGCACACCCAC |
| R | TGTCCCGACCATTGCTGTTT | |
| rTNFα | F | ATCCGAGATGTGGAACTGGC |
| R | ACTGATGAGAGGGAGCCCAT | |
| rGAPDH | F | GCGAGATCCCGCTAACATCA |
| R | CTCGTGGTTCACACCCATCA |
r, Rat; F, forward primer; R, reverse primer.
Western blotting
Whole kidney tissues were homogenized using a lysis buffer, and the protein content in the lysate was estimated. Proteins were separated by SDS PAGE using ten percent gel. Subsequently, proteins were transferred to the PVDF membrane and incubated with a protein block (5% BSA). Following protein block, the membrane was incubated overnight with primary antibodies anti-SIRT1 and anti-DNMT1 (Affinity Biosciences, USA), at 1 : 1000 dilution. Furthermore, the membrane was washed, incubated with an anti-rabbit secondary antibody (Elabscience, Texas, USA), and the blots were developed in ChemiDoc (Thermo Fischer Scientific, Waltham, USA) using substrate ECL. The blots were quantified using Image J (NIH, USA).
Statistical analysis
GraphPad Prism 8.0 (CA, USA) was used to analyze all the data. The data were represented as Mean ± SEM. Data involving two groups were analyzed using the unpaired Student t-test. In contrast, the data with multiple groups (more than two) were analyzed using one-way ANOVA, followed by post hoc Tukey's test. A P-value less than 0.05 value was considered statistically significant.
Results
EECQ maintains body weight and attenuates kidney weight, kidney hypertrophy
After twelve weeks of the experiment, DN group rats exhibited a significant decrease in body weight in comparison to control rats (Figure 1A). Kidney weight (Figure 1B) and kidney hypertrophy index (Figure 1C) was significantly increased as compared to the control group rats. Nonetheless, treatment with EECQ maintains body weight similar to the control group
Effect of EECQ on (A) body weight, (B) kidney weight, (C) kidney hypertrophy, (D) fasting blood glucose, (E) fasting insulin level, (F) OGTT, (G) corresponding AUC, (H) IPITT, (I) corresponding AUC. Data are shown as Mean ± S.E.M (n = 6). *: control versus DN; #: DN versus DN + EECQ; **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.
EECQ alleviates fasting blood glucose, insulin and insulin resistance
The administration of HFD/STZ for twelve weeks led to an increase in fasting blood glucose, insulin level and substantially enhanced insulin resistance in the DN group rats. On the other hand, the administration of EECQ significantly decreased the level of fasting blood glucose (Figure 1D) and insulin (Figure 1E), along with alleviating the glucose (Figure 1F and G) and insulin (Figure 1H and I) tolerance tests.
EECQ ameliorates lipid profile
The level of HDL in the DN group was significantly decreased while the LDL, TG, VLDL and TC level were significantly increased as compared to the control group. On the administration of EECQ, the HDL level was significantly increased, whereas LDL, TG, VLDL and TC level was markedly decreased as compared to the DN group rats (Figure 2A–E).
Effect of EECQ on (A) HDL, (B) LDL, (C) TG, (D) VLDL, (E) TC and (F) H & E staining of kidney sections. Data are shown as Mean ± S.E.M (n = 6). *: control versus DN; #: DN versus DN + EECQ; **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.
EECQ protects against glomerular mesangial expansion in diabetic nephropathy rats
Histological examination of kidney tissue of DN group rats revealed the presence of mesangial expansion of glomerulus along with the thickening of the basement membrane. Nevertheless, control and EECQ rats do not demonstrate such abnormalities (Figure 2F), indicating the renoprotective effect of EECQ.
EECQ rescues from AGEs instigated SIRTs inhibition
The AGEs level in kidney tissue was found to be significantly elevated in the DN group rats but not in control rats. In contrast, extract-treated rats revealed a significantly lower level of AGEs (Figure 3A) as compared to the DN group rats. Moreover, the expression of SIRT1 mRNA (Figure 3B) and protein (Figure 3D and E) determined by qRT-PCR and immunoblotting was reduced in DN group rats than in control. At the same time, diabetic nephropathy rats administered with EECQ demonstrated elevated expression compared to only DN group rats, propounded protective effect of EECQ against SIRT1 deregulation induced by AGE aggravated level in DN group rats.
Effect of EECQ on (A) AGEs concentration, (B) SIRT1 mRNA expression, (C) DNMT1 mRNA expression, (D–F) SIRT1 and DNMT1 protein expression (n = 3), (G) SOD activity, (H and I) SOD1 and 3 mRNA expression, (J) ROS fluorescence intensity, (K) MDA level, (L) GSH level. mRNA and protein expressions were normalized by corresponding GAPDH expression. Data are shown as Mean ± S.E.M (n = 6). *: Control versus DN; #: DN versus DN + EECQ; *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.
EECQ hedges the kidney against DNMTs persuaded downregulation of SODs and subsequent oxidative stress and ROS production in DN rats
The mRNA (Figure 3C) and protein (Figure 3D and F) expression of DNMT1, demonstrated to be substantially enhanced in the kidney of diabetic nephropathy rats. In line with this, the nephropathy rats showed a significantly reduced activity (Figure 3F) and expression of antioxidant enzyme SOD1 and SOD3 (Figure 3G and H). Parallelly, the DN group demonstrated an increased level of ROS (Figure 3I), MDA (Figure 3J) and decreased level of GSH (Figure 3K), attributed to the reduced SODs. Contrarily, treatment with EECQ ameliorated ROS production, MDA level and enhanced the level of GSH by increasing the expression of SODs and reducing the expression of DNMTs.
EECQ safeguards from TGF-β induced renal fibrosis in DN rats
Enhanced fibrosis in DN rats was reflected by PSR staining of kidney sections, which depicted elevated collagen content around the glomerulus. However, the collagen content in EECQ rats was equivalent to control rats (Figure 4A and B), thereby propounding protection against renal fibrosis by EECQ. Furthermore, the mRNA expression of TGF-β ascertained in kidney tissue, revealed enhanced expression in DN rats but not in control (Figure 4C). Contrarily, EECQ exposure to the DN rats exhibited protection against elevated TGF-β expression. Subsequently, the downstream molecules of TGF-β activation, including Smad2, Smad3, col1 and col3 (Figure 3D–G), were determined to be deregulated in diabetic nephropathy rats. Nevertheless, EECQ encountered DN rats and control rats unveiled regular expression of the aforementioned fibrosis markers.
Effect of EECQ on (A and B) PSR staining of kidney sections, (C) corresponding collagen content, (D) TGFβ, (E and F) Smad2 and 3, (G and H) Col1 and 3 mRNA expression. mRNA expression was normalized by GAPDH expression. Data are shown as Mean ± S.E.M (n = 6). *: control versus DN; #: DN versus DN + EECQ; *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.
EECQ ameliorates renal dysfunction in DN rats
Serum level of creatinine and blood urea nitrogen was significantly increased, whereas the albumin level and creatinine clearance were significantly decreased in DN group rats as compared to the control. On the administration of EECQ, it decreased the creatinine level (Figure 5A) as well as blood urea nitrogen (Figure 5B) and enhanced the level of albumin (Figure 5C) and creatinine clearance (Figure 5D) as compared to the DN group rats. Furthermore, the GFR was enhanced in diabetic nephropathy rats but not in control. Contrarily, treatment with EECQ ameliorated the elevated GFR in DN rats (Figure 5E).
Effect of EECQ on (A) serum creatinine, (B) BUN, (C) albumin, (D) creatinine clearance, (E) GFR, (F) TNFα, (G) IL6, (H) IL1β plasma level, (I) TNFα, (J) IL6 and (K) IL1β mRNA expression. mRNA expression was normalized by GAPDH expression. Data are shown as Mean ± S.E.M (n = 6). *: control versus DN; #: DN versus DN + EECQ; *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001.
EECQ alleviates renal inflammation by downregulating the overexpressed inflammatory cytokines in DN rats
The renal inflammation was assessed by the estimation of inflammatory cytokines in the circulation and kidney tissue. The level inflammatory cytokines, including IL6, IL1β and TNFα, were significantly elevated in DN rats but not in control rats. Interestingly, EECQ encountered rats depicted significant protection from the soared inflammatory cytokines (Figure 5F–H). Moreover, the mRNA expression of the aforementioned cytokines (Figure 5I–K) analyzed by qPCR further bolstered the above outcome.
Discussion
In the current study, we administered the EECQ to HFD/STZ-induced diabetic nephropathy rats to determine its renoprotective effects by attenuating renal dysfunction, fibrosis and inflammation. By the end of the experiment, the EECQ extract demonstrated a dramatic reduction in hyperglycemia, hyperlipidemia, proinflammatory cytokines and renal function biomarkers in DN rats. Thus, our data indicated that EECQ protected against HFD/STZ-induced nephropathy. Besides, hyperglycemia and hyperlipidemia result in enhanced serum levels of AGEs, which plays a vital role in the progression of DN.[28] The EECQ ameliorated the AGEs level in the treated diabetic rats.
Furthermore, to scrutinize the molecular mechanism behind the renoprotective activity of EECQ, we explored its effect on the SIRT1 pathway. Previous studies suggested SIRT1 acts as a critical factor in metabolism and inflammation in diabetes-induced kidney injury,[29] and plays a crucial role in the treatment of diabetic nephropathy.[30] As described above, the current model depicted enhanced AGEs level, which is known to downregulate the SIRT1 expression.[9] Parallelly our model manifested the reduced expression of SIRT1. Astonishingly, EECQ exposure to diabetic rats substantially restored the reduced SIRT1 expression. Moreover, SIRT1 controls DNA methylation by modulating the DNMT1.[10, 11] Besides, DNMT1 is a principal epigenetic regulator that regulates the expression of genes involved in insulin signaling, thereby facilitates insulin resistance,[31] suggesting involvement in DN. The current study exhibited elevated expression of DNMT1 in the kidney of diabetic nephropathy rats; however, safeguarded by EECQ therapy. Apart from facilitating insulin resistance, DNMT1 additionally exacerbates ROS production and oxidative stress through encouraging DNA methylation of the antioxidant SOD genes.[12] Accordingly, the diabetic nephropathy rats showed a significantly reduced activity and expression of SOD1 and SOD3 genes, decreased level of GSH while the increased level of MDA and ROS, indicating potentially high oxidative stress. Interestingly, treatment with EECQ ameliorated ROS production, MDA level and augmented the level of GSH by increasing the expression of SODs, possibly due to reduced DNMT1 expression. Subsequently, the elevated ROS and oxidative stress predispose the tissues to fibrosis by overexpressing fibrosis marker TGF-β.[13] Several in vitro and in vivo studies have examined the effect of flavonoids in DN. Most of the findings showed that they improve renal function, as evidenced by lower serum creatinine and urinary albumin levels.[32, 33] Flavonoids inhibit DN by inhibiting ROS formation, thereby acting as an antioxidant by decreasing MDA level and enhancing the level of SOD and sirtuin1.[34–37] In diabetic patients, AGEs may accumulate in the podocytes, glomerular basement membrane and mesangial cells, compromising renal function via oxidative stress, inflammation and fibrosis. As a result, blocking AGEs and their receptor system (RAGE), as well as downstream associated pathways, is one of the potential treatments for DN.[38] Flavonoids/extract decreases AGE-induced oxidative stress by increasing SOD activity and decreasing MDA levels and ROS production.[39] Furthermore, this flavonoid inhibits an increase in collagen and TGF-1 levels.[39] Furthermore, flavonoid inhibits increase in collagen and TGF-1 levels.39] Other flavonoids have been shown to have an anti-fibrotic effect by inhibiting AGEs-induced expression of collagens and MMPs in mesangial cells and preventing collagen and AGEs aggregation.[40] Consequently, we checked the expression of TGF-β and their downstream molecules, including Smad2, Smad3, col1 and col3 in the kidney of DN and EECQ exposed rats. DN rats manifested enhanced levels; nevertheless, EECQ encountered DN rats unveiled regular expression of the aforementioned fibrosis markers. Furthermore, we performed PSR staining of kidney sections to visualize fibrotic changes in the context of collagen content. DN group rats kidney sections displayed enhanced collagen content area, which is attributed to increased fibrosis produced by HFD consumption. Contrarily, the collagen content was significantly lower in EECQ encountered DN rats that further bolstered its antifibrotic property.
Conclusion
To conclude, our study revealed that the EECQ mitigates renal fibrosis, dysfunction and inflammation in diabetic nephropathy rats by altering SIRT1/DNMT1 pathway may be due to their phytoconstituents present such as quercetin and resveratrol. EECQ thus exhibits protective behaviour in diabetic nephropathy caused by HFD/STZ. Therefore, it may be useful in the early treatment of diabetic nephropathy. However, our analysis further needs thorough research on EECQ to establish the active principles responsible for its nephroprotective action in diabetes.
Acknowledgments
We acknowledge the Indian Council of Medical Research for providing research fellowship to A.A.S and U.K.; Council of Scientific & Industrial Research for providing research fellowship to R.G. CSIR-CDRI communication number for this article is 10237.
Author Contributions
A.A.S. and J.R.G. designed the research work; A.A.S., M.I.R., R.G. and U.K., performed the research experiments; A.A.S., M.I.R. and J.R.G. analyzed and compiled the data; A.A.S. and J.R.G. prepared the manuscript.
Funding
The current work was financially assisted by the Council of Scientific and Industrial Research, Indian Council of Medical Research, University Grant Commission, and Department of Biotechnology, Govt of India.
Conflict of Interest
All authors declared that there is no conflict of interest.
References
