Chebulagic Acid Alleviates Inflammation Via Regulation of Skeletal Muscle IR/IRS-1/AKT/GLUT4 Signaling Pathway in Diabetic Rats

Chebulagic Acid Alleviates Inflammation Via Regulation of Skeletal Muscle IR/IRS-1/AKT/GLUT4 Signaling Pathway in Diabetic Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chebulagic Acid Alleviates Inflammation Via Regulation of Skeletal Muscle IR/IRS-1/AKT/GLUT4 Signaling Pathway in Diabetic Rats Ganesh Vasu, Sundaram Ramalingam, Karuppiah Muthu, Sundaram Ramalingam Tutor, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3859769/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Examining the contribution of chebulagic acid in high fat diet/streptozotocin (HFD/STZ)-induced diabetic nephropathy was the main goal of this investigation. Wistar male rats were fed HFD for two weeks before receiving a 35 mg/kg STZ intraperitoneal dosage. During 30 days, diabetic rats were fed metformin and chebulagic acid (50 mg/kg b.w./day each). Blood and kidney samples were also taken following the study for biochemical and histological analysis. Chebulagic acid was administered orally to diabetic rats, considerably lowering blood sugar, serum creatinine, urea, and homeostasis model assessment of insulin resistance (HOMA-IR) levels while simultaneously increasing plasma insulin. In addition, diabetic rats had elevated levels of renal pro-inflammatory cytokines with concurrently increased levels of anti-inflammatory cytokines. They also had lower lipid peroxidation product and increased renal enzymatic and non-enzymatic antioxidant enzyme status. Moreover, chebulagic acid therapy increased the amounts of mRNA for the insulin signaling components GLUT4 and Akt in the gastrocnemius muscles of diabetic rats as well as insulin receptor (IR), insulin receptorsubstrate-1 (IRS-1), and Akt. According to these findings, chebulagic acid has anti-diabetic nephropathy actions that are attenuated. Diabetes Hyperglycaemia Insulin resistant Metformin Molecular Targets Tannin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction The metabolic disorder known as diabetes mellitus affects the metabolism of carbohydrates, lipids, and proteins by causing insulin resistance and reduced insulin secretion by pancreatic β-cells (Kahn 2008; Wang et al. 2018; Syed 2022). Lifelong type 2 diabetes mellitus (T2DM) causes an increase in morbidity and mortality, which lowers overall quality of life (Teck 2022; Hulsizer et al. 2023). Persistent hyperglycemia is associated with long-term harm to and functioning of the kidneys, nerves, eyes, and cardiovascular system (Wild et al. 2004). Since it causes end-stage renal failure in diabetic individuals, diabetic nephropathy (DN) is the most hazardous of all T2DM side effects (Rehman et al., 2019; Vaz de Castro et al.2022). A diabetic patient's risk of developing diabetic nephropathy ranges from 15 to 40% throughout the course of their lifetime (Shang et al.2013) As extracellular matrix accumulates, glomerular basement membranes degrade, and glomeruli shrink, the kidney's structural and functional characteristics are affected, and this condition is known as diabetic nephropathy (Piccoli et al. 2015; Xu et al.2018). The generation of reactive oxygen species (ROS), which in turn triggers oxidative stress and inflammation, has been shown to occur as a result of persistent hyperglycemia, according to current studies. Also, a rise in ROS generation can activate a number of cellular pathways that aid in the course of illness due to the weakening of antioxidant defense mechanisms (Abou-Hany et al.2018). The two primary therapeutic modalities that treat or prevent the onset and progression of diabetic nephropathy are blood glucose control and renin-angiotensin system blockade. The low effectiveness and numerous untoward side effects of current treatments, however, have not reduced the disease's incidence rate (Johnson and Spurney 2015). Recently, there has been a lot of focus on the development of innovative, effective, and risk-free therapeutic alternatives that target several pathways (glycemic management, oxidative stress inhibition, and inflammatory pathways) implicated in the development of diabetic nephropathy (Abou-Hany 2018). Because of this, utilizing a novel medication combining hypoglycemic, anti-inflammatory, and antioxidant qualities may shield against diabetic nephropathy. (Kooti et al.2016). Given these details, chebulagic acid, an organic compound present in the fruits of Terminalia chebula Retz., a Combretaceae species, was chosen. Chebulagic acid possesses anti-inflammatory, anti-tumor, anti-angiogenic, anti-hyperglycemic, neuroprotective, and gastroprotective properties, according to Shanmuganathan and Angayarkanni (2018), Wang et al. (2018), Athira et al. (2017), Huang et al. (2012), and other studies (Kim et al. 2014; Liu et al. 2017). Chebulagic acid, which we recently discovered increases PPAR-γ and GLUT-4, decreases HFD/streptozotocin-induced poor glucose metabolism and insulin resistance in type 2 diabetic rats (Ganesh et al. 2021). The objective of the current study was to investigate the potential anti-inflammatory, anti-oxidant, and anti-hyperglycemic effects of chebulagic acid in rats that underwent an HFD/streptozotocin-induced modification in renal function. Materials and Methods Drugs and Chemicals Streptozotocin, metformin, total RNA isolation reagent (TRIR), and chebulagic acid (98% purity Catalog No. CFN92295, respectively, were acquired from Chem Faces, China; Invitrogen, USA; Sisco Research Laboratories, India; and Merck, Germany. Additional substances of an analytical grade were employed in this investigation. Experimental Animals Male adult albino Wistar rats weighing between 200 and 220 g were acquired from the Sri Muthukumaran Medical College Hospital & Research Institute in Mangadu, Chennai, Tamil Nadu, India. Rats were kept in sterile, clean polypropylene cages in accredited animal facilities with a 12-h light/dark cycle, constant temperature (25 ± 2°C), and free access to commercial rat food (Pranav Agro Industries Ltd., Pune, Maharashtra, India) and water. Type 2 Diabetes Induction Four groups of six animals each were formed from the total number of animals. 84.3% ordinary laboratory chow, 5% lard, 10% yolk powder, 0.2% cholesterol, and 0.5% bile salt were used in the rats' high-fat diet for 2 weeks (Xie et al. 2005 ). During a 2-week period, the animals were kept fasting all night before receiving a low dosage of streptozotocin (35 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH 4.5) (Wu et al. 2012 ). After 3 + days of induction, blood was drawn from the animals and evaluated for fasting blood glucose levels using a glucometer (ACCU-Check Active, Roche, Switzerland). Rats with fasting blood glucose levels more than 300 mg/dl were deemed to have diabetes. For a further 4 weeks, the diabetic rats were fed a high-fat diet. Experimental design The experimental animals, which included 18 diabetic and 6 normal rats, were split into four groups, each of which had at least six rats (n = 6). In a prior work, we employed three different doses of chebulagic acid (25, 50, and 100 mg/kg b.w.) that were dissolved in 0.5 ml of 1% CMC (Carboxyl methylcellulose) in distilled water and given orally to diabetic rats using an intragastric tube for a duration of 30 days. Chebulagic acid at 50 mg/kg body weight had a substantial, metformin-comparable impact in lowering blood sugar. Because of this, the 25 mg and 100 mg/kg bw administered groups were left out of this study.Group I: normal control rats; group II: diabetic control rats; group III: diabetic + chebulagic acid (50 mg/kg bw) (Narasimhan et al. 2015 ); and group IV: diabetic + metformin (50 mg/kg bw). The animals underwent therapy for 30 days before being denied food overnight, given a deep anesthetic with diethyl ether, and then killed by cervical decapitation. For the purposes of separating the plasma and serum, blood samples were taken both with and without anticoagulants. Kidneys were quickly excised and washed in ice-cold saline to remove the blood. A portion of kidney tissue was minced and homogenized (10%, w/v) with 0.1 M Tris -HCl buffer (pH 7.4) and centrifuged at 3000× g for 10 min at 4 o C by a REMI cooling centrifuge. The resulting supernatant was used for enzyme assays. For histological analysis, another kidney fragment was stored in 10% buffered formalin solution. All biochemical calculations were finished within 24 h of the animal's death. Serum Biochemistry Assessment The manufacturer's instructions were followed when standard kits (Agappe Diagnostic Pvt. Ltd., India) were used to calorimetrically measure the serum parameters of kidney function (urea and creatinine). According to Trinder 1969 , the available enzymic colorimetric test was used to determine the glucose levels. Following the manufacturer's instructions, rat insulin ELISA Millipore kit was used to measure the amount of serum insulin. Lipid peroxidation and hydroperoxides were estimated in the kidney tissues by the method of Niehius and Samuelsson ( 1968 ) and Jiang et al. ( 1992 ), respectively. Catalase (CAT) superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), ascorbic acid (vitamin C), α-tocopherol (vitamin E), GSH, and protein were determined by the method of Sinha ( 1972 ). Stringer et al. ( 1989 ), Rotruck et al. ( 1973 ) Habig et al. ( 1974 ), Omaye et al. ( 1979 ), Baker et al. ( 1980 ), Ellman ( 1959 ), and Lowry et al. ( 1951 ), respectively. According to the methodology of Matthews et al., the homeostasis model assessment of basal insulin resistance (HOMA-IR) was computed based on the fasting blood insulin and glucose concentrations (1985). HOMA-IR index = fasting glucose (mg/dl) × fasting insulin (mU/ml)/405. Inflammatory Cytokines Plasma concentrations of interleukin 1β, interleukin 6 (IL6), interleukin 10 (IL10), tumor necrosis factor-alpha (TNF-α) and NF-kB p65 were determined using commercial rat ELISA kits (Ray Biotech, Inc., Norcross, GA, USA), following the instructions in respective kit manuals. Spectrophotometry at 450 nm was used to calculate the proinflammatory cytokine levels. With the use of standard cytokines, standard plots were created, and from these plots, concentrations for unidentified samples could be determined. Renal Tissue Immunohistochemistry The immunohistochemical staining of renal tissues to determine the expression of NF-κB and TNF-α was as described by Sambrook et al. ( 1989 ) with slight modification. Five millimeter sections of the paraffin-embedded kidney tissue were made. The sections underwent xylene-based deparaffinization before being rehydrated in ethanol series (100, 90, 70, 50, and 30%). The slides were then washed in phosphate buffered saline (PBS; 50 mM mono and di phosphate, 100 mM NaCl, pH 7.4) before being exposed to 3% H 2 O 2 at room temperature for 15 min to inhibit endogenous peroxidase activity. Antigen retrieval on the slides was place during a 15-min incubation with citrate buffer (10 mM) at 60°C. Then, the slides were incubated with blocking buffer 3% BSA for 3 h at room temperature and then, the slides were incubated overnight at 4°C with monoclonal anti NF-kB and TNF-α (dilution 1:200 and 1:200, Santa Cruz, CA, USA). The slides were then treated with anti-rabbit and anti-mouse detection secondary antibodies for 2 h at 4°C, rinsed with PBS, and incubated with DAB solution (3, 3 diaminobenzidine tetrahydrochloride 0.05%, 1X PBS-10 ml, and H 2 O 2 0.01%). Following that, the pieces were dried using ethanol concentrations of 30, 50, 70, 90, and 100%. After counterstaining the parts with hematoxyline and mounting them with DPX, they were finished. The immunoreactive positive expression of NF-κB and TNF-α were viewed under microscope and taken photograph. Histological changes in the stained sections were viewed interpreted by a pathologist. Real-Time PCR With the use of a TRIR (total RNA isolation reagent) kit, total RNA was extracted from reference and test samples. In order to fully allow the dissociation of nucleoprotein complexes, 100 mg of fresh tissue was homogenized with 1 ml TRIR, and the homogenate was then immediately transferred to a microfuge tube. This was done for 60 min at 80°C. After adding 0.2 ml of chloroform, the mixture was vortexed for 1 min before being chilled for 5 min at 4°C. For 15 min at 4°C, the homogenates were centrifuged at 12000 ×g. The aqueous phase (top layer) was carefully transferred to a brand-new microfuge tube, to which an equal volume of isopropanol was added, vortexed for 15 s, and then stored at 4°C for 10 min. At 4°C and 12000 ×g for 10 min, the samples were centrifuged. After discarding the supernatant, a vortex was used to wash the RNA pellet in 1 ml of 75% ethanol. The isolated RNA was estimated spectrometrically by the method of Fourney et al. ( 1988 ). The RNA concentration was expressed in microgram (µg). According to the manufacturer's instructions, complementary DNA (cDNA) was created from 2 µg of total RNA using the reverse transcriptase kit from Eurogentec (Seraing, Belgium).To perform real-time PCR, the reaction mixture containing 2x reaction buffer (Takara SyBr green master mix), Forward and reverse primers of the target gene and house-keeping gene, water and β-actin (the primer sequences were listed in Table S1 as supporting information) in total volume of 45 µl expect the cDNA was made, mixed intensively and spun down. In individual PCR vials, about 5 µl of control DNA for positive control, 5µl of water for negative control and 5 µl of template cDNA for samples were taken and reaction mixture (45 µl) were added. 40 cycles (95°C for 5 min, 95°C for 5 s, 60°C for 20 s and 72°C for 40 s) was set up for the reaction and obtained results were plotted by the PCR machine (Stratagene MX 3000P, Agilent Technologies, 530l, Stevens Creek Blvd, Santa Clara CA, 95051) on a graph. Relative quantification was calculated from the melt and amplification curves analysis Kidney Histopathological Examination In order to conduct a histological examination, a sample of renal tissue was obtained and stored in 10% formalin solution. The samples from each group were embedded in paraffin wax, cut into sections that were 5 µm thick, and stained with hematoxylin and eosin (H &E stain). A light microscope (Olympus Optical, Japan) with a 40X magnification was used to view the stained sections. Transmission Electron Microscopic Analysis Electron microscopy was used to examine the ultra-structure of the kidneys of normal and diabetic rats. The tissue sample is immediately submerged in the principal fixative, which is 2.5% glutaraldehyde made in a 0.1 M sodium cacodylate buffer with a pH 7.4 solution. The specimen is then cut into small bits of the size 1 mm × 1 mm on a glass slide using a sharp blade or scalpel. The fixative was gently reintroduced to the little parts, and they were left submerged there for 4 to 8 h at 8°C. After that, the bits were washed three times for a total of 10 min each time using the same buffer. The secondary fixative was then added, which is 0.1% Osmium tetroxide (OsO 4 ) produced in the same buffer. This post fixation was conducted for 2 h at 8°C. The surplus fixative was once again rinsed three times for a total of 10 min with the same buffer. The specimen pieces were then subjected to a graded series of treatments for 10 min each using 30, 50, 70, 80, and 90% acetone and again for 10 min each using 100% acetone, followed by two treatments of 10 min each using propylene oxide. The tissue bits were then infiltrated with the resin mixture consisting of Epon 812 resin, DDSA (dodecenyl succinic anhydride) and NMA (Nadic® methyl anhydride) starting with 25, 50, 75, and finally with 100% resin mixture for 2 h at each concentration. Finally, they were inserted in Easy molds at 60°C for 48 h using the same resin mixture with additional catalyst (DMP 30). The Leica Ultra cut R Ultra microtome with diamond or glass blades was used to extract the resin blocks from the mold, trim them, and segment them. Toludine blue was used to stain the semi-thin slices, which were then examined under a light microscope to look for any areas of interest in that specific block. After that, extremely thin pieces were cut, assembled on copper grids, and stained with lead citrate and uranyl acetate. A transmission electron microscope (BioTem H-7650 Hitachi, Japan) was used to analyze the grids at a 100 K-fold enlargement. Statistical analysis All data were expressed as mean ± SD. One-way ANOVA followed by least significant difference (LSD) test; p- value of less than 0.05 were considered to indicate statistical significance between the groups. Results Plasma Glucose, Insulin, and HOMA-IR index Rats with diabetes had much higher blood glucose levels than control rats, but the latter had significantly lower plasma insulin levels. Chebulagic acid was provided to diabetic rats, and as compared to the diabetic rats that were not treated, the blood glucose levels were dramatically reduced and the plasma insulin levels were significantly enhanced. HOMA-IR significantly increased in diabetic rats compared to control rats in this situation. In comparison to control rats, diabetic rats' HOMA-IR index was markedly decreased by chelbulagic acid + metformin administration. The outcome from diabetic rats treated with metformin and the impact that was seen were equivalent (Fig. 1). kidney function Serum urea and creatinine values for control and experimental rats are summarized in Table 1. As comparison to untreated diabetic rats, the levels of urea and creatinine were considerably lower after 30 days of therapy with chebulagic acid and metformin. Lipid Peroxidation Figure 2 depicts the concentrations of TBARS and HP in the kidneys of rats with and without diabetes. When compared to normal rats, diabetic rats had higher amounts of TBARS and HP in their kidneys. These values nearly returned to normal following therapy with chelbulagic acid and metformin. Antioxidant activities The SOD, CAT, GPx, and GST activities were dramatically reduced in diabetic rats as compared to healthy control rats. When treated with chebulagic acid plus metformin, diabetic rats' SOD, CAT, GPx, and GST activity increased, indicating that the antioxidant enzyme systems had been nearly fully recovered (Fig. 3). Non-enzymatic Antioxidants Figure 4 displays the concentrations of non-enzymatic antioxidants in the kidney of diabetic and control rats, including vitamin C, tocopherol, and reduced glutathione. When compared to normal control rats, diabetic rats' levels of vitamin C, tocopherol, and reduced glutathione were considerably lower. Chebulagic acid plus metformin treatment of diabetic rats resulted in a significant rise in the levels of non-enzymatic antioxidants. Inflammatory Biomarkers In comparison to control rats, there was a considerable increase in the levels of pro-inflammatory cytokines such TNF-a, IL-1, IL-6, and NF-kB p65, while a significant decrease in the levels of the anti-inflammatory cytokine IL-10. When oral doses of chelagic acid and metformin were administered to diabetic rats, these levels were markedly lower than those of untreated diabetic rats, and they eventually returned to near-normal levels (Fig.5). NF-κB Expression The kidney immunohistochemical expression of NF-κB in control and experimental rats is shown in Fig. 6. Nuclear translocation of NF-κB expression was found to be increased in the kidney of diabetic rats (Fig. 6B) when compared to control (6A). Chebulagic acid and metformin treatment reduced NF-κB nuclear translocation in the kidney of diabetic rats (Fig. 6 C and 6 D). TNF-α Expression Kidney immunohistochemical expression of TNF-α in control and experimental rats is depicted in Fig.7. The proinflammatory cytokine TNF-α expression was enhanced in the kidney of diabetic rats (Fig. 7B) when compared to normal control rats (Fig. 7A). TNF expression was reduced in diabetic rats treated with chebulagic acid and metformin (Fig. 7C and 7D). IR/IRS-1/Akt/ GLUT4 Expression GLUT4, which is a vital glucose transporter, is regulated by the Akt signaling system in the muscle. When diabetic rats were compared to control rats, the mRNA levels of IR, IRS-1, AKT, and GLUT4 were significantly decreased. These gene expression levels were increased in diabetic rats after receiving metformin and chelbulagic acid. Figures 8A–8D show the outcomes as they were obtained. Renal Histological Changes The control rats had normal glomeruli and tubules (Fig. 9A), while diabetic rats had necrotic and deteriorated renal tubules, as well as inflammatory cell infiltration in renal glomeruli and peritubular capillary congestion (Fig. 9B). Interestingly, treatment of diabetic rats with chebulagic acid and metformin reduced the histological alterations in the kidney, demonstrating better patterned renal architecture with reasonably normal glomeruli and tubules and minor inflammatory cell infiltration (Fig. 9C and 9D). Electron microscopic Examination The ultrastructure of the kidney was studied by transmission electron microscopy in addition to the standard hematoxylin and eosin-stained study. There are no lipid droplets seen in the kidneys of normal control rats, and their architecture is normal (Fig. 10A). The glomerular basement membrane thickened in the diabetic rats, and there were few lipid droplets. They also had considerable foot process fusion and broadening (Fig. 10B). Yet, administration of chebulagic acid and metformin to diabetic mice improved these changes (Fig. 10C and 10D). Discussion Diabetes mellitus is more common than ever, and it frequently leads to serious metabolic disorders and other serious consequences (Cloete 2022 ). A serious global health issue is the progression of diabetes complications. These problems are fatal, but due to a lack of efficient therapies, it is still challenging to stop their development and progression. In order to effectively combat the danger of diabetes sequelae, notably diabetic nephropathy, novel pharmaceutical agents are urgently required. Recent research has concentrated on the utilization of natural substances as workable substitutes for the creation of novel anti-diabetic and reno-protective medications (Borgohain et al. 2017 ; Governa et al.2018). The majority of polyphenols from plants contain antioxidant and anti-inflammatory properties, and they act as preventative agents by reducing or preventing the oxidative stress and inflammatory response that hyperglycemia causes (Serafini and Peluso 2016 ). As a result, the current work investigates how chebulagic acid protects the kidneys in diabetic rats produced by HFD/STZ. Combining the HFD diet with the administration of a small dose of STZ can result in the development of insulin resistance and a significant rise in blood glucose levels (Asrafuzzaman et al.2017). This model causes hyperglycemia and metabolic profiles that are very similar to those seen in humans with type 2 diabetes (Skovsø 2014 ). The body's vital tissues and organs might suffer harm from hyperglycemia. For instance, it has the ability to specifically obliterate renal glomerulus mesangial cells and capillary endothelial cells of the retina. (Ni et al. 2019 ). The significant reductions in blood glucose and HOMA-IR, together with a concurrent rise in insulin levels in diabetic rats treated with chebulagic acid and metformin, demonstrate the tested tannin's antidiabetic potential. Chebulagic acid therapy improved the glycogen content of diabetic rat liver and muscle, indicating improved insulin production and sensitivity, which encouraged the tissues to absorb glucose and store it as glycogen in the liver and muscle, according to our prior research. The enhanced pancreatic histology provided more evidence in favor of this (Ganesh et al. 2021). The diabetic rats displayed notable rises in serum urea and creatinine levels; this is consistent with former reports (Ni et al. 2019 ; Othman et al.2021). Blood urea and creatinine levels are reliable indicators of kidney function, and any increase in any of these values is a sign of impaired kidney function (Othman et al.2021). The detected histological alterations in the kidney were consistent with the decrease in renal function seen in rats with diabetes produced by HFD/STZ. When chebulagic acid and metformin were administered to diabetic rats, the levels of urea and creatinine dropped to levels that were equivalent to those of normal control rats, indicating improvement in the kidneys' impaired function. Generally speaking, lipid peroxidation may be defined as the process whereby oxidants like free radicals destroy lipids that contain carbon-carbon double bonds, particularly polyunsaturated fatty acids (Ayala et al.2014). The increased lipid peroxidation during diabetes could be related to an ineffective antioxidant system (Rastogi et al. 2008 ). Lipid peroxidation caused by free radicals has been linked to a variety of diseases, including diabetes (Kumar et al.2007). Free radicals can also be produced through the auto-oxidation of unsaturated lipids found in membrane and plasma lipids. When the produced free radical interacts with the polyunsaturated fatty acids in the cell membrane, lipid peroxidation might result. Lipid peroxidation will result in an increase in free radical production (Prabakaran and Ashokkumar 2013 ). Increased lipid peroxidation reduces membrane fluidity and alters the activity of membrane-bound enzymes and receptors, impairing membrane functions (Bhagavathy and Sumathi 2012 ). Its compounds are poisonous to the majority of bodily cells and have been connected to a number of diseases. Despite the fact that they are secondary products of oxidative stress and are generated as a result of the detrimental effect of ROS produced during lipid peroxidation, TBARS and HP levels were found to be considerably higher in HFD/STZ-induced diabetic rats. Recent studies have discovered a strong correlation between lower levels of lipid peroxidation and supplementation with phytochemicals having antioxidant capacity (Fidan and Dündar 2008 ). Chebulagic acid and metformin were given orally to diabetic rats in the current study to considerably reduce elevated TBARS and HP levels to levels that were close to normal. According to the current study, chebulagic acid also prevents oxidative damage by scavenging free radicals generated by prolonged hyperglycemia. The detoxification of harmful oxygen radicals is carried out by endogenous antioxidant enzymes (SOD, CAT, GPx, and GST). Increased levels of lipid peroxidation may indicate that the body's enzymatic antioxidant defense mechanism is becoming less effective. Molecular oxygen and hydrogen peroxide, two reactive molecules rather than free radicals, are produced as a result of the catalytic action of the antioxidant enzyme SOD. Other enzyme-based antioxidants, such as CAT, hasten the breakdown of hydrogen peroxide while shielding tissues from dangerous hydroxyl radicals. GPx, an enzyme that fights free radicals, aids in the reduction of potentially hazardous substances including lipid peroxides and other chemical components (Brigelius-Flohé et al.2003). In the conjugation of lethal electrophiles to GSH, a set of isoenzymes known as GST is active. In the present investigation, a reduced level of GST activity was observed, which may be due to a deficiency in GSH. The production of oxygen free radicals in STZ-treated -cells has been demonstrated in numerous studies, and it is hypothesized that reactive oxygen free radicals may inactivate and reduce the activities of antioxidant enzymes like SOD, CAT, GPx, and GST that are crucial for defending cells from oxidative damage (Cemek et al.2008). The low levels of SOD, CAT, GPx, and GST activity observed in diabetic rats in this study suggested stress brought on by the disease, whereas diabetic rats treated with chebulagic acid showed a marked increase in these antioxidant enzymes' activities, which may be related to the substance's scavenging ability. Free radicals are instantly detoxified by vitamins C and E, which are created by the body through nutrition. The results of this study showed that diabetic rats have reduced levels of vitamins C and E. Hydroxyl radicals, singlet oxygen radicals, and the active state of depleted vitamin E are all suppressed by the hydrophilic antioxidant vitamin C, which is also a key component of the diet. Even when there are other antioxidants present, vitamin C deficiency causes the production of hydroperoxides (Prabakaran and Ashokkumar 2013 ). Due to its greater role in oxyradical scavenging, vitamin C levels may be lower in diabetics. As a lipophilic antioxidant, vitamin E adds phenolic hydrogen to a polyunsaturated fatty acid free radical that has been peroxidized. This stops the chain process that would otherwise result in membrane lipid peroxidation (Opara 2002 ). On the other hand, administering chebulagic acid to a group of diabetic rats significantly restored the changed levels to close to normal, illuminating the antioxidant potential of chebulagic acid. GSH is one of the essential compounds for maintaining cell integrity against ROS, as it can scavenge free radicals and reduce H 2 O 2 (Masella et al. 2004 ;Wu et al.2004). Rats with HFD/STZ-induced diabetes had significantly lower tissue GSH concentrations than normal rats. According to certain research, diabetic rats who were given STZ had considerably lower tissue GSH concentrations than the rats in the control group (Gothandam et al.,2019; Ramalingam et al.2022). When GSH is reduced below its baseline level, ROS and oxidative stress are encouraged, which has a cascading impact on the structural and functional integrity of cell and organelle membranes (Franco and Cidlowski 2012 ). Vitamins E and C must be recycled, and GSH serves as a co-substrate for the enzymes GPx and GST, which guard against the harmful effects of oxygen radicals. NADPH must be present in order to keep the amount of oxidized glutathione stable. When there is inadequate glucose oxidation during the pentose phosphate cycle due to insulin deficiency, there is less intracellular NADPH, which lowers GSH levels (Maritim et al.2003). Rats with diabetes that have lower GSH levels in their kidneys may be more vulnerable to oxidative injury. It has been hypothesized that antioxidants that maintain GSH concentrations can restore cellular defense mechanisms, reduce lipid peroxidation, and shield tissue from oxidative damage. Tissue GSH levels have likely decreased due to increased oxidative stress brought on by a significant increase in aldehydic lipid peroxidation products (Prabakaran and Ashok kumar 2013). In the current study, GSH level s in the kidneys were found to be higher in the chebulagic acid -treated rats. Chebulagic acid is hypothesized to have anti-inflammatory effects that contribute to its ability to prevent diabetic nephropathy. Many proinflammatory mediators, namely anti-inflammatory cytokines like IL-10, which seem to control inflammatory processes, have been related to the development of diabetic nephropathy. When IL-10 prevented NF-B from acting, less proinflammatory cytokine was produced and macrophage death was inhibited. Excessive production of reactive oxygen species (ROS) activates nuclear factor-kappa B (NF-κB), which increases the release of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6. The release of pro-inflammatory cytokines promotes the production of reactive oxygen species (ROS) (Mason and Wahab 2003 ). TNF-α plays an important role in diabetic nephropathy by increasing the production of ROS, inducing renal cell apoptosis, increasing albumin permeability in the glomerulus, and activating the release of vasoconstrictive mediators in the mesangial cell, resulting in reduced flood flow (Ni et al. 2019 ). Chebulagic acid and metformin supplementation was successful in lowering the levels of renal TNF-α, IL-1β, and IL-6 and increasing the levels of IL-10 (anti-inflammatory cytokine) in diabetic rats, implying that chebulagic acid supplementation could be used to avoid inflammatory responses in diabetic rats. Similar to this, the immunohistochemistry expression levels of NF-B and TNF-α were examined in the renal tissue of diabetic rats, and their increased expression levels were nearly normalized by the injection of chebulagic acid. The mRNA levels of IR, IRS-1, Akt, and GLUT 4 in the gastrocnemius muscle of diabetic rats were considerably lower, indicating heightened oxidative stress and inflammatory markers, which in turn reduced these insulin signaling pathways. These findings are consistent with earlier publications (Babu et al.2020; Deenadayalan et al.2021; Mahmoud et al.2021). Surprisingly, giving diabetic rats chebulagic acid plus metformin enhanced their ability to produce insulin, which might be attributed to the activation of the IR/IRS-1/Akt pathway in the gastrocnemius muscle. Also, after administering chebulagic acid to diabetic rats, better histological and ultrastructural alterations were seen in the kidney tissue, providing additional evidence to corroborate the biochemical results. Conclusion Because of their accessibility, lack of side effects, and cost-effectiveness, natural products high in polyphenols, such as plant extracts and their bioactive constituents, are intriguing therapeutic candidates that require greater research. These products may be used to treat and prevent type 2 diabetes. Chebulagic acid comes from plants, thus the current research reveals that it has an antihyperglycemic impact on HFD/STZ-induced diabetic nephropathy by lowering oxidative stress and inflammation through up-regulation of (IR/IRS-1/Akt and GLUT4) genes implicated in the insulin signaling cascade. For future possibilities of the chemical being employed as an anti-diabetic medication, more human clinical trials are advised. Declarations Author contributions RS: paper draft and experimental supervision; VG: experiments conducted; KM: draft correction and editing. PJ: Editing and final correction. All authors have read and approved the final submission. Ethics approval The experimental protocol was approved by the Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (IAEC No: 16/2016). Acknowledgements The First author gratefully acknowledges the DST-SERB (Early Career Research Award- File No ECR/2016/001693), New Delhi, India, for providing financial support to purchase chebulagic acid to carry out this research work. References Abou-Hany HO, Atef H, Said E, Elkashef HA, Salem HA (2018) Crocin mediated amelioration of oxidative burden and inflammatory cascade suppresses diabetic nephropathy progression in diabetic rats. Chem Biol Interact 284:90-100. https://doi.org/10.1016/j.cbi.2018.02.001 Asrafuzzaman M, Cao Y, Afroz R, Kamato D, Gray S, Little PJ (2017) Animal models for assessing the impact of natural products on the aetiology and metabolic pathophysiology of Type 2 diabetes. Biomed Pharmacother 89:1242-1251. https://doi.org/10.1016/j.biopha.2017.03.010 Athira AP, Abhinand CS, Saja K, Helen A, Reddanna P, Sudhakaran PR (2017) Anti-angiogenic effect of chebulagic acid involves inhibition of the VEGFR2- and GSK-3β-dependent signaling pathways. Biochem. Cell Biol 95(5):563-570. https://doi.org/10.1139/bcb-2016-0132 Ayala A, Muñoz MF, Argüelles S (2014) Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev 2014:360438. https://doi.org/10.1155/2014/360438 Babu S, Krishnan M, Rajagopal P, Periyasamy V, Veeraraghavan V, Govindan R, Jayaraman S (2020) Beta-sitosterol attenuates insulin resistance in adipose tissue via IRS-1/Akt mediated insulin signaling in high fat diet and sucrose induced type-2 diabetic rats. Eur. J. Pharmacol 873:173004. https://doi.org/10.1016/j.ejphar.2020.173004 Baker H, Frank O, DeAngelis B, Feingold S (1980) Plasma tocopherol in man at various times after ingesting free or acetylated tocopherol. Nutr Rep Int 21:531–536. Bhagavathy S, Sumathi P (2012) Stabilization of membrane bound ATPases and lipid peroxidation by carotenoids from Chlorococcum humicola in Benzo(a)pyrene induced toxicity. Asian Pac J Trop Biomed (5):380-4. https://doi.org/10.1016/S2221-1691(12)60060-4. Borgohain MP, Chowdhury L, Ahmed S, Bolshette N, Devasani K, Das TJ, Mohapatra A, Lahkar M (2017) Renoprotective and antioxidative effects of methanolic Paederia foetida leaf extract on experimental diabetic nephropathy in rats. J. Ethnopharmacol 198:451-459. https://doi.org/10.1016/j.jep.2017.01.035 Brigelius-Flohé R, Banning A, Schnurr K (2003) Selenium-dependent enzymes in endothelial cell function. Antioxid Redox Signal. 2:205-215. https://doi.org/10.1089/152308603764816569. Cemek M, Kağa S, Simşek N, Büyükokuroğlu ME, Konuk M (2008) Antihyperglycemic and antioxidative potential of Matricaria chamomilla L. in streptozotocin-induced diabetic rats. J. Nat. Med. 62(3):284-93. https://doi.org/10.1007/s11418-008-0228-1. Cloete L (2022). Diabetes mellitus: an overview of the types, symptoms, complications and management. Nurs Stand.37(1):61-66. https://doi.org/10.7748/ns.2021.e11709. Danesh FR, Sadeghi MM, Amro N, Kanwar YS (2002) 3-Hydroxy-3-methylglutaryl CoA reductase inhibitors prevent high glucose-induced proliferation of mesangial cells via modulation of Rho GTPase/ p21 signaling pathway: Implications for diabetic nephropathy. Proc. Natl. Acad. Sci. U S A. 99(12):8301-8305. https://doi.org/10.1073/pnas.122228799 Deenadayalan A, Subramanian V, Paramasivan V, Veeraraghavan VP, Rengasamy G, Sadagopan JC, Jayaraman S (2021) Stevioside Attenuates Insulin Resistance in Skeletal Muscle by Facilitating IR/IRS-1/Akt/GLUT 4 Signaling Pathways: An In Vivo and In Silico Approach. Molecules. 26(24):7689. https://doi.org/10.3390/molecules26247689 Ellman, GL (1959). Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70–77. https://doi.org/10.1016/0003-9861(59)90090-6 Fidan AF, Dündar Y, (2008) The effects of Yucca schidigera and Quillaja saponaria on DNA damage, protein oxidation, lipid peroxidation, and some biochemical parameters in streptozotocin-induced diabetic rats. J. Diabetes Complications 22(5):348-356. https://doi.org/10.1016/j.jdiacomp.2007.11.010 Franco R, Cidlowski JA (2012) Glutathione efflux and cell death. Antioxid Redox Signal. 17(12):1694-713. https://doi.org/10.1089/ars.2012.4553. Fourney RM, Miyakoshi J, Day III, RS, Paterson MC (1988) Northern blotting: efficient RNA staining and transfer. Bethesda Res. Lab. Focus 10(1):5,6. Ganesh V, Ramalingam S, Karuppiah, M (2022) Chebulagic acid attenuates HFD/streptozotocin induced impaired glucose metabolism and insulin resistance via up regulations of PPAR γ and GLUT 4 in type 2 diabetic rats. Toxicology Mechanisms and Methods 32(3):159-170. https://doi.org/10.1080/15376516.2021.1976333 Gothandam K, Ganesan VS, Ayyasamy T, Ramalingam S (2019) Antioxidant potential of theaflavin ameliorates the activities of key enzymes of glucose metabolism in high fat diet and streptozotocin - induced diabetic rats. Redox Rep 1:41-50. https://doi.org/10.1080/13510002.2019.1624085 Governa P, Baini G, Borgonetti V, Cettolin G, Giachetti D, Magnano AR, Miraldi E, Biagi M (2018). Phytotherapy in the Management of Diabetes: A Review. Molecules. 23(1):105. https://doi.org/10.3390/molecules23010105 Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione-S-transferase: the first step in mercapturic acid formation. J. Biol. Chem 249:7130–7139. https://doi.org/10.1016/S0021-9258(19)42083-8 Huang YN, Zhao DD, Gao B, Zhong K, Zhu RX, Zhang Y, Xie WJ, Jia LR, Gao H (2012) Anti-hyperglycemic effect of chebulagic acid from the fruits of Terminalia chebula Retz. Int. J. Mol. Sci., 13(5):6320-33. https://doi.org/10.3390/ijms13056320 Hulsizer AL, Witte AP, Attridge RL, Urteaga EM. (2023) Hyperglycemia Post-Influenza Vaccine in Patients with Diabetes. Ann Pharmacother 57(1):51-54. https://doi.org/10.1177/10600280221098101 Jiang, ZY, Hunt JV, Wolff SP (1992) Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal. Biochem. 202:384–389. https://doi.org/10.1016/0003-2697(92)90122-N Johnson SA, Spurney RF (2015) Twenty years after ACEIs and ARBs: emerging treatment strategies for diabetic nephropathy. Am. J. Physiol. Renal. Physiol 309(10):F807-820 https://doi.org/10.1152/ajprenal.00266.2015 Kahn SE (2008) The relative contributions of insulin resistance and beta cell dysfunction in the pathophysiology of type-2 diabetes. Diabetologia 46:3–19. https://doi.org/10.1007/s00125-002-1009-0 Kim HJ, Kim J, Kang KS, Lee KT, Yang HO (2014) Neuroprotective Effect of Chebulagic Acid via Autophagy Induction in SH-SY5Y Cells. Biomol Ther 22(4):275-81. https://doi.org/10.4062/biomolther.2014.068 Kooti W, Farokhipour M, Asadzadeh Z, Ashtary-Larky D, Asadi-Samani M (2016) The role of medicinal plants in the treatment of diabetes: a systematic review. Electron Physician 8(1):1832-42. https://doi.org/ 10.19082/1832. Kumar G, Sharmila Banu G, Murugesan AG, Rajasekara Pandian M (2007) Effect of Helicteres isora . Bark Extracts on Brain Antioxidant Status and Lipid Peroxidation in Streptozotocin Diabetic Rats, Pharmaceutical Biology 45(10):753-759. https://doi.org/10.1080/13880200701585782 Liu W, Shang P, Liu T, Xu H, Ren D, Zhou W, Wen A, Ding Y (2017) Gastroprotective effects of chebulagic acid against ethanol-induced gastric injury in rats. Chem. Biol. Interact 278:1-8. https://doi.org/10.1016/j.cbi.2017.09.019 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with folinphenol reagent. J. Biol. Chem., 193:265–275. https://doi.org/10.1016/S0021-9258(19)52451-6 Mahmoud MF, Abdelaal S, Mohammed HO, El-Shazly AM, Daoud R, El Raey MA, Sobeh M (2021) Syzygium jambos extract mitigates pancreatic oxidative stress, inflammation and apoptosis and modulates hepatic IRS-2/AKT/GLUT4 signaling pathway in streptozotocin-induced diabetic rats. Biomed. Pharmacother 142:112085. https://doi.org/10.1016/j.biopha.2021.112085 Maritim AC, Sanders RA, Watkins JB (2003). 3rd. Diabetes, oxidative stress, and antioxidants: a review. J. Biochem. Mol. Toxicol., 17(1):24-38. https://doi.org/10.1002/jbt.10058 Mason RM, Wahab NA (2003) Extracellular matrix metabolism in diabetic nephropathy. J. Am. Soc. Nephrol.5:1358-73. https://doi.org/ 10.1097/01.ASN.0000065640. 77499.D7 Masella R, Varì R, D'Archivio M, Di Benedetto R, Matarrese P, Malorni W, Scazzocchio B, Giovannini C (2004) Extra virgin olive oil biophenols inhibit cell-mediated oxidation of LDL by increasing the mRNA transcription of glutathione-related enzymes. J Nutr 134(4):785-791. https://doi.org/ 10.1093/jn/134.4.785. Matthews D.R, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC (1985) Homeostasis model assessment: Insulin resistance and beta‑cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412‑9. https://doi.org/10.1007/BF00280883 Murli R, Saravanan R (2012) Antidiabetic effect of D-limonene, a monoterpene in streptozotocin-induced diabetic rats. Biomed. Prev. Nutr 2:269–275. https://doi.org/10.1016/j.bionut.2012.08.008 Narasimhan A, Chinnaiyan M, Karundevi B (2015) Ferulic acid exerts its antidiabetic effect by modulating insulin-signalling molecules in the liver of high-fat diet and fructose-induced type-2 diabetic adult male rat. Appl Physiol Nutr Metab 40(8):769-81. https://doi.org/10.1139/apnm-2015-0002 Ni Z, Guo L, Liu F, Olatunji OJ, Yin M (2019) Allium tuberosum alleviates diabetic nephropathy by supressing hyperglycemia-induced oxidative stress and inflammation in high fat diet/streptozotocin treated rats. Biomed Pharmacother 112:108678. https://doi.org/10.1016/j.biopha.2019.108678 Niehius WG, Samuelsson D (1968) Formation of malondialdehyde from phospholipids arachidonate during microsomal lipid peroxidation. Eur. J. Biochem., 6:26–130. https://doi.org/10.1111/j.1432-1033.1968.tb00428.x Omaye ST, Turnbull TD, Sauberlich HE, (1979) Selected method for the determination of ascorbic acid in animal cells, tissues and fluid. In:McCormic DB, Wright DL, editors. Methods enzymol. Vol. 62.New York: Academic Press; p. 3–11. https://doi.org/ 10.1016/0076-6879(79)62181-x Opara EC (2002) Oxidative stress, micronutrients, diabetes mellitus and its complications. J. R. Soc. Promot. Health.122(1):28-34. https://doi.org/10.1177/146642400212200112 Othman MS, Khaled AM, Al-Bagawi AH, Fareid MA, Ghany RA, Habotta OA, Abdel Moniem AE (2021) Hepatorenal protective efficacy of flavonoids from Ocimum basilicum extract in diabetic albino rats: A focus on hypoglycemic, antioxidant, anti-inflammatory and anti-apoptotic activities. Biomed. Pharmacother 144:112287. https://doi.org/10.1016/j.biopha.2021.112287 Piccoli GB, Grassi G, Cabiddu G, Nazha M, Roggero S, Capizzi I, De Pascale A, Priola AM, Vico CD, Maxia S, Loi V, Asunis AM, Pani A, Veltri A (2015) Diabetic Kidney Disease: A Syndrome Rather Than a Single Disease. Rev. Diabet. Stud 12(1-2):87-109. https://doi.org/10.1900/RDS.2015.12.87 Prabakaran D, Ashokkumar N (2013). Protective effect of esculetin on hyperglycemia-mediated oxidative damage in the hepatic and renal tissues of experimental diabetic rats. Biochimie. 95(2):366-373. https://doi.org/10.1016/j.biochi.2012.10.008 Ramalingam S, Karuppiah M, Thiruppathi M, Palanivelu S, Panchanatham S (2020) Antioxidant potential of biflavonoid attenuates hyperglycemia by modulating the carbohydrate metabolic enzymes in high fat diet/streptozotocin induced diabetic rats. Redox Rep. 1:1-10. https://doi.org/10.1080/13510002.2020.1722914 Rastogi M, Ojha RP, Rajamanickam GV, Agrawal A, Aggarwal A, Dubey GP (2008) Curcuminoids modulates oxidative damage and mitochondrial dysfunction in diabetic rat brain. Free Radic. Res. 42(11-12):999-1005. https://doi.org/10.1080/10715760802571988 Rehman MU, Rashid SM, Rasool S, Shakeel S, Ahmad B, Ahmad SB (2019. Zingerone (4-(4-hydroxy-3-methylphenyl)butan-2-one) ameliorates renal function via controlling oxidative burst and inflammation in experimental diabetic nephropathy. Arch. Physiol. Biochem., 125(3):201-209. https://doi.org/10.1080/13813455.2018.1448422 Remuzzi G, Ruggenenti P, Perna A, Dimitrov BD, de Zeeuw D, Hille DA, Shahinfar S, Carides GW, Brenner BM (2004) Continuum of renoprotection with losartan at all stages of type 2 diabetic nephropathy: a post hoc analysis of the RENAAL trial results. J. Am. Soc.Nephrol., (12):3117-3125. https://doi.org/10.1097/01.ASN.0000146423.71226.0C Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179:588–590. https://doi.org/10.1126/science.179.4073.588 Sambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Serafini M, Peluso I (2016) Functional Foods for Health: The Interrelated Antioxidant and Anti-Inflammatory Role of Fruits, Vegetables, Herbs, Spices and Cocoa in Humans. CurrPharmDes.22(44):67016715. https://doi.org/ 10.2174/1381612823666161123094235 Shang G, Gao P, Zhao Z, Chen Q, Jiang T, Zhang N, Li H (2013) 3,5-Diiodo-l-thyronine ameliorates diabetic nephropathy in streptozotocin-induced diabetic rats. Biochim Biophys Acta 1832(5):674-84. https://doi.org/10.1016/j.bbadis.2013.01.023 Shanmuganathan S, Angayarkanni N (2018) Chebulagic acid Chebulinic acid and Gallic acid, the active principles of Triphala, inhibit TNFα induced pro-angiogenic and pro-inflammatory activities in retinal capillary endothelial cells by inhibiting p38, ERK and NFkB phosphorylation. Vascul Pharmacol 108:23-35. https://doi.org/10.1016/j.vph.2018.04.005 Sinha KA (1972) Colorimetric assay of catalase. Anal. Biochem., 47:389–394. https://doi.org/10.1016/0003-2697(72)90132-7 Skovsø S (2014) Modeling type 2 diabetes in rats using high fat diet and streptozotocin. J. Diabetes Investig. 5(4):349-58. https://doi.org/10.1111/jdi.12235 Stringer MD, Gorog PG, Freeman A, Kakkar VV (1989) Lipid peroxides and atherosclerosis. British Medical Journal 298:281–284. https://doi.org/10.1136/bmj.298.6669.281 Syed FZ (2022) Type 1 Diabetes Mellitus. Ann Intern Med.175(3):33-48. https://doi.org/10.7326/AITC202203150 Teck J (2022) Diabetes-Associated Comorbidities. Prim Care 49(2):275-286. https://doi.org/10.1016/j.pop.2021.11.004 Trinder P (1969). Determination of glucose in blood using glucose oxidase with an alternative oxygen receptor. Ann. Clin. Biochem., 6:24–27. https://doi.org/10.1177/000456326900600108 Vaz de Castro PAS, Bitencourt L, de Oliveira Campos JL, Fischer BL, Soares de Brito SBC, Soares BS, Drummond JB, Simões E Silva AC (2022) Nephrogenic diabetes insipidus: a comprehensive overview. J Pediatr Endocrinol Metab 35(4):421-434. https://doi.org/10.1515/jpem-2021-0566 Wang K, Wang H, Liu Y, Shui W, Wang J, Cao P, Wang H, You R, Zhang Y (2018) Dendrobium officinale polysaccharide attenuates type 2 diabetes mellitus via the regulation of PI3K/Akt-mediated glycogen synthesis and glucose metabolism, J. Funct. Foods 40:261–271. https://doi.org/10.1016/j.jff.2017.11.004 Wang M, Li Y, Hu X, Wang M, (2018) Chebulinic acid derived from triphala is a promising antitumour agent in human colorectal carcinoma cell lines. BMC Complement Altern Med. 18(1):342. https://doi.org/10.1186/s12906-018-2412-5 Wild S, Roglic G, Green A, Sicree R, King H (2004) Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27(5):1047-53. https://doi.org/10.2337/diacare.27.5.1047 Wu D, Wen W, Li C, Qi R, Xia Zhao J, Hua Lu C, Yan Zhong Y (2012) Ameliorative effect of berberine on renal damage in rats with diabetes induced by high-fat diet and st streptozotocin. Phytomedicine 19:712–718. https://doi.org/10.1016/j.phymed.2012 .03.003 Wu G Fang YZ, Yang S, Lupton JR, Turner ND (2004) Glutathione metabolism and its implications for health1 J. Nutr 134(3):489-92. https://doi.org/10.1093/jn/134.3.489 Xie W, Xing D, Sun H, Wang W, Ding Y, Du L (2005) The effects of Ananas comosus L. leaves on diabetic-dyslipidemic rats induced by alloxan and a high fat/ high-cholesterol diet. Am. J. Chinese Med 33:95–105. https://doi.org/10.1142/S0192415X05002692 Xu Y, Bai L, Chen X, Li Y, Qin Y, Meng X, Zhang Q (2018) 6-Shogaol ameliorates diabetic nephropathy through anti-inflammatory, hyperlipidemic, anti-oxidative activity in db/db mice. Biomed.Pharmacother.97:633-641. https://doi.org/10.1016/j.biopha.2017.10.084 Table Table 1. Effects of chebulagic acid on the levels of urea and creatinine in control and experimental animals Parameters Control Diabetes Induced Diabetes + Chebulagic acid (50mg/kg b.w) Diabetes + Metformin (50mg/kg b.w) Urea (mg/dl) 25.40±1.80 80.21±6.89* 38.79±3.29 # 36.9±2.98 Creatinine (mg/dl) 0.61±0.03 1.9±0.07* 0.9±0.05 # 0.87±0.04 Values are given as mean ± S.D for six animals in each group. Values are considered significantly different at P < 0.05 with post hoc LSD test P < 0.05. statistical differences are expressed as (* # †) * Control vs Diabetic rats # Diabetic rats vs Diabetic rats treated with chebulagic acid (100mg/kg b.w) † Diabetic rats treated with chebulagic acid (50mg/kg b.w ) vs Diabetic rats treated with Metformin (50mg/kg b.w) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3859769","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267354577,"identity":"a9b04e67-dfc7-4625-ba0b-e5be1baaa25d","order_by":0,"name":"Ganesh Vasu","email":"","orcid":"","institution":"Meenakshi Academy of Higher Education and Research","correspondingAuthor":false,"prefix":"","firstName":"Ganesh","middleName":"","lastName":"Vasu","suffix":""},{"id":267354578,"identity":"d240b650-a65c-466e-94ed-9fc37116af82","order_by":1,"name":"Sundaram Ramalingam","email":"","orcid":"","institution":"Tagore Medical College \u0026 Hospital Rathinamangalam","correspondingAuthor":false,"prefix":"","firstName":"Sundaram","middleName":"","lastName":"Ramalingam","suffix":""},{"id":267354579,"identity":"ff7fb9a1-4763-42b2-84d4-8639125321e1","order_by":2,"name":"Karuppiah Muthu","email":"","orcid":"","institution":"Manomaniam Sundaranar University","correspondingAuthor":false,"prefix":"","firstName":"Karuppiah","middleName":"","lastName":"Muthu","suffix":""},{"id":267354580,"identity":"6fecde54-7f74-4720-9c8d-b96a62df7b92","order_by":3,"name":"Sundaram Ramalingam Tutor","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYJACCRDBxnD++48PIAY7sVr4GA8YSM4AaWEmVosc8wEDaR4Qi5AW+Rm5B2/83GMXzcZ2IMHY5tc2eT5mBsYPH3NwazG4kZds2fMsObeN58CB5Ny+24ZtzAzMkjO34dEikWMmwXOAObdN4mDD4dye24xALWzMvHi0yM/IMZP8c6A+t03+MWOzZc9te4JaGG7kmEnzHDic28ZwjJmZ4cftRIJaDM68MbaWOXAcqOUMG2Nvw+3kNmbGZrx+kW/PMbz55kB17vyGM2wMP/7ctp3f3nzww0d8DkMBjG1gsoFY9SDwhxTFo2AUjIJRMFIAAAKsUZd2/4yUAAAAAElFTkSuQmCC","orcid":"","institution":"Tagore Medical College \u0026 Hospital Rathinamangalam","correspondingAuthor":true,"prefix":"","firstName":"Sundaram","middleName":"Ramalingam","lastName":"Tutor","suffix":""},{"id":267354581,"identity":"5a05645f-1f8e-4ad9-81ea-09889490a983","order_by":4,"name":"Prabhakaran J","email":"","orcid":"","institution":"Tagore Medical College \u0026 Hospital Rathinamangalam","correspondingAuthor":false,"prefix":"","firstName":"Prabhakaran","middleName":"","lastName":"J","suffix":""}],"badges":[],"createdAt":"2024-01-13 10:14:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3859769/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3859769/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49746587,"identity":"0082299e-d9b4-4c56-9760-7b585c31953a","added_by":"auto","created_at":"2024-01-17 11:04:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A, B \u0026amp; C Effects of chebulagic acid on plasma glucose, insulin levels and HOMA-IR index in control and experimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues are given as mean ± S.D for six animals in each group. Values are considered significantly different at \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 with post hoc LSD test \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05. statistical differences are expressed as \u003cstrong\u003e(* # †)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e* Control vs Diabetic rats\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e# \u003c/strong\u003e\u0026nbsp;Diabetic rats vs Diabetic rats treated with chebulagic acid (100mg/kg b.w)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e†\u003c/strong\u003e Diabetic rats treated with chebulagic acid (50mg/kg b.w ) vs Diabetic rats treated with\u003c/p\u003e\n\u003cp\u003eMetformin (50mg/kg b.w)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/35e40e1c567d4b76bd6ca0da.png"},{"id":49746586,"identity":"17ae9ce3-4d81-4335-b5a1-88ed8fd4a877","added_by":"auto","created_at":"2024-01-17 11:04:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of chebulagic acid on the levels of TBARS and HP in the renal tissues of control and experimental rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues are given as mean ± S.D for six animals in each group. Values are considered significantly different at P \u0026lt; 0.05 with post hoc LSD test P \u0026lt; 0.05. statistical differences are expressed as (* # †)\u003c/p\u003e\n\u003cp\u003e* Control vs Diabetic rats\u003c/p\u003e\n\u003cp\u003e# Diabetic rats vs Diabetic rats treated with chebulagic acid (100mg/kg b.w)\u003c/p\u003e\n\u003cp\u003e† Diabetic rats treated with chebulagic acid (50mg/kg b.w ) vs Diabetic rats treated with\u003c/p\u003e\n\u003cp\u003eMetformin (50mg/kg b.w)\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/cc738851bfdf06af1f0ca8bc.png"},{"id":49746741,"identity":"76f6dc23-ff71-4f7c-b76d-f09e8a53a4f5","added_by":"auto","created_at":"2024-01-17 11:12:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":17077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of chebulagic acid on enzymatic antioxidants activities (SOD, CAT, GPX and GST) in the renal tissue of control and experimental rats.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues are given as mean ± S.D for six animals in each group. Values are considered significantly different at P \u0026lt; 0.05 with post hoc LSD test P \u0026lt; 0.05. statistical differences are expressed as (* # †)\u003c/p\u003e\n\u003cp\u003e* Control vs Diabetic rats\u003c/p\u003e\n\u003cp\u003e#\u0026nbsp; Diabetic rats vs Diabetic rats treated with chebulagic acid (100mg/kg b.w)\u003c/p\u003e\n\u003cp\u003e† Diabetic rats treated with chebulagic acid (50mg/kg b.w ) vs Diabetic rats treated with\u003c/p\u003e\n\u003cp\u003eMetformin (50mg/kg b.w)The activities of enzymes are expressed as follows: SOD – One unit of activity was taken as the enzyme quantity, which gave 50% inhibition of nitroblue tetrazolium reduction in 1 min/mg protein; CAT – μmoles of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e consumed/minute; GPx – μg of glutathione consumed/minute/mg protein; GST- μmoles of 1-chloro 2,4-dinitrobenzene-GSH conjugate formed/minute/mg protein.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/542bf7b495fa22487a50b108.png"},{"id":49746745,"identity":"d546df49-1881-4682-b566-914170e9aba2","added_by":"auto","created_at":"2024-01-17 11:12:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14386,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of chebulagic acid on the levels of non-enzymatic antioxidants in renal tissues of control and experimental rats\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues are given as mean ± S.D for six animals in each group. Values are considered significantly different at P \u0026lt; 0.05 with post hoc LSD test P \u0026lt; 0.05. statistical differences are expressed as (* # †)\u003c/p\u003e\n\u003cp\u003e* Control vs Diabetic rats\u003c/p\u003e\n\u003cp\u003e# Diabetic rats vs Diabetic rats treated with chebulagic acid (100mg/kg b.w)\u003c/p\u003e\n\u003cp\u003e† Diabetic rats treated with chebulagic acid (50mg/kg b.w ) vs Diabetic rats treated with\u003c/p\u003e\n\u003cp\u003eMetformin (50mg/kg b.w)\u003c/p\u003e\n\u003cp\u003eUnits are expressed as: μM/mg tissue of Vitamin C, Vitamin E and Reduced glutathione (GSH)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/bfb6021628a5ad4130c12fe7.png"},{"id":49746742,"identity":"964d4daa-e023-417f-9d35-438b390ab9fb","added_by":"auto","created_at":"2024-01-17 11:12:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":13019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of chebulagic acid on the levels of IL-1β, IL-6, TNF-α, NF-α, p65 subunit of NF-κβ, and IL-10 in plasma of control and experimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUnits are expressed as: \u003c/strong\u003eIL-1β, IL-6, IL-10, TNF-α pg/ml and NF-KB p65 – ng/ml\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues are given as mean ± S.D for six animals in each group. Values are considered significantly different at P \u0026lt; 0.05 with post hoc LSD test P \u0026lt; 0.05. statistical differences are expressed as (* # †)\u003c/p\u003e\n\u003cp\u003e* Control vs Diabetic rats\u003c/p\u003e\n\u003cp\u003e# Diabetic rats vs Diabetic rats treated with chebulagic acid (100mg/kg b.w)\u003c/p\u003e\n\u003cp\u003e† Diabetic rats treated with chebulagic acid (50mg/kg b.w ) vs Diabetic rats treated with\u003c/p\u003e\n\u003cp\u003eMetformin (50mg/kg b.w)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/1d42b297e28649fd68c4f6c9.png"},{"id":49746589,"identity":"83e73d31-08f2-4f95-8f8e-7aacfbfc1a9d","added_by":"auto","created_at":"2024-01-17 11:04:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":217666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical localization of NF-κB in kidney of control and experimental animals (40×).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Control; (B)Diabetes Induced rats (C) Diabetes + Chebulagic acid (D) Diabetes + Metformin\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/f5742215e12edac2fb4259af.png"},{"id":49746743,"identity":"7d47a097-6201-4528-a00b-f2e4171293d1","added_by":"auto","created_at":"2024-01-17 11:12:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":245059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical localization of TNF-α in kidney of control and \u0026nbsp;experimental animals (40×).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Control; (B)Diabetes Induced rats (C) Diabetes + Chebulagic acid (D) Diabetes + Metformin\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/8b5aaf659a23fb72c9ea08f8.png"},{"id":49746593,"identity":"659d8170-f4ce-4819-95e6-be1e5f7efa44","added_by":"auto","created_at":"2024-01-17 11:04:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":72970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of chebulagic acid on the mRNA expression of IR/IRS-1/Akt/ GLUT4 in skeletal muscle of control and experimental animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues are given as mean ± S.D for six animals in each group. Values are considered significantly different at P \u0026lt; 0.05 with post hoc LSD test P \u0026lt; 0.05. statistical differences are expressed as (* # †)\u003c/p\u003e\n\u003cp\u003e* Control vs Diabetic rats\u003c/p\u003e\n\u003cp\u003e# Diabetic rats vs Diabetic rats treated with chebulagic acid (100mg/kg b.w)\u003c/p\u003e\n\u003cp\u003e† Diabetic rats treated with chebulagic acid (50mg/kg b.w) vs Diabetic rats treated with\u003c/p\u003e\n\u003cp\u003eMetformin (50mg/kg b.w)\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/695c2de1054d8c98209a73df.png"},{"id":49746744,"identity":"35f1458d-bb94-42c4-b409-7462f322ec05","added_by":"auto","created_at":"2024-01-17 11:12:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":239486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological section of renal tissue of control and experimental rats (40X).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Control; (B)Diabetes Induced rats (C) Diabetes + Chebulagic acid (D) Diabetes + Metformin\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/67cd0d7554f80958bacb4e8c.png"},{"id":49746594,"identity":"7a60e8cd-bb55-45e1-9f3b-91209ed70d36","added_by":"auto","created_at":"2024-01-17 11:04:31","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":228301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUltra structure o renal tissue of control and experimental rats (100k).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Control; (B)Diabetes Induced rats (C) Diabetes + Chebulagic acid (D) Diabetes + Metformin\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/e66fff83185985689f779d9a.png"},{"id":50230308,"identity":"58380599-e8c8-4643-93e3-b6abf462b4df","added_by":"auto","created_at":"2024-01-26 20:37:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1721288,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3859769/v1/f0d05342-4167-4295-a2be-7b962bce98fc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chebulagic Acid Alleviates Inflammation Via Regulation of Skeletal Muscle IR/IRS-1/AKT/GLUT4 Signaling Pathway in Diabetic Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe metabolic disorder known as diabetes mellitus affects the metabolism of carbohydrates, lipids, and proteins by causing insulin resistance and reduced insulin secretion by pancreatic β-cells (Kahn 2008; Wang et al. 2018; Syed 2022). Lifelong type 2 diabetes mellitus (T2DM) causes an increase in morbidity and mortality, which lowers overall quality of life (Teck 2022; Hulsizer et al. 2023). Persistent hyperglycemia is associated with long-term harm to and functioning of the kidneys, nerves, eyes, and cardiovascular system (Wild et al. 2004). Since it causes end-stage renal failure in diabetic individuals, diabetic nephropathy (DN) is the most hazardous of all T2DM side effects (Rehman et al., 2019; Vaz de Castro et al.2022). A diabetic patient's risk of developing diabetic nephropathy ranges from 15 to 40% throughout the course of their lifetime (Shang et al.2013)\u003c/p\u003e\n\u003cp\u003eAs extracellular matrix accumulates, glomerular basement membranes degrade, and glomeruli shrink, the kidney's structural and functional characteristics are affected, and this condition is known as diabetic nephropathy (Piccoli et al. 2015; Xu et al.2018). The generation of reactive oxygen species (ROS), which in turn triggers oxidative stress and inflammation, has been shown to occur as a result of persistent hyperglycemia, according to current studies. Also, a rise in\u0026nbsp;ROS generation can activate a number of cellular pathways that aid in the course of illness due to the weakening of antioxidant defense mechanisms (Abou-Hany et al.2018).\u003c/p\u003e\n\u003cp\u003eThe two primary therapeutic modalities that treat or prevent the onset and progression of diabetic nephropathy are blood glucose control and renin-angiotensin system blockade. The low effectiveness and numerous untoward side effects of current treatments, however, have not reduced the disease's incidence rate (Johnson and \u0026nbsp; Spurney 2015). Recently, there has been a lot of focus on the development of innovative, effective, and risk-free therapeutic alternatives that target several pathways (glycemic management, oxidative stress inhibition, and inflammatory pathways) implicated in the development of diabetic nephropathy (Abou-Hany 2018). Because of this, utilizing a novel medication combining hypoglycemic, anti-inflammatory, and antioxidant qualities may shield against diabetic nephropathy. (Kooti et al.2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGiven these details, chebulagic acid, an organic compound present in the fruits of\u0026nbsp;\u003cem\u003eTerminalia chebula\u003c/em\u003e Retz., a Combretaceae species, was chosen. Chebulagic acid possesses anti-inflammatory, anti-tumor, anti-angiogenic, anti-hyperglycemic, neuroprotective, and gastroprotective properties, according to Shanmuganathan and Angayarkanni (2018), Wang et al. (2018), Athira et al. (2017), Huang et al. (2012), and other studies (Kim et al. 2014; Liu et al. 2017). Chebulagic acid, which we recently discovered increases PPAR-γ and GLUT-4, decreases HFD/streptozotocin-induced poor glucose metabolism and insulin resistance in type 2 diabetic rats (Ganesh et al. 2021). The objective of the current study was to investigate the potential anti-inflammatory, anti-oxidant, and anti-hyperglycemic effects of chebulagic acid in rats that underwent an HFD/streptozotocin-induced modification in renal function.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDrugs and Chemicals\u003c/h2\u003e \u003cp\u003eStreptozotocin, metformin, total RNA isolation reagent (TRIR), and chebulagic acid (98% purity Catalog No. CFN92295, respectively, were acquired from Chem Faces, China; Invitrogen, USA; Sisco Research Laboratories, India; and Merck, Germany. Additional substances of an analytical grade were employed in this investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Animals\u003c/h2\u003e \u003cp\u003eMale adult albino Wistar rats weighing between 200 and 220 g were acquired from the Sri Muthukumaran Medical College Hospital \u0026amp; Research Institute in Mangadu, Chennai, Tamil Nadu, India. Rats were kept in sterile, clean polypropylene cages in accredited animal facilities with a 12-h light/dark cycle, constant temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C), and free access to commercial rat food (Pranav Agro Industries Ltd., Pune, Maharashtra, India) and water.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eType 2 Diabetes Induction\u003c/h2\u003e \u003cp\u003eFour groups of six animals each were formed from the total number of animals. 84.3% ordinary laboratory chow, 5% lard, 10% yolk powder, 0.2% cholesterol, and 0.5% bile salt were used in the rats' high-fat diet for 2 weeks (Xie et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). During a 2-week period, the animals were kept fasting all night before receiving a low dosage of streptozotocin (35 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH 4.5) (Wu et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). After 3\u0026thinsp;+\u0026thinsp;days of induction, blood was drawn from the animals and evaluated for fasting blood glucose levels using a glucometer (ACCU-Check Active, Roche, Switzerland). Rats with fasting blood glucose levels more than 300 mg/dl were deemed to have diabetes. For a further 4 weeks, the diabetic rats were fed a high-fat diet.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eThe experimental animals, which included 18 diabetic and 6 normal rats, were split into four groups, each of which had at least six rats (n\u0026thinsp;=\u0026thinsp;6). In a prior work, we employed three different doses of chebulagic acid (25, 50, and 100 mg/kg b.w.) that were dissolved in 0.5 ml of 1% CMC (Carboxyl methylcellulose) in distilled water and given orally to diabetic rats using an intragastric tube for a duration of 30 days. Chebulagic acid at 50 mg/kg body weight had a substantial, metformin-comparable impact in lowering blood sugar. Because of this, the 25 mg and 100 mg/kg bw administered groups were left out of this study.Group I: normal control rats; group II: diabetic control rats; group III: diabetic\u0026thinsp;+\u0026thinsp;chebulagic acid (50 mg/kg bw) (Narasimhan et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); and group IV: diabetic\u0026thinsp;+\u0026thinsp;metformin (50 mg/kg bw).\u003c/p\u003e \u003cp\u003eThe animals underwent therapy for 30 days before being denied food overnight, given a deep anesthetic with diethyl ether, and then killed by cervical decapitation. For the purposes of separating the plasma and serum, blood samples were taken both with and without anticoagulants. Kidneys were quickly excised and washed in ice-cold saline to remove the blood. A portion of kidney tissue was minced and homogenized (10%, w/v) with 0.1 M Tris -HCl buffer (pH 7.4) and centrifuged at 3000\u0026times; g for 10 min at 4\u003csup\u003eo\u003c/sup\u003eC by a REMI cooling centrifuge. The resulting supernatant was used for enzyme assays. For histological analysis, another kidney fragment was stored in 10% buffered formalin solution. All biochemical calculations were finished within 24 h of the animal's death.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSerum Biochemistry Assessment\u003c/h2\u003e \u003cp\u003eThe manufacturer's instructions were followed when standard kits (Agappe Diagnostic Pvt. Ltd., India) were used to calorimetrically measure the serum parameters of kidney function (urea and creatinine). According to Trinder \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1969\u003c/span\u003e, the available enzymic colorimetric test was used to determine the glucose levels. Following the manufacturer's instructions, rat insulin ELISA Millipore kit was used to measure the amount of serum insulin. Lipid peroxidation and hydroperoxides were estimated in the kidney tissues by the method of Niehius and Samuelsson (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1968\u003c/span\u003e) and Jiang et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), respectively. Catalase (CAT) superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST), ascorbic acid (vitamin C), α-tocopherol (vitamin E), GSH, and protein were determined by the method of Sinha (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1972\u003c/span\u003e). Stringer et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), Rotruck et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1973\u003c/span\u003e) Habig et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1974\u003c/span\u003e), Omaye et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1979\u003c/span\u003e), Baker et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1980\u003c/span\u003e), Ellman (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1959\u003c/span\u003e), and Lowry et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1951\u003c/span\u003e), respectively. According to the methodology of Matthews et al., the homeostasis model assessment of basal insulin resistance (HOMA-IR) was computed based on the fasting blood insulin and glucose concentrations (1985).\u003c/p\u003e \u003cp\u003eHOMA-IR index\u0026thinsp;=\u0026thinsp;fasting glucose (mg/dl) \u0026times; fasting insulin (mU/ml)/405.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInflammatory Cytokines\u003c/h2\u003e \u003cp\u003ePlasma concentrations of interleukin 1β, interleukin 6 (IL6), interleukin 10 (IL10), tumor necrosis factor-alpha (TNF-α) and NF-kB p65 were determined using commercial rat ELISA kits (Ray Biotech, Inc., Norcross, GA, USA), following the instructions in respective kit manuals. Spectrophotometry at 450 nm was used to calculate the proinflammatory cytokine levels. With the use of standard cytokines, standard plots were created, and from these plots, concentrations for unidentified samples could be determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRenal Tissue Immunohistochemistry\u003c/h2\u003e \u003cp\u003eThe immunohistochemical staining of renal tissues to determine the expression of NF-κB and TNF-α was as described by Sambrook et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1989\u003c/span\u003e) with slight modification. Five millimeter sections of the paraffin-embedded kidney tissue were made. The sections underwent xylene-based deparaffinization before being rehydrated in ethanol series (100, 90, 70, 50, and 30%). The slides were then washed in phosphate buffered saline (PBS; 50 mM mono and di phosphate, 100 mM NaCl, pH 7.4) before being exposed to 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e at room temperature for 15 min to inhibit endogenous peroxidase activity. Antigen retrieval on the slides was place during a 15-min incubation with citrate buffer (10 mM) at 60\u0026deg;C. Then, the slides were incubated with blocking buffer 3% BSA for 3 h at room temperature and then, the slides were incubated overnight at 4\u0026deg;C with monoclonal anti NF-kB and TNF-α (dilution 1:200 and 1:200, Santa Cruz, CA, USA). The slides were then treated with anti-rabbit and anti-mouse detection secondary antibodies for 2 h at 4\u0026deg;C, rinsed with PBS, and incubated with DAB solution (3, 3 diaminobenzidine tetrahydrochloride 0.05%, 1X PBS-10 ml, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 0.01%). Following that, the pieces were dried using ethanol concentrations of 30, 50, 70, 90, and 100%. After counterstaining the parts with hematoxyline and mounting them with DPX, they were finished. The immunoreactive positive expression of NF-κB and TNF-α were viewed under microscope and taken photograph. Histological changes in the stained sections were viewed interpreted by a pathologist.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eReal-Time PCR\u003c/h2\u003e \u003cp\u003eWith the use of a TRIR (total RNA isolation reagent) kit, total RNA was extracted from reference and test samples. In order to fully allow the dissociation of nucleoprotein complexes, 100 mg of fresh tissue was homogenized with 1 ml TRIR, and the homogenate was then immediately transferred to a microfuge tube. This was done for 60 min at 80\u0026deg;C. After adding 0.2 ml of chloroform, the mixture was vortexed for 1 min before being chilled for 5 min at 4\u0026deg;C. For 15 min at 4\u0026deg;C, the homogenates were centrifuged at 12000 \u0026times;g. The aqueous phase (top layer) was carefully transferred to a brand-new microfuge tube, to which an equal volume of isopropanol was added, vortexed for 15 s, and then stored at 4\u0026deg;C for 10 min. At 4\u0026deg;C and 12000 \u0026times;g for 10 min, the samples were centrifuged. After discarding the supernatant, a vortex was used to wash the RNA pellet in 1 ml of 75% ethanol. The isolated RNA was estimated spectrometrically by the method of Fourney et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). The RNA concentration was expressed in microgram (\u0026micro;g).\u003c/p\u003e \u003cp\u003e According to the manufacturer's instructions, complementary DNA (cDNA) was created from 2 \u0026micro;g of total RNA using the reverse transcriptase kit from Eurogentec (Seraing, Belgium).To perform real-time PCR, the reaction mixture containing 2x reaction buffer (Takara SyBr green master mix), Forward and reverse primers of the target gene and house-keeping gene, water and β-actin (the primer sequences were listed in Table S1 as supporting information) in total volume of 45 \u0026micro;l expect the cDNA was made, mixed intensively and spun down. In individual PCR vials, about 5 \u0026micro;l of control DNA for positive control, 5\u0026micro;l of water for negative control and 5 \u0026micro;l of template cDNA for samples were taken and reaction mixture (45 \u0026micro;l) were added. 40 cycles (95\u0026deg;C for 5 min, 95\u0026deg;C for 5 s, 60\u0026deg;C for 20 s and 72\u0026deg;C for 40 s) was set up for the reaction and obtained results were plotted by the PCR machine (Stratagene MX 3000P, Agilent Technologies, 530l, Stevens Creek Blvd, Santa Clara CA, 95051) on a graph. Relative quantification was calculated from the melt and amplification curves analysis\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eKidney Histopathological Examination\u003c/h2\u003e \u003cp\u003eIn order to conduct a histological examination, a sample of renal tissue was obtained and stored in 10% formalin solution. The samples from each group were embedded in paraffin wax, cut into sections that were 5 \u0026micro;m thick, and stained with hematoxylin and eosin (H \u0026amp;E stain). A light microscope (Olympus Optical, Japan) with a 40X magnification was used to view the stained sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransmission Electron Microscopic Analysis\u003c/h2\u003e \u003cp\u003eElectron microscopy was used to examine the ultra-structure of the kidneys of normal and diabetic rats. The tissue sample is immediately submerged in the principal fixative, which is 2.5% glutaraldehyde made in a 0.1 M sodium cacodylate buffer with a pH 7.4 solution. The specimen is then cut into small bits of the size 1 mm \u0026times; 1 mm on a glass slide using a sharp blade or scalpel. The fixative was gently reintroduced to the little parts, and they were left submerged there for 4 to 8 h at 8\u0026deg;C. After that, the bits were washed three times for a total of 10 min each time using the same buffer. The secondary fixative was then added, which is 0.1% Osmium tetroxide (OsO\u003csub\u003e4\u003c/sub\u003e) produced in the same buffer. This post fixation was conducted for 2 h at 8\u0026deg;C. The surplus fixative was once again rinsed three times for a total of 10 min with the same buffer. The specimen pieces were then subjected to a graded series of treatments for 10 min each using 30, 50, 70, 80, and 90% acetone and again for 10 min each using 100% acetone, followed by two treatments of 10 min each using propylene oxide. The tissue bits were then infiltrated with the resin mixture consisting of Epon 812 resin, DDSA (dodecenyl succinic anhydride) and NMA (Nadic\u0026reg; methyl anhydride) starting with 25, 50, 75, and finally with 100% resin mixture for 2 h at each concentration. Finally, they were inserted in Easy molds at 60\u0026deg;C for 48 h using the same resin mixture with additional catalyst (DMP 30). The Leica Ultra cut R Ultra microtome with diamond or glass blades was used to extract the resin blocks from the mold, trim them, and segment them. Toludine blue was used to stain the semi-thin slices, which were then examined under a light microscope to look for any areas of interest in that specific block. After that, extremely thin pieces were cut, assembled on copper grids, and stained with lead citrate and uranyl acetate. A transmission electron microscope (BioTem H-7650 Hitachi, Japan) was used to analyze the grids at a 100 K-fold enlargement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. One-way ANOVA followed by least significant difference (LSD) test; \u003cem\u003ep-\u003c/em\u003evalue of less than 0.05 were considered to indicate statistical significance between the groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePlasma Glucose, Insulin, and HOMA-IR index\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats with diabetes had much higher blood glucose levels than control rats, but the latter had significantly lower plasma insulin levels. Chebulagic acid was provided to diabetic rats, and as compared to the diabetic rats that were not treated, the blood glucose levels were dramatically reduced and the plasma insulin levels were significantly enhanced. HOMA-IR significantly increased in diabetic rats compared to control rats in this situation. In comparison to control rats, diabetic rats' HOMA-IR index was markedly decreased by chelbulagic acid + metformin administration. The outcome from diabetic rats treated with metformin and the impact that was seen were equivalent\u0026nbsp;(Fig. 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ekidney function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum urea and creatinine values for control and experimental rats are summarized in Table 1. As comparison to untreated diabetic rats, the levels of urea and creatinine were considerably lower after 30 days of therapy with chebulagic acid and metformin. \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid Peroxidation\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 2 depicts the concentrations\u0026nbsp;of TBARS and HP in the kidneys of\u0026nbsp;rats with and without diabetes. When compared to normal rats,\u0026nbsp;diabetic\u0026nbsp;rats had higher amounts\u0026nbsp;of TBARS and HP in their kidneys. These values nearly returned\u0026nbsp;to normal\u0026nbsp;following therapy with chelbulagic\u0026nbsp;acid and metformin.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAntioxidant activities\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SOD, CAT, GPx, and GST activities were dramatically reduced in diabetic rats as compared to healthy control rats. When treated with chebulagic acid plus metformin, diabetic rats' SOD, CAT, GPx, and GST activity increased, indicating that the antioxidant enzyme systems had been nearly fully recovered\u0026nbsp;(Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eNon-enzymatic Antioxidants\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 4 displays the concentrations of non-enzymatic antioxidants in the kidney of diabetic and control rats, including\u0026nbsp;vitamin C, tocopherol, and reduced glutathione. When compared to normal control rats, diabetic rats' levels of vitamin C, tocopherol, and reduced glutathione were considerably lower. Chebulagic acid plus metformin treatment of diabetic rats resulted in a significant rise in the levels of non-enzymatic antioxidants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInflammatory Biomarkers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn comparison to control rats, there was a considerable increase in the levels of pro-inflammatory cytokines such TNF-a, IL-1, IL-6, and NF-kB p65, while a significant decrease in the levels of the anti-inflammatory cytokine IL-10. When oral doses of chelagic acid and metformin were administered to diabetic rats, these levels were markedly lower than those of untreated diabetic rats, and they eventually returned to near-normal levels (Fig.5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNF-κB Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe kidney immunohistochemical expression of NF-κB in control and experimental rats is shown in Fig. 6. \u0026nbsp;Nuclear translocation of NF-κB expression was found to be increased in the kidney of diabetic rats (Fig. 6B) when compared to control (6A). Chebulagic acid and metformin treatment reduced NF-κB nuclear translocation in the kidney of diabetic rats (Fig. 6 C and 6 D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTNF-α Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKidney immunohistochemical expression of TNF-α in control and experimental rats is depicted in Fig.7. \u0026nbsp;The proinflammatory cytokine TNF-α expression was enhanced in the kidney of diabetic rats (Fig. 7B) when compared to normal control rats (Fig. 7A). TNF expression was reduced in diabetic rats treated with chebulagic acid and metformin (Fig. 7C and 7D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIR/IRS-1/Akt/ GLUT4 Expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGLUT4, which is a vital glucose transporter, is regulated by the Akt signaling\u0026nbsp;system in the muscle. When diabetic rats were compared to control rats, the mRNA levels of IR, IRS-1, AKT, and GLUT4 were significantly decreased. These gene expression levels were increased in diabetic rats after receiving metformin and chelbulagic acid.\u0026nbsp;Figures\u0026nbsp;8A–8D\u0026nbsp;show the outcomes as they were obtained.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRenal Histological Changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;The control rats had normal glomeruli and tubules (Fig. 9A), while diabetic rats had necrotic and deteriorated renal tubules, as well as inflammatory cell infiltration in renal glomeruli and peritubular capillary congestion (Fig. 9B). Interestingly, treatment of diabetic rats with chebulagic acid and metformin reduced the histological alterations in the kidney, demonstrating better patterned renal architecture with reasonably normal glomeruli and tubules and minor inflammatory cell infiltration (Fig. 9C and 9D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectron microscopic Examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ultrastructure of the kidney was studied by transmission electron microscopy in addition to the standard hematoxylin and eosin-stained study. There are no lipid droplets seen in the kidneys of normal control rats, and their architecture is normal (Fig. 10A).\u0026nbsp;The glomerular basement membrane thickened in the diabetic rats, and there were few lipid droplets. They also had considerable foot process fusion and broadening\u0026nbsp;(Fig. 10B). Yet, administration of chebulagic acid and metformin to diabetic mice improved these changes (Fig. 10C and 10D).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDiabetes mellitus is more common than ever, and it frequently leads to serious metabolic disorders and other serious consequences (Cloete \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A serious global health issue is the progression of diabetes complications. These problems are fatal, but due to a lack of efficient therapies, it is still challenging to stop their development and progression. In order to effectively combat the danger of diabetes sequelae, notably diabetic nephropathy, novel pharmaceutical agents are urgently required. Recent research has concentrated on the utilization of natural substances as workable substitutes for the creation of novel anti-diabetic and reno-protective medications (Borgohain et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Governa et al.2018). The majority of polyphenols from plants contain antioxidant and anti-inflammatory properties, and they act as preventative agents by reducing or preventing the oxidative stress and inflammatory response that hyperglycemia causes (Serafini and Peluso \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As a result, the current work investigates how chebulagic acid protects the kidneys in diabetic rats produced by HFD/STZ.\u003c/p\u003e \u003cp\u003eCombining the HFD diet with the administration of a small dose of STZ can result in the development of insulin resistance and a significant rise in blood glucose levels (Asrafuzzaman et al.2017). This model causes hyperglycemia and metabolic profiles that are very similar to those seen in humans with type 2 diabetes (Skovs\u0026oslash; \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The body's vital tissues and organs might suffer harm from hyperglycemia. For instance, it has the ability to specifically obliterate renal glomerulus mesangial cells and capillary endothelial cells of the retina. (Ni et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The significant reductions in blood glucose and HOMA-IR, together with a concurrent rise in insulin levels in diabetic rats treated with chebulagic acid and metformin, demonstrate the tested tannin's antidiabetic potential. Chebulagic acid therapy improved the glycogen content of diabetic rat liver and muscle, indicating improved insulin production and sensitivity, which encouraged the tissues to absorb glucose and store it as glycogen in the liver and muscle, according to our prior research. The enhanced pancreatic histology provided more evidence in favor of this (Ganesh et al. 2021).\u003c/p\u003e \u003cp\u003eThe diabetic rats displayed notable rises in serum urea and creatinine levels; this is consistent with former reports (Ni et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Othman et al.2021). Blood urea and creatinine levels are reliable indicators of kidney function, and any increase in any of these values is a sign of impaired kidney function (Othman et al.2021). The detected histological alterations in the kidney were consistent with the decrease in renal function seen in rats with diabetes produced by HFD/STZ. When chebulagic acid and metformin were administered to diabetic rats, the levels of urea and creatinine dropped to levels that were equivalent to those of normal control rats, indicating improvement in the kidneys' impaired function.\u003c/p\u003e \u003cp\u003eGenerally speaking, lipid peroxidation may be defined as the process whereby oxidants like free radicals destroy lipids that contain carbon-carbon double bonds, particularly polyunsaturated fatty acids (Ayala et al.2014). The increased lipid peroxidation during diabetes could be related to an ineffective antioxidant system (Rastogi et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Lipid peroxidation caused by free radicals has been linked to a variety of diseases, including diabetes (Kumar et al.2007). Free radicals can also be produced through the auto-oxidation of unsaturated lipids found in membrane and plasma lipids. When the produced free radical interacts with the polyunsaturated fatty acids in the cell membrane, lipid peroxidation might result. Lipid peroxidation will result in an increase in free radical production (Prabakaran and Ashokkumar \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Increased lipid peroxidation reduces membrane fluidity and alters the activity of membrane-bound enzymes and receptors, impairing membrane functions (Bhagavathy and Sumathi \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Its compounds are poisonous to the majority of bodily cells and have been connected to a number of diseases. Despite the fact that they are secondary products of oxidative stress and are generated as a result of the detrimental effect of ROS produced during lipid peroxidation, TBARS and HP levels were found to be considerably higher in HFD/STZ-induced diabetic rats. Recent studies have discovered a strong correlation between lower levels of lipid peroxidation and supplementation with phytochemicals having antioxidant capacity (Fidan and D\u0026uuml;ndar \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Chebulagic acid and metformin were given orally to diabetic rats in the current study to considerably reduce elevated TBARS and HP levels to levels that were close to normal. According to the current study, chebulagic acid also prevents oxidative damage by scavenging free radicals generated by prolonged hyperglycemia.\u003c/p\u003e \u003cp\u003eThe detoxification of harmful oxygen radicals is carried out by endogenous antioxidant enzymes (SOD, CAT, GPx, and GST). Increased levels of lipid peroxidation may indicate that the body's enzymatic antioxidant defense mechanism is becoming less effective. Molecular oxygen and hydrogen peroxide, two reactive molecules rather than free radicals, are produced as a result of the catalytic action of the antioxidant enzyme SOD. Other enzyme-based antioxidants, such as CAT, hasten the breakdown of hydrogen peroxide while shielding tissues from dangerous hydroxyl radicals. GPx, an enzyme that fights free radicals, aids in the reduction of potentially hazardous substances including lipid peroxides and other chemical components (Brigelius-Floh\u0026eacute; et al.2003). In the conjugation of lethal electrophiles to GSH, a set of isoenzymes known as GST is active. In the present investigation, a reduced level of GST activity was observed, which may be due to a deficiency in GSH. The production of oxygen free radicals in STZ-treated -cells has been demonstrated in numerous studies, and it is hypothesized that reactive oxygen free radicals may inactivate and reduce the activities of antioxidant enzymes like SOD, CAT, GPx, and GST that are crucial for defending cells from oxidative damage (Cemek et al.2008). The low levels of SOD, CAT, GPx, and GST activity observed in diabetic rats in this study suggested stress brought on by the disease, whereas diabetic rats treated with chebulagic acid showed a marked increase in these antioxidant enzymes' activities, which may be related to the substance's scavenging ability.\u003c/p\u003e \u003cp\u003eFree radicals are instantly detoxified by vitamins C and E, which are created by the body through nutrition. The results of this study showed that diabetic rats have reduced levels of vitamins C and E. Hydroxyl radicals, singlet oxygen radicals, and the active state of depleted vitamin E are all suppressed by the hydrophilic antioxidant vitamin C, which is also a key component of the diet. Even when there are other antioxidants present, vitamin C deficiency causes the production of hydroperoxides (Prabakaran and Ashokkumar \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Due to its greater role in oxyradical scavenging, vitamin C levels may be lower in diabetics. As a lipophilic antioxidant, vitamin E adds phenolic hydrogen to a polyunsaturated fatty acid free radical that has been peroxidized. This stops the chain process that would otherwise result in membrane lipid peroxidation (Opara \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). On the other hand, administering chebulagic acid to a group of diabetic rats significantly restored the changed levels to close to normal, illuminating the antioxidant potential of chebulagic acid.\u003c/p\u003e \u003cp\u003eGSH is one of the essential compounds for maintaining cell integrity against ROS, as it can scavenge free radicals and reduce H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (Masella et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2004\u003c/span\u003e;Wu et al.2004). Rats with HFD/STZ-induced diabetes had significantly lower tissue GSH concentrations than normal rats. According to certain research, diabetic rats who were given STZ had considerably lower tissue GSH concentrations than the rats in the control group (Gothandam et al.,2019; Ramalingam et al.2022). When GSH is reduced below its baseline level, ROS and oxidative stress are encouraged, which has a cascading impact on the structural and functional integrity of cell and organelle membranes (Franco and Cidlowski \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Vitamins E and C must be recycled, and GSH serves as a co-substrate for the enzymes GPx and GST, which guard against the harmful effects of oxygen radicals. NADPH must be present in order to keep the amount of oxidized glutathione stable. When there is inadequate glucose oxidation during the pentose phosphate cycle due to insulin deficiency, there is less intracellular NADPH, which lowers GSH levels (Maritim et al.2003). Rats with diabetes that have lower GSH levels in their kidneys may be more vulnerable to oxidative injury. It has been hypothesized that antioxidants that maintain GSH concentrations can restore cellular defense mechanisms, reduce lipid peroxidation, and shield tissue from oxidative damage. Tissue GSH levels have likely decreased due to increased oxidative stress brought on by a significant increase in aldehydic lipid peroxidation products (Prabakaran and Ashok kumar 2013). In the current study, GSH level s in the kidneys were found to be higher in the chebulagic acid -treated rats.\u003c/p\u003e \u003cp\u003eChebulagic acid is hypothesized to have anti-inflammatory effects that contribute to its ability to prevent diabetic nephropathy. Many proinflammatory mediators, namely anti-inflammatory cytokines like IL-10, which seem to control inflammatory processes, have been related to the development of diabetic nephropathy. When IL-10 prevented NF-B from acting, less proinflammatory cytokine was produced and macrophage death was inhibited.\u003c/p\u003e \u003cp\u003eExcessive production of reactive oxygen species (ROS) activates nuclear factor-kappa B (NF-κB), which increases the release of pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6. The release of pro-inflammatory cytokines promotes the production of reactive oxygen species (ROS) (Mason and Wahab \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). TNF-α plays an important role in diabetic nephropathy by increasing the production of ROS, inducing renal cell apoptosis, increasing albumin permeability in the glomerulus, and activating the release of vasoconstrictive mediators in the mesangial cell, resulting in reduced flood flow (Ni et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Chebulagic acid and metformin supplementation was successful in lowering the levels of renal TNF-α, IL-1β, and IL-6 and increasing the levels of IL-10 (anti-inflammatory cytokine) in diabetic rats, implying that chebulagic acid supplementation could be used to avoid inflammatory responses in diabetic rats. Similar to this, the immunohistochemistry expression levels of NF-B and TNF-α were examined in the renal tissue of diabetic rats, and their increased expression levels were nearly normalized by the injection of chebulagic acid.\u003c/p\u003e \u003cp\u003eThe mRNA levels of IR, IRS-1, Akt, and GLUT 4 in the gastrocnemius muscle of diabetic rats were considerably lower, indicating heightened oxidative stress and inflammatory markers, which in turn reduced these insulin signaling pathways. These findings are consistent with earlier publications (Babu et al.2020; Deenadayalan et al.2021; Mahmoud et al.2021). Surprisingly, giving diabetic rats chebulagic acid plus metformin enhanced their ability to produce insulin, which might be attributed to the activation of the IR/IRS-1/Akt pathway in the gastrocnemius muscle. Also, after administering chebulagic acid to diabetic rats, better histological and ultrastructural alterations were seen in the kidney tissue, providing additional evidence to corroborate the biochemical results.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBecause of their accessibility, lack of side effects, and cost-effectiveness, natural products high in polyphenols, such as plant extracts and their bioactive constituents, are intriguing therapeutic candidates that require greater research. These products may be used to treat and prevent type 2 diabetes. Chebulagic acid comes from plants, thus the current research reveals that it has an antihyperglycemic impact on HFD/STZ-induced diabetic nephropathy by lowering oxidative stress and inflammation through up-regulation of (IR/IRS-1/Akt and GLUT4) genes implicated in the insulin signaling cascade. For future possibilities of the chemical being employed as an anti-diabetic medication, more human clinical trials are advised.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRS: paper draft and experimental supervision; VG: experiments conducted; KM: draft correction and editing. \u0026nbsp;PJ: Editing and final correction.\u0026nbsp;All authors have read and approved the final submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental protocol was approved by the Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (IAEC No: 16/2016).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe First author gratefully acknowledges the DST-SERB (Early Career Research Award- File No ECR/2016/001693), New Delhi, India, for providing financial support to purchase chebulagic acid to carry out this research work.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbou-Hany HO, Atef H, Said E, Elkashef HA, Salem HA (2018) Crocin mediated amelioration of oxidative burden and inflammatory cascade suppresses diabetic nephropathy progression in diabetic rats. Chem Biol Interact 284:90-100. https://doi.org/10.1016/j.cbi.2018.02.001\u003c/li\u003e\n \u003cli\u003eAsrafuzzaman M, Cao Y, Afroz R, Kamato D, Gray S, Little PJ (2017) Animal models for assessing the impact of natural products on the aetiology and metabolic pathophysiology of Type 2 diabetes. Biomed Pharmacother 89:1242-1251. https://doi.org/10.1016/j.biopha.2017.03.010\u003c/li\u003e\n \u003cli\u003eAthira AP, Abhinand CS, Saja K, Helen A, Reddanna P, Sudhakaran PR (2017) Anti-angiogenic effect of chebulagic acid involves inhibition of the VEGFR2- and GSK-3β-dependent signaling pathways. Biochem. Cell Biol 95(5):563-570. https://doi.org/10.1139/bcb-2016-0132\u003c/li\u003e\n \u003cli\u003eAyala A, Muñoz MF, Argüelles S (2014) Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxid Med Cell Longev 2014:360438. https://doi.org/10.1155/2014/360438\u003c/li\u003e\n \u003cli\u003eBabu S, Krishnan M, Rajagopal P, Periyasamy V, Veeraraghavan V, Govindan R, Jayaraman S (2020) Beta-sitosterol attenuates insulin resistance in adipose tissue via IRS-1/Akt mediated insulin signaling in high fat diet and sucrose induced type-2 diabetic rats. Eur. J. Pharmacol 873:173004. https://doi.org/10.1016/j.ejphar.2020.173004\u003c/li\u003e\n \u003cli\u003eBaker H, Frank O, DeAngelis B, Feingold S (1980) Plasma tocopherol in man at various times after ingesting free or acetylated tocopherol. Nutr Rep Int 21:531–536.\u003c/li\u003e\n \u003cli\u003eBhagavathy S, Sumathi P (2012) Stabilization of membrane bound ATPases and lipid peroxidation by carotenoids from Chlorococcum humicola in Benzo(a)pyrene induced toxicity. Asian Pac J Trop Biomed (5):380-4. https://doi.org/10.1016/S2221-1691(12)60060-4.\u003c/li\u003e\n \u003cli\u003eBorgohain MP, Chowdhury L, Ahmed S, Bolshette N, Devasani K, Das TJ, Mohapatra A, Lahkar M (2017) Renoprotective and antioxidative effects of methanolic Paederia foetida leaf extract on experimental diabetic nephropathy in rats. J. Ethnopharmacol 198:451-459. https://doi.org/10.1016/j.jep.2017.01.035\u003c/li\u003e\n \u003cli\u003eBrigelius-Flohé R, Banning A, Schnurr K (2003) Selenium-dependent enzymes in endothelial cell function. Antioxid Redox Signal. 2:205-215. https://doi.org/10.1089/152308603764816569.\u003c/li\u003e\n \u003cli\u003eCemek M, Kağa S, Simşek N, Büyükokuroğlu ME, Konuk M (2008) Antihyperglycemic and antioxidative potential of \u003cem\u003eMatricaria chamomilla\u003c/em\u003e L. in streptozotocin-induced diabetic rats. J. Nat. Med. 62(3):284-93. https://doi.org/10.1007/s11418-008-0228-1.\u003c/li\u003e\n \u003cli\u003eCloete L (2022). Diabetes mellitus: an overview of the types, symptoms, complications and management. Nurs Stand.37(1):61-66. https://doi.org/10.7748/ns.2021.e11709.\u003c/li\u003e\n \u003cli\u003eDanesh FR, Sadeghi MM, Amro N, Kanwar YS (2002) 3-Hydroxy-3-methylglutaryl CoA reductase inhibitors prevent high glucose-induced proliferation of mesangial cells via modulation of Rho GTPase/ p21 signaling pathway: Implications for diabetic nephropathy. Proc. Natl. Acad. Sci. U S A. 99(12):8301-8305. https://doi.org/10.1073/pnas.122228799\u003c/li\u003e\n \u003cli\u003eDeenadayalan A, Subramanian V, Paramasivan V, Veeraraghavan VP, Rengasamy G, Sadagopan JC, Jayaraman S (2021) Stevioside Attenuates Insulin Resistance in Skeletal Muscle by Facilitating IR/IRS-1/Akt/GLUT 4 Signaling Pathways: An In Vivo and In Silico Approach. Molecules. 26(24):7689. https://doi.org/10.3390/molecules26247689\u003c/li\u003e\n \u003cli\u003eEllman, GL (1959). Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70–77. https://doi.org/10.1016/0003-9861(59)90090-6\u003c/li\u003e\n \u003cli\u003eFidan AF, Dündar Y, (2008) The effects of Yucca schidigera and Quillaja saponaria on DNA damage, protein oxidation, lipid peroxidation, and some biochemical parameters in streptozotocin-induced diabetic rats. J. Diabetes Complications 22(5):348-356. https://doi.org/10.1016/j.jdiacomp.2007.11.010\u003c/li\u003e\n \u003cli\u003eFranco R, Cidlowski JA (2012) Glutathione efflux and cell death. Antioxid Redox Signal. 17(12):1694-713. https://doi.org/10.1089/ars.2012.4553.\u003c/li\u003e\n \u003cli\u003eFourney RM, Miyakoshi J, Day III, RS, Paterson MC (1988) Northern blotting: efficient RNA staining and transfer. \u003cem\u003eBethesda Res. Lab. Focus\u003c/em\u003e10(1):5,6.\u003c/li\u003e\n \u003cli\u003eGanesh V, Ramalingam S, Karuppiah, M (2022) Chebulagic acid attenuates HFD/streptozotocin induced impaired glucose metabolism and insulin resistance via up regulations of PPAR γ and GLUT 4 in type 2 diabetic rats. Toxicology Mechanisms and Methods 32(3):159-170. https://doi.org/10.1080/15376516.2021.1976333\u003c/li\u003e\n \u003cli\u003eGothandam K, Ganesan VS, Ayyasamy T, Ramalingam S (2019) Antioxidant potential of theaflavin ameliorates the activities of key enzymes of glucose metabolism in high fat diet and streptozotocin - induced diabetic rats. Redox Rep 1:41-50. https://doi.org/10.1080/13510002.2019.1624085\u003c/li\u003e\n \u003cli\u003eGoverna P, Baini G, Borgonetti V, Cettolin G, Giachetti D, Magnano AR, Miraldi E, Biagi M (2018). Phytotherapy in the Management of Diabetes: A Review. Molecules. 23(1):105. https://doi.org/10.3390/molecules23010105\u003c/li\u003e\n \u003cli\u003eHabig WH, Pabst MJ, Jakoby WB (1974) Glutathione-S-transferase: the first step in mercapturic acid formation. J. Biol. Chem 249:7130–7139. https://doi.org/10.1016/S0021-9258(19)42083-8\u003c/li\u003e\n \u003cli\u003eHuang YN, Zhao DD, Gao B, Zhong K, Zhu RX, Zhang Y, Xie WJ, Jia LR, Gao H (2012) Anti-hyperglycemic effect of chebulagic acid from the fruits of \u003cem\u003eTerminalia chebula\u003c/em\u003e Retz. Int. J. Mol. Sci., 13(5):6320-33. https://doi.org/10.3390/ijms13056320\u003c/li\u003e\n \u003cli\u003eHulsizer AL, Witte AP, Attridge RL, Urteaga EM. (2023) Hyperglycemia Post-Influenza Vaccine in Patients with Diabetes. Ann Pharmacother 57(1):51-54. https://doi.org/10.1177/10600280221098101\u003c/li\u003e\n \u003cli\u003eJiang, ZY, Hunt JV, Wolff SP (1992) Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Anal. Biochem. 202:384–389. https://doi.org/10.1016/0003-2697(92)90122-N\u003c/li\u003e\n \u003cli\u003eJohnson SA, Spurney RF (2015) Twenty years after ACEIs and ARBs: emerging treatment strategies for diabetic nephropathy. Am. J. Physiol. Renal. Physiol 309(10):F807-820 https://doi.org/10.1152/ajprenal.00266.2015\u003c/li\u003e\n \u003cli\u003eKahn SE (2008) The relative contributions of insulin resistance and beta cell dysfunction in the pathophysiology of type-2 diabetes. Diabetologia 46:3–19. https://doi.org/10.1007/s00125-002-1009-0\u003c/li\u003e\n \u003cli\u003eKim HJ, Kim J, Kang KS, Lee KT, Yang HO (2014) Neuroprotective Effect of Chebulagic Acid via Autophagy Induction in SH-SY5Y Cells. Biomol Ther 22(4):275-81. https://doi.org/10.4062/biomolther.2014.068\u003c/li\u003e\n \u003cli\u003eKooti W, Farokhipour M, Asadzadeh Z, Ashtary-Larky D, Asadi-Samani M (2016) The role of medicinal plants in the treatment of diabetes: a systematic review. Electron Physician 8(1):1832-42. https://doi.org/ 10.19082/1832.\u003c/li\u003e\n \u003cli\u003eKumar G, Sharmila Banu G, Murugesan AG, Rajasekara Pandian M (2007) Effect of \u003cem\u003eHelicteres isora\u003c/em\u003e. Bark Extracts on Brain Antioxidant Status and Lipid Peroxidation in Streptozotocin Diabetic Rats, Pharmaceutical Biology 45(10):753-759. https://doi.org/10.1080/13880200701585782\u003c/li\u003e\n \u003cli\u003eLiu W, Shang P, Liu T, Xu H, Ren D, Zhou W, Wen A, Ding Y (2017) Gastroprotective effects of chebulagic acid against ethanol-induced gastric injury in rats. Chem. Biol. Interact 278:1-8. https://doi.org/10.1016/j.cbi.2017.09.019\u003c/li\u003e\n \u003cli\u003eLowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951). Protein measurement with folinphenol reagent. J. Biol. Chem., 193:265–275. https://doi.org/10.1016/S0021-9258(19)52451-6\u003c/li\u003e\n \u003cli\u003eMahmoud MF, Abdelaal S, Mohammed HO, El-Shazly AM, Daoud R, El Raey MA, Sobeh M (2021) \u003cem\u003eSyzygium jambos\u003c/em\u003e extract mitigates pancreatic oxidative stress, inflammation and apoptosis and modulates hepatic IRS-2/AKT/GLUT4 signaling pathway in streptozotocin-induced diabetic rats. Biomed. Pharmacother 142:112085. https://doi.org/10.1016/j.biopha.2021.112085\u003c/li\u003e\n \u003cli\u003eMaritim AC, Sanders RA, Watkins JB (2003). 3rd. Diabetes, oxidative stress, and antioxidants: a review. J. Biochem. Mol. Toxicol., 17(1):24-38. https://doi.org/10.1002/jbt.10058\u003c/li\u003e\n \u003cli\u003eMason RM, Wahab NA (2003) Extracellular matrix metabolism in diabetic nephropathy. J. Am. Soc. Nephrol.5:1358-73. https://doi.org/ 10.1097/01.ASN.0000065640. 77499.D7\u003c/li\u003e\n \u003cli\u003eMasella R, Varì R, D'Archivio M, Di Benedetto R, Matarrese P, Malorni W, Scazzocchio B, Giovannini C (2004) Extra virgin olive oil biophenols inhibit cell-mediated oxidation of LDL by increasing the mRNA transcription of glutathione-related enzymes. J Nutr 134(4):785-791. https://doi.org/ 10.1093/jn/134.4.785.\u003c/li\u003e\n \u003cli\u003eMatthews D.R, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC (1985) Homeostasis model assessment: Insulin resistance and beta‑cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412‑9. https://doi.org/10.1007/BF00280883\u003c/li\u003e\n \u003cli\u003eMurli R, Saravanan R (2012) Antidiabetic effect of D-limonene, a monoterpene in streptozotocin-induced diabetic rats. Biomed. Prev. Nutr 2:269–275. https://doi.org/10.1016/j.bionut.2012.08.008\u003c/li\u003e\n \u003cli\u003eNarasimhan A, Chinnaiyan M, Karundevi B (2015) Ferulic acid exerts its antidiabetic effect by modulating insulin-signalling molecules in the liver of high-fat diet and fructose-induced type-2 diabetic adult male rat. Appl Physiol Nutr Metab 40(8):769-81. https://doi.org/10.1139/apnm-2015-0002\u003c/li\u003e\n \u003cli\u003eNi Z, Guo L, Liu F, Olatunji OJ, Yin M (2019) \u003cem\u003eAllium tuberosum\u003c/em\u003e alleviates diabetic nephropathy by supressing hyperglycemia-induced oxidative stress and inflammation in high fat diet/streptozotocin treated rats. Biomed Pharmacother 112:108678. https://doi.org/10.1016/j.biopha.2019.108678\u003c/li\u003e\n \u003cli\u003eNiehius WG, Samuelsson D (1968) Formation of malondialdehyde from phospholipids arachidonate during microsomal lipid peroxidation. Eur. J. Biochem., 6:26–130. https://doi.org/10.1111/j.1432-1033.1968.tb00428.x\u003c/li\u003e\n \u003cli\u003eOmaye ST, Turnbull TD, Sauberlich HE, (1979) Selected method for the determination of ascorbic acid in animal cells, tissues and fluid. In:McCormic DB, Wright DL, editors. Methods enzymol. Vol. 62.New York: Academic Press; p. 3–11. https://doi.org/ 10.1016/0076-6879(79)62181-x\u003c/li\u003e\n \u003cli\u003eOpara EC (2002) Oxidative stress, micronutrients, diabetes mellitus and its complications. J. R. Soc. Promot. Health.122(1):28-34. https://doi.org/10.1177/146642400212200112\u003c/li\u003e\n \u003cli\u003eOthman MS, Khaled AM, Al-Bagawi AH, Fareid MA, Ghany RA, Habotta OA, Abdel Moniem AE (2021) Hepatorenal protective efficacy of flavonoids from Ocimum basilicum extract in diabetic albino rats: A focus on hypoglycemic, antioxidant, anti-inflammatory and anti-apoptotic activities. Biomed. Pharmacother 144:112287. https://doi.org/10.1016/j.biopha.2021.112287\u003c/li\u003e\n \u003cli\u003ePiccoli GB, Grassi G, Cabiddu G, Nazha M, Roggero S, Capizzi I, De Pascale A, Priola AM, Vico CD, Maxia S, Loi V, Asunis AM, Pani A, Veltri A (2015) Diabetic Kidney Disease: A Syndrome Rather Than a Single Disease. Rev. Diabet. Stud 12(1-2):87-109. https://doi.org/10.1900/RDS.2015.12.87\u003c/li\u003e\n \u003cli\u003ePrabakaran D, Ashokkumar N (2013). Protective effect of esculetin on hyperglycemia-mediated oxidative damage in the hepatic and renal tissues of experimental diabetic rats. Biochimie. 95(2):366-373. https://doi.org/10.1016/j.biochi.2012.10.008\u003c/li\u003e\n \u003cli\u003eRamalingam S, Karuppiah M, Thiruppathi M, Palanivelu S, Panchanatham S (2020) Antioxidant potential of biflavonoid attenuates hyperglycemia by modulating the carbohydrate metabolic enzymes in high fat diet/streptozotocin induced diabetic rats. Redox Rep. 1:1-10. https://doi.org/10.1080/13510002.2020.1722914\u003c/li\u003e\n \u003cli\u003eRastogi M, Ojha RP, Rajamanickam GV, Agrawal A, Aggarwal A, Dubey GP (2008) Curcuminoids modulates oxidative damage and mitochondrial dysfunction in diabetic rat brain. Free Radic. Res. 42(11-12):999-1005. https://doi.org/10.1080/10715760802571988\u003c/li\u003e\n \u003cli\u003eRehman MU, Rashid SM, Rasool S, Shakeel S, Ahmad B, Ahmad SB (2019. Zingerone (4-(4-hydroxy-3-methylphenyl)butan-2-one) ameliorates renal function via controlling oxidative burst and inflammation in experimental diabetic nephropathy. Arch. Physiol. Biochem., 125(3):201-209. https://doi.org/10.1080/13813455.2018.1448422\u003c/li\u003e\n \u003cli\u003eRemuzzi G, Ruggenenti P, Perna A, Dimitrov BD, de Zeeuw D, Hille DA, Shahinfar S, Carides GW, Brenner BM (2004) Continuum of renoprotection with losartan at all stages of type 2 diabetic nephropathy: a post hoc analysis of the RENAAL trial results. J. Am. Soc.Nephrol., (12):3117-3125. https://doi.org/10.1097/01.ASN.0000146423.71226.0C\u003c/li\u003e\n \u003cli\u003eRotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG (1973) Selenium: biochemical role as a component of glutathione peroxidase. Science 179:588–590. https://doi.org/10.1126/science.179.4073.588\u003c/li\u003e\n \u003cli\u003eSambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.\u003c/li\u003e\n \u003cli\u003eSerafini M, Peluso I (2016) Functional Foods for Health: The Interrelated Antioxidant and Anti-Inflammatory Role of Fruits, Vegetables, Herbs, Spices and Cocoa in Humans. CurrPharmDes.22(44):67016715. \u003cu\u003ehttps://doi.org/\u003c/u\u003e10.2174/1381612823666161123094235\u003c/li\u003e\n \u003cli\u003eShang G, Gao P, Zhao Z, Chen Q, Jiang T, Zhang N, Li H (2013) 3,5-Diiodo-l-thyronine ameliorates diabetic nephropathy in streptozotocin-induced diabetic rats. Biochim Biophys Acta 1832(5):674-84. https://doi.org/10.1016/j.bbadis.2013.01.023\u003c/li\u003e\n \u003cli\u003eShanmuganathan S, Angayarkanni N (2018) Chebulagic acid Chebulinic acid and Gallic acid, the active principles of Triphala, inhibit TNFα induced pro-angiogenic and pro-inflammatory activities in retinal capillary endothelial cells by inhibiting p38, ERK and NFkB phosphorylation. Vascul Pharmacol 108:23-35. https://doi.org/10.1016/j.vph.2018.04.005\u003c/li\u003e\n \u003cli\u003eSinha KA (1972) Colorimetric assay of catalase. Anal. Biochem., 47:389–394. https://doi.org/10.1016/0003-2697(72)90132-7\u003c/li\u003e\n \u003cli\u003eSkovsø S (2014) Modeling type 2 diabetes in rats using high fat diet and streptozotocin. J. Diabetes Investig. 5(4):349-58. https://doi.org/10.1111/jdi.12235\u003c/li\u003e\n \u003cli\u003eStringer MD, Gorog PG, Freeman A, Kakkar VV (1989) Lipid peroxides and atherosclerosis. British Medical Journal 298:281–284. https://doi.org/10.1136/bmj.298.6669.281\u003c/li\u003e\n \u003cli\u003eSyed FZ (2022) Type 1 Diabetes Mellitus. Ann Intern Med.175(3):33-48. https://doi.org/10.7326/AITC202203150\u003c/li\u003e\n \u003cli\u003eTeck J (2022) Diabetes-Associated Comorbidities. Prim Care 49(2):275-286. https://doi.org/10.1016/j.pop.2021.11.004\u003c/li\u003e\n \u003cli\u003eTrinder P (1969). Determination of glucose in blood using glucose oxidase with an alternative oxygen receptor. Ann. Clin. Biochem., 6:24–27. https://doi.org/10.1177/000456326900600108\u003c/li\u003e\n \u003cli\u003eVaz de Castro PAS, Bitencourt L, de Oliveira Campos JL, Fischer BL, Soares de Brito SBC, Soares BS, Drummond JB, Simões E Silva AC (2022) Nephrogenic diabetes insipidus: a comprehensive overview. J Pediatr Endocrinol Metab 35(4):421-434. https://doi.org/10.1515/jpem-2021-0566\u003c/li\u003e\n \u003cli\u003eWang K, Wang H, Liu Y, Shui W, Wang J, Cao P, Wang H, You R, Zhang Y (2018) Dendrobium officinale polysaccharide attenuates type 2 diabetes mellitus via the regulation of PI3K/Akt-mediated glycogen synthesis and glucose metabolism, J. Funct. Foods 40:261–271. https://doi.org/10.1016/j.jff.2017.11.004\u003c/li\u003e\n \u003cli\u003eWang M, Li Y, Hu X, Wang M, (2018) Chebulinic acid derived from triphala is a promising antitumour agent in human colorectal carcinoma cell lines. BMC Complement Altern Med. 18(1):342. https://doi.org/10.1186/s12906-018-2412-5\u003c/li\u003e\n \u003cli\u003eWild S, Roglic G, Green A, Sicree R, King H (2004) Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27(5):1047-53. https://doi.org/10.2337/diacare.27.5.1047\u003c/li\u003e\n \u003cli\u003eWu D, Wen W, Li C, Qi R, Xia Zhao J, Hua Lu C, Yan Zhong Y (2012) Ameliorative effect of berberine on renal damage in rats with diabetes induced by high-fat diet and st streptozotocin. Phytomedicine 19:712–718. https://doi.org/10.1016/j.phymed.2012 .03.003\u003c/li\u003e\n \u003cli\u003eWu G Fang YZ, Yang S, Lupton JR, Turner ND (2004) Glutathione metabolism and its implications for health1 J. Nutr 134(3):489-92. https://doi.org/10.1093/jn/134.3.489\u003c/li\u003e\n \u003cli\u003eXie W, Xing D, Sun H, Wang W, Ding Y, Du L (2005) The effects of Ananas comosus L. leaves on diabetic-dyslipidemic rats induced by alloxan and a high fat/ high-cholesterol diet. Am. J. Chinese Med 33:95–105. https://doi.org/10.1142/S0192415X05002692\u003c/li\u003e\n \u003cli\u003eXu Y, Bai L, Chen X, Li Y, Qin Y, Meng X, Zhang Q (2018) 6-Shogaol ameliorates diabetic nephropathy through anti-inflammatory, hyperlipidemic, anti-oxidative activity in db/db mice. Biomed.Pharmacother.97:633-641. https://doi.org/10.1016/j.biopha.2017.10.084\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1.\u0026nbsp;Effects of chebulagic acid on the levels of urea and creatinine in\u0026nbsp;control and experimental animals\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"662\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.971342383107089%\" valign=\"top\"\u003e\n \u003cp\u003eParameters\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.082956259426847%\" valign=\"top\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.155354449472096%\" valign=\"top\"\u003e\n \u003cp\u003eDiabetes Induced\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.339366515837103%\" valign=\"top\"\u003e\n \u003cp\u003eDiabetes + Chebulagic acid (50mg/kg b.w)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.45098039215686%\" valign=\"top\"\u003e\n \u003cp\u003eDiabetes + Metformin (50mg/kg b.w)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.971342383107089%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.082956259426847%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.155354449472096%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.339366515837103%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.45098039215686%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.971342383107089%\" valign=\"top\"\u003e\n \u003cp\u003eUrea (mg/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.082956259426847%\" valign=\"top\"\u003e\n \u003cp\u003e25.40\u0026plusmn;1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.155354449472096%\" valign=\"top\"\u003e\n \u003cp\u003e80.21\u0026plusmn;6.89*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.339366515837103%\" valign=\"top\"\u003e\n \u003cp\u003e38.79\u0026plusmn;3.29\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.45098039215686%\" valign=\"top\"\u003e\n \u003cp\u003e36.9\u0026plusmn;2.98\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.971342383107089%\" valign=\"top\"\u003e\n \u003cp\u003eCreatinine\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(mg/dl)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.082956259426847%\" valign=\"top\"\u003e\n \u003cp\u003e0.61\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.155354449472096%\" valign=\"top\"\u003e\n \u003cp\u003e1.9\u0026plusmn;0.07*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.339366515837103%\" valign=\"top\"\u003e\n \u003cp\u003e0.9\u0026plusmn;0.05\u003cstrong\u003e\u003csup\u003e#\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.45098039215686%\" valign=\"top\"\u003e\n \u003cp\u003e0.87\u0026plusmn;0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eValues are given as mean \u0026plusmn; S.D for six animals in each group. Values are considered significantly different at \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05 with post hoc LSD test \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05. statistical differences are expressed as\u0026nbsp;\u003cstrong\u003e(* # \u0026dagger;)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e* Control vs Diabetic rats\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e#\u0026nbsp;\u003c/strong\u003e Diabetic rats vs Diabetic rats treated with chebulagic acid (100mg/kg b.w)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026dagger;\u003c/strong\u003e Diabetic rats treated with chebulagic acid (50mg/kg b.w ) vs Diabetic rats treated with\u003c/p\u003e\n\u003cp\u003eMetformin (50mg/kg b.w)\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diabetes, Hyperglycaemia, Insulin resistant, Metformin, Molecular Targets, Tannin","lastPublishedDoi":"10.21203/rs.3.rs-3859769/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3859769/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExamining the contribution of chebulagic acid in high fat diet/streptozotocin (HFD/STZ)-induced diabetic nephropathy was the main goal of this investigation. Wistar male rats were fed HFD for two weeks before receiving a 35 mg/kg STZ intraperitoneal dosage. During 30 days, diabetic rats were fed metformin and chebulagic acid (50 mg/kg b.w./day each). Blood and kidney samples were also taken following the study for biochemical and histological analysis. Chebulagic acid was administered orally to diabetic rats, considerably lowering blood sugar, serum creatinine, urea, and homeostasis model assessment of insulin resistance (HOMA-IR) levels while simultaneously increasing plasma insulin. In addition, diabetic rats had elevated levels of renal pro-inflammatory cytokines with concurrently increased levels of anti-inflammatory cytokines. They also had lower lipid peroxidation product and increased renal enzymatic and non-enzymatic antioxidant enzyme status. Moreover, chebulagic acid therapy increased the amounts of mRNA for the insulin signaling components GLUT4 and Akt in the gastrocnemius muscles of diabetic rats as well as insulin receptor (IR), insulin receptorsubstrate-1 (IRS-1), and Akt. According to these findings, chebulagic acid has anti-diabetic nephropathy actions that are attenuated.\u003c/p\u003e","manuscriptTitle":"Chebulagic Acid Alleviates Inflammation Via Regulation of Skeletal Muscle IR/IRS-1/AKT/GLUT4 Signaling Pathway in Diabetic Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 11:04:26","doi":"10.21203/rs.3.rs-3859769/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ce46af35-ad78-4013-ba91-34b9f3e75bf4","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-26T20:29:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-17 11:04:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3859769","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3859769","identity":"rs-3859769","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}