Modulazione sinergica della via dello stress del reticolo endoplasmatico, del danno ossidativo al DNA e dell'apoptosi da parte della β-amirina e della metformina nell'attenuare il danno renale indotto dall'iperglicemia utilizzando un modello di zebrafish adulto

Introduction

Diabetes is a metabolic disorder characterized by inadequate insulin production by the pancreas in response to elevated blood glucose levels [ ]. Diabetic kidney disease, a microvascular complication of diabetes, is a pressing global health concern. High blood glucose levels can lead to renal arterial calcification and an increase in the thickness of the arterial medial layer [ ]. A recent study estimated that 20–50% of the global diabetic population is at risk of developing diabetic kidney disease, which may progress to end-stage renal disease (ESRD) [ ].

Pentacyclic triterpenoids, a class of phytochemicals widespread in plants, have garnered attention for their potential medicinal properties, particularly their hypothesized hypoglycemic effects [ , ]. Among these compounds, β-amyrin has undergone extensive investigation, displaying promising antihyperglycemic effects. However, the intricate molecular mechanisms underlying its actions are still not fully elucidated. In vivo studies conducted using hyperglycemic animal models have demonstrated anti-inflammatory, antihyperglycemic, and hepatoprotective properties of β-amyrin [ ]. It is believed that β-amyrin exerts its effects through modulating key inflammatory pathways such as NF-κB and reducing oxidative stress, which contributes to its anti-inflammatory and glucose-lowering effects. Further, histopathological studies have revealed that β-amyrin can protect beta cells, while diabetes-induced animal models have shown a controlled increase in serum total cholesterol (TC) and triglyceride (TG) levels [ ]. Additionally, bioavailability and pharmacokinetic studies on β-amyrin have demonstrated moderate absorption and stability, though its bioavailability may not match that of metformin.

Metformin, the preferred treatment option in diabetes management for over five decades [ ], exerts its effects by inhibiting hepatic gluconeogenesis and antagonizing glucagon action in hyperglycemic conditions, independent of insulin secretion regulation [ ]. In cases where metformin monotherapy proves inadequate in achieving target glycated hemoglobin levels, combination therapy is often employed to optimize treatment outcomes [ ].

In this study, we assessed the potential of β-amyrin, both alone and in combination with metformin, in mitigating diabetes and its associated renal complications. Our research was carried out using a hyperglycemic Zebrafish (ZF) model, which has been gaining popularity in recent times due to its comparable basic renal anatomy to mammalian kidneys, characterized by glomerular filtration and tubular filtration processing. ZF, known scientifically as Danio rerio , is a highly advantageous model for drug screening applications due to its abbreviated reproductive cycle, diminutive size and capacity to yield hundreds of offspring every week. Additionally, these organisms have proven to be highly effective in studying acute kidney injury and expediting the induction of diabetic conditions, rendering them a promising candidate for diabetes research [ – ].

The novelty of this study lies in exploring the synergistic effects of β-amyrin in combination with metformin, a combination that has not been extensively studied, particularly in the context of diabetic nephropathy. While β-amyrin has demonstrated promising antihyperglycemic effects individually, its combined action with metformin may offer a unique therapeutic strategy for managing diabetes and its associated renal complications.

Experimental

### Materials

β-amyrin (Merck), metformin (Merck), and naringenin were obtained from Thamil Kumaran Enterprises, (Chennai, Tamil Nadu, India). Glucose 111 mM was obtained from Sigma Aldrich (Mumbai, India). The glucometer and strips were from GluNEO Lite® (Chandigarh, India). Total cholesterol (TC), triglycerides (TG), and total protein measurement kits were from Dialab (Wiener Neudorf, Austria). The insulin Enzyme Linked Immunosorbent Assay (ELISA) kit was from Ray Biotech (Norcross, GA, USA). Antibodies including β-Actin (housekeeping gene), 78-kDa glucose-regulated protein (GRP-78), protein kinase RNA-like ER kinase (PERK), eukaryotic initiation factor 2 alpha (eIF2α), activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP), inositol-requiring enzyme 1 (IRE1α), B-cell lymphoma protein 2 (Bcl-2), Bcl-2-associated X-protein (Bax), Caspase-3, Nuclear factor kappa B (NF-kB), tumor necrosis factor (TNF-α) and interleukin-16 (IL-6) and Protein A/G PLUS Sepharose beads and anti-rabbit FITC conjugated secondary antibody were obtained from Santa Cruz Biotechnology (St. Louis, MO, UA). All the other chemicals and reagents used in the experiments were procured from Sigma-Aldrich and were of high purity and analytical grade.

### Animals

Adult wild-type ZF ( Danio rerio ; 7–9 months old; 3–5 cm; both sexes) were procured from an aquarium shop, 777 Aquatics India Pvt. Ltd, Bhopal, Madhya Pradesh (MP), India. These ZF were acclimatized for 07 days in 40 L tanks and fed twice a day with micro pellets. The aquarium tank was maintained with a photoperiod of 14 h light and 10 h darkness at a constant temperature (25 ± 2 °C) [ ]. Tank water was changed daily. This study was approved by the Institutional Animal Ethics Committee of the Central Animal Facility, JSS Academy of Higher Education & Research (JSSAHER), Mysuru, Karnataka, India (approval number: JSSAHER/CPT/IAEC/179/2023).

### Establishing hyperglycemia model

Six batches of 25 ZF were placed in 20 L fish tanks, with one batch designated as the Normal group. The Normal group ZF were maintained in regular fish water without glucose. The remaining five tanks were filled with a 111 mM glucose solution [ ]. The glucose solutions were changed every third day to prevent microbial contamination. The fish were fed twice daily. Before initiating glucose immersion on Day 0, baseline blood glucose levels were assessed in randomly selected fish from each batch. The ZF were observed for 14 days, during which the fish were continuously monitored from 9:00 am to 3:00 pm for signs of stress, such as increased gill movement and swimming difficulties. At the end of the 14-day period, blood glucose levels were measured to assess the glucose induction in ZF immersed in glucose solution [ ]. Fish were randomly selected from each glucose-immersed batch and tested to confirm the diabetic status and ensure that hyperglycemia was maintained throughout the experiment.

### Procedure for measuring blood glucose levels

The caudal vein procedure was followed to measure blood glucose levels in ZF (Fig. ) [ ]. Prior to the procedure, all animals were fasted for 12 h and placed in an aquarium containing glucose-free water for 15 min to prevent glucometer strip contamination [ , ]. The ZF were anesthetized using ice-cold water. The fish was then positioned on a damp cloth and stabilized for the procedure, and the caudal vein near the tail fin was identified using magnification. A small incision was made near the vein and blood was carefully drawn by inserting and aspirating using a needle. This blood sample was then transferred to a glucose meter by placing a glucometer test strip (GluNEO Lite®) to measure glucose levels. Post-procedural care involved monitoring the fish until it recovered from anesthesia, ensuring its well-being throughout the process.

### Experimental groups

The ZF in a diabetic state were randomly assigned to five groups, each comprising 20 diabetic ZF and housed in separate 15 L tanks. The groups were as follows: Group 1 (Normal) consisted of normoglycemic fish with no glucose and no treatment; Group 2 (Control) consisted of glucose-induced diabetic fish with no treatment; Group 3 (Standard) consisted of glucose-induced diabetic fish treated with naringenin at a concentration of 25 mg/L; Group 4 (Treatment 1) consisted of glucose-induced diabetic fish treated with β-amyrin at a concentration of 50 mg/L; Group 5 (Treatment 2) consisted of glucose-induced diabetic fish treated with metformin at a concentration of 25 mM; and Group 6 (Treatment 3) consisted of glucose-induced diabetic fish treated with a combination of β-amyrin and metformin at concentrations of 50 mg/L and 12.5 mM, respectively. The experimental groups are summarized in Table .

The different treatments were prepared and administered daily for a period of 14 days. The β-amyrin, metformin, and β-amyrin+metformin combination solutions were prepared and added to the tanks to achieve final concentrations of 50 mg/L, 25 mM, and 50 mg/L + 12.5 mM, respectively. Naringenin (25 mg/L) was used as the standard treatment, while the Normal and Control group received no treatment. The doses of β-amyrin and metformin were determined based on prior in vitro studies, which identified these concentrations as optimal for enhancing cell viability.

For the combination therapy, β-amyrin and metformin were administered simultaneously. Metformin chloridate was dissolved in water and Naringenin and β-amyrin solutions were prepared by dissolving the drugs in DMSO (final concentration 0.0005%). During the treatment period, the fish were closely monitored for any signs of stress or abnormal behavior. On the 14th day post-treatment, blood glucose levels, and other serum parameters were measured to assess the efficacy of the treatments. After the termination of the treatment on the 15th day after treatment, the ZF were anesthetized and sacrificed. Blood samples were collected, and kidneys were isolated from each ZF for further analysis. The experimental design is summarized in Fig. .

### Anaesthesia and sacrifice

In this study, euthanasia of Zebrafish was performed using a two-step process involving hypothermic anesthesia followed by tail decapitation. The fish were first anesthetized by immersion in ice-cold water (4 °C) to induce hypothermia, a widely accepted method for fish anesthesia due to its rapid and humane onset with minimal physiological stress. The fish were carefully monitored for signs of Stage III anesthesia, including loss of equilibrium, cessation of operculum (gill cover) movements, and lack of response to external stimuli, to ensure they were fully anesthetized and unconscious before proceeding with euthanasia [ ].

Once the fish were confirmed to be fully anesthetized, euthanasia was conducted by tail decapitation. A cross-sectional cut was made at the base of the tail using a sterile scalpel to obtain blood for various analyses (Fig. ) [ , ]. This method was chosen for its effectiveness in quickly severing the spinal cord, ensuring a swift and humane death while allowing for efficient blood collection for various analyses. Following blood collection, kidneys were carefully extracted through dissection for further study (Fig. ).

### Assessment of ZF kidney and body weight ratio

This involved placing the ZF in a container (without water) on a precise scale and recording its weight before performing the anesthesia and sacrifice procedures. After sacrificing ZF and collecting blood, the kidneys from the ZF were carefully extracted through dissection and weighed separately from the rest of the body. The ZF kidney and body weight ratio were calculated using the following formula.

\documentclass[12pt]{minimal}

\usepackage{amsmath}

\usepackage{wasysym}

\usepackage{amsfonts}

\usepackage{amssymb}

\usepackage{amsbsy}

\usepackage{mathrsfs}

\usepackage{upgreek}

\setlength{\oddsidemargin}{-69pt}

\begin{document}$$Weight~ratio=\frac{{Kidney~weight}}{{Body~weight~\% }} \times 100$$\end{document}

### Biochemical estimations of serum parameters

On the 14th day, after the usual morning treatment, fasting blood glucose was monitored. Subsequently, ZF were fed as per the regular schedule and, blood glucose levels were measured two hours post-feeding to assess the postprandial blood glucose levels to evaluate the efficacy of the treatments in controlling blood glucose spikes after feeding. To measure various biochemical parameters, the ZF were fasted for 12 h and then transferred to fish tanks with glucose-free water for 15 min. Afterward, they were anesthetized and sacrificed (as shown in the anesthesia and sacrifice section) to obtain blood. The serum was separated by centrifuging blood at 10× g rpm for 15 min. The serum sample was stored at −80 °C for the quantitative estimation of various biochemical parameters, including serum albumin, serum creatinine, serum total proteins, serum urea, serum TC and serum TG, which were measured using respective commercially available assay kits and by an automated biochemical analyzer (Beckman Coulter-AU480, CA, USA) [ ].

### Serum insulin ELISA assay

The insulin levels in the serum samples of ZF were measured using an insulin ELISA assay kit (Cat. No. PG6642R, Puregene, Genetix Biotech Asia Pvt. Ltd) as per the manufacturer’s protocol. The absorbance was read at 450 nm on a Varioskan Flash microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The insulin concentration in the serum was calculated using the standard values provided by the kit. The results are expressed as insulin levels in ng/mL blood.

### Analysis of endogenous antioxidant markers in kidney tissues

At the end of the study period, after sacrificing the ZF and collecting blood, their kidneys were removed immediately (Fig. ) [ ]. The kidney tissues of ZF were minced and homogenized at a concentration of 10% w/v in a cold phosphate buffer with a pH of 7.4 and with 1.15% KCl, followed by centrifugation in a microtube at 2500 rpm (4 °C) for 15 min. The soluble supernatant was used for further analysis [ , ]. Protein content was estimated using Bradford reagent (Sigma–Aldrich, USA). Reduced Glutathione (GSH) activity in the kidney tissue supernatant was determined according to the method of Sedlak and Lindsay [ ]. Catalase (CAT) activity in the kidney tissue supernatant was determined according to Sinha’s method [ ]. Superoxide dismutase (SOD) activity was measured by the protocol developed by Kakkar et al. [ ].

### Analysis of the degree of lipid peroxidation in kidney tissues

The degree of lipid peroxidation (LPO) in kidney tissues was determined by measuring the absorbance at 532 nm of the color formed by the malondialdehyde (MDA) reaction with thiobarbituric acid (TBA). MDA levels were measured in the supernatant obtained after centrifuging the kidney homogenate (homogenized in 1.15% KCl) using the method developed by Placer et al. [ ].

### Determination of DNA damage (8-OHdG level)

8-OHdG analysis in kidney tissue homogenates was done with a ready-to-use commercial kit (8-OHdG) using the competitive binding of the 8-OHdG monoclonal antigen (anti-8-OHdG) to the sample, standard, or 8-OHdGs that have not been previously bound to the wells of the plate: (201-00-0041/SunRed). The kidney samples stored at −80 °C samples were dissolved and mixed with chitin solutions and standards. After incubating for 10 min in a plate shaker at 37 °C in the dark, Stop Solution (50 µl) was added to the plate wells. The absorbance of samples was measured within 10 min at 450 nm using an ELISA plate reader [ ].

### Gene expression analysis by quantitative real-time RT-qPCR for the assessment of proinflammatory cytokines markers in ZF kidney tissues

The expression of isoforms of NF-κB, TNF-α, and IL-6 genes was evaluated by a quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) assay. Total RNA was isolated from ZF renal tissues using TRIzol™ reagent (Invitrogen, Carlsbad, CA, USA) and quantified by spectrophotometry. After that, cDNA was synthesized from 1 µg of total RNA using the ImProm-II™ Reverse Transcription System (Promega). RT-qPCR was performed with diluted cDNA, SYBR® Green I, Platinum® Taq DNA polymerase, and specific primers for NF-κB, TNF-α, and IL-6 (Table ).

The cycling conditions were 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 35 s, and 72 °C for 15 s A melting curve analysis confirmed amplification specificity. The efficiency for each sample was computed using LinRegPCR version 2012.3 software ( http://LinRegPCR.nl ) , and the stability of NF-κB, TNF-α, and IL-6 genes (M-value) as well as the optimal number of reference genes based on pairwise variation (V) was assessed using GeNorm 3.5 software ( http://medgen.ugent.be/genorm/ ). Relative RNA expression levels were determined using the 2−ΔΔCT method [ , ].

### Western blot analysis of ZF kidney tissues for assessment of ER stress and apoptosis markers

Tissue samples of ZF kidneys were homogenized to extract proteins using radioimmunoprecipitation assay (RIPA) buffer containing phosphatase and protease inhibitors. The kidney tissue lysates were centrifuged to collect the supernatant, and the concentration of proteins was determined using the bicinchoninic acid assay (BCA). Laemmli sample buffer containing equal amounts of lysate proteins were heated at 65 °C. Poly (vinylidene fluoride) (PVDF) membranes were utilized to transfer the proteins of interest, and the membranes were blocked with 5% non-fat milk in Tris-buffered saline with 0.1% Tween® 20 Detergent (TBST) buffer at room temperature for an hour.

Next, they were incubated overnight at 4 °C with corresponding primary antibodies: anti-cleaved caspase-3, anti-Bax, anti-Bcl-2, anti-PERK, anti-ATF4, anti-CHOP, anti-phosphorylated PERK (1:1000, Boosen Biotechnology Co., Ltd. Beijing, China) or β-actin (1:2000, Zhongshan Golden Bridge Biotechnology Co., Ltd. Beijing, China). After that, the membranes were washed with TBST thrice and incubated with Horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000, Zhongshan Golden Bridge Biotechnology Co., Ltd. Beijing, China) at room temperature. The antibody-antigen complexes were visualized using an enhanced chemiluminescence reagent (Bridgen Co., Ltd, Beijing, China) and analyzed quantitatively by densitometry with Image J software (NIH, Bethesda, MD, USA). The relative density of immunoreactive bands was normalized to the density of the corresponding bands of β-actin [ , ].

### Apoptosis assay

A terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nickend-labeling (TUNEL) assay detected apoptotic nuclei in kidney sections. TUNEL assay was performed using the TUNEL Detection Kit (Roche Diagnostics. Mannheim, Germany) according to the manufacturer’s method. Positive staining, appearing as dark brown, was observed in a minimum of five random fields under 400× magnification in each section. For quantitative analysis, the Image-Pro Plus 6.0 (IPP6) image analysis software was used to quantify the TUNEL-staining intensity in positive cells and expressed as the ratio of the mean of Normal group [ ].

### Immunohistochemistry of ZF kidney tissues

The 3-µm thick paraffin sections of kidneys were processed using a standard immunostaining protocol. The sections were deparaffinized and rehydrated. Immunoperoxidase staining was performed using the primary antibody: anti-glucose-regulated protein 78 (GRP78) (1:200, Wanlei Biological Co., Ltd. Shenyang, China). Immunostaining procedures were conducted according to the manufacturer’s methods. Positive staining, appearing as dark brown, was observed in a minimum of five random fields under 400× magnification in each section. The IPP6 image analysis software was used to quantify the immunostaining intensity in positive areas and expressed as the ratio of the mean of the normal group [ ].

### Histopathological examination of ZF kidney

For histopathology examination, the kidneys from each group were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections after harvesting. The sections were stained with H&E, PAS, and Masson for routine renal histopathological examination by light microscopy. Positive staining was observed in a minimum of five random fields under 400x magnification in each section. The IPP6 image analysis software (Media Cybernetics, Rockville, MD, USA) was used to quantify the PAS and Masson staining intensity in positive areas and expressed as the ratio of the mean of the Normal group [ , ].

### Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0.2. The results were presented as mean ± SEM ( n = 15). The statistical significance of the differences between various groups was determined by one-way ANOVA followed by Tukey post hoc multiple comparison test and two-way ANOVA followed by Bonferroni post hoc multiple comparison tests, which were performed for weekly body weight and blood glucose level measurements. The p ≤ 0.05 was considered statistically significant. The degree of statistical significance was indicated in figure legend as p a when compared to normal (normoglycemic ZF), p b when compared to control (hyperglycemic ZF), and p c when compared to standard.

Results

### Establishing hyperglycemia model using 111 mM glucose solution

On Day 14, the assessment of blood glucose levels in the normal water tank and glucose tanks showed a significantly higher blood glucose level in ZF exposed to the glucose tanks (Normal Water ZF: 60.56 ± 1.91 mg/dL; Glucose Tank ZF: 388.75 ± 4.32 mg/dL, p ≤ 0.05) (Fig. ).

### Effect of test compounds on blood glucose levels, body weight, and kidney/body weight ratio in glucose-induced diabetic ZF

The study compared blood glucose levels, body weight, and kidney body weight ratio (KBWR) of hyperglycemic ZF treated with Standard and test compounds (β-amyrin, metformin, and their combination) (Fig. ). The blood glucose level of Control ZF was significantly higher than of Normal group ZF. However, treatment with β-amyrin, metformin, and the β-amyrin+metformin combination successfully managed to control these levels. The β-amyrin+metformin combination was the most effective, reducing blood glucose levels to 104.54 ± 1.63 mg/dL, compared to 151.06 ± 1.93 mg/dL by β-amyrin and 111.41 ± 2.50 mg/dL by metformin.

Moreover, the study revealed that hyperglycemic ZF had a significantly higher ( p ≤ 0.05) body weight (569.63 ± 19.63 mg) than normal ZF (305.01 ± 9.51 mg). However, treatment with the test compounds significantly reduced ( p ≤ 0.05) the body weight of the hyperglycemic ZF, with the ZF treated with the β-amyrin+metformin combination showing an average weight of 306.93 ± 20.43 mg, which was lower than the average body weight of ZF treated with β-amyrin alone (369.41 ± 27.06 mg) or metformin alone (377.40 ± 22.77 mg).

Our research also revealed that the induction of hyperglycemia in ZF resulted in an increased KBWR. However, the ZF treated with test compounds showed significantly lower KBWR ( p ≤ 0.05) than Control, with β-amyrin+metformin combination showing lower values (2.89 ± 0.27%), as compared to ZF that received treatment with β-amyrin (3.22 ± 0.22%) and metformin (2.95 ± 0.25%) individually.

### Effect test compounds on inhibition of ER stress avert metabolic aberrations and renal injuries in glucose-induced diabetic ZF

The effect of β-amyrin and metformin, alone and combined were studied on metabolic and renal parameters (Fig. ). The combination of β-Amyrin+Metformin exhibited significantly ( p ≤ 0.05) lower serum urea levels (19.57 ± 1.61 mg/dL) compared to the individual drugs, β-amyrin (27.02 ± 0.96 mg/dL) and metformin (24.53 ± 1.29 mg/dL). Serum creatinine values were also significantly ( p ≤ 0.05) lower in ZF treated with the combination of β-amyrin+metformin (0.56 ± 0.02 mg/dL) than those receiving the individual treatment of β-amyrin (0.7 ± 0.01 mg/dL) and metformin (0.6 ± 0.02 mg/dL). Conversely, albumin levels were significantly ( p ≤ 0.05) higher with the combination of β-amyrin+metformin (2.61 ± 0.15 mg/dL) than observed with individual treatments of β-amyrin (2.44 ± 0.12 mg/dL) and metformin (2.17 ± 0.19 mg/dL). Similarly, total protein levels were significantly ( p ≤ 0.05) higher when treated with the combination of β-amyrin+metformin (2.03 ± 0.07 mg/dL) than when treated with β-amyrin (1.85 ± 0.1 mg/dL) and metformin (1.89 ± 0.08 mg/dL) alone.

Moreover, the combination of β-amyrin+metformin resulted in significantly ( p ≤ 0.05) lower serum TC (110.4 ± 3.28 mg/dL) and serum TG (203.66 ± 2.65 mg/dL) compared to individual drugs, with β-amyrin treatment showing 112.73 ± 4.42 mg/dL serum TC and 256.06 ± 4.35 mg/dL serum TG, and metformin showing 111.13 ± 3.86 mg/dL serum TC and 262.86 ± 315 mg/dL serum TG.

Further, the results revealed that the combination of β-amyrin and metformin resulted in a statistically significant decrease ( p ≤ 0.05) in both serum insulin and fasting blood glucose levels. Specifically, the serum insulin levels were found to be significantly lower with β-amyrin+metformin (12.50 ± 0.83 mg/dL) treatment as compared to treatment with β-amyrin (16.10 ± 0.87 mg/dL) and metformin (13.77 ± 0.75 mg/dL) alone. Similarly, the fasting blood glucose levels were also significantly lower with β-amyrin+metformin (72.6 ± 4.39 mg/dL) treatment compared to individual treatments with β-amyrin (127.87 ± 7.67 mg/dL) and metformin (77.87 ± 3.42 mg/dL) alone.

Moreover, the postprandial glucose levels were significantly ( p ≤ 0.05) lower upon treatment with the combination of β-amyrin+metformin (136.53 ± 13.55 mg/dL) compared to β-amyrin (207.93 ± 10.99 mg/dL) and metformin (189.13 ± 14.41 mg/dL) alone.

### Effect of test compounds on DNA damage and antioxidant capacity in glucose-induced diabetic ZF

The effect of test compounds on 8-hydroxy-2′ -deoxyguanosine (8-OHdG) (an indicator of DNA damage), the antioxidant enzymes (SOD, CAT, and GSH), and LPO was examined in glucose-induced diabetic ZF (Fig. ). The raised 8-OHdG level in hyperglycemic ZF decreased to a greater extent when treated with metformin (0.24 ± 0.02 ng/mL) or β-Amyrin+Metformin combination (0.24 ± 0.01 ng/mL) than when treated with β-amyrin alone (0.33 ± 0.01 ng/mL). Similarly, SOD, CAT, and GSH levels decreased in hyperglycemic ZF as compared to normal ZF. Treatment with test compounds increased these levels, with the β-amyrin+metformin combination showing higher SOD, CAT, and GSH levels than β-amyrin or metformin alone.

Moreover, the levels of thiobarbituric acid reactive substances (TABRS) were significantly higher in hyperglycemic ZF (10.72 ± 0.21 nmol/mg) than in normal ZF (4.34 ± 0.20 nmol/mg). Treatment with test compounds reduced raised TBARS levels in these hyperglycemic ZF, with β-amyrin+metformin combination resulting in maximum reduction of (5.63 ± 0.15 nmol/mg) of protein, compared to β-amyrin (6.06 ± 0.19 nmol/mg) and metformin (5.98 ± 0.15 nmol/mg).

### β-amyrin and metformin combination abrogates hyperglycemia-induced ER stress and apoptosis in glucose-induced diabetic ZF

The expression of various ER stress was measured in hyperglycemic ZF after treatment with test compounds (Fig. ). The levels of GRP78 expression were found to be significantly elevated in hyperglycemic ZF (4.35 ± 0.10). However, the test compounds reduced these elevated levels, with the combination of β-amyrin+metformin showing the maximum reduction (1.22 ± 0.08 unit), as compared to β-amyrin (3 ± 0.19) unit or metformin (1.98 ± 0.13) alone. Similarly, the p-PERK/PERK ratio decreased with the test compounds, with the lowest value observed in the β-amyrin+metformin group (1.18 ± 0.09), followed by metformin (1.32 ± 0.12) and β-amyrin (2.93 ± 0.16) against diseased ZF (5.98 ± 0.28).

Furthermore, the elevated IRE1α values in diseased ZF (4.58 ± 0.18) were reduced to 1.02 ± 0.11 in the β-amyrin+metformin treated group, 1.21 ± 0.01 for the metformin treated group, and 2.21 ± 0.43 for the β-amyrin treated group. Similarly, the p-eIF2α/eIF2α ratios were lowered to 1.55 ± 0.13 for β-amyrin+metformin, 2.11 ± 0.23 for metformin, and 3 ± 0.09 for β-amyrin from 6.12 ± 0.15 in diseased ZF. The ATF4 values were found to be 0.98 ± 0.21 for β-Amyrin+Metformin, 1.01 ± 0.23 for metformin, and 1.58 ± 0.11 for β-amyrin against 4.78 ± 0.38 in untreated hyperglycemic ZF. Finally, the raised CHOP values of 5.27 ± 0.39 were lowered to 1.12 ± 0.19 for β-amyrin+metformin and 1.75 ± 0.14 for metformin.

The expressions of apoptosis markers in normal, hyperglycemic, and test compound-treated ZF are summarized below (Fig. ). The study revealed that the levels of apoptotic proteins were significantly elevated in hyperglycemic ZF as compared to normal ZF. Bax, Bcl-2, and Cleaved Caspase-3 levels in hyperglycemic ZF were 6.78 ± 0.45, 5.95 ± 0.65, and 4.78 ± 0.45, respectively. Upon treatment with test compounds, the values reduced significantly with the maximum reduction observed with β-amyrin+metformin; the levels of BAX, BCL-2, and Cleaved Caspase-3 being 1.02 ± 0.10, 1.01 ± 0.29 and 1.11 ± 0.11, respectively.

### Effect of test compounds on proinflammatory cytokines in glucose-induced diabetic ZF

The findings revealed that the combination of β-amyrin+metformin resulted in significantly lower values of proinflammatory cytokines markers in hyperglycemic ZF compared to the other treatment groups ( p ≤ 0.05) (Fig. ). The mRNA expression levels of NF-ĸB were 1.00 ± 0.27 with β-amyrin+metformin, 1.54 ± 0.31 with metformin, 2.63 ± 0.19 with β-amyrin and 3.84 ± 0.18 with naringenin (NAR). The Levels of TNF-α were 1.35 ± 0.07 with β-amyrin+metformin, 1.53 ± 0.26 with metformin, 2.66 ± 0.07 with β-amyrin, and 2.10 ± 0.21 with Standard. IL-6 values were 1.09 ± 0.37 with β-amyrin+metformin, 1.44 ± 0.32 with metformin, 2.99 ± 0.18 with β-amyrin, and 2.89 ± 0.41 with Standard.

### Effect of test compounds on immunohistochemistry of kidney tissues in glucose-induced diabetic ZF

In the study of the relative expression of the GRP78 protein in the renal tissue of hyperglycemic ZF, it was found that the combination of β-amyrin and metformin (1.15 ± 0.13) showed significantly lower values ( p ≤ 0.05) compared to β-amyrin (2.21 ± 0.38) or metformin (1.52 ± 0.15), alone and the standard treatment (2.25 ± 0.31) (Fig. ).

### Effect of test compounds on apoptosis (TUNEL staining) assay of kidney tissues in glucose-induced diabetic ZF

The TUNEL staining was used to quantitatively analyze apoptotic renal tubular epithelial cells (Fig. ). The rate of tissue damage was significantly lowest ( p ≤ 0.05), with the combination of β-amyrin and metformin showing an apoptosis score of 1.63 ± 0.14. This was lower than in the Control (7.10 ± 0.52) and the Standard groups (3.57 ± 0.35). The groups treated with individual metformin (1.91 ± 0.28) and β-amyrin (2.48 ± 0.30) showed higher apoptosis scores than the combination.

### Effect of test compounds on histopathology of kidney tissues in glucose-induced diabetic ZF

The kidney photomicrographs were studied using the H&E stain (Fig. ). The stain revealed degeneration of the glomerulus and vacuolation of tubules in the Control. In contrast, the groups treated with NAR, β-amyrin, metformin, and the combination of β-amyrin metformin showed normal histoarchitecture.

The histopathology of renal tissues of normal and hyperglycemic ZF treated with test compounds was studied following PAS staining (Fig. ). The renal tubular injury scores were significantly ( p ≤ 0.05) lower with the combination β-amyrin+metformin (0.66 ± 0.13) than the untreated group (2.33 ± 0.12) and when individually treated with metformin (0.83 ± 0.09) or β-amyrin (1.16 ± 0.11).

The effect of test compounds on the histopathology of ZF-kidney tissues following the Masson Trichome stain is captured below (Fig. ). The Masson staining of positive areas showed significantly ( p ≤ 0.05) lower scores with a combination of β-amyrin+metformin (1.18 ± 0.25) than the Standard (2.08 ± 0.27) or β-amyrin (1.9 ± 0.17) and metformin (1.52 ± 0.15) individually.

Discussion

Diabetes is a chronic disease that often leads to various comorbidities, including chronic kidney disease (CKD). Animal models have been widely used for screening and assessing the efficacy of multiple drugs to treat CKD in diabetic patients. Among these animal models, ZF have several advantages, including being biologically sustainable for use as animal models, simpler replication of human disease, and sensitivity towards endocrine gland functions and glucose metabolism [ , ]. This study investigated the potential therapeutic benefits of β-amyrin alone and combined with metformin in managing diabetes and associated renal complications using diabetic ZF. The results demonstrated significant improvements in various metabolic and renal parameters, highlighting the synergistic effects of the combination therapy.

In our study, ZF were induced with hyperglycemia using the glucose immersion method for 14 days. The 111 mM glucose solution was chosen based on previous literature establishing high survival of ZF with this method [ ]. The results showed that the blood glucose levels of glucose-induced hyperglycemic ZF were five times higher than their baseline blood glucose levels and compared to the uninduced normoglycemic ZF. The group of ZF that was treated with a combination of β-amyrin and metformin showed the most efficient control of rising blood glucose levels than any other treated groups of hyperglycemic ZF, indicating a synergistic effect in the treatment of hyperglycemia. This aligns with previous studies suggesting that β-amyrin may interact with the cannabinoid system to improve glycemia [ , ], while metformin reduces glucose production in the liver and improves insulin sensitivity in peripheral tissues [ ].

The ZF were monitored for body weight gain after 14 days of treatment. The high glucose (HG)-induced ZF gained much more weight than the normoglycemic ZF, of which the combination of β-amyrin and metformin showed the lowest weight gain compared to those treated with Standard drug or β-amyrin and metformin individually, highlighting the potential of the β-amyrin+metformin combination in managing weight in hyperglycemic conditions. Previous research has shown that treatment with amyrins reduced the high-fat diet-induced increase in body weight and visceral fat content [ ]. Similarly, metformin also demonstrated weight loss in a prior study in mice by affecting neuronal activity in the appetite-regulating brain regions [ ]. Additionally, the KBWR of the group treated with the combination of β-amyrin and metformin reduced significantly, indicating the potential of these compounds in managing kidney function.

The study further investigated the impact of test compounds on metabolic and renal parameters in HG-induced ZF. The test compounds significantly decreased elevated serum urea and creatinine levels, well-recognized markers of renal function [ , ]. The combination therapy was more effective than using the drugs individually, indicating the nephroprotective potential of the combination. This is consistent with the known effects of metformin in reducing renal inflammation and fibrosis [ ] and the antioxidant and anti-inflammatory properties of β-amyrin, which ultimately results in improved kidney function [ ].

Hyperglycemia significantly reduced albumin and total protein levels in ZF, which is likely due to compromised insulin regulation of albumin production. High glucose levels in the blood may worsen beta cell dysfunction, leading to a decrease in insulin secretion. This, in turn, can cause a decline in hepatic albumin synthesis [ ]. In our study, the administration of test compounds raised these albumin levels, with a tremendous increase observed when treated with the combination of β-amyrin+metformin compared to individual treatments, suggesting their potential renoprotective effect. This finding is supported by clinical studies linking low serum albumin concentrations to the development of ESRD and the progression of kidney failure in newly diagnosed diabetic patients [ , ].

Furthermore, the combination of β-amyrin and metformin showed significantly lower serum total cholesterol and triglyceride levels than the individual treatments. This could be attributed to the potential lipid-lowering effects of β-amyrin and metformin, as both have been reported to exhibit hypolipidemic activity [ , ]. Many prior studies have established that high plasma triglycerides were associated with diabetic kidney disease [ – ].

The study findings also revealed that the combination therapy resulted in a statistically significant decrease in serum insulin and fasting blood glucose levels, indicating a potentially improved glucose homeostasis and insulin sensitivity. This synergistic effect is likely due to β-amyrin’s potential role in reducing inflammation [ ], complementing metformin’s inhibition of hepatic gluconeogenesis, and improving insulin sensitivity [ ]. The postprandial glucose levels were also significantly lowered upon treatment with the combination of β-amyrin and metformin, further highlighting their potential as a combination therapy for hyperglycemia management.

The study also investigated the effects of test compounds on DNA damage and antioxidant enzyme levels in glucose-induced diabetic ZF. The results showed that hyperglycemic ZF displayed elevated levels of 8-OHdG, an indicator of DNA damage [ , ], and decreased levels of antioxidant enzymes, including SOD, CAT, and GSH [ ]. However, treatment with β-amyrin and metformin, either alone or in combination, decreased the levels of 8-OHdG, indicating the repair of damaged DNA. Moreover, treatment with combination therapy resulted in higher levels of SOD, CAT, and GSH than treatment with either compound alone, suggesting higher efficacy in reducing oxidative stress and restoring antioxidant capacity in diabetic ZF. The findings were consistent with previous studies demonstrating the antioxidant properties of β-amyrin and metformin [ , ]. In addition, the reduced levels of thiobarbituric acid reactive substances (TBARS; byproduct of LPO as degradation products of fats) in the test compounds-treated fish indicated a decrease in LPO, leading to improved protection of cell membranes [ ], and further supporting the antioxidant effects of the test compounds.

Next, the study demonstrated that treatment with β-amyrin and metformin, alone and in combination, led to substantial reductions in the expression of pro-apoptotic (Bax, cleaved caspase-3) and ER stress markers (PERK, p-PERK, IRE1α, eIF2α, p-eIF2α, ATF4, CHOP) in the renal tissues of diabetic ZF. The combination therapy exhibited a more pronounced reduction compared to the individual drugs. Additionally, diabetic ZF treated with the test compounds showed lowered levels of the p-PERK/PERK ratio, IRE1α, and p-eIF2α/eIF2α ratios. These findings suggest that targeting ER stress with these compounds could prevent renal fibrosis and glomerular sclerosis in diabetic nephropathy, as ER stress-induced inflammation exacerbates renal damage, perpetuating a cycle of tissue injury and dysfunction [ ]. The reduction in pro-apoptotic markers further supported the potential of these compounds in reducing renal inflammation. Prior research has also established a connection between Caspase-3 and apoptosis, inflammation, and fibrosis in chronic renal scarring [ ].

The reduction in mRNA expression levels of NF-κB, TNF-α, and IL-6 in renal tissues of diabetic fish supports the potent anti-inflammatory effect of the test compounds in mitigating inflammation associated with hyperglycemic renal toxicity in vivo. The combination therapy demonstrated superior results to therapy alone, indicating a synergistic effect. This finding is consistent with earlier studies reporting the anti-inflammatory effects of β-amyrin and metformin in various models of inflammation and diabetes [ , ].

Our findings were further corroborated by the results of the TUNEL staining assay, which revealed a significant reduction in apoptotic renal tubular epithelial cells in glucose-induced diabetic ZF treated with a combination of β-amyrin and metformin compared to individual treatments and the Control. This finding aligns with an earlier study demonstrating the synergistic effects of combining natural compounds with conventional medications in mitigating apoptosis in diabetic conditions [ ].

In parallel, the histopathological analysis of kidney tissues using H&E staining and PASM staining further supported the efficacy of the β-amyrin+metformin combination in reducing renal tubular injury scores compared to individual treatments and the untreated group. These results are consistent with research highlighting the protective effects of both β-amyrin and metformin on renal histology in diabetic models [ , ].

Moreover, the Masson Trichome staining results demonstrated a significant decrease in fibrotic changes in ZF kidney tissues treated with β-amyrin+metformin compared to NAR, β-amyrin, and metformin alone. This outcome underscores the potential of combining β-amyrin and metformin to attenuate fibrosis in diabetic kidney disease, corroborating findings from studies on the anti-fibrotic properties of these compounds [ , ].

Conclusion

The study demonstrated that a combination of β-amyrin and metformin had a synergistic effect in preserving renal resilience in a hyperglycemic ZF model. Compared to either drug alone, the combination therapy was more effective in reducing blood glucose levels, restoring kidney function, reducing oxidative stress and inflammation, and improving renal architecture. These findings suggest that β-amyrin and metformin combination therapy could be a promising approach for managing diabetic kidney disease in humans.

The study has limitations owing to variations in the immune system and drug metabolism between Zebrafish and mammals and because Zebrafish do not entirely replicate the complex metabolic and physiological characteristics of human diabetes and kidney function, which may impact the extrapolation of findings to human applications. Despite these limitations, the study establishes a strong foundation for subsequent research and validation in other animal models and, eventually, in human subjects. Further studies are warranted to investigate the clinical relevance of these results.

Electronic supplementary material

Below is the link to the electronic supplementary material.