Saroglitazar ameliorates alcoholic liver disease and is associated with enhanced ethanol metabolism

Abstract

BACKGROUND

Alcoholic liver disease (ALD), or alcohol-related liver disease, is a major global cause of morbidity and mortality, encompassing a spectrum of conditions from simple steatosis to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Alcohol consumption contributes to approximately 5.3% of global deaths and 5.1% of the worldwide burden of disease and injury. With approximately 43% of the global population consuming alcohol, it remains a key driver of liver damage, accounting for nearly 60% of cirrhosis cases in Europe, North America, and Latin America. There remains an unmet need for effective treatments that can reverse or prevent alcohol-induced liver disease.

AIM

To determine whether saroglitazar could prevent or improve alcohol-induced liver injury in the widely used National Institute on Alcohol Abuse and Alcoholism model using C57 mice.

METHODS

Under the preventive study design, male C57BL/6 mice (8-10 weeks old) were fed on a Lieber-DeCarli diet containing 5% (v/v) ethanol were orally administered saroglitazar at 0.4 mg/kg and 0.8 mg/kg doses for 10 days. In the therapeutic study design, animals were fed with a liquid ethanolic (4%) diet for 4 weeks and after confirming the presence of ALD, the mice were administered saroglitazar at doses of 0.4 mg/kg or 0.8 mg/kg orally once daily for 4 weeks.

RESULTS

In both study designs, saroglitazar significantly attenuated ethanol-induced elevations in plasma alanine aminotransferase and plasma ethanol concentration. Changes in plasma ethanol were accompanied by significantly elevated plasma acetate levels, a non-toxic alcohol metabolite, increased hepatic aldehyde dehydrogenase 2 activity and upregulated expression of alcohol dehydrogenase genes. In the therapeutic study design saroglitazar resulted in complete reversal of hepatic lipid accumulation and significant improvement in liver histology. The comparator hepatoprotective reference compound, ursodeoxycholic acid, did not produce any significant improvement in ethanol metabolism, although it significantly reduced liver injury markers such as plasma alanine aminotransferase and aspartate aminotransferase.

CONCLUSION

Saroglitazar is associated with altered alcohol metabolism, significantly improves plasma and liver biomarkers associated with ALD and highlights its potential as a promising therapeutic candidate for the treatment of ALD.

Key Words: Dual-peroxisome proliferator-activated receptor alpha/gamma agonist; Alcoholic liver disease; Saroglitazar; Alcohol metabolism; National Institute on Alcohol Abuse and Alcoholism mouse model

Core Tip: Alcoholic liver disease is one of the major causes of morbidity worldwide and there is an unmet need for a drug to prevent or treat this condition. Saroglitazar, which is approved in India for the treatment of metabolic dysfunction-associated steatotic liver disease and metabolic dysfunction-associated steatohepatitis, showed beneficial effect like reduction in alanine aminotransferase, aspartate aminotransferase and hepatic triglyceride in animal model of alcoholic liver disease. Saroglitazar effectively altered the alcohol metabolism markers, thus reducing the alcohol-induced liver damage.

INTRODUCTION

Alcoholic liver disease (ALD), also called alcohol-related liver disease (ARLD), is a major cause of morbidity and mortality worldwide. According to Global Status Report on Alcohol and Health 2024, about 2.5 billion people drink alcohol worldwide, and more than half of the population in the United States, Europe, and the Western Pacific regions consumes alcohol. Chronic heavy drinking is the etiology or risk factor for many diseases, such as ARLD, acute pancreatitis, and alcohol-related cardiomyopathy. According to the World Health Organization data, global number of deaths caused by alcohol was approximately 2.6 million in 2016. ARLD has become one of the major causes of alcohol-related death[1]. Almost all heavy drinkers develop fatty liver, but only 20%-40% of them progress to more severe forms of ALD, and the underlying mechanisms that contribute to disease progression remain largely unknown. Chronic alcohol consumption is a leading cause of chronic liver disease worldwide, leading to cirrhosis and hepatocellular carcinoma.

Following ingestion, more than 90% of ingested alcohol get metabolized by the liver, and this burden remains the principal cause of liver damage in ALD. The oxidative metabolism involves the conversion of ethanol into acetaldehyde in presence of alcohol dehydrogenase (ADH) and then the acetaldehyde gets converted to acetate in the presence of aldehyde dehydrogenase (ALDH). Acetaldehyde is considered to be the key toxin in alcohol mediated liver injury, inflammation and fibrosis[2] whereas the acetate is considered to be non-toxic and further broken down into carbon dioxide and water for elimination, according to the National Institute on Alcohol Abuse and Alcoholism (NIAAA).

Looking at growing rate of alcohol related death, there is an unmet need for an effective treatment which can reverse or prevent the alcohol-induced liver injury. Previous studies have demonstrated that peroxisome proliferator-activated receptor gamma activation have beneficial effect on alcohol-induced liver injury in rats[3,4]. Peroxisome proliferator-activated receptor alpha (PPARα) activation improves steatosis and prevent the pathological progression of hepatic diseases caused by chronic alcohol exposure in C57 mice[5]. Saroglitazar is a novel PPARα/γ dual agonist having predominant PPARα activity. In various preclinical and clinical studies saroglitazar has shown efficacy as a treatment of metabolic dysfunction-associated steatotic liver disease (MASLD), metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma[6,7]. Saroglitazar has been approved in India for the treatment of MASLD and MASH.

Considering the role of PPAR agonists in alcohol-induced liver injury and role of saroglitazar in management of MASH, we evaluated the efficacy of saroglitazar in the mouse model for alcoholic liver injury developed by NIAAA[8,9].

MATERIALS AND METHODS

Chemicals

Saroglitazar was synthesized by Zydus Lifesciences Limited, Ahmedabad, India. Ursodeoxycholic acid (UDCA) was procured from Tokyo Chemical Industry Co. Ltd, India. Saroglitazar was the compound evaluated in this study, while UDCA was included as a hepatoprotective reference compound based on its established cytoprotective and anti-inflammatory effects in liver diseases, as well as its prior evaluation in experimental models of hepatic injury.

Animal experiments

Healthy male C57BL/6J mice aged 8-10 weeks (Strain: 000664; originally sourced from The Jackson Laboratory), bred at the Animal Research Facility of Zydus Lifesciences Limited, supplied by Zydus Lifesciences Limited, and used in this experiment. All animals were housed in individually ventilated cages (3-4 mice per cage) and maintained on a standard laboratory rodent diet (Teklad 2018C, Harlan Laboratories, United States) and water ad libitum in a temperature (25 ± 3 °C) and humidity (50%-70%)-controlled environment with a 12-hour/12-hour dark-light cycle in a facility accredited by Association for Assessment and Accreditation of Laboratory Animal Care International and all animal experiments were approved by Institutional Animal Ethics Committee of Zydus Research Centre, Zydus Lifesciences Limited, under protocol No. ZRC/PH/BP/049/02-2K23 . Then the animals were provided with Lieber-DeCarli’82 liquid ethanolic diet (F1258SP) or control liquid diet (F1259SP) in a specified 50 mL feeding tube procured from Bio-Serv, Flemington, NJ. The diet was prepared as per manufacturer instruction using the feeding protocol as reported in the NIAAA model[8].

In the preventive study design, a total of 36 animals with comparable body weights were selected and randomized into four treatment groups (n = 9/group) based on their body weight. Animals were group-housed, with three animals housed per cage throughout the study period. The animals were acclimatized with the liquid diet for 3 days, then all the animals were pair-fed with disease control group with 5% liquid ethanolic diet or control liquid diet for a period of 10 days along with daily oral administration of vehicle or saroglitazar at 0.4 mg/kg and 0.8 mg/kg dose (n = 9/group). The recommended clinical dose of saroglitazar for the treatment of MASH is 4 mg. Based on body surface area-based dose conversion, the corresponding human equivalent dose in mice was calculated as 0.8 mg/kg, and a lower dose of 0.4 mg/kg (half of the human equivalent dose) was also evaluated. Animals fed a control liquid diet in combination with vehicle were assigned to the normal control group, whereas animals administered a 5% (v/v) ethanolic liquid diet together with vehicle constituted the disease control group. On day 11, just after the dose administration, mice receiving the ethanolic diet were administered an acute ethanol binge [5 g/kg, orally (PO); equivalent to 31.5% (v/v)] and the normal control group will be PO administered with water in the morning at 08:30. Blood samples were collected from retro-orbital sinus under isoflurane anaesthesia, at 2-hour post-binge to measure plasma ethanol and acetate levels, and again at 9-hour post-binge to assess plasma alanine aminotransferase (ALT) concentrations.

In the therapeutic study design, 70 mice were gradually acclimatized to the ethanol diet, starting with 1% ethanol and increasing to 4% over one week and 8 animals were acclimatized with control liquid diet. After this acclimatization period, the mice were maintained on a 4% ethanol diet or control diet ad libitum for 8 weeks. Diet was prepared and provided fresh each day; body weight and feed consumption were monitored regularly. After 4 weeks of ethanol diet feeding, body weight of all the animals were recorded. All mice received a dose of ethanol binge [5 g/kg, PO (i.e., 31.5% (v/v)] in the morning at 08:30 to assess the initial damage. Blood collection was performed under isoflurane anaesthesia from retro-orbital sinus, at 2-hour post binge for plasma ethanol level and 9-hour post binge for estimation of plasma ALT and aspartate aminotransferase (AST) level. Animals with plasma ethanol level > 400 mg/dL and a significantly high level of plasma ALT were selected for the study and randomized to different treatment groups such that the mean plasma ethanol level and plasma ALT level were not significantly different within groups (Supplementary Table 1). Animals were grouphoused, with three animals housed per cage throughout the study period. Saroglitazar and UDCA were formulated in Tween 80 and 0.5% sodium carboxymethyl cellulose (0.5:99.5) to achieve a dosing volume of 10 mL/kg. Mice received either vehicle (disease control), saroglitazar (0.4 mg/kg or 0.8 mg/kg, once daily), or UDCA (250 mg/kg, twice daily) via oral gavage for 4 weeks (n = 9/group) and maintained on 4% ethanol diet till completion of the study. The dose of saroglitazar was selected as described in the above for preventive study design. In multiple murine studies, UDCA has been administered at 0.5% (w/w) in the diet, corresponding to an effective dose of approximately 500 mg/kg/day. Therefore, UDCA was administered at 250 mg/kg twice daily to achieve the target daily dose[10,11]. A normal control group (n = 8) received vehicle with a control diet. To minimize differences in caloric and ethanol intake, animals in the treatment groups were pair-fed with the disease control group. All treatments were administered in the morning, with UDCA animals receiving an additional evening dose. At the end of the 4-week period, all animals were given an oral ethanol binge (5 g/kg) in the morning at 08:30. Blood samples were collected under isoflurane anaesthesia from retro-orbital sinus, 2 hours post-binge to assess plasma alcohol and acetate levels, and again at 9 hours to evaluate liver toxicity biomarkers. Following this, animals were euthanized and livers were harvested. Portions of liver tissue were snap-frozen in liquid nitrogen for gene expression, biochemical analysis, and ALDH2 activity, while other sections were preserved in 10% neutral buffered formalin for histological examination using hematoxylin and eosin (HE) staining.

Biochemical analysis

Plasma ethanol, ALT, AST and triglyceride (TG) levels were measured using commercial kits on a Mindray BS-240 analyser (Shenzhen Mindray Bio-Medical Electronics Co., Ltd, China). Plasma acetate level was measured using acetate colorimetric assay kit (Cat No. MAK086 from SIGMA-ALDRICH). The hepatic ALDH2 activity was measured as per the manufacturer’s instruction using mitochondrial ALDH2 activity assay kit (Cat No. ab115348 from Abcam). Total liver lipids were extracted, and hepatic TG and total cholesterol (TC) content was quantified using TG and TC test kits from Agappe Diagnostics, India.

Liver histology

Liver tissue samples were collected and immediately fixed in 10% neutral buffered formalin to preserve tissue integrity. Following fixation, the samples were processed using a routine paraffin embedding technique, ensuring optimal preservation for histopathological evaluation. The paraffin-embedded tissues were precisely sectioned into 5 µm thick slices and mounted onto slides for further histopathological evaluation.

Subsequently, these slides were stained using HE stain, a widely used staining method that highlights cellular structures and morphological details under light microscopy. Further, the collagen specific (type-I and type-III) special staining of Sirius Red was performed to confirm the evidence of fibrosis in liver parenchyma. The HE-stained sections were meticulously evaluated for the presence and severity of steatosis, hepatocellular degeneration, inflammation and fibrosis. The use of severity grade to characterize the histopathological changes to meet the international standard was performed and criteria of histopathological changes are as follows: (1) Grade 0: Absence of steatosis; (2) Grade 1: Minimal steatosis, occupying < 20% of the hepatic parenchyma; (3) Grade 2: Mild steatosis, occupying 21%-50% of the hepatic parenchyma; (4) Grade 3: Moderate steatosis, occupying 51%-75% of the hepatic parenchyma; and (5) Grade 4: Severe steatosis, occupying 76%-100% of the hepatic parenchyma[12]. Histology analysis was performed by an experienced board-certified pathologist (Diplomat of Indian Board of Toxicologic Pathology) and peer reviewed by a senior pathologist, a Diplomat of Indian College of Veterinary Pathologists to ensure accuracy and consistency.

The quantitative polymerase chain reaction analysis

Liver samples were homogenized in RNA-Xpress reagent (HiMedia, India), and total RNA was extracted from the tissues according to the supplier’s instructions. One microgram of total RNA from each sample was used for firststrand cDNA synthesis using the Verso cDNA synthesis kit (Thermo Fisher Scientific, India). Equal amounts of cDNA were then subjected to quantitative real-time polymerase chain reaction (PCR) using the ABI Prism 7500 system (Applied Biosystems). Gene expression was assessed using gene-specific primers for CYP2E1, ADH1, and ALDH2 with Kapa SYBR FAST (KK4618, KAPA, United States). β-actin was validated prior to analysis and confirmed to be a stable reference gene under ethanol exposure conditions. The PCR conditions included an initial denaturation at 95 °C for 5 minutes, followed by 40 cycles of denaturation at 95 °C for 10 seconds and annealing/extension at 60 °C for 32 seconds. Primer specificity for all genes was verified by single-peak melt curve analysis, confirming amplification of a single PCR product with primer efficiency exceeding 90%. Relative gene expression was calculated using the 2^-ΔΔCt method, and data are presented as fold changes with corresponding statistical significance. The primer sequences used were: (1) For CYP2E1, forward (5’-CTGAGATATGGGCTCCTGATTC-3’) and reverse (5’-ATCTCATGCACTACAGCGTC-3’); (2) For Adh1, forward (5’-GGAGGGGTGGACTTTTCGTT-3’) and reverse (5’-TACGACGACGCTTACACCAC-3’); and (3) For Aldh2, forward (5’-AGACCATCGAGGAGGTTGTG-3’) and reverse (5’-CCCCAAACACATCGTAGCAG-3’). Gene expression levels were normalized to beta-actin, amplified using the forward primer (5’-CACTGTCGAGTCGCGTCC-3’) and reverse primer (5’-TCATCCATGGCGAACTGGTG-3’) as mentioned in Supplementary Table 2.

Statistical analysis

We used a sample size of n = 9, which lies within the commonly accepted range for preclinical studies (n = 6-12) and is generally sufficient to achieve statistical significance. The data were analysed using one-way analysis of variance followed by Dunnett multiple comparison method. Error bars represent SEM. Statistical analyses were performed with Prism version 9 (Graph Pad Software, La Jolla, CA, United States). P values for each comparison were calculated, and P ≤ 0.05 was considered statistically significant.

RESULTS

Preventive study design

Saroglitazar significantly prevents the ethanol induced elevation in marker of liver injury: Ten days of chronic ethanol feeding followed by an acute binge on day 11 resulted in marked hepatocellular injury, evidenced by a 7.9 ± 1.5-fold increase in plasma ALT levels compared with normal controls (253.9 ± 48.4 U/L vs 32.1 ± 3.1 U/L). Treatment with saroglitazar produced a robust hepatoprotective effect, lowering ALT concentrations by 70.3% ± 3.4% and 70.0% ± 1.5% at doses of 0.4 mg/kg and 0.8 mg/kg, respectively, relative to the disease control group (Figure 1A). These findings indicate that saroglitazar effectively mitigates ethanol-induced liver injury in this experimental model.

Figure 1 Effect of saroglitazar on plasma alanine aminotransferase, ethanol, and acetate levels in the preventive study design. Mice were administered saroglitazar or vehicle during a 10-day ethanol-feeding protocol. Blood samples were collected 2 hours after the ethanol binge to measure plasma ethanol and acetate levels, and 9 hours after the binge to assess plasma alanine aminotransferase. A: It shows plasma alanine aminotransferase concentrations; B: It shows plasma ethanol levels; C: It shows plasma acetate levels. Data are presented as mean ± SEM (n = 9). Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s multiple comparison test. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs disease control group. ALT: Alanine aminotransferase; PO: Orally; OD: Once daily.

Saroglitazar significantly reduced the plasma ethanol level and increase the acetate level: Plasma concentrations of ethanol and its non-toxic metabolite, acetate were quantified 2 hours after the ethanol binge. Saroglitazar treatment significantly enhanced ethanol clearance, reducing plasma ethanol levels by 31.9% ± 1.3% and 36.2% ± 3.5% at doses of 0.4 mg/kg and 0.8 mg/kg, respectively, compared with the disease control group (Figure 1B). Correspondingly, acetate levels increased by 81.1% ± 21.2% and 68% ± 23.3% at the same doses, indicating a shift toward accelerated detoxification through altered ethanol metabolism to non-toxic by-products (Figure 1C). These findings collectively suggest that saroglitazar facilitates more efficient ethanol biotransformation under conditions of acute-on-chronic exposure.

Therapeutic study design

Saroglitazar significantly reduced the markers of liver injury and plasma lipid levels: This data underscores the therapeutic potential of saroglitazar and UDCA in mitigating elevated plasma ALT, AST, and TG levels – key indicators of liver dysfunction and metabolic imbalance. Notably, in the disease control group, plasma ALT and AST levels were elevated by 5.3 ± 2.6-fold and 1.9 ± 0.8-fold, respectively, signaling significant hepatic stress or damage. Saroglitazar treatment reduced plasma ALT by 81.6% ± 1.4% and 57.1% ± 8.2%, and AST by 68.9% ± 3.0% and 44.0% ± 10.8 %, at doses of 0.4 mg/kg and 0.8 mg/kg doses, respectively. UDCA treatment led to a 77% reduction in ALT and a 65% reduction in AST compared to disease controls (Figure 2A and B).

Figure 2 Effect of saroglitazar on liver injury markers and plasma triglyceride in therapeutic study design. Mice were treated with saroglitazar or vehicle for 4 weeks during ethanol feeding. Blood samples were collected 9 hours after the ethanol binge to measure plasma alanine aminotransferase, aspartate aminotransferase, and triglyceride levels. A: It shows plasma alanine aminotransferase concentrations; B: It shows plasma aspartate aminotransferase levels; C: It shows plasma triglyceride levels. Data are presented as mean ± SEM (n = 8 for normal control and n = 9 disease control and treatments). Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s multiple comparison test. ^aP < 0.05 vs disease control group. ALT: Alanine aminotransferase; PO: Orally; OD: Once daily; UDCA: Ursodeoxycholic acid; AST: Aspartate aminotransferase; TG: Triglyceride.

Saroglitazar showed dose-dependent reduction in the plasma TG levels which was 25.3% ± 12.9% at 0.4 mg/kg dose and 40.5% ± 10.7% at 0.8 mg/kg dose while the UDCA treatment did not show any significant reduction in the plasma TG levels (Figure 2C).

Saroglitazar reduces the plasma ethanol level by increasing the markers of alcohol metabolism: To assess saroglitazar’s impact on alcohol metabolism, plasma ethanol and its metabolite acetate were measured 2 hours post-ethanol binge. Saroglitazar significantly reduced plasma ethanol levels by 43.5% ± 2.2% and 44.9% ± 2.8% at 0.4 mg/kg and 0.8 mg/kg doses, respectively (Figure 3A). In contrast, UDCA showed a non-significant 7.4% ± 7.2% reduction after 4 weeks of treatment. The non-toxic by-product of ethanol metabolism, acetate levels increased by 2.3 ± 0.4-fold and 3.4 ± 0.2-fold at 0.4 mg and 0.8 mg dose respectively and are significant as compared to disease control group (Figure 3B). UDCA did not show any significant change in the plasma acetate level. To support this mechanism, hepatic ALDH2 activity – responsible for converting toxic aldehydes to non-toxic acetate – was measured. Saroglitazar significantly increased ALDH2 activity by 1.7 ± 0.1-fold at 0.4 mg/kg and 2.0 ± 0.1-fold at 0.8 mg/kg compared to the disease control group (Figure 4). UDCA treatment resulted in minimal 1.3 ± 0.1-fold rise in hepatic ALDH2 activity as compared to the disease control group. Despite a significant reduction in plasma ethanol and an increase in ALDH2 enzymatic activity, acetaldehyde concentrations remained unchanged across the measured conditions (Supplementary Table 3), suggesting stable steady-state levels without detectable systemic accumulation.

Figure 3 Effect of saroglitazar on plasma ethanol and acetate levels in the therapeutic study design. Mice received saroglitazar or vehicle during a 4-week ethanolfeeding protocol. Blood samples were collected 2 hours after the ethanol binge to determine plasma ethanol and acetate levels. A: It shows plasma ethanol concentrations; B: It shows plasma acetate levels. Data are presented as mean ± SEM (n = 8 for normal control and n = 9 disease control and treatments). Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s multiple comparison test. ^bP < 0.01, ^cP < 0.001 vs disease control group. PO: Orally; OD: Once daily; BID: Twice daily; UDCA: Ursodeoxycholic acid.

Figure 4 Effect of saroglitazar on liver aldehyde dehydrogenase 2 activity in therapeutic study design: Mice received saroglitazar or vehicle during a 4-week ethanol-feeding protocol. Liver samples were collected 9 hours after the ethanol binge to determine aldehyde dehydrogenase 2 activity using an enzyme-linked immunosorbent assay-based assay. It shows hepatic aldehyde dehydrogenase 2 activity expressed as mean ± SEM (n = 8 for normal control and n = 9 disease control and treatments). Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s multiple comparison test. ^aP < 0.05, ^cP < 0.001 vs disease control group. PO: Orally; OD: Once daily; BID: Twice daily; UDCA: Ursodeoxycholic acid; ALDH2: Aldehyde dehydrogenase 2.

Saroglitazar up-regulated the hepatic genes involved in alcohol metabolism: The major hepatic genes that contribute to alcohol metabolism include Cyp2E1, Adh and Aldh2[9]. Saroglitazar treatment at both 0.4 mg/kg and 0.8 mg/kg doses upregulated the hepatic expression of Adh and Cyp2E1 genes as compared to disease control (Figure 5). There was no significant alteration in the hepatic gene expression of aldh2 in the saroglitazar treated animals.

Figure 5 Effect of saroglitazar on hepatic genes involved in alcohol metabolism. Liver mRNA expression of genes involved in alcohol metabolism was assessed by quantitative polymerase chain reaction. It shows the expression levels of key metabolic genes, including Cyp2E1, Adh and Aldh2, in liver tissue from mice treated with saroglitazar or vehicle. Data are presented as mean ± SEM (n = 8 for normal control and n = 9 disease control and treatments). Statistical analysis was performed using one-way analysis of variance followed by Dunnett’s multiple comparison test. ^cP < 0.001 vs disease control group. PO: Orally; OD: Once daily; BID: Twice daily; UDCA: Ursodeoxycholic acid.

Significant alteration in hepatic lipid accumulation was observed by saroglitazar treatment: Liver histology using HE staining showed that liquid ethanolic diet feeding resulted in significantly high steatosis score which is one of the common features in ALD. The disease control group exhibited a steatosis score of 2.1 ± 0.4, which was reduced to 0.8 ± 0.2 following treatment with saroglitazar at 0.4 mg/kg. Notably, a complete reversal of steatosis was observed by saroglitazar at a dose of 0.8 mg/kg. While saroglitazar showed significant improvement in the hepatic steatosis, UDCA failed to show any improvement in the steatosis score vs disease control 2.6 ± 0.3 vs 2.1± 0.4) (Figure 6A). We have assessed the fibrotic changes in HE -stained sections, but no significant differences in fibrosis-related features were observed across the treatment groups. To further validate these findings, Sirius red staining was performed to specifically assess collagen deposition. Consistent with the HE analysis, Sirius Red staining did not reveal any significant differences across the groups. The corresponding data are presented in Supplementary Table 4. Biochemical analysis of hepatic TG and TC levels showed trends consistent with the steatosis scores observed in HE stained liver sections. The dose-dependent reductions in liver lipid levels observed with saroglitazar were 18.8% ± 7.2% and 41.6% ± 5.0% for liver TGs (Figure 6B), and 22% ± 8.1% and 50.2% ± 4.4% for liver TC (Figure 6C) at 0.4 mg/kg and 0.8 mg/kg doses respectively. The comparator hepatoprotective reference compound, UDCA used in this study did not show any reduction in the liver lipid levels.

Figure 6 Effect of saroglitazar on hepatic steatosis and lipid accumulation in therapeutic study design. A: Liver histology was evaluated using hematoxylin and eosin staining to assess the degree of steatosis. ^bP < 0.01, ^cP < 0.001; B and C: Liver tissue samples were homogenized to quantify hepatic triglyceride and total cholesterol levels. Data are presented as mean ± SEM (n = 8 for normal control and n = 9 disease control and treatments). Statistical analysis was performed using oneway analysis of variance followed by Dunnett’s multiple comparison test. ^bP < 0.01 vs disease control group. HE: Hematoxylin and eosin; PO: Orally; OD: Once daily; BID: Twice daily; UDCA: Ursodeoxycholic acid.

DISCUSSION

Alcoholic liver disease poses a significant global health challenge, with few treatments beyond abstinence and supportive care. This study presents strong preclinical evidence that saroglitazar, a dual PPARα/γ agonist, offers hepatoprotective benefits in alcohol-induced liver injury by altering the markers of ethanol metabolism, reducing hepatocellular damage, and modulating key metabolic enzymes – effects that go beyond lipid regulation. A key novel observation from our study is that saroglitazar significantly reduces plasma ethanol levels following binge administration while concomitantly increasing plasma acetate levels. This change is consistent with accelerated ethanol clearance, likely mediated by saroglitazar-induced upregulation of hepatic ADH and ALDH2 activities. Ethanol metabolism in the liver occurs primarily via sequential oxidation of ethanol to acetaldehyde by ADH, followed by rapid conversion of acetaldehyde to acetate by ALDH2[13]. Acetaldehyde is considered as the most toxic metabolite, implicated in mitochondrial dysfunction, protein adduct formation, oxidative stress, and fibrogenesis[14]. Conversely, acetate is relatively benign and readily utilized in peripheral tissues for energy metabolism. By enhancing ALDH2 activity and increasing acetate formation, saroglitazar appears to accelerate the detoxification of acetaldehyde, thereby reducing its hepatotoxic burden. Although we observed a reduction in plasma ethanol and an increase in acetate levels, no significant change in plasma acetaldehyde was detected. This likely reflects rapid hepatic clearance of acetaldehyde, as high ALDH2 activity efficiently converts acetaldehyde to acetate, thereby limiting its accumulation and systemic spillover into the blood stream[15]. Previous studies have demonstrated that impaired ALDH2 activity predisposes to greater ALD severity and carcinogenesis[16]. Genetic polymorphisms in ALDH2 (e.g., ALDH2*2 common in East Asians) confer reduced enzymatic activity and heightened susceptibility to alcohol-induced liver injury[17]. Pharmacological upregulation of ALDH2 has therefore been proposed as a therapeutic strategy in ALD[18]. Our findings highlight saroglitazar’s translational potential, demonstrated by its significant enhancement of hepatic ALDH2 activity, upregulation of ADH gene expression and a trend in increasing CYP2E1 gene expression – indicating a comprehensive remodelling of ethanol-metabolizing pathways. CYP2E1 is responsible for approximately 10% of ethanol oxidation and is classically associated with reactive oxygen species generation and hepatotoxicity[19]. In the present study, although CYP2E1 expression showed an increasing trend, the change did not reach statistical significance and was not accompanied by a significant increase in oxidative stress or inflammatory markers. Specifically, no significant changes were observed in hepatic malondialdehyde levels (data presented in Supplementary Table 3). Therefore, contribution of CYP2E1 to the observed changes in ethanol metabolism markers remains uncertain. The above-mentioned changes were accompanied by substantial reductions in plasma ALT and AST levels, reflecting decreased hepatocellular injury. Histological analysis revealed marked improvement in hepatic steatosis, with near-complete resolution at higher doses. Concurrent reductions in hepatic TG and cholesterol levels further support saroglitazar’ s role in restoring lipid homeostasis.

The pathogenesis of ALD involves complex interactions among ethanol metabolism, lipid accumulation, oxidative stress, and inflammatory signalling[20,21]. PPARα plays a central role in hepatic fatty acid oxidation, while PPARγ regulates lipid storage and insulin sensitivity[22]. Agonism of these receptors has been shown to reduce hepatic steatosis in MASLD and MASH. For example, clofibrate (a PPARα agonist) attenuates steatosis and inflammation in ethanol-fed mice[23], and pioglitazone (a PPARγ agonist) improves insulin sensitivity and steatosis in metabolic dysfunction-associated fatty liver disease[24]. Saroglitazar, with dual PPARα/γ agonism, may therefore exert synergistic benefits by enhancing fatty acid oxidation, reducing de novo lipogenesis, and modulating adiponectin signaling[25,26]. Indeed, saroglitazar has demonstrated efficacy in multiple rodent models of MASH and hepatocellular carcinoma[6,7]. Our findings extend these benefits to alcohol-induced steatosis, thereby broadening its therapeutic spectrum.

In the present study, UDCA was included for exploratory purposes for the comparative assessment of hepatoprotective effects. Although not a standard therapy for ALD, UDCA is widely used in other liver disorders and has been reported to exert cytoprotective effects, including improvement in liver enzyme levels and attenuation of hepatic injury. In this study, saroglitazar outperformed UDCA by not only reducing ALT and AST levels more effectively but also significantly improving steatosis, lowering TG accumulation, and enhancing markers of ethanol clearance. These results are aligned with clinical data showing limited efficacy of UDCA in ALD[27]. UDCA primarily exerts cytoprotective and anti-apoptotic effects by stabilizing hepatocyte membranes and reducing bile acid toxicity[28], but lacks robust metabolic effects. In contrast, saroglitazar directly targets the lipid dysregulation and ethanol metabolism markers central to ALD pathogenesis.

ALDH2 is a mitochondrial NAD⁺-dependent enzyme that catalyzes the conversion of acetaldehyde to acetate. The activity of such enzymes is influenced by intracellular NAD⁺ availability, which can enhance catalytic function independent of changes in gene expression. Therefore, the observed increase in ALDH2 activity without a corresponding change in mRNA levels may reflect cofactor-driven regulation and/or post-transcriptional mechanisms of PPARα mediated mechanism[29]. Our findings underscore the potential of saroglitazar as a promising therapeutic candidate for ALD rather than merely a hepatoprotective agent. Saroglitazar appears to mitigate ALD progression by altering ethanol metabolism markers like enhancing ADH/ALDH2 activity, reducing acetaldehyde toxicity, correcting ethanol-induced dyslipidemia via PPARα/γ activation and enhancing antioxidant defences, as previously observed in MASH models[6]. Saroglitazar may also have additional benefits in ALD patients. Elevated plasma ethanol contributes not only to hepatic injury but also to pancreatitis, cardiomyopathy, and neurotoxicity[30]. By accelerating ethanol elimination, saroglitazar may reduce these extrahepatic complications. Furthermore, acetate, the end-product elevated by saroglitazar, serves as a metabolic substrate for the tricarboxylic acid cycle and may support energy homeostasis in peripheral tissues[31].

Despite its promising findings, our study has certain limitations. First, the murine NIAAA model, although widely used, does not fully capture the chronicity and complexity of human ALD such as fibrosis, alcoholic hepatitis, and/or cirrhosis. Consistent with the known characteristics of this model, our experimental setup did not reveal histological features of hepatic fibrosis or more advanced liver injury. Currently, pharmacological interventions for ALD are limited and largely unsatisfactory. Corticosteroids remain the mainstay for severe alcoholic hepatitis but are associated with high relapse rates and infectious complications. Other agents such as pentoxifylline, N-acetylcysteine, and antioxidants have yielded inconsistent results[32,33]. Our data suggest that saroglitazar has potential not only as a hepatoprotective therapy but also as a metabolic modulator and ethanol detoxifier, offering a multi-pronged benefit in ALD. This study provides a strong rationale for exploring saroglitazar for ALD.

CONCLUSION

Saroglitazar demonstrated significant protective and therapeutic efficacy against alcohol-induced liver injury in the NIAAA mouse model. Saroglitazar markedly improved plasma and hepatic biomarkers of liver injury, accompanied by normalization of liver histology and reversal of hepatic steatosis. The beneficial effects were associated with altered ethanol metabolism markers, as evidenced by increased ALDH2 activity, upregulation of ADH gene expression, and elevated plasma acetate levels. Notably, saroglitazar improved ethanol clearance, a mechanism not observed with UDCA treatment. These findings suggest that saroglitazar not only mitigates liver injury but also targets key metabolic pathways involved in ALD pathogenesis. Overall, saroglitazar holds promise as a novel therapy targeting key pathogenic factors for the prevention and treatment of alcoholic liver disease.

ACKNOWLEDGEMENTS

All the authors acknowledge the support received from Zydus Lifesciences Limited, Ahmedabad, India.