Sodium Orthovanadate Mitigates Nonalcoholic Fatty Liver Disease by Enhancing Autophagy

Abstract

Sodium orthovanadate (SOV) has been investigated in recent research for its therapeutic efficacy in treating metabolic disorders. Considering the rising prevalence of non-alcoholic fatty liver disease (NAFLD), the effects of SOV on NAFLD remain to be further investigated. The aim of this study was to investigate the role and mechanism of SOV in NAFLD. Two mouse models were established by induction with high fat diet (HFD) and Western diet supplemented with the sugar in drinking water (WDS), respectively. We searched for the downstream molecules of SOV by RNA sequencing, followed by rescue experiments with an autophagy inhibitor (3-MA) in HepG2 cells as well as in animal models. The results showed that in HFD and WDS-induced NAFLD models, SOV significantly reduced body weight, inhibited lipid deposition, lowered serum triglyceride and cholesterol levels. Then RNA sequencing and RT-PCR found that the effect of SOV might be related to the activation of autophagy coregulated by hypoxia-inducible factor 1 and autophagy-related gene 5. The protective effects of SOV-activated autophagy were blocked by 3-MA, leading to the restoration of lipid deposition in vitro and in vivo. We conclude that SOV could activate liver cell autophagy, thereby improving lipid deposition and metabolism during the course of NAFLD. Our findings revealed the potential of SOV for controlling NAFLD.

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is a prevalent health care concern and an increasing epidemic with an estimated global prevalence of 25%.¹⁾ It spans a continuum of liver conditions from non-alcoholic fatty liver to non-alcoholic steatohepatitis (NASH), advancing to liver cirrhosis and potentially leading to hepatocellular carcinoma (HCC), thus posing a substantial public health burden linked to contemporary lifestyle factors.²⁾ The complex pathogenic mechanisms of NAFLD have not been completely elucidated. Previous studies have reported the multiple factors, such as biotoxicity, oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction. Current therapeutic strategies for NAFLD are limited and primarily focus on lifestyle modifications. Pharmacological interventions are still under investigation, with drugs targeting various aspects of NAFLD pathophysiology, such as insulin resistance, lipid metabolism, and inflammation.^3,4) However, drug research for NAFLD still has a long way to go before it can be widely used in clinical practice.^5,6)

Autophagy is an evolutionarily conserved degradation process for cytosolic macromolecules and damaged organelles. The potential role of autophagy in hepatic lipid metabolism has been recognized, while dysfunction of hepatic autophagy has been found to contribute to NAFLD.^7,8) Mitochondrial dysfunction is a crucial pathogenesis involved in NAFLD due to the significant impact of mitochondria on cellular lipid metabolism and oxidative stress.^9,10) Recently, autophagy is considered a vital means of sustaining mitochondrial homeostasis in NAFLD. Several medicines and signaling pathways are involved in regulating autophagy in NAFLD. If manipulated properly, these drugs could potentially restore autophagy in hepatocytes, ultimately improving metabolic outcomes. For example, pterostilbene effectively alleviated oxidative stress damage induced by excessive lipid accumulation in hepatocytes through the autophagy-related gene (ATG) family and activation of autophagy.¹¹⁾ The pathological role of deregulated autophagy in the development of NAFLD and suggest the therapeutic potential of enhancing autophagy for designing a new curative therapy for NAFLD.¹²⁾

Sodium orthovanadate (SOV) is a commonly used laboratory compound for preserving proteins by inhibiting alkaline phosphatase and ATPase. As an inorganic compound of vanadium, SOV has been investigated in medical research for its therapeutic efficacy across a diverse array of pathological conditions, including oncological, metabolic, and neurodegenerative disorders.^13–16) Gupta et al. checked the beneficial effect of SOV in diabetic and suggested that SOV may be an additional antidiabetic agent in preventing diabetic complications.¹⁷⁾ Additionally, empirical evidence from the rodent model elucidated that SOV could attenuate hepatic stellate cell activation and fibrogenesis.¹⁸⁾ Moreover, its efficacy in modulating cellular proliferation and promoting apoptosis in neoplastic cells, as observed in oral squamous cell carcinoma and hepatocellular carcinoma, underscores its broad biological impact.^19–21) However, the explicit role and included signaling pathway of SOV in NAFLD progression has not been experimentally demonstrated.

Considering the current deficit in efficacious pharmacological interventions and the therapeutic potential of SOV within NAFLD, in this study, animal models and cellular assays were designed to elucidate the role and mechanism of SOV in NAFLD pathophysiology, particularly focusing on its impact on mitochondrial function. This research also aims to provide an insight into the therapeutic potential of SOV for NAFLD intervention.

MATERIALS AND METHODS

Husbandry, Diets, and Intervention in Mice

The animal experiments conducted in this study received approval from the Nanjing Medical University Institutional Animal Care and Use Committee and performed in accordance with the guiding principles of institutional animal ethics committee. All mice were housed in a specific-pathogen-free facility on a 12h light-dark cycle, and the environment was well controlled for temperature and humidity. Eight-week-old male C57BL/6J mice were obtained from Model Animal Research Center of Yangzhou University. The diet groups were as followed: Standard diet (SD, Research Diets, D10012G, New Brunswick, NJ, U.S.A.), High-fat diet (HFD, fat contented 60 kcal%, Research Diets, D12492) with ordinary water and Western diet (Research Diets, D12079B) accompanied by the sugar solution (WDS) (23.1 g/L fructose (Sigma, G8270, St. Louis, MO, U.S.A.) and 18.9 g/L glucose (Sigma, F0127)) (WDS). Mice models were conceptualized based on prior research,^22,23) in which the basic early NAFLD model was first established with HFD/WDS for 8 weeks, and then randomly assigned to the corresponding different intervention groups (n = 6 per group).

SOV (NaVO₃) was sourced from Sigma (S6508) and administered to the mouse in the SOV and SOV + autophagy inhibitor (3-MA) groups via intraperitoneal injection at a dosage of 2.5 mg/kg daily. For the 3-MA groups, mouse received an additional intraperitoneal injection of 3-MA (30 mg/kg/d) (Selleck, S2767, Houston, TX, U.S.A.) in phosphate buffer saline (PBS). Mouse in control groups received intraperitoneal injections of PBS vehicle. To simulate the dietary treatment and as a negative control group, we also set up an SD group which received intraperitoneal vehicle administration on SD. Body weight and food intake metrics were recorded on a weekly basis. After the final treatment, mice were anesthetized with ether after a 12-h fasting period, and blood and liver tissue samples were collected for further analysis.

Cell Culture and Free Fatty Acids (FFA) Treatment

HepG2 cells were obtained from Cellbank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in the Dulbecco’s modified Eagle’s medium (DMEM; Gibco, U.S.A.) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin and 100 µg/mL streptomycin in the incubator at 37 °C with 5% CO₂ (Thermo Fisher, Waltham, MA, U.S.A.). FFA treatment (oleic acid and palmitic acid at a ratio of 2 : 1, Selleck, S4707 and S3794) involved incubating cells with 0.5 mM FFA in the presence of free bovine serum albumin for 24 h. The concentration of SOV (5 µM) used in this experiment was determined based on previous in vitro studies and our preliminary experiment.^13,24) The cells treated with 3-MA were at a concentration of 5 mM with serum-free media for 24 h before FFA and (or) SOV treated.²⁵⁾ After 24 h of incubation with FFA and (or) SOV, hepatocytes were scraped from the plate and harvested for further analysis.

Real-Time Quantitative PCR (RT-PCR)

RNA was isolated from tissues by using TRIzol according to the manufacturer’s protocol (Invitrogen, CA, U.S.A.). Following reverse transcription and RT-PCR was performed using RT-PCR kit (TaKaRa, Shiga, Japan). The 2^−ΔΔCt method was applied to evaluate the expression of target gene. Each mRNA expression level was normalized to β-actin mRNA expression level. The sequences of primers used in this study are listed in Supplementary Table S1.

Oil Red O Staining and Transmission Electron Microscopy (TEM)

To assess hepatic lipid accumulation, frozen liver sections and formaldehyde-washed cell plates were prepared and stained with 0.5% Oil Red O (Sigma) for 10 min. Afterward wash off the excess dye with isopropyl alcohol, and counterstain with Mayer’s hematoxylin (Sigma) for 40 s. The sections were visualized under the microscope and pictured (Olympus, BX43F, Tokyo, Japan). The average IOD values of Oil Red were obtained by analyzing five random fields per slide using Image-Pro Plus (6.0).

For TEM to observe the state of mitochondria in the cytoplasm, tissues were fixed in 4% formaldehyde and 1% glutaraldehyde. Subsequently, they were processed for transmission electron microscopy using a Hitachi HT-7800 electron microscope (Magnification = 4000, Accelerating Voltage = 80000, U.S.A.). Every index was detected three times.

RNA Sequencing

Tissues were extracted total RNA with TRIzol respectively (HFD + vehicle and HFD + SOV, 3 vs. 3). Then RNA quality was tested using 2100 Bioanalyzer (Agilent, Santa Clara, CA, U.S.A.) and quantified with the NanoDrop-2000 (Thermo Scientific). Paired-end RNA-seq sequencing library were sequenced on Novaseq 6000 platform and 150 bp paired-end reads. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Differential expression analysis was performed using the DESeq.^2,26) p-Value <0.05 and foldchange >2 or foldchange <0.5 was set as the threshold for significantly differential expression gene (DEGs). Then the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were carried out by KOBAS (http://kobas.cbi.pku.edu.cn/home.do),²⁷⁾ and hierarchical cluster analysis of DEGs was performed using R (v 3.2.0) to demonstrate the expression pattern of genes in different groups.

Enzyme-Linked Immunosorbent Assay (ELISA) and Serum Biochemistry Assay

ELISA was performed on tissue homogenate collected from mice livers using the Mouse microtubule-associated protein 1 light chain 3B (LC3B) ELISA kit according to the manufacturer’s instructions (EM3479M, Wellbio, Korea). Cholesterol (CHO), triglycerides (TG) and alanine aminotransferase (ALT) were measured in Clinical Laboratory (Cobas 8000, Switzerland, GPO-PAP Kit for cell TG) of Changzhou No.2 People’s Hospital.

Statistical Analysis

The software of SPSS (26.0) and GraphPad Prism (9.0) were used for statistical analysis and plotting. All data were expressed as mean ± standard deviation. Unpaired Student t-test was used for comparison between two groups following normal distribution. p-Values less than 0.05 were considered statistically significant.

RESULTS

SOV Inhibits NAFLD Progression in Animal Models

The effectiveness of SOV therapy in inhibiting the pathological progression of NAFLD was evaluated in mice that were fed 8 weeks of high-fat diet, with SOV administration commencing after the initiation of the HFD. The selected administration dose of 2.5 mg/kg was based on previous literature from animal studies and our preliminary experiment.^14,20) Concurrently, we also set up an SD group to serve as a negative control, and a group that received intraperitoneal vehicle administration on HFD acted as a positive control (Fig. 1A).

Fig. 1. HFD-Induced Hepatic Steatosis Was Attenuated by SOV in Mice

A. The protocol for HFD treatment; B. Comparison of the physical appearance of mice in each group; C. Representative liver phenotypes and photomicrographs of liver sections stained with H&E or Oil Red O (Scale bar: 200 µm) between the HFD+ Vehicle (HFD) and HFD + SOV groups (n = 6 per group, HFD + SD was not shown as a negative control). This study involved the analysis of at least 10 fields per slide); D. Trends in body weight changes (HFD vs. HFD + SOV compared and followed the same way, data were analyzed using unpaired t-test; mean ± standard deviation, p < 0.001, n = 6), liver tissue specimen weights (mean ± standard deviation, p < 0.001), and serum liver function indicator (ALT level) in three groups (HFD vs. HFD + SOV, mean ± standard deviation, p > 0.05); E. Serum cholesterol (HFD vs. HFD + SOV, mean ± standard deviation, p = 0.007) and triglyceride (mean ± standard deviation, p = 0.001) levels, as well as the expression of lipid metabolism-related factors PPARα (mean ± standard deviation, p = 0.004) and CPT1A (mean ± standard deviation, p = 0.003) in liver tissues of three groups.

At baseline and upon initiating diet restructuring, no significant differences in body weight were observed among the three groups. The mice were humanely sacrificed, and data were collected at the end of the 18-week feeding period. Experimental findings indicated that mice in the HFD + Vehicle group (HFD) exhibited the largest body size, followed by the HFD + SOV group (SOV), while the negative control group (SD) mice displayed the smallest body size (Fig. 1B). Notably, compared with the HFD group, the results demonstrated SOV reduced hepatocellular fat deposition, as evidenced by hematoxylin–eosin (H&E) and Oil Red O staining (Fig. 1C). With a non-significant difference in daily food intake, the trend of body weight changes in mice and the weight of liver specimens indicated that the results of the HFD + SOV group were superior to those of the HFD group (Figs. 1C, D, Supplementary A). While serum ALT levels did not differ significantly, analyses of CHO, TG, and lipid metabolic pathway markers (PP1Rα and CPT1A) via RT-PCR indicated milder fatty liver lesions in the SOV group than in the positive control group (Figs. 1D, E).

Considering the multifactorial etiology of NAFLD, we concurrently developed the animal model exposed to the Western diet and accompanied by the sugar solution (WDS). The process of constructing WDS animal model was similar to that of HFD (Fig. 2A), 14 weeks after starting the WDS feeding, a notable increase in body size was noted in the WDS + vehicle group (WDS), as opposed to both the WDS + SOV and SD groups (Fig. 2B). Utilizing methodologies similar to those of the HFD model, mice treated with WDS + SOV demonstrated improved outcomes in terms of weight, hepatocyte steatosis, and lipid metabolism compared to the WDS + Vehicle group (Figs. 2C–E). However, we also found that SOV could not fully alleviate fat deposition in hepatocytes compared to the negative control group (SD). Collectively, these two animal models elucidated the role of SOV in mitigating the progression of NAFLD in mice.

Fig. 2. WDS-Induced Hepatic Steatosis Was Attenuated by SOV in Mice

A. The protocol for WDS treatment; B. Analyze the physical features of mice in each group; C. Examine representative liver phonotypes and photomicrographs of liver sections stained with H&E or Oil Red O (Scale bar: 200 µm) between the WDS + Vehicle (WDS) and WDS + SOV groups (n = 6 per group, WDS + SD was not shown as a negative control); D. Trends in body weight changes (WDS vs. WDS + SOV compared and followed the same way, data were analyzed using unpaired t-test; mean ± standard deviation, p < 0.001, n = 6), liver tissue specimen weights (mean ± standard deviation, p = 0.003) and serum ALT levels in three groups (WDS vs. WDS + SOV, mean ± standard deviation, p > 0.05); E. Serum cholesterol (mean ± standard deviation, p = 0.006) and triglyceride (mean ± standard deviation, p = 0.005) levels and expression of PPARα (mean ± standard deviation, p = 0.002) and CPT1A (mean ± standard deviation, p = 0.002) in liver tissues.

Screening the Downstream Mitochondrial Autophagy Pathway Using RNA Sequencing

To elucidate the signaling pathway effects of SOV, transcriptomic analyses were conducted on liver tissues obtained from the HFD + vehicle and HFD + SOV groups. Data were analyzed using Heat map (Fig. Supplementary B), and the analysis detected differentially expressed genes. SOV treatment reversed 203 upregulated genes and 337 downregulated genes (Fig. 3A) in HFD. Gene set enrichment analysis indicated that the genes affected by SOV treatment were enriched in the cell cycle and P53 (Figs. 3B, C). Literature has shown the role of SOV as an ATPase inhibitor in modulating the cell cycle and apoptosis within the condition of liver fibrosis and cancer, which partly corresponds to our results.^19,28) To explore other potential effects of SOV, we performed PCR validation on other pathway factors related to the cell cycle in HFD and WDS tissues. The results confirmed the above observations, and also highlighting modifications in the ATG5, hypoxia-inducible factor-1α (HIF1α), CDK1, and LC3B genes after SOV treated, which are crucial for autophagy (Figs. 3C, D). Considering the crucial role of autophagy in the progression of NAFLD, these findings indicate the therapeutic potential of SOV in NAFLD probably caused by its promoting autophagy in hepatic cells.

Fig. 3. Effects of SOV on Differentially Expressed Genes of Mice Models

A. Volcano plot showed differentially expressed genes between the HFD and HFD + SOV groups by RNA-seq (203 upregulated genes (red) and 337 downregulated genes (blue), * p < 0.05); B. Bubble diagram for the KEGG pathway analysis of differentially expressed transcripts identified by RNA-seq; C. The expression levels of screened genes were validated in WDS and WDS + SOV-treated mice groups (Data were analyzed using unpaired t-test; mean ± standard deviation, p < 0.001, n = 6); D. The expression levels of screened genes were validated in HFD and HFD + SOV-treated mice (mean ± standard deviation, ATG5: p = 0.006, HIF1α: p < 0.001; LC3B: p = 0.002, n = 6).

The Autophagy Inhibitor (3-MA) Suppressed the Lipid-Lowering Effect of SOV in HepG2 Cells

Next, the role of SOV in mediating the autophagy in FFA-treated hepatocytes was examined. Previous studies often typically utilized FFA cultured HepG2 cells to explore the NAFLD disease process in vitro.¹⁰⁾ The optimal concentration of SOV for experimentation was determined based on prior literature and preliminary tests²¹⁾ (Fig. Supplementary C). Subsequently, the HepG2 cells were exposed to a high-fat condition by using FFA for 24 h. Oil Red O staining revealed a significant decrease in lipid droplets within the SOV-treated group compared to the control group. Conversely, the application of an 3-MA reversibly resulted in an increased presence of lipid droplets with SOV²⁵⁾ (Figs. 4A, B, Fig. Supplementary D). The SOV treatment was associated with an elevated mRNA level of HIF1α, an effect that remained unchanged with the addition of 3-MA (Fig. 4C). Furthermore, the expression of key autophagy and mitophagy factors, including ATG5, LC3B and PINK1, was significantly increased by SOV but reversed by 3-MA intervention (Figs. 4C, D). These hepatocyte-based experiments demonstrate that inhibiting autophagy compromises the protective effect of SOV against NAFLD progression.

Fig. 4. Effects of SOV on Autophagy and Lipid Accumulation in FFA-Treated HepG2 Cells

A, B. Oil Red staining analysis in HepG2 cells across four groups: FFA control, FFA + SOV, FFA + 3-MA, and FFA + SOV + 3-MA, illustrating lipid accumulation differences (Date were analyzed using one-way ANOVA followed by t test, mean ± standard deviation, FFA control vs. FFA + SOV: p = 0.004, FFA + SOV vs. FFA + SOV + 3-MA: p = 0.037, n = 5) ; C. RT-PCR analysis showcasing the expression levels of autophagy-related factors (ATG5, HIF1α, LC3B and PINK1) in cells subjected to different treatments (FFA + SOV vs. FFA + SOV + 3-MA: mean ± standard deviation, p < 0.001, n = 3); D. LC3B levels tested by ELISA under different treatments (mean ± standard deviation, FFA control vs. FFA + SOV: p = 0.008, FFA + SOV vs. FFA + SOV + 3-MA: p = 0.033, n = 3).

Treatment with 3-MA Alleviated the Suppressive Effects of SOV on HFD-Induced Hepatic Steatosis in Mice

In order to explore the impact of autophagy caused by SOV in the progress of NAFLD, the mice model of HFD was conducted, and hypothetically, the intraperitoneal administration of 3-MA was utilized to counteract the effect of SOV treatment. Following 8 weeks of HFD feeding, mice were categorized into four distinct groups for comparative assessment: positive control, 3-MA control, SOV, and SOV + 3-MA (n = 6 per group fed with high-fat). Subsequently, no significant differences were noted in terms of body weight and fat accumulation among mice post administration of 3-MA, compared with the positive control group (Figs. 5A, B, Fig. Supplementary F). These findings are in line with previous research indicating the inhibition of mitochondrial autophagy during NAFLD.²⁹⁾ In comparison to the group treated with SOV, the co-administration of 3-MA, which hinders autophagy, led to an increase in body weight and intracellular lipid droplets within hepatic cells (Figs. 5C, D). TEM observation revealed a higher number of mitochondria and mitochondrial autophagosomes in the SOV-treated group. However, the addition of 3-MA resulted in a decrease in autophagosomes, and reduced ratio of LC-3B level (Figs. 5D, E). There was no significant change in serum ALT levels, but the levels of TG and CHO indicated a partial reversal of the protective effects of SOV against hepatocellular steatosis upon 3-MA administration (Fig. 5F, Fig. Supplementary G). This implied that the use of 3-MA partially counteracted the protective effect of SOV against hepatocellular steatosis, but it remains difficult to control liver function impairment. The in vivo HFD animal experiment demonstrated that the protective effect of SOV against the progression of NAFLD may be impeded by the autophagy inhibitor (3-MA).

Fig. 5. SOV Ameliorated HFD-Induced Lipid Accumulation via Autophagy Activation in Mice

A. Comparison of body size among the groups: HFD+ Vehicle (HFD), HFD + 3-MA, HFD + SOV, and HFD + SOV + 3-MA; B. Line graph depicting the body weight in each group (Date were analyzed using one-way ANOVA followed by unpaired t test, HFD + SOV vs. HFD + SOV + 3-MA compared and followed the same way; mean ± standard deviation, p < 0.001); C. Representative liver sections of liver tissues with H&E (Scale bar: 200 µm) or Oil Red O (Scale bar: 100 µm), illustrating lipid accumulation differences among groups; D. The ultrastructure of liver tissues was observed via TEM. HFD treatment induced abnormal mitochondrial morphology (e.g., abnormal shape, swelling, lacking cristae) (white arrows). SOV increased the auto-lysosomal structures (black arrows) were decreased in the cells of HFD + SOV + 3-MA (L, lipid drops, M, mitochondria, Scale bar: 1 µm). E. Quantitative analysis comparing of the proportion of Oil-Red-stained cells (HFD + SOV vs. HFD + SOV + 3-MA: mean ± standard deviation, p = 0.002) and LC3B levels of ELISA among groups (HFD + SOV vs. HFD + SOV + 3-MA, mean ± standard deviation, p = 0.004). F. Comparative analysis of serum ALT (HFD + SOV vs. HFD + SOV + 3-MA: mean ± standard deviation, p > 0.05), CHO (HFD + SOV vs. HFD + SOV + 3-MA: mean ± standard deviation, p = 0.001), and TG (HFD + SOV vs. HFD + SOV + 3-MA: mean ± standard deviation, p = 0.003) levels in each group, indicating metabolic changes.

DISCUSSION

With societal advancement, the prevalence of obesity and associated metabolic disorders, such as insulin resistance and NAFLD, has escalated, posing significant public health challenges.³⁰⁾ NAFLD, a complex clinicopathological condition, is characterized by hepatocellular steatosis and lipid accumulation, with the potential progression from simple steatosis to NASH and HCC. Therefore, there is a pressing need to develop effective and safety treatments for NAFLD. Although the FDA recently approved the first drug for NAFLD, Resmetirom, there is still a strong interest in exploring the feasibility of other drugs.³¹⁾ The unexpected therapeutic efficacy of drugs such as sorafenib and disulfiram in NAFLD has sparked further research into repurposing established medications for this condition.^22,32)

SOV has been recognized as a potential agent for managing diabetes in previous studies.¹⁵⁾ Research has shown that the combined treatment of SOV and fenugreek in diabetic animal models can prevent high blood sugar levels and modify lipid profiles in both blood and tissues.²⁴⁾ Moreover, as a competitive inhibitor of PTP, SOV has been found to influence the fatty acid composition in macrophages by increasing the levels of certain fatty acids.¹⁵⁾ The lack of PTP1B may confer protection against cardiac abnormalities resulting from endoplasmic reticulum stress by modulating ATG5/LC3B and autophagy.³³⁾ Additionally, PTP1B exhibits a dual function in both the advancement and regression of NASH within the context of NAFLD.³⁴⁾ This underscores the importance of examining the effectiveness of SOV in the treatment of NAFLD. Despite indications from these studies suggesting that SOV may impact lipid metabolism, there is a lack of definitive animal and cellular experiments elucidating the specific role of SOV in NAFLD. Therefore, this study proposed SOV as a potential therapeutic candidate for treating NAFLD. Through the use of animal models exposed to high-fat and high-fructose diet, we demonstrated the therapeutic effects of SOV in NAFLD. We anticipate elucidating the shared mechanisms by which SOV regulates NAFLD through the validation of findings obtained from two distinct animal models. Subsequent sequencing analyses have identified changes in downstream signaling pathways, with RT-PCR validation of liver tissues focusing on alterations in the cell cycle and autophagy pathway.

Autophagy plays a pivotal role in NAFLD development, characterized by increased reactive oxygen species production, lipid peroxidation, reduced cellular energy levels, heightened pro-inflammatory responses, and structural damage to mitochondria, as well as the formation of autophagosomes. Mitophagy, the targeted removal of dysfunctional mitochondria, is also essential for maintaining cellular oxidative balance under normal circumstances.²⁹⁾ Recent studies have increasingly highlighted the role of autophagy in the treatment of liver disease³⁵⁾: Yan et al. reported that Schisandrin B inhibited hepatic steatosis and promoted fatty acid oxidation by activating autophagy through the AMP activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway.³⁶⁾ Kim et al. found that Evogliptin may also alleviate steatohepatitis in HFD-fed mice by ameliorating steatosis and oxidative stress through modulating autophagy in the liver.³⁷⁾ Despite PTP1B being identified as a critical promoter of NAFLD progression, SOV, as a PTP inhibitor, is anticipated to play a role in mitigating NAFLD.^34,38) The impact of SOV on the apoptosis and P53 has been widely acknowledged, and we have expanded our horizons to other potential signaling pathways. Through HepG2 cells culture and animal experiments with and without the addition of an autophagy inhibitor (3-MA), we demonstrated that SOV inhibits the progression of NAFLD by enhancing autophagy, possibly through HIF1α/ATG5 regulation.

ATG5 plays a pivotal role in membrane Atg8ylation, influencing downstream processes including canonical mitophagy and noncanonical processes.^39,40) Tang et al. reported that the activation of ATG5 inhibits pyroptosis to improve hepatic steatosis.⁴¹⁾ Research has established that HIF1α-induced autophagy within the BNIP3 pathway plays a significant role in liver injury. Furthermore, it has been demonstrated that HIF1α can also activate autophagy via ATG5 under hypoxic conditions, indicating a multifaceted regulatory role of HIF1α in autophagy processes.^42,43) Notably, instances of HIF1α-mediated mitophagy have been documented in various diseases, underscoring its broader implications.⁴⁴⁾ The unique attributes of mitophagy position it as a critical mechanism for mitigating hepatocyte damage caused by the abnormal accumulation of dysfunctional organelles.^45,46) The alterations observed in the fatty acid oxidation and mitophagy pathway in this study may be attributed to the influence of HIF1α. The progression of NAFLD to cirrhosis and even HCC is a long-term outcome of multicellular interactions. Although there have been individual studies on SOV for liver fibrosis and hepatocellular carcinoma,^18,20) further investigation is needed to determine the specific role of SOV in regulating autophagy and fatty acid oxidation within this dynamic process. When comparing the weight change and serum biochemical indexes between the SOV group and the SD group, we observed that the improvement in body weight and liver function after taking the drug was not as effective as that achieved through dietary modification. Therefore, early dietary modification remains the most effective approach to treating NAFLD at present. This phenomenon was also observed in the studies of disulfiram and sorafenib, where the self-regulation of the organism after a healthy diet was superior to the intervention of exogenous drugs in the early course of NAFLD. However, dietary and lifestyle changes are hard to maintain. As a result, there are more stringent requirements for the efficacy and safety of drugs used in the treatment of NAFLD.⁵⁾ It was observed that the liver tissue of the SOV group had infiltration of inflammatory cells. The levels of ALT did not decrease in conjunction with the reduction of lipid deposition. This phenomenon may be associated with the activation of hepatic inflammatory pathways by SOV/HIF1α, and further research is warranted to elucidate this observation. SOV could influence the apoptosis via P53 in cancers and potentially offering antidepressant-like benefits or mitigating stress-induced anhedonia,^16,47,48) and in our preliminary experiments, high SOV concentration could induce transient locomotor excitability in mice, leading us to utilize lower SOV concentrations in subsequent studies to minimize adverse effects. The regulation of SOV-autophagy in NAFLD is intricate, with autophagy acting as a double-edged sword, eliciting varied effects across different disease stages.^49,50) The broad spectrum of effects and side effects of SOV likely played a role in the observed remission of fatty liver and minimal alterations in liver function, as evidenced by ALT levels. Our further study will explore the underlying mechanism of this phenomenon.

In conclusion, this research has identified a new relationship between SOV-induced autophagy and its lipid-lowering impact on liver cells. SOV notably boosted the autophagy process in liver cells and animal models. Additionally, inhibiting the autophagy pathway diminished the efficacy of SOV. These results provide a pharmacological rationale for considering the use of SOV in clinical settings as a potential preventive or therapeutic intervention for NAFLD.

Acknowledgments

We thank Prof. Beicheng Sun and Nanjing Drum Tower Hospital for laboratory equipment help.

Funding

This work was supported by a Grant from Changzhou High-level Medical Talent Training Project (GW2023019 and 2022CZBJ064), Changzhou Sci & Tech Program (CM20223008, 2022CZLJ017, and CJ20220127). Changzhou Medical Center of Nanjing Medical University Program (PCMCB202212, PCMCM202204, and BSH202209).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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