α-Lipoic Acid Ameliorates Arsenic-Induced Lipid Disorders by Promoting Peroxisomal β-Oxidation and Reducing Lipophagy in Chicken Hepatocyte

Abstract

Liver disease poses a significant threat to global public health, with arsenic (As) recognized as a major environmental toxin contributing to liver injury. However, the specific mechanisms and the protective effects of α-lipoic acid (LA) remain unclear. Therefore, this study employs network toxicology and network pharmacology to comprehensively analyze the hepatotoxic mechanism of As and the hepatoprotective mechanism of LA, and further verifies the mechanisms of peroxisomal β-oxidation and lipophagy in the process. The network analysis results show that As induces liver damage mainly through autophagy, apoptosis, lipid metabolism, and oxidative stress, whereas LA exerts its hepatoprotective properties mainly by regulating lipid metabolism. Further verifications find that As inhibits SIRT1 expression, activates the P53 and Notch pathways, damages mitochondria, inhibits peroxisomal β-oxidation, increases lipid accumulation, and enhances lipophagy in the liver, while LA intervention alleviates As-induced lipid accumulation and enhances lipophagy by targeting SIRT1, ameliorating mitochondrial damage, enhancing peroxisomal β-oxidation, thereby alleviating As-induced liver damage. This study further clarifies the mechanism of As hepatotoxicity and provides a theoretical basis for LA as a potential hepatoprotective agent.

Keywords: alpha‐lipoic acid; arsenic; hepatotoxicity; lipophagy; peroxisomal β‐oxidation.

Figure 1

As network toxicology analysis results. A) Venn diagram for screening As‐induced liver damage related genes. B) KEGG enrichment results of As‐induced liver damage related genes. C) PPI network diagram of arsenic‐induced liver damage related genes. D) GO enrichment circle diagram of As‐induced liver damage related genes.

Figure 2

Effects of As exposure and LA intervention on growth status, liver structure and function of chickens. A) Body weight, n = 10. B) Liver organ coefficient, n = 10. C,D) Detection results of liver function index (AST and ALT), n = 6. E) Liver appearance observation. F) HE staining results (400×, red star: disordered hepatic cords; black arrows: lipid droplets; green arrows: enlarged intercellular spaces). ^* P < 0.05, ^** P < 0.01, versus Ctrl; ^# P < 0.05, ^## P < 0.01, versus As.

Figure 3

LA network pharmacology analysis results. A) Potential target genes network diagram of LA. B) GO enrichment network map of target genes. C) Analysis of the proportion of each biological process (GO enrichment). D) GO enrichment network map of lipid metabolism‐related terms. E) KEGG enrichment network map of target genes. F) Molecular docking results of LA and SIRT1. G) Detection results of SIRT1 protein expression level, n = 3. ^* P < 0.05, versus Ctrl; ^# P < 0.05, versus As.

Figure 4

Effects of As exposure and LA intervention on lipid metabolism in the chicken liver. A–D) Detection results of the liver lipid metabolism index (T‐CHO, TG, LDL‐C, and HDL‐C), n = 6. E) Detection results of the liver As content, n = 6. F) Oil red O staining results (400×, green arrows: lipid droplets). G) Statistical results of Oil Red O staining, n = 6. H,I) Detection results of P53 and Notch1 protein expression levels, n = 3. ^* P < 0.05, ^** P < 0.01, versus Ctrl; ^# P < 0.05, ^## P < 0.01, versus As.

Figure 5

Effects of As exposure and LA intervention on the peroxisomal β‐oxidation in the chicken liver. A–C) The mRNA (n = 6)and protein (n = 3) expression levels of the lipid metabolism key genes detection results. D) Acetyl CoA detection results, n = 6. E) Results of mtDNA detection, n = 6. F) The mRNA expression levels of the fatty acid β‐oxidation key genes detection results, n = 6. G) The protein expression levels of the peroxisomal β‐oxidation key genes detection results, n = 3. H) Peroxisome immunofluorescence labeling results (400×). ^* P < 0.05, ^** P < 0.01, versus Ctrl; ^# P < 0.05, ^## P < 0.01, versus As.

Figure 6

Effects of As exposure and LA intervention on the lipophagy in the chicken liver. A) Immunofluorescence colocalization lipophagy detection results (400×, ATGL: lipid droplets; LC3: lysosomes, yellow arrows: lipolysosome). B) Detection results of the mRNA expression levels of the lipophagy key genes, n = 6. C,D) Detection results of the protein expression levels of the lipophagy key genes in the lipid droplets, n = 3. ^* P < 0.05, ^** P < 0.01, versus Ctrl; ^# P < 0.05, ^## P < 0.01, versus As.

Figure 7

Establishment of the mechanism research model of chicken liver cell line (LMH cell line). A) Statistical results of cell proliferation rates under As treatment at different concentrations and for different times, n = 6. B) Statistical results of cell proliferation rate under treatment with different concentrations of LA, n = 6. C) Statistical results of cell proliferation rate under combined treatment with As and different concentrations of LA, n = 6. D) The protein expression levels of SIRT1 detection results, n = 3. E) Statistical results of the mRNA expression levels of the lipid metabolism key genes under As and/or LA treatment, n = 6. F) Statistical results of the protein expression level of LIPA under As and/or LA treatment, n = 3. G–I) Statistical results of ACOX1 protein expression levels in the different treatment groups, n = 3. ^* P < 0.05, ^** P < 0.01, versus Ctrl; ^# P < 0.05, ^## P < 0.01, versus As.

Figure 8

Effects of TRCDA treatment on hepatocyte function, lipid metabolism, ROS content, and cell death under As and/or LA exposure. A) Detection results of hepatocyte function indexes, n = 6. B) Detection results of lipid metabolism indexes, n = 6. C) PI fluorescence staining detection results (400×). D,E) PI and H2DCFDA fluorescence staining flow cytometry detection results, n = 6. F) OCR and ECAR detection results, n = 4. G) Mitochondrial membrane potential detection results, n = 6. H) JC‐1 fluorescence staining detection results.^* P < 0.05, ^** P < 0.01, versus Ctrl; ^# P < 0.05, ^## P < 0.01, versus As.

Figure 9

Effects of TRCDA treatment on the hepatocyte lipophagy under As and/or LA exposure. A) Fluorescence colocalization lipophagy detection results (400×, Nile red: lipid droplets; Lyso‐Tracker: Lysosome, yellow box: lipolysosome). B–E) Detection results of the protein expression levels of lipophagy key genes in the lipid droplets under different treatment groups, n = 3. ^* P < 0.05, versus Ctrl; ^# P < 0.05, versus As.