Abstract:

Objective To explore the ameliorative effect of puerarin （Pue） on non-alcoholic fatty liver disease （NAFLD） induced by high-fat diet in the mice， and to clatrify its possible mechanism. Method A total of 36 C57BL/6J mice were randomly divided into control group， model group， low dose of Pue group （20.0 mg·kg^-1）， medium dose of Pue group （40.0 mg·kg^-1）， high dose of Pue group （80.0 mg·kg^-1）， and positive drug group ［atorvastatin calcium （AT） group］ （10.0 mg·kg^-1 AT）， with 6 mice in each group. Except for control group， in which the mice were given normal diet， all of the mice in the other groups were fed with high-fat diet to establish the NAFLD models. The body weights of the mice in various groups were measured， and the liver indexes was calculated； the pathomorphology of liver tissue of the mice in various groups was observed by HE staining method. The total cholesterol （TC） and triglyceride （TG） levels in serum and liver tissue， alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels in serum， the malondialdehyde （MDA） levels， superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） activities in liver tissue， as well as interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） levels in liver tissue of the mice in various groups were detected by commercial biochemical assay kits. Within the framework of network pharmacology， the potential therapeutic targets of Pue were predicted by integrating multiple databases， including PubChem and Swiss Target Prediction， and other publicly available resources. These targets were cross-referenced with NAFLD-associated disease targets retrieved from GeneCards and other platforms， enabling the construction of a high-confidence protein-protein interaction （PPI） network. Subsequently， Gene Ontology （GO） function and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway enrichment analyses were performed to elucidate the therapeutic mechanisms of Pue in NAFLD intervention； Western blotting method was used to detect the protein expression levels of Kelch-like ECH-associated protein 1 （Keap1）， nuclear factor erythroid 2-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1）， and quinone oxidoreductase 1 （NQO1） in liver tissue of the mice in various groups. Additionally， 30 mice were randomly divided into control group，?model group， Pue group （80.0 mg·kg^-1 Pue gavage）， model+Nrf2 inhibitor ML385 group （30.0 mg·kg^-1 ML385 intraperitoneal administration）， and Pue+ML385 group?（80.0 mg·kg^-1 Pue gavage， 30 mg·kg^-1 ML385 intraperitoneal administration）. After treatment， the oxidative stress markers， inflammatory cytokines， and protein expression levels metioned above were detected. Results Compared with control group， the body weight and liver index of the mice in model group were significantly increased （P<0.01）； compared with model group， the body weight and liver index of the mice in low， medium， and high doses of Pue groups and AT group were significantly decreased （P<0.05 or P<0.01）. The HE staining results showed compared with control group there was significant lipid accumulation and pathological damage in liver tissue of the mice in model group； compared with model group， the accumulation of lipid droplets and pathological damage in liver tissue of the mice in low， medium， and high doses of Pue groups and AT group were improved to varying degrees. Compared with control group， the levels of TC， TG， IL-6， IL-1β， TNF-α， and MDA in liver tissue of the mice in the model group， as well as the serum TC， TG， ALT， and AST levels were significantly increased （P<0.01）， while the activities of SOD and GSH-Px in liver tissue were significantly decreased （P<0.01）； compared with model group， the levels of TC， TG， IL-6， IL-1β， TNF-α， and MDA in liver tissue， as well as the levels of TC， TG， ALT， and AST in serum of the mice in low， medium， and high doses of Pue groups and AT group were significantly decreased （P<0.05 or P<0.01）， while the activities of SOD and GSH-Px in liver tissue were significantly increased （P<0.05 or P<0.01）. The Network pharmacology analysis results identified the core targets of Pue in treating NAFLD， including Nrf2， TNF， and IL-6， and predicted that Pue may exert its effects through regulating oxidative stress， inflammation， and apoptosis pathways. The Western blotting results showed that compared with control group， the expression level of Keap1 protein in liver tissue of the mice in model group was increased （P<0.01）， while the expression levels of Nrf2， HO-1， and NQO1 proteins were decreased （P<0.01）； compared with model group， the expression levels of Keap1 protein in liver tissue of the mice in low， medium， and high doses of Pue groups and AT group were decreased （P<0.05 or P<0.01）， while the expression levels of Nrf2， HO-1， and NQO1 proteins were increased （P<0.05 or P<0.01）. After intervention with Nrf2 inhibitor ML385， compared with Pue group， the levels of IL-6， IL-1β， TNF-α， and MDA in liver tissue of the mice in Pue+ML385 group were significantly increased （P<0.05）， while the activities of SOD and GSH-Px were significantly decreased （P<0.05 or P<0.01）， the expression level of Keap1 protein in liver tissue was increased （P<0.01）， and the expression levels of Nrf2， HO-1， and NQO1 proteins were decreased （P<0.01）. Conclusion Pue can improve the lipid metabolism and liver function in the NAFLD mice， alleviate the liver damage， enhance the liver tissue antioxidant capacity， and reduce the release of inflammatory factors. Its mechanism may be related to the activation of the Keap1/Nrf2/HO-1 signaling pathway.

Key words: Puerarin, Non-alcoholic fatty liver disease, Oxidative stress, Inflammation, Nuclear factor erythroid 2-related factor 2