| Objective: To explore the potential pharmacodynamic mechanisms of anti-steatotic effects of Jiang Zhi Granules(JZG)against non-alcoholic fatty liver disease(NAFLD),thereby providing experimental evidence for its clinical application.Methods: 1.Systematic pharmacology was used in this research to screen candidate compounds,predict drug targets and conduct network analyses for JZG on NAFLD,while enrichment analyses were performed next for biological processes,molecular functions,celelular components and signaling pathways to systematically explore the potential mechanisms of anti-steatotic effects of JZG against NAFLD.2.HepG2 cells and primary hepatocytes were harvested and incubated in culture medium containing palmitic acid(PA)in the presence or absence of either JZG or rapamycin.The ALT levels in cell supernatants were detected by ELISA,while the accumulation of lipid droplets were observed by oil red O and BODIPY staining.Furthermore,JC-1 and ROS staining were performed here to examine the mitochondrial integrity and the reactive oxygen species production.The autophagic flux was evaluated by stable fluorescentexpressing cell lines,and the binding of lipid droplets and lysosomes were observed by double fluorescent staining.Western blot analyses were performed finally to examine the IRS-1,PI3 K,AKT,mTOR,LC3 and P62 expression in vitro.3.A murine model of NAFLD induced by high-fat diets was employed in this study,which was treated with intragastric administration of JZG.The general conditions and the body weight changes were recorded for the duration of this trial,while the liver weights and the hepatic morphology were observed at the end of the experiment.Furthermore,HE and oil red O staining were performed to evaluate the pathological changes in liver tissues,and the contents of serumTC,TG,ALT and AST were detected by biochemical detection.Finally,western blot analyses were performed to examine the IRS-1,PI3 K,AKT,mTOR,LC3 and P62 expression in vivo.Results: 1.Systematic pharmacological analyses revealed 96 potential ingredients and 338 related drug targets in JZG.Enrichment analyses showed that the pharmacodynamic mechanisms of JZG against NAFLD contained 368 putative targets,mainly involving the metabolic processes related to the PI3K-Akt and MAPK signaling pathways.2.The stimulation of PA induced the increase of ALT levels in the supernatants of HepG2 cells and primary hepatocytes,while JZG significantly reduced the high ALT levels.In the meantime,JZG effectively improved the PA induced lipid droplets accumulation,mitochondrial integrity damage and reactive oxygen species production,which suggested that JZG could improve the cell trauma induced by PA.3.The stimulation of PA up-regulated the protein expression of LC3 II,down-regulated the protein expression of P62,and inhibited the protein phosphorylation levels of IRS-1,PI3 K,AKT and mTOR.JZG aggravated the changes of mTOR,LC3 and P62 induced by PA,whereas reversed the changes of IRS-1,PI3 K and AKT,thus activating the autophagic flux,promoting the combination of lipid droplets and lysosomes,and finally degrading the lipid droplets.4.Western blot analyses displayed that high-fat diets administration induced the increase of LC3 II expression,while reduced the phosphorylation levels of IRS-1,PI3 K,AKT and mTOR.JZG exacerbated the changes of mTOR and LC3,whereas reversed the changes of IRS-1 and AKT induced by high-fat diet,hence alleviating the raise of body weights,liver weights and serum indicators.Meanwhile,JZG also improved the accumulation of lipid droplets,the formation of fat vacuoles and the infiltration of inflammatory cells in liver tissues.Conclutions: 1.The potential pharmacodynamic mechanisms of JZG on NAFLD mainly involves the metabolic processes of organisms related to PI3K-Akt and MAPK signaling pathways.2.JZG could active autophagy by regulating the mTOR signaling pathway to improve the disturbance of lipid metabolism in NAFLD. |
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