Mechanism Of The High Lipid Microenvironment Inhibiting Hepatic Stellate Cell Activation Via Chaperone-mediated Autophagy

Background and objectiveAlcoholic fatty liver disease(Nonalcoholic fatty liver disease,NAFLD)can develop from liver simple steatosis to liver fibrosis,in which the activation of hepatic stellate cells(hepatic stellate cell,HSCs)is closely related to the occurrence and development of liver fibrosis.HSCs in the resting state in the liver mainly play the role of storage lipids,with retinol being the most predominant lipid.After activation,HSCs transdifferentiate into myofibroblast-like cells,which secrete extracellular matrix and collagen and promote the development of liver fibrosis.The most obvious marker of HSCs activation is the loss of lipid droplets within the cytoplasm,which is closely associated with the lipid autophagy that occurs within the cells.It has been shown that in vitro administration of lipids such as retinol can increase lipid droplets in HSCs and inhibit or even reverse the activation of HSCs,and this reversal may be associated with the inhibition of lipid autophagy.Perilipin5(PLIN5)has been confirmed to play an important role in the activation of HSCs and collagen generation.PLIN5 is also found to be one of the substrates of molecular chaperone-mediated autophagy(CMA),and recent studies show that the participation of CMA in the degradation of lipid droplet protein may be a prerequisite step for lipid autophagy in cells.Therefore,this study concluded that the high lipid microenvironment may induce the accumulation of lipid droplet protein PLIN5 through the influence on HSCs molecular chaperone-mediated autophagy,which promotes the increase of lipid droplets and the reactivation of HSCs.Methods1.Establishment of short-term high-fat diet rat model and histological detection: Twenty-one adult male SD rats with SPF grade were randomly divided into two groups: normal group(n = 10,normal diet)and high fat group(n = 11,high-fat diet),which were fed free water.After feeding for 1 day,portal venous blood and liver were extracted from 3 rats at the same time for 7 consecutive days.The content of Free fatty acid(FFA)in serum was detected by the kit,and the changes of tissue morphology and lipid droplet were observed by histological staining of liver.2.Effects of short-term high-fat diet on hepatic fibrosis and CMA participation in PLIN5 autophagy degradation pathway in rats: RNA and protein were extracted from rat liver tissues,and the gene and protein expressions of HSCs activation related molecules,collagen,CMA,PLIN5 and other molecules in rat liver were detected by RT-q PCR and Western blotting experiments.3.Establishing in vitro high-fat microenvironment and detecting the effect of high-fat microenvironment on HSCs activation: Rat hepatic stectin cell line(HSC-T6)was cultured with Palmitic acid(PA)PA solution at different concentrations to construct high-fat microenvironment in vitro.RNA and protein were extracted from the cells,and the changes of HSCs activation related molecules and collagen gene and protein expression after PA treatment were detected by RT-q PCR,Western blotting and other experiments.4.Effects of in vitro high-fat microenvironment on CMA’s participation in the autophagy degradation pathway of PLIN5 in HSC-T6: HSC-T6 cells were treated with different concentrations of PA,and the expression changes of PLIN5 and CMArelated proteins were detected by Western blotting.5.To verify the relationship between degradation of PLIN5 in CMA and activation of HSCs: Hsc70 inhibitor VER155008 was used alone or mixed with PA to treat HSC-T6 cells.The expression changes of Collagen III,PLIN5,Hsc70 and other related proteins were detected by Western blotting.Results1.FFA content in hepatic portal vein of rats was detected by FFA kit.The results showed that the portal vein FFA content of rats in high fat group was significantly higher than that in normal group.Through histological staining,we observed that the cell structure in the liver tissue of rats in the ordinary group was clear and dense,and only a few red dotted lipid droplets were distributed in the liver cells,while the structure of the liver tissue of rats in the high lipid group was more loose,and the cytoplasm of the liver cells showed distinct deep red bulbous lipid droplets The results showed that the short-term high-fat rat model was established successfully,and the content of FFA in liver of rats was significantly increased after short-term high-fat diet,and the accumulation of lipid in liver cells was obvious.2.The results of RT-q PCR and Western blotting showed that Col3a1 m RNA in the liver tissues of rats in the high fat group was significantly decreased compared with that in the normal group,but Collagen Ⅲ protein expression in the liver tissues of rats in the high fat group was significantly increased.Compared with the normal group,the lipid drop protein PLIN5 and the CMA-related protein Hsc70 in the liver tissue of rats in the high fat group were significantly increased,while the LAMP-2A showed a significant increase trend.These results indicated that collagen production and lipid drop protein expression increased in the liver of rats fed a short-term high fat diet,and high fat diet induced changes in molecular chaperon-mediated autophagy in liver cells.3.Rat hepatic stellate cell line HSC-T6 cells were treated with different concentrations of PA.RT-q PCR was used to detect the effects of PA on genes related to activation and collagen generation in HSCs.The results showed that,compared with the control group,after PA treatment,The gene and protein expression levels of α-SMA,Collagen Ⅰ and Collagen Ⅲ in HSCs were significantly decreased.These results indicated that PA inhibited the activation of HSCs and the production of collagen.4.Hsc-T6 of rat hepatic stellate cell line were treated with different concentrations of PA,and Western blotting was used to detect the effects of PA on proteins involved in the autophagy degradation pathway of PLIN5 in HSCs.The results showed that,compared with the control group,The CMA-related molecule Hsc70 and Lamp-2A in HSCs showed a significantly decreased trend,and correspondingly,the protein expression of CMA substrate lipid drop protein PLIN5 was significantly increased.The immunofluorescence staining results showed that PLIN5 in the cytoplasm of HSCs was significantly increased after PA treatment.The results of oil red O staining showed that PA promoted the deposition of lipid droplets in HSCs,and the content of lipid droplets in the cytoplasm of HSCS increased with the increase of PA concentration.These results indicated that PA inhibited molecular chaperonmediated autophagy in HSCs and promoted the increase of PLIN5 protein and lipid droplet in HSCs.5.Hsc70 molecular inhibitor VER155008 and its combination with PA were used to treat HSC-T6 cells.Western blotting was used to detect the effects of Hsc70 inhibitor on the lipid drop protein and collagen of HSCs.The results showed that VER155008 significantly increased the expression of PLIN5 protein and inhibited the expression of Collagen Ⅲ protein in HSCs compared with the control group.VER155008 combined with PA showed more significant effects on the up-regulation of PLIN5 protein expression in HSCs and the inhibition of Collagen Ⅲ protein expression than the use of inhibitors alone.This further confirmed that PA inhibited the PLIN5 degradation pathway of CMA in HSCs,resulting in increased PLIN5 protein accumulation in the cytoplasm,increased lipid droplets,inhibited HSCs activation,and reduced collagen production.Conclusions1.Short-term high-fat diet induces increased FFA content in liver and significant lipid deposition in liver cells,which successfully establishes a short-term high-fat microenvironment rat model.2.The high-fat microenvironment promotes the expression of Collagen Ⅲprotein and lipid drop protein PLIN5 in the liver,and inhibites the molecular chaperonmediated autophagy in the liver.3.The In Vitro high-fat microenvironment induced by palmitic acid inhibites the activation of HSCs and the production of collagen,and increases the expression of PLIN5 protein and lipid droplets in the cytoplasm.4.After the inhibition of molecular chaperone-mediated autophagy,lipid droplets and PLIN5 protein increases,but collagen production decreases in HSCs.