Mechanism Of High-fat Dietary NAFL Fibrosis Induced By Mechanical Stress-activated Hepatic Stellate Cells

Objective:1.To explore the mechanism of hepatic stellate cells（HSCs）induced fibro sis induced by high-fat dietary NAFL stress microenvironment;2.To explore the role of YAP in the activation of HSCs in NAFL stage.Method:1.NAFL fibrosis,HSCs activation and stress microenvironment:（1）In vivo experiment: The expression of α-SMA,Collagen Ⅲ and YAP in liver tissue was detected by immunohistochemical staining using wax block specimens of high-fat dietary NAFL rat model constructed in our laboratory;（2）In vitro experiments: Rat primary HSCs and HSC-T6 cells were used as the research objects,and the mechanical pressure microenvironment cytology model was established under mechanical pressure for 24 h.Experimental group: 2 mm Hg,4mm Hg,6 mm Hg;Control group: 0 mm Hg.Cell proliferation was detected by flow cytometry;western blot was used to detect the protein expressions of α-SMA,Collagen Ⅲ and YAP in HSCs;RT-q PCR was used to detect the m RNA expression levels of Acta2,Col3a1 and Yap genes.2.The role of YAP in the activation of HSCs by mechanical stress:（1）The expression and localization of YAP and p-YAP were observed by immunofluorescence and Confocal;the nucleoplasmic proteins of HSC-T6 cells were isolated,and the expression of p-YAP was detected by western blot;（2）Under stress conditions,the YAP inhibitor Verteporfin was treated with HSC-T6 cells for 24 h,and the effects on the gene expression of Yap,Acta2,Col3a1 and Ctgf were detected;（3）HSC-T6 cells were cultured and treated with mechanical stress for 24 h.RT-q PCR was used to detect the expression of YAP-regulated target genes Ctgf and Ankrd1.Result:1.NAFL fibrosis,stress microenvironment and HSCs activation:（1）NAFL rats significantly induced hepatocyte steatosis without obvious hepatocyte necrosis and inflammatory cell infiltration;（2）The expression of Collagen Ⅲ in the liver tissue of NAFL rats was significantly increased in the Disse space of the hepatic lobule,indicating non-inflammatory fibrosis;（3）NAFL rat hepatocytes and HSCs YAP showed strong nuclear positive staining,suggesting the stressful microenvironment of NAFL;（4）Mechanical pressure of 2 mm Hg for 24 h promoted the proliferation of HSC-T6 cells（P < 0.05）,but the effects of 4 mm Hg and 6 mm Hg on cell proliferation were not statistically significant（P > 0.05）;（5）Mechanical pressure（2 mm Hg,4 mm Hg,6 mm Hg,24 h）inhibited the expression of α-SMA in HSC-T6 cells（P < 0.05）,inhibited the expression of Collagen Ⅲ,and had no significant effect on the expression of α-SMA in primary HSCs（P > 0.05）,but promoted the expression of Collagen Ⅲ in primary HSCs（2mm Hg,24 h）（P < 0.05）;（6）Mechanical stress promoted YAP expression in HSC-T6 cells（4 mm Hg,24 h）and primary HSCs（2 mm Hg and 4 mm Hg,24 h）（P < 0.05）.2.The role of YAP in the activation of HSCs by mechanical stress:（1）Mechanical stress induces the nuclear translocation and activation of YAP in HSC-T6 cells,and promotes the expression of cytoplasmic p-YAP;（2）The YAP inhibitor Verteporfin significantly inhibited the expression of Yap,Acta2,Col3a1 and Ctgf genes;（3）Mechanical stress had no effect on the m RNA expressions of YAP target genes Ctgf and Ankrd1 in HSC-T6 cells（P > 0.05）.Conclusion:（1）In the NAFL stage,there are fibrosis,HSCs activation and stress microenvironment;（2）Mechanical stress promotes HSCs activation,proliferation,and YAP activation and nuclear translocation;（3）YAP regulates the expression of Acta2,Col3a1 and Ctgf genes in HSCs.