Effect And Mechanism Of Activation Of Hepatic Stellate Cells In Hypoxia Microenvironment

Background and PurposeThe development process of non-alcoholic fatty liver disease（NAFLD）includes non-alcoholic fatty liver（NAFL）,non-alcoholic steatohepatitis（NASH）,and liver fibrosis and liver cirrhosis,in which liver fibrosis is significantly related to the mortality of NAFLD patients,and the activation of hepatic stellate cells（HSCs）is a key link in the development of liver fibrosis.It is generally believed that the activation and fibrosis process of HSCs is secondary to the NASH stage.We found that liver fibrosis exists in the early stage of NAFLD（NAFL）,but its pathogenesis is still unclear.In the NAFL stage,due to steatosis and increased volume of hepatocytes,the intra-sinusoidal pressure increases,the hepatic blood flow decreases,and hypoxia occurs in the Disse space where HSCs are located.Studies have shown that HSCs are activated in a hypoxic microenvironment,so is the occurrence of liver fibrosis in the NAFL stage caused by hypoxia?In this paper,the effect of hypoxic microenvironment on HSCs activation and liver fibrosis was investigated by observing the liver histology of the NAFL rat model and establishing an in vitro hypoxic cytology model.Methods1.Detection of hepatic hypoxia microenvironment in NAFL rat model（1）Observation of hepatic steatosis in NAFL rat model by HE staining;（2）Observation of liver fibrosis in NAFL rat model by Masson staining;（3）The activation of HSCs and the hepatic hypoxia microenvironment were observed in the NAFL rat model by IHC experiments.2.Establishment of an in vitro cytological model of hypoxia（1）In hypoxia（1%O₂）culture,the morphology of HSC-T6 and primary rat HSCs showed a spindle-shaped transformation;q PCR detection of HSC-T6 HIF-1αm RNA expression increased,confirming that the hypoxia microenvironment model was successfully established.（2）The growth of HSC-T6 under hypoxia was observed under a microscope,and the expression of HIF-1αwas observed by immunofluorescence;3.The effect of hypoxic microenvironment on HSCs activation and liver fibrosis（1）Research objects and experimental groups:1)Research objects:HSC-T6,primary HSCs2)Experimental grouping:Control group:hypoxia time 0 h;Experimental group:hypoxia time was 4 h,12 h,24 h,respectively（2）The effects of hypoxic microenvironment on the proliferation and migration of HSC-T6 were studied through CCK-8 and scratch experiments;（3）The effects of hypoxia microenvironment on the m RNA and protein expression levels of HSC-T6,primary HSCs HIF-1α,α-SMA,CollagenⅠ、CollagenⅢwere studied by q PCR and Western Blotting experiments;（4）The effects of hypoxic microenvironment on the m RNA and protein expression levels of HSC-T6 YAP were studied by q PCR and Western Blotting experiments.Results1.There is a hypoxic microenvironment in the liver of the NAFL rat model and the activation of HSCs（1）HE staining:a large number of fat vacuoles were seen in the hepatocytes of the NAFL rat model group（2）Masson staining:the hepatocytes in the NAFL rat model group were surrounded by blue collagen fibers（3）IHC staining:HIF-1αandα-SMA were positively expressed in HSCs of NAFL rat model2.Establishment of an in vitro cytological model of hypoxia（1）After 12 h of hypoxia（1%O₂）culture,the morphology of HSC-T6 and primary HSCs showed a spindle-shaped transformation;q PCR detected the increase of HIF-1αm RNA expression in HSC-T6.（2）Compared with the 0 h group,HSC-T6 cultured in hypoxia for 12 h significantly promoted the expression of HIF-1αmolecules（P<0.05）;immunofluorescence staining confirmed that HIF-1αmainly existed in the nucleus of HSC-T6 cells,confirming that The hypoxic microenvironment model was successfully established.3.The effect of hypoxic microenvironment（1%O₂）on HSCs activation and liver fibrosis（1）Compared with the 0 h group,HSC-T6 cultured under hypoxia for 4 h,12 h,and 24 h promoted cell proliferation（P<0.05）,while culturing for 24 h inhibited cell proliferation（P<0.05）;Hypoxia culture of HSC-T6 for 12 h and 24 h inhibited cell migration（P<0.05）.（2）Compared with the 0 h group,culturing HSC-T6 and primary rat HSCs for 4h,12 h and 24 h in hypoxia significantly promoted the m RNA expression of HSC-T6,primary rat HSCsα-SMA,Collagen I and Collagen III（P<0.05）.（3）Compared with the 0 h group,hypoxia promoted the expression level of HSC-T6 YAP m RNA（P<0.05）.Conclusions1.There is a hypoxic microenvironment and the activation of HSCs in the liver of the NAFL rat model;2.Successfully established an in vitro hypoxic microenvironment cell model;3.Hypoxic microenvironment activates HSCs and participates in NAFL fibrosis;Hypoxic microenvironment activates HSCs through Hippo/YAP signaling pathway.