Effects Of Gremlin On Proliferation,Apoptosis And Extracellular Matrix Secretion Of Primary Hepatic Stellate Cells In Mice

Objective:To investigate the effects of Gremlin on proliferation,apoptosis and extracellular matrix secretion of primary hepatic stellate cells in mice,and to explore the role of Gremlin in the development and progression of liver fibrosis,and to provide experimental theoretical basis for the pathogenesis of liver fibrosis.Methods:After using the classic liver two-step enzyme circulation perfusion method to remove the liver capsule components of mice,the mouse HSC contained rich adipose tissue,which was lower than other intrahepatic cells.Therefore,the mouse primary HSC was isolated by density gradient centrifugation.Then,the cells were cultured in a37°C,5%CO₂ incubator^[1].The mouse HSC obtained by circulating perfusion were randomly set into four groups,namely,a control group,a 0.05%DMSO group,a Gremlin100μg/L group（low dose group）,and a Gremlin 200μg/L group（high dose group）.DMEM complete medium was added as a control group,DMEM medium containing 0.05%DMSO was set to 0.05%DMSO group,100μg/L Gremlin DMEM medium was set to low dose group,and 200μg/L Gremlin DMEM medium was set to high dosage group.The cells of each group were placed in a 37°C,5%CO ₂ incubator for 24 hours,and the proliferation rate and apoptosis rate of each group were determined by CCK-8 method and flow cytometry respectively.Western blotting and RT-qPCR method were used.The expression of proteins and mRNA such as Collagen I,Collagen III and a-SMA in each group of cells was examined.Result:1)The cell proliferation rate of Gremlin 100μg/L and Gremlin 200μg/L was higher than that of the control group and DMSO group（P<0.05）.There was no significant difference between the Gremlin 100μg/L dose group and the Gremlin 200μg/L dose group（P>0.05）.The apoptosis rate of Gremlin 100μg/L and Gremlin 200μg/L was lower than that of the control group and DMSO group（P<0.05）.There was no significant difference in the apoptosis rate between Gremlin 100μg/L and Gremlin 200μg/L（P>0.05）.2)The relative expression levels of Collagen I,Collagen III and a-SMA mRNA in Gremlin 100μg/L and Gremlin 200μg/L groups were higher than those in the control group and DMSO group（P<0.05）,and the relative expression of mRNA in the Gremlin 200μg/L dose group was higher than Gremlin 100μg/L.The dose group（P<0.05）,but the control group and DMSO group were not statistically significant（P>0.05）.The relative expression levels of Collagen I,Collagen III and a-SMA in the Gremlin 200μg/L dose group were higher than those in the Gremlin 100μg/L dose group,the control group and the DMSO group（P<0.05）,but the Gremlin 100μg/L dose group was compared with the control group and the DMSO group,The difference was not statistically significant（P>0.05）.Conclusion:Gremlin is involved in the induction of the activation of primary hepatic stellate cells in mice,which promotes the proliferation of primary hepatic stellate cells in mice,inhibits the apoptosis of mouse hepatic stellate cells,and promotes the secretion of extracellular matrix by primary hepatic stellate cells.There by participating in the occurrence and development of liver fibrosis.