Study Of The Mechanism Of Follistatin Like 1 In The Senesence Of Hepatic Stellate Cells

Liver fibrosis is an abnormal proliferation of connective tissue in the liver caused by various pathogenic factors.Its long-term continuous development will develop into cirrhosis and even liver cancer.At present,there is still no effective treatment.In normal liver,hepatic stellate cells（HSCs）are at rest state.Under pathological conditions,HSCs are activated（activated HSCs,aHSCs）,which are the main producer of the extracellular matrix（ECM）during liver fibrosis.Studies have found that liver fibrosis can be reversed after removing the pathogenic factors.During this process,aHSCs transdifferentiate into inactivated HSCs,undergo cell senescence or apoptosis and thenare eliminated by immune cells in the body.Follistatin-like 1（Fstl1）has a variety of biological functions.Our previous study found that Fstl1 mainly expresses in aHSCs and modulates their trans-differentiation,as well as plays an important role in the process of hepatic fibrosis.However,its role in the resolution of live fibrosis needs further investigation.In this study,we investigated the role of Fstl1 in the senescence of aHSCs in vitro and in the regression of liver fibrosis in vivo.The main findings and conclusions are as follows:（1）At first,we study the expression of Fstl1 during the resolution of liver fibrosis in mice.We established a murine liver fibrosis model by intraperitoneal injection of carbon tetrachloride（CCl₄）twice a week which lasts four consecutive weeks.After the last injection of CCl₄,the mice were recovered for 30 days.We found aHSCs activation markerα-smooth muscle actin（α-SMA,encoded by Acta2）and ECM main protein Collagen type I（Col1）gene expression was significantly down-regulated,close to the normal group level,and found that Fstl1 expression was down-regulated during this resolution process.Haplodeficiency of Fstl1in mice(Fstl1^+/-)had less liver fibrosis and recovered faster than wild-type mice(Fstl1^+/+).The senescence-related genes,i.e.Tumor protein P53（TP53,P53）,cyclin-dependent kinase inhibitor 2A（P16）,Cyclin Dependent Kinase Inhibitor 1B（P27）and Cyclin Dependent Kinase Inhibitor 1A（P21）were inhibited in the liver of mice which were intravenous injection of adenovirus over-expressing FSTL1 in the tail vein.These results indicate that Fstl1 is down-regulated during the resolution of liver fibrosis and may participate in the process of liver fibrosis regression and modulate cellular senescence.（2）We further examined the effect of Fstl1 on HSC senescence in vitro.We used the cell cycle-specific anti-tumor drug Etoposide to induce human hepatic stellate cell line LX-2 or mouse primary HSCs to establish senescent cell models.Cell senescence-specific staining（β-gal staining）confirmed the successful model establishment.Etoposide stimulation up-regulated the expression of senescence-related genes,such as P53,P21,P27,P16,SIRT1（sirtuin1）and others,while down-regulated the expression of fibrosis-related genesα-SMA and COL1 genes,as well as inhibited FSTL1 expression.Knocking down the expression of FSTL1 using siRNA showed increased cellular senescence compared with the control group byβ-gal staining in LX-2 cells in vitro.Si-Fstl1 down-regulated the expression of fibrosis-related genesα-SMA and COL1,but increased the expression of senescence-related genes P53,P21,P27,P16,and SIRT1.On the other hand,over expression of FSTL1 showed the reverse effect.Furthermore,knocking down Fstl1 inhibited cell migration,and reduced the expression of fibrosis-related genes in primary HSCs,while overexpressing Fstl1 showed the reverse effect.These data demonstrated that Fstl1 promotes the activation of HSCs and inhibits their senescence in vitro.（3）In order to study the possible mechanism of Fstl1 on HSCs,we analyzed the effect of knockdown expression of Fstl1 on the primary HSCs after 5 days of culture by droplet microfluidic single cell transcriptome sequencing technology.As a result,we found that the expression levels of genes related to liver fibrosis and ECM deposition,such as Collagen Type II Alpha 1 Chain（Col2a1）,Col5a3,Col5a1,Col1a11 was significantly reduced in the Fstl1 low-expression cell cluster.In contrast,the expression levels of genes related to senescence and apoptosis such as CXC Motif Chemokine Ligand 10（Cxcl10）,Growth Arrest and DNA Damage Inducible Gamma（Gadd45g）,TNF Superfamily Member 15（Tnfsf15）,Interleukin 16（Il6）were up-regulated in the Fstl1 low-expression cell cluster.The enrichment analysis of differential expression genes indicates that Fstl1 may affect the senescence and apoptosis of HSCs through PI3K-AKT and MAPK signaling pathways,thus affecting the process of liver fibrosis.In conclusion,Fstl1 may play an important role in the regression of liver fibrosis and inhibits the senescence process of aHSCs.Targeting Fstl1 may serve as a potential therapeutic approach in the treatment for liver fibrosis.