Study Of The Mechanism Of Fstl1 In Liver Fibrosis

Liver fibrosis is characterized by changes in tissue architecture and widespread scar formation resulting from chronic,nonresolving inflammation,ultimately leading to liver cirrhosis and its complications,liver failure and even hepatocellular carcinoma.Recent study shows that the activation of hepatic stellate cells?HSCs?plays a significant role in liver fibrosis.Follistatin-like 1?Fstl1?is a secreted glycoprotein,also named TGF-?1-stimulated clone 36?TSC-36?,has extensive biological functions.However,the role of Fstl1 on liver fibrogenesis and development remains unclear.MicroRNA?miRNA?is a class of non-coding single stranded RNA molecules encoded by about 22 nucleotides.They are involved in the regulation of gene expression after transcription in plants and animals.MiR29 is associated with a variety of tissue fibrosis,and the deep sequencing of myotube cells reveals that Fstl1 may be the downstream target of mi R29.Therefore,based on the database research and experiments in vivo and in vitro,we found Fstl1 was associated with liver fibrosis and hepatic stellate cells activation.Further more,Fstl1 may regulate the pathogenesis and development of liver fibrosis through the TGF-?1-miR29a-Fstl1 regulatory circuit according to the in vivo and in vitro experiments.The main conclusions are as follows:?1?The expression of Fstl1 in liver fibrosis tissues and activated hepatic stellate cells was determined.We conducted a database investigation and used the CCl₄ induced murine liver fibrosis model and TGF-?1 activated of human hepatic stellate cell LX-2,rat hepatic stellate cell HSC-T6 and CFSC-8B in vitro.The HSCs activation marker?-SMA and one of the most important ECM components Collagen I?Col1?were significantly up-regulated after TGF-?1stimulation in all three HSCs cell lines,which demonstrated the validity of the model.From the database research we found that FSTL1 m RNA was increased by 11%in patients with liver fibrosis stage 3-4 compared with stage 1-2.Compared with 0d,Fstl1 was increased by 60%,90%and 137%in mice liver fibrosis tissues of CCl₄ treatment for 7 d,14 d and 28 d.Fstl1 was up-regulated in three liver fibrotic cell models.Immunofluorescence staining showed that the expression of Fstl1 and?-SMA in hepatic fibrosis mice induced by CCl₄ was colocalized.?2?The effect of Fstl1 gene and protein on liver fibrosis.Haplodeficiency of Fstl1 in mice or knockdown of FSTL1 on LX-2 cells reduced the liver fibrosis degree and inhibited the activation of hepatic stellate cells and ECM deposition through Smad2/JNK pathway.Blockage of Fstl1 with a neutralizing antibody in mice and LX-2 cells alleviated the degree of liver fibrosis induced by CCl₄ and reduced the activation and migration of LX-2 induced by TGF-?1through inhibiting phosphorylation of Smad2/JNK.Fstl1 recombinant adenovirus infections of mice or LX-2 increased the degree of liver fibrosis induced by CCl₄ and promoted the activation and migration of LX-2 by faciliting Smad2/JNK signaling pathway.In addition,the conditional medium containing cell culture medium from Ad-Fstl1 infected LX-2 cells promoted?-SMA and COL1 mRNA expression.The results indicated that Fstl1 promoted the degree of hepatic fibrosis through Smad2/JNK pathway and may be a potential therapeutic target for hepatic fibrosis.?3?The study of Fstl1 regulation pathways.By detecting the expression of miR29a in hepatic stellate cells and liver fibrosis tissues together with miR29a Mimics or Inhibitor transfection experiment,we found that Fstl1 regulated the degree of hepatic fibrosis by inhibiting miR29a expression in hepatic stellate cells in vitro and in CCl₄ induced hepatic fibrotic mouse models in vivo.The 3'UTR plasmid validation experiment results showed that FSTL1 was a direct target of miR29a,which further proved that TGF-?1-miR29a-Fstl1regulation pathway may exist in hepatic fibrosis.In conclusion,our study found that Fstl1 may regulate the genesis and development of hepatic fibrosis through TGF?1-miR29a-Fstl1.Targeting Fstl1 may become a new method for the treatment of liver fibrosis in the future.