Study On The Effect And Mechanism Of New Proline Hydroxylase Inhibitor BT1001 And BT1011 On Liver Fibrosis

A variety of etiologies can lead to liver fibrosis,such as viruses,alcohol,and parasites.Liver fibrotic progression can be partly inhibited by removing or controlling the causes,but the fibrosis persists in some exceptions.Better understanding of the fibrosis mechanism will afford opportunities to directly target the fibrotic process and develop the therapies of inhibiting or reversing liver disease progression.Liver fibrosis is the result of an imbalance of liver injury and repair.Despite some progress have been made in the clinical research of anti fibrosis,but there are no exact treatment effect in clinical.Even for the cause of antiviral treatment can not solve all the patients,especially in the advanced liver cirrhosis.Therefore,the treatment of key target for liver fibrosis may be promising in the basic research.The hydroxyproline in collagen does not exist in other proteins.It does not participate in the biosynthesis of collagen in the form of ready-made.Instead,it is transformed from the proline of collagen chain synthesized by hydroxylase.Therefore,the intervention of 4-hydroxyl-proline hydroxylase activity has been recognized as an effective way to inhibit the excess collagen(fibrosis).In this study,10 cases of healthy liver tissues were collected in the department of hepatobiliary surgery of the First Affiliated Hospital of Jilin University.The primary hepatic stellate cells were isolated by perfusion in vitro.We selected LX2 and primary hepatic stellate cells HSC as target cells in vitro to test two new hydroxyproline inhibitors BT1001 and BT1011 function.We apply immunohistochemistry,flow cytometry,immunoprecipitation,Real-Time PCR and Elisa and other methods to study the role and mechanism of BT1001 and BT1011 on liver fibrosis.The results showed that BT1001 and BT1011 could significantly inhibit the level of ?-SMA secreted by LX2 cells.PCR results showed that the high dose group of BT1001 and BT1011 could significantly inhibit the Col1A1 expression of LX2 cells,and increase the MMP-2 expression in LX2 cells obviously.Three are no difference in the expression of TIMP-1 and TIMP-2.Compared with the LX2 cell line,the human primary hepatic stellate cell HSC cells secrete higher level of ?-SMA under the same stimulation.The results were similar to that of LX2 cell lines.BT1001 and BT1011 could significantly inhibit the Col1A1 expression in HSC cells,and obviously improve the MMP-2 expression in HSC cells.Cell proliferation was inhibited after BT1001 and BT1011 treated on LX2 cells.Compared with blank control group,there was significant difference(P<0.05),and the highest inhibition rate was(42.56±5.21%).The results of absolute count of cells were the same as that of MTT result.We found that the apoptosis of LX2 cells caused by BT1001 and BT1011 was induced by the activation of the apoptotic gene Caspase-3.It was found that the apoptosis of LX2 cells caused by BT1001 and BT1011 was obviously inhibited after the Caspase-3 gene was silenced.We followed up the experimental observation of rat liver fibrosis model in vivo.A rat liver fibrosis model was induced by common bile duct ligation.The animals were randomly divided into 6 groups: sham operation group,model control group,low dose test group,middle dose test group and high dose test group.After successful establishment of each group,blood biochemical indicators,viscera index,liver histopathology,liver collagen and enzyme detection were used to demonstrate the success of modeling.Under the experimental conditions,the prevention effects on bile duct ligation liver fibrosis was observed after intragastric administration of BT1001 and BT1011.To summary,we first demonstrated that BT1001 and BT1011,two new hydroxylproline inhibitor,can inhibit collagen synthesis and collagen degradation in hepatic stellate cells and inhibit the process of liver fibrosis in vivo and in vitro.On the other hand,they can also induce the apoptosis of LX2 cells through Caspase-3 signaling pathway and inhibit the process of liver fibrosis.It also points out a new direction that can be used to target or treat hepatic fibrosis.