Protective Mechanism And Application Of Fibroblast Growth Factor 21 In Mice With Cholestatic Hepatic Fibrosis

Objective:Studies have shown that FGF21 has a protective effect on the liver,but its role in the development of liver fibrosis is still unclear.Therefore,we performed common bile duct ligation(BDL)on wild-type and FGF21-knockout C57BL/6J mice to induce cholestasis to simulate cholestatic liver fibrosis,and explore the protective mechanism and application value of FGF21 in liver fibrosis induced by BDL.Methods:Healthy male wild-type and FGF21 KO C57BL/6J mice aged 6-8 weeks were selected for sham operation(sham)and BDL respectively.The abdominal cavity of the mice in the sham group was opened and the common bile duct was freed but without ligation,and the abdominal cavity of the mice in the BDL group was opened and the common bile duct was ligated.The experimental period was 2 weeks.Wild-type mice were injected with FGF21 adenovirus through the tail vein,followed by sham and BDL.Wild-type and FGF21 KO C57BL/6J mice were administered intragastrically by sodium butyrate(NaB)for one month,and then sham and BDL operation were performed.The general state of mice in each group after operation was observed,then on the 14th day after the operation mice were euthanized and the liver and spleen were collected and weighed.The organ index(the percentage of organ weight/body weight)was calculated,and serum total bile acids(TBA),serum total bilirubin(TBIL)were measured.The pathological changes of liver were observed by H&E and Masson staining.Western blot was used to quantitatively detect the expressions of liver fibrosis,inflammation and apoptosis related proteins in each group.The mRNA expressions of α-smooth muscle actin(α-SMA),transforming growth factor β1(TGF-β1)and other fibrotic markers in mice liver were detected by real-time PCR,L02 cells were cultured and treated with taurocholic acid(TCA)and Taurochenodeoxycholic acid(TCA),cell necrosis was then detected using a cell necrosis test kit.At the same time,L02 cells were treated with NaB to determine whether NaB could relieve the bile acid-induced hepatocyte injury.Results:1.After BDL operation,the weight and activity of mice were decreased,accompanied by yellow skin and urine color,and TBA and TBIL were significantly increased(p<0.05).Liver histopathology showed inflammatory cell infiltration in the liver tissue of BDL group mice,bile duct epithelioid cell hyperplasia,bile duct dilatation,and peribile duct fibrous tissue hyperplasia in the portal area,large necrotic hepatocytes,disordered hepatic lobular structure,and collagen fiber hyperplasia,suggesting successful construction of cholestatic hepatic fibrosis model.Protein and gene levels showed that the expressions of FGF21,fibrosis,apoptosis,inflammation and other related proteins in the liver were significantly increased after BDL induction.2.Compared with WT-BDL group,the yellow staining degree of peritoneum and intestinal tract of mice in FGF21 KO-BDL group was increased,nodules on liver surface were increased,liver and spleen indexes were significantly increased(p<0.05),liver pathology showed that inflammatory cell infiltration,liver cell necrosis,bile duct dilation,collagen fiber deposition and other aspects were increased.The expressions of Timpl,α-Sma,Tgf-β and other liver fibrosis related mRNA were significantly increased,while the expressions of fibrosis and apoptosis related proteins were up-regulated.3.Compared with mice in WT-BDL group,the yellow stain of intestinal tract and peritoneum of mice after adenovirus overexpression(WT-BDL+AdFGF21 group)was reduced,the nodules of liver were reduced,the indexes of liver and spleen were decreased,and the content of bile acid in gallbladder was increased.H&E and Masson staining showed that the liver of mice in WT-BDL+AdFGF21 group was relieved in inflammatory cell infiltration,bile duct dilation,hepatocyte necrosis,collagen fiber deposition and other aspects.Protein and gene levels showed that compared with the WT-BDL group,the WT-BDL+AdFGF21 group had less liver fibrosis and bile duct damage.4.After one-month gavage of normal saline or NaB in WT mice,there was no significant difference in AST and ALT of mice given NaB and normal saline(p>0.05),indicating that sodium butyrate did not damage the liver,while WB test showed that sodium butyrate can induce the expression of FGF21 in liver.In addition,Intragastric administration of sodium butyrate can relieve liver fibrosis caused by BDL,which is manifested as reduced yellowing degree of peritoneum and liver and reduced nodules of liver.Liver pathology showed reduced bile duct dilation,inflammatory cell infiltration and collagen fiber production.At the same time,liver function was improved,and the expression of inflammation,fibrosis,apoptosis,oxidative stress,cell proliferation related protein in the liver were decreased.5.Compared with WT-BDL+NaB group,the weight of mice in FGF21 KO-BDL+NaB group was decreased significantly,the degree of yellow staining of peritoneum was aggravated,the liver was enlarged and the texture became hard,and there were obvious granular yellow-green nodules on the surface.Liver pathology showed that compared with mice in WT-BDL+NaB group,hepatocyte necrosis,bile duct dilation,collagen fiber deposition were increased in mice in FGF21 KO-BDL+NaB group.Meanwhile,liver function deteriorates,and the expression of inflammation,fibrosis,apoptosis,oxidative stress,cell proliferation related protein in the liver was increased or no significant changes.The results suggest that after FGF21 knockout,sodium butyrate may not be able to play an anti-inflammatory and anti-fibrosis role,resulting in liver fibrosis can not be alleviated or even aggravated.6.Sodium butyrate alleviates bile acid-induced inflammation and apoptosis of LO2 cells:The expressions of inflammatory and apoptotic factors such as p-NF-κB,p-JNK,NLRP3 and BCL2 were increased after treatment with bile acid.Meanwhile,NaB treatment could reduce the inflammation and apoptosis induced by bile acid.The necrotic staining of LO2 cells showed that the necrosis of LO2 cells increased significantly after treatment with bile acid,and the hepatocyte injury induced by bile acid could be alleviated after treatment with NaB.Conclusion:1.FGF21 deficiency can aggravate cholestatic hepatic fibrosis in mice.2.Exogenous FGF21 alleviates liver fibrosis caused by BDL.3.NaB is dependent on FGF21 to alleviate cholestatic hepatic fibrosis in mice.