The Inhibitory Effect And Mechanism Of Dehydroandrographolide On Liver Fibrosis Induced By Bile Duct Ligation In Mice

ObjectiveThe main purpose of this study is to investigate the protective effect of dehydroandrographolide(DA)on liver fibrosis induced by cholestasis in mice by using the model of bile duct ligation(BDL)in mice,and to explore the mechanism of DA in the process of liver fibrosis by culturing human LX-2 cells in vitro.MethodsAnimal experiment: Forty C57BL/6J male mice were randomly divided into four groups: Carboxy Methyl Cellulose(CMC)+sham group,DA+sham group,CMC+BDL group and DA+BDL group,with 10 mice in each group.The mice in the BDL group underwent double ligation and interruption of common bile duct.The mice in the sham group underwent sham operation,and the common bile duct was only threaded without ligation.The mice in the DA+sham group and DA+BDL group were given DA orally(100mg/kg)once a day,while the mice in the CMC+sham group and CMC+BDL group were given the same dose of CMC.After 5 days of continuous intragastric administration,the mice in each group underwent corresponding surgery,and then continued intragastric administration for 12 hours after operation.7 days later,the mice were sacrificed to collect serum and liver samples for the following tests: alanine aminotransferase(ALT),total bilirubin(TBIL)and total bile acid(TBA)kits was used to detect the concentration of ALT,TBIL and TBA in the serum;paraffin sections of mice liver were stained with HE,and pathological changes of liver tissues were observed under microscope.Sirius red staining was used to observe the degree of liver fibrosis.Quantitative real-time fluorescence polymerase chain reaction(qRT-PCR)was used to detect the relative expression of liver fibrosis-related genes,including transforming growth factor-beta(TGF-?)and collagen type I alpha1(Col1a1),and Western blot was used to detect the relative expression of alpha-smooth muscle actin(?-SMA)in liver tissue.Cell experiment in vitro: Human LX-2 cells were cultured in vitro and activated by TGF-?.Divided into blank solvent(DMSO)control group,DA group(20?M/L),TGF-? group(2ng/ml),low-dose DA(5?M/L)+TGF-?(2ng/ml)group,medium-dose DA(10?M/L)+TGF-?(2ng/ml)group and high-dose DA(20?M/L)+TGF-?(2ng/ml)group.The mRNA level of Col1a1 in LX-2 cells was detected by qRT-PCR.Western blot was used to detect the relative expression of ?-SMA protein in each group.ResultsAnimal experiment: In mice experiment,we found that the levels of ALT,TBIL and TBA in serum of mice in CMC+BDL group were increased significantly(P<0.01),but after DA treatment,the levels of these biochemical indicators were decreased significantly(P<0.01),and the liver injury was significantly alleviated.Morphological analysis of liver showed that HE staining showed that compared with CMC+sham group,the structure of hepatic lobules of mice in CMC+BDL group was damaged and the liver cord was broken.There were a large number of inflammatory cells infiltrating in the portal area,the bile duct wall was significantly thicker,and there was obvious proliferation.There were a large number of diffuse,punctate hepatocyte necrosis foci.Compared with the CMC+BDL group,the pathological damage,inflammatory cell infiltration and necrosis of the DA+BDL group were significantly improved.Sirius red staining showed that collagen fibers deposited around the portal area in the liver tissue of CMC+BDL mice compared with the CMC+sham group.Compared with the CMC+BDL group,collagen fibers deposited around the portal area in the liver tissue of DA+BDL mice were decreased significantly,and the degree of collagen fibers decreased(P<0.01).At the same time,the results of qRT-PCR and Western blot showed that compared with CMC+sham group,the expression levels of TGF-?,Col1a1 and ?-SMA in liver tissue of mice in CMC+BDL group were increased,while DA significantly decreased the expression levels of TGF-?,Col1a1 and ?-SMA induced by BDL in liver tissue of mice,with significant difference(P<0.05 or P<0.01).In vitro cell experiments: qRT-PCR and Western blot results showed that DA treatment could significantly inhibit the expression of Col1a1 and ?-SMA protein in human LX-2 cells stimulated by TGF-?(P<0.05 or P<0.01).ConclusionsDA has protective effect on liver injury induced by BDL in mice;DA may inhibit the activation of hepatic stellate cells(HSC)by inhibiting the expression of fibrosis-related factors,thus achieving anti-fibrosis effect.