Study On Protective Effect And Mechanism Of Hepatocyte Growth-Promoting Factor On Chemical Liver Injury In Mice And LO2 Cells Induced By H₂O₂

OBJECTIVE To study the protective effects and mechanism of hepatocyte growth-promoting factor on mice of chemical liver injury and LO2 cells induced by H2O2. It is hope that can provide some theoretical basis for clinical applicable disease of PHGF. METHODS The test is divided into two kinds between in vivo and in vitro:(1) In vivo. First, SDS-PAGE is used to verify the molecular weight of PHGF. then using volume 9 of national drug standards (WS-10001 (HD-0828)-2002) to test the activity. After that, the test used CCl4 lavage to establish the model of chemical liver injury in mice. the experiment are divided into 6 groups. Blank control group, model group, three dose PHGF group (12.5 mg·kg-1、25 mg·kg-1、50 mg·kg-1) and Positive control group(Yongning capsule). The models are made after 7 days. then take the blood to test ALT and AST value; Take the mice liver tissues histologic observation and immunohistochemical method to detect the Bcl-2 and Bax expression. Using western blot method to detect the Bcl-2 and Bax protein expression of mouse liver. (2) In vitro. First, the effects of PHGF on LO2 cells are determined by MTT, then LO2 cells are treated with H2O2 DMEM nutrient solution as apoptosis model. Test is divided into 5 groups. Blank control group, model group and three concentration PHGF group(50、100、200 μg·mL-1). MTT method is used to determine the proliferation of different groups induced by PHGF. The cell apoptosis rate of different groups is assured by flow cytometry. Using Western blot method to detect the Bcl-2 and Bax protein expression of LO2 cells of different groups. RESULTS In vivo tests show:The ALT and AST value of each dose group of PHGF significantly lower than the model group(P<0.05);Through the pathological observation, each dose group of liver cell necrosis situation is obviously better than the model group; Immunohistochemical experiments showed that the expression of Bcl-2 protein significantly stronger than the model group (P<0.05), while the expression of Bax protein significantly weaker than model group (P<0.05);Western blot results show that the PHGF group, according to the dose from high to low, the expression of Bcl-2 protein significantly stronger than the model group (P<0.01), while the expression of Bax protein significantly weaker than model group (P<0.01).In vitro tests show:The effect of PHGF on LO2 cells within the concentration range (500～2.5 μg·mL-1) are no obvious proliferation and cytotoxicity; The best model concentration of H2O2 is 0.25 mmoL·L-1. The optical density of each concentration PHGF group is significantly higher than H2O2 model group (P<0.05);Flow cytometry results show that the LO2 cell apoptosis rate of each concentration PHGF group significantly lower than in H2O2 model group (P<0.01). Western blot results show that the PHGF group, according to the concentration from high to low, the expression of Bcl-2 protein significantly stronger than the H2O2 model group (P<0.01), while the expression of Bax protein significantly weaker than H2O2 model group (P<0.01). CONCLUSION PHGF can prevent the chemical hepatic injury induced by CCl4 in mice and apoptosis of LO2 cells induced by H2O2. The mechanism of action may up the expression of Bcl-2 protein and down expression of Bax protein.