The Protective Effect Of EGCG On Immunological Liver Injury Induced By TP And The Relevant Mechanism Research

ObjectiveEpigallocatechin-3-gallate(EGCG) is a polyphenolic compound extracted from green tea, which possesses remarkable anti-inflammatory and antioxidant property. In this study, we established a model of immunological liver injury in mice to investigate the protective effect of EGCG on triptolide(TP)-induced immunological liver injuries and the relevant mechanisms of action。Methods1. Construction and identification of a model of immunological liver injury induced by TP in mice. A total of 20 female C57BL/6 mice were randomly divided into two groups: control group and TP group. The mice in TP group were orally administered with TP 500 μg/kg, the mice in normal group were given equal amount of normal saline, and blood was taken after 22 h, then all the mice were sacrificed. The alanine aminotransaminase(ALT) level in serum in the two group was examined by Reitman Frankel method. HE staining was used to observed the changes of the hepatic histopathology. The hepatic interleukin(IL)-17 and IL-10 levels were examined by ELISA. The m RNA expression of hepatic retinoid related orphan receptor(ROR-γt) and forkhead/winged-helix family transcriptional repressor p3(Foxp3) were examined by RT-PCR.2. The protective effect of EGCG on immunological liver injury induced by TP and the relevant mechanism research. A total of 40 female C57BL/6 mice were randomly divided into four groups: control group,EGCG group, TP group and TP+EGCG group. In TP group, mice were administered by gavage orally with 500 μg/kg TP at the end of EGCG administration. In EGCG group, mice were administered by oral gavage of5 mg/kg EGCG twice daily for 10 days. In TP+EGCG group, mice were treated with EGCG plus TP on day 10. In control group, mice were given the same volume of normal saline. The ALT level in serum was examined by Reitman Frankel method. The activity of hepatic malondialdehyde(MDA), superoxide dismutase(SOD) and restored glutathione(GSH)were examined by spectrophotometry. HE staining was used to observed the changes of the hepatic histopathology. The hepatic IL-17 and IL-10 levels were examined by ELISA. The m RNA expression of hepatic ROR-γt,Foxp3, nuclear factor(NF)-κB and myeloid differentiation factor 88(My D88) were examined by RT-PCR. The expression of hepatic T cell Ig and mucin domain protein 3(Tim3) and Toll-like receptor4(TLR4) protein were examined by Western blotting.Results1. Compared with the mice in normal group, the serum ALT levels of TP group were obviously elevated(P<0.005). Meanwhile, liver pathological sections of TP group showed more hepatic necrosis and neutrophil infiltration than control group, these results suggested that TP caused acute liver injury; Furthermore, in TP group, the concentration of hepatic IL-17 and the m RNA expression of ROR-γt closely related to the change of Th17 were higher than control group(P<0.005). On the contrary,the contentration of hepatic IL-10 and the m RNA expression of Foxp3 closely related to the change of Treg were significantly lower than control group(SOD P<0.05; GSH, Foxp3, Tim3, IL-10 P<0.005). These showed that TP induced Th17/Treg imbalance and successfully constructed the immunological liver injury model induced by TP.2. The serum ALT levels in TP+EGCG group mice were obviously reduced, compared to TP group(P <0.005); Meanwhile, the degree of inflammatory necrosis of liver cells in the TP+EGCG group was significantly lower than that in the TP group, these results suggested that EGCG possessed protective effect on TP-induced acute liver injury. There were no significantly differences between EGCG group and control group(P>0.05). Furthermore, in TP group, the activity of MDA, the expression of TLR4 protein, the m RNA expression of ROR-γt, NF-κB and My D88 and the contentration of hepatic IL-17 were higher than control group(P<0.005). On the contrary, the activity of SOD, GSH, the m RNA expression of Foxp3, the expression of Tim3 protein and the contentration of hepatic IL-10 were significantly lower than control group(SOD P<0.05;GSH, IL-10 P<0.005). In TP+EGCG group, activity of MDA, the expression of TLR4 protein, the m RNA expression of ROR-γt, NF-κB and My D88 and the contentration of hepatic IL-17 were lower than TP group(TLR4, P<0.05; MDA, IL-17, ROR-γt, NF-κB, My D88 P<0.005). On the contrary, the activity of SOD, GSH, the m RNA expression of Foxp3, the expression of Tim3 protein and the concentration of hepatic IL-10 in TP+EGCG group were significantly higher than TP group(SOD, Tim3P<0.05; GSH, Foxp3, IL-10 P<0.005). Among them, the changes of oxidation indexes of MDA, SOD and GSH showed that EGCG had antioxidant effect; the changes of IL-17, IL-10, ROR-γt and Foxp3 suggested that EGCG corrected TP-induced Th17/Treg imbalance; the changes of tim3, TLR4, NF-κB and My D88 displayed that the mechanism of EGCG correcting the imbalance between Th17 and Treg cells.Conclusions1. The immunological liver injury model in mice induced by TP was successfully constructed.2. EGCG possessed hepatoprotective effect against TP-induced immunological liver injury through its antioxidant actions and regulatingTh17/Treg imbalance actions.3. EGCG regulated Th17/Treg imbalance through enhancing Tim3 activation and inhibiting TLR4-NF-κB-My D88 signal pathway.