柴胡皂苷d通过调控Rev-erbα-NLRP3通路抗肝纤维化的机制研究OA

Objective Saikosaponin-d(SSd)is a bioactive compound derived from Chaihu(Bupleuri Radix),which has been proved to inhibit the expression of NLRP3 inflammasome,thus showing anti fibrosis properties.The liver circadian clock gene was found to regulate NLRP3 by Rev-erbα(nuclear receptor subfamily/group D member 1)to reduce inflammatory diseases.This study aims to explore the specific regulatory mechanism of SSd on Rev-erbα-NLRP3 pathway in liver fiber model.Methods(1)The liver fibrosis model of C57BL/6 mice was established with carbon tetrachloride(CCl4)mixture in vivo.The mice were intraperitoneally injected with SSd for drug intervention,and the liver tissues were collected after 6 weeks.The degree of liver fibrosis in each group was observed by Masson,Sirius red staining and immunohistochemistry.RT-PCR and Western blot were used to detect the expression of Rev-erbα,NLRP3 inflammasome and IL-18,IL-1β in liver tissue.(2)In vitro,LX-2 cells were activated by transforming growth factor-β1(TGF-β1)for 24 hours.The activity of LX-2 cells was detected by CCK8 assay.The expression of α-SMA was detected by immunofluorescence.The expression of Rev-erbα,NLRP3 inflammasome and downstream inflammatory factors IL-18 and IL-1β in liver tissue were detected by RT-PCR and Western blot.Results(1)Masson,Sirius Red staining and immunohistochemistry results showed that the liver collagen fiber deposition in the model group was obvious,and the expressions of α-smooth muscle actin(α-SMA),collagen 1(COL-1),TGF-β1 were increased,and SSd intervention could reverse the above effects(P<0.001),with obvious anti fibrosis effect.RT-PCR and Western blot showed that the expression of Rev-erbα in the liver tissue of the model group was decreased,and the expression of NLRP3 inflammasome and downstream inflammatory factors IL-18 and IL-1β were increased.After SSd intervention,the expression of Rev-erbα was significantly increased,and the NLRP3 inflammasome pathway was inhibited(P<0.01).(2)CCK8 assay showed that the cell viability in the model group was increased,while that in the SSd group was decreased(P<0.01);Immunofluorescence detection showed that the expression of green fluorescence and α-SMA increased in the model group,while the expression of green fluorescence and α-SMA decreased in the SSd group;Western blot and RT-PCR showed that the expression of Rev-erbα in the model group was decreased,and the expression of NLRP3 inflammasome and downstream inflammatory cytokines IL-18 and IL-1 β were increased(P<0.05);SSd intervention can significantly up regulate the expression of Rev-erbα and inhibit the NLRP3 inflammasome pathway(P<0.01).Conclusion SSd can inhibit the NLRP3 inflammasome pathway by up regulating the expression of Rev-erbα,thus playing an anti-hepatic fibrosis role.