Inhibition Of Heat Shock Protein 90 Ameliorates Murine Primary Biliary Cholangitis By Inhibiting Necroptosis

Objective: Primary biliary cholangitis(PBC)is an autoimmune liver disease characterized by non suppurative destructive cholangitis,lymphocytic infiltration in the portal tracts,and chronic cholestasis.It has been reported that necroptosis plays an important role in the pathogenesis of PBC,but its regulatory mechanism remains to be elucidated.Heat shock protein 90(HSP90)regulates necroptosis and plays a key role in the pathogenesis of various liver diseases.We aimed to determine the expression level of HSP90 in PBC and explore the effect of inhibiting HSP90 on the progression of PBC.Furthermore,whether HSP90 affected the progression of PBC by regulating necroptosis was elucidated.Method: Female C57BL/6J 6-week-old mice were randomly divided into 5 groups: control group,PBC group,PBC+2.5 mg/Kg 17-DMAG group,PBC+5 mg/Kg 17-DMAG group and PBC+10 mg/Kg 17-DMAG group.For PBC mice,100 ?g of 2OA-BSA was injected intraperitoneally at the first and the fifteenth day.Poly I:C was injected intraperitoneally at a dose of 5 mg/kg once every 3 days from the third day.The treatment group was injected intraperitoneally with 17-DMAG every other day.The control group was injected intraperitoneally with an equal volume of PBS.After 8 weeks,all mice were sacrificed.Besides,their serum and liver were collected.Anti-PDC-E2 antibodies,IL-6,IFN-? and TNF-? expression levels in serum were measured with enzyme-linked immunosorbent assay(ELISA).The expression levels of IL-6,IFN-?,and MCP-1 in the liver were detected by RT-PCR.The expression levels of HSP90,RIP1,RIP3,p-RIP3,MLKL and p-MLKL proteins were detected by Western blotting.The degree of liver inflammation was evaluated by HE staining.Immunohistochemistry(IHC)was used to detect the expression level of HSP90.Results:(1)We observed infiltration of lymphocytes or mononuclear cells in portal tracts,particularly surrounding damaged intralobular bile ducts.The production of anti-PDC-E2 antibody in serum was significantly increased.The above results indicated that the PBC mouse model was successfully established.(2)IHC and Western blotting results showed that the expression level of HSP90 of PBC mice was higher than that of the control group.(3)Inhibition of HSP90 decreased the level of anti-PDC-E2 antibody in serum of PBC mice.Besides,the expression levels of IL-6,IFN-?,TNF-? and MCP-1 were decreased in serum and liver.Furthermore,the degree of inflammation in the liver was significantly alleviated in PBC mice.(4)The expression levels of RIP1,RIP3,p-RIP3,MLKL and p-MLKL proteins were increased in the liver of PBC mice,and all of which were reduced by inhibiting HSP90.Conclusion: We constructed the mouse model of PBC successfully and found that the expression level of HSP90 was increased in the liver of PBC mice.Inhibition of HSP90 ameliorates murine PBC by inhibiting necroptosis.HSP90 might be a promising target for PBC treatment.