Anti-hepatic Fibrotic Mechanism Of Acanthus Ilicifolius Alkaloid A Involving In High Mobility Group Box 1

Objective: To observe the role of high mobility group box 1?HMGB1?in the formation of hepatic fibrosis and its relation with the extracellular receptor TLR4 receptor activation,investigating the anti-hepatic fibrotic mechanism of Acanthus ilicifolius alkaloid A?4-hydroxy-2-benzoxazolone,HBOA?.Methods: 60 Sprague Dawley?SD?male rats were randomly divided into hepatic fibrosis model group?n=50?and normal control group?n=10?.The rats of hepatic fibrosis model group were administered intragastrically 40 % CCl₄ olive oil solution with the volume of 3 ml·kg-1,the first dose of CCl₄ olive oil solution was double,two times per week for a duration of 12 weeks so as to induce liver fibrosis.The normal control group were administered intragastrically normal saline.In the 8th week,the successful hepatic fibrosis model rats were randomly divided into model control group?MC?,colchicine group?Colchicine,0.4 mg·kg-1?,HBOA high-dose group?HBOA-H,100 mg·kg-1?and HBOA low-dose group?HBOA-L,50 mg·kg-1?.Colchicine group and HBOA group were treated with different drugs daily for 4 weeks,model control group and normal control group?NC?were administered 0.6%CMC-Na solution 10mg·kg-1.24 hours after the last administration,blood was drawn from inferior caval vein and serum was separated to measure the levels of alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,total bilirubin?T-BIL?,superoxide dismutase?SOD?,malondialdehyde?MDA?,glutathione peroxidase?GSH-Px?.Moreover,Enzyme linked immunosorbent assay?ELISA?method was used to measure the level of HMGB1,interleukin-1??IL-1??,interleukin-6?IL-6?,tumor necrosis factor-??TNF-??,hyaluronic acid?HA?,laminin?LN?,Collagen type IV?Col-IV?,procollagen type ??PC??.Then,liver tissue was excised and weighed for calculating liver index,and the change of liver histopathological and collagen was observed by HE dyeing and Masson dyeing.Moreover,the HMGB1,TLR4,smooth muscle actin ???-SMA?protein expression of liver tissues were examined by immunohistochemistry.The HMGB1,TLR4,myeloid differentiation factor 88?My D88?,nuclear factor-kappa B?NF-?B?,transforming growth factor ?1?TGF-?1?m RNA expression were measured by real-time fluorescence quantitative PCR.Results: Serological experimental results showed that,compared with the model control group,the levels of ALT,AST,T-BIL,MDA,HMGB1,IL-1?,IL-6,TNF-?,HA,LN,Col-IV,PC? were significantly decreased?P<0.05 or P<0.01?in groups of HBOA high-dose and HBOA low-dose.Nevertheless,the levels of SOD,GSH-Px were increased?P<0.05 or P<0.01?in the HBOA groups.Interstingly,serum HMGB1 level had significant positive correlation to other factors.Pathological results showed that HBOA could markedly attenuate the fibrotic degree induced by CCl₄.Compared with normal control group,immunohistochemical experimental results showed that the protein expression of HMGB1,TLR4,?-SMA in model control group were increased?P<0.01?;compared with model control group,the protein expressions of HMGB1,TLR4,?-SMA in HBOA high-dose and HBOA low-dose group were decreased?P<0.01?.RT-PCR experiments showed that the expressions of HMGB1,TLR4,My D88,NF-?B and TGF-?1 m RNA in model control group rats were increased significantly compared with normal control group?P<0.01?;the expressions of HMGB1,TLR4,My D88,NF-?B and TGF-?1 m RNA in HBOA HBOA high-dose and HBOA low-dose group rats were decreased significantly compared with model control group?P<0.01?.Conclusion: HBOA has beneficial effects on liver fibrosis induced by CCl₄ in rats,the inhibition of inflammatory response to HMGB1,TLR4-My D88-NF-?B signaling pathway may account for its anti-fibrosis mechanism.