| By observing the activation of High mobility group box protein 1 to Hepatic stellate cells, and observing the role of RAGE in the process of activation. to explore the effect and related mechanism of HMGB1 to HSC, and to provide ideas and experimental basis for cirrhosis prevention and cure.Methods:The first part of the experiment:The recovery HSC-T6 cells were cultured in DMEM at 37℃5% CO2 incubator. HSC-T6 cells were digested by 0.25% trypsin, after being covered with 80%. And the cells on logarithmic phase were prepared for the experiment. HSC-T6 cells were seeded on 6-well cell culture plates(1×105/ml) and cultured at 37℃5% CO2 incubator, after being covered with 80%, then switched to serum-free culture medium of DMEM for 2h to make all of the cells in Go period for the experiment. The trials are divided into three groups:the first one:Control group (DMEM culture medium 1ml); the second one:HMGB1 group (HMGB1 0.1ug/ml); the third one: HMGB1 antibody group (after cultivated with HMGB1 antibody 20ug/ml for 2 hours+HMGB1 0.1ug/ml), each group cells were stimulated for 24h, then the expression of a-SMA and TGF-βin HSCs-T6 tested by immunohistochemistry, the levels of HA, PIIIP, CIV and TGF-β1 in the supernatant were measured by ELISAThe second part of the experiment:The recovery HSC-T6 cells were cultured in DMEM at 37℃5% CO2 incubator. After fusing 80%, HSC-T6 cells were digested by 0.25% trypsin. And the cells on logarithmic phase were prepared for the experiment. The trials are divided into three groups:the first one:(A) Control group (DMEM culture medium 1ml);(B) HMGB1 group (HMGB1 0.1ug/ml); (C) anti-RAG E+HMGB1 group (after cultivated with RAGE antibody 20ug/ml for 2 hours+HMGB1 0.1ug/ml); the second one:(A) Control group (DMEM culture medium1ml); (B) HMGB1 group (HMGB1 0.1ug/ml); (C) sRAGE+HMGB1 group (combined sRAGE 20ug/ml with HMGB1 0.1ug/ml for 2 hours + DMEM culture medium):the third one:(A) Control group(DMEM culture medium 1ml); (B) HMGB1 group (HMGB1 0.1ug/ml):(C) AGE+HMGB1 group (after cultivated with AGE 160ug/ml for 2 hours+HMGB1 0.1ug/ml); The expression of a-SMA and TGF-β1 in HSCs-T6 using immunohistochemistry, the levels of HA, PIIIP, CIV and TGF-β1 in the supernatant were measured by ELISA. 1. HMGB1 could activate HSC significantly:The levels of a-SMA. TGF-βin HSCs without stimulation were very low by immunohistochemistry, however, the expression of them increased obviously after been stimulated by HMGB1(P<0.05). Pretreated with anti-HMGB1, the contents of a-SMA and TGF-β1 reduced markedly compared with HMGB1 group (P<0.05), but the contents of them have no significant difference compared with control group(P>0.05). The levels of HA, PIIIP, CIV and TGF-β1 of contral group was very low in serum measured by ELISA. however, The levels of them in HMGB1 group were much higher than in contral group(P<0.05). Pretreated with anti-HMGB1, the levels of HA. PIIIP. PIVP and TGF-β1 reduced significantly compared with HMGB1 group (P<0.05). but the levels of them have no significant difference compared with control group(P>0.05).2. RAGE antibody could inhibit the activation of HMGB1 to HSC:In Anti-RAGE+HMGB1 group, the levels of a-SMA, TGF-β1 in HSC was lower than HMGB1 group by immunohistochemistry(P<0.05). The levels of HA, PIIIP, CIV and TGF-β1 in serum of contral group was very low compared with HMGB1 group by ELISA, however, The levels of them in HMGB1 group were much higher than in contral group(P<0.05). In Anti-RAGE+HMGB1 group, the expression of a-SMA. TGF-β1 by immunohistochemistry and The levels of HA, PIIIP. CIV TGF-β1 by ELISA were increased relatively compared with control group, but the differences was not statistically significant (P>0.05).3. sRAGE could antagonist the activation of HMGB1 to HSC:In sRAGE+HMGB1 group, the levels of a-SMA, TGF-β1 in HSC decreased apparently compared with HMGBl group by immunohistochemistry(P<0.05). the content of HA, PIIIP, PIVP and TGF-β1 in supernatant fluid decreased markedly compared with HMGB1 group(P<0.05). the expression of a-SMA, TGF-β1 by immunohistochemistry and the levels of HA, PIIIP, CIV TGF-β1 by ELISA were increased relatively compared with control group, but the differences was not statistically significant (P> 0.05).4. AGE could not enhance the activated effect of HMGB1 to HSC:In AGE+HMGB1 group, the levels of a-SMA, TGF-β1 in HSC increased relatively compared with HMGB1 group by immunohistochemistry, but the differences was not statistically significant (P>0.05); the content of HA. PIIIP. CIV and TGF-β1 in supernatant fluid increased relatively compared with HMGB1 group by ELISA. but the differences was not statistically significant (P>0.05):the expression of a-SMA. TGF-β1 by immunohistochemistry and the levels of HA, PIIIP, CIV TGF-β1 by ELISA were increased markedly compared with control group, the differences was statistically significant (P< 0.05). 1. HMGB1 could promote HSC-T6 express a-SMA, TGF-β1, increase the secretion of HA. PIIIP, CIV and TGF-β1, and play an important role in the process of hepatic fibrosis.2. Anti-RAGE and sRAGE could inhibited the activation of HMGBlto HSC, decrease the expression of a-SMA, TGF-β, reduce the secretion of HA, PIIIP, CIV, TGF-β1 in HSC. and ameliorate the development of hepatic fibrosis. HMGB1 combined the same receptor with AGE, but AGE did not inhibited the activation of HMGB1 to HSC.HMGB1 as a target in above results provided new ideas and experimental basis for liver fibrosis and cirrhosis treatment. |
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