The Role Of High Mobility Group Box Protein 1 In The Progress Of Hepatic Fibrosis

Objective: To investigate the role of High Mobility Group Box Protein 1 in the progress of hepatic fibrosis through the establishment of rat liver fibrosis model and cultured hepatic stellate cell .Methods: The present experiments included animal experiments and cell experiments: (1) Animal experiments: Thirty wistar male rats were completely randomly divided into control group (n=6) and four model groups (n=24 2, 4, 6, 8 week), model groups were established by CCL4. Twenty-four model rats were sacrificed separately on the 2, 4, 6, 8 week. The serum ALT, HA and the expression of HMGB1 in liver tissue were detected by automatic biochemical analyzer, RIA, immunohistochemitry respectively. Liver tissue specimens were stained with HE for Pathomorphological observation. (2) Cell experiments: 1. HSC-T6 cells cultured: HSC-T6 cells were cultured in DMEM (containing 100 U / ml penicillin, 100 U / ml streptomycin, 10% fetal bovine serum, 3mmol / L glutamyl amine) at 37℃5% CO₂ incubator cultivate. HSC-T6 cells were digested by 0.25% trypsin, 0.02% EDTA 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 cultivate for 24 h, then switched to serum-free culture medium of DMEM for 2h to make all of the cells in G0 period for the experiment. 2. The relationship doses of HMGB1 and the ECM secretion of the HSC-T6 cells. HSC-T6 cells were divided into six groups (0 mg / L, 0.1mg / L, 1.0mg / L, 10mg / L,20mg / L,40mg / L ), each group cells were stimulated by different concentrations of HMGB1 for 6h, then the Pâ…¢P and Câ…£of the culture supernatant in each group were tested by ELISA method. 3. The relationship stimulated time of HMGB1 and secretion of ECM in HSC-T6 cells. The cells were divided into control groups and stimulated groups (HMGB1 10mg /L), the culture supernatant in each group were obtained at 0h, 6h, 12h,18h, 24h five time points respectively . There were three wells for each group. The Pâ…¢P and Câ…£of culture supernatant were detected by ELISA method.Results: 1. Histopathological features of liver tissue: Compared with normal groups, lobules of liver in the rats of model groups of 2 week, 4 week, 6 week were disorder with a pile of deposition of fibrous tissue, inflammatory cell infiltration and fatty degeneration of hepatocytes ,Fibroplasias formed at 8 week .The content of HMGB1 in liver tissue were increased obviously accompanied by inflammatory.2. The data of animal experiments suggested that the ALT, HA and HMGB1 of model groups were significantly higher than normal control groups (P<0.05). Compared with normal control group ,the ALT of model groups remarkbably increased (229.82±17.68, 42.85±19.919, P<0.05), reaching a peak at the 2 week and then declining,which suggested that the inflammation and necrosis of liver cells at 2 week were more serious than the other time points. The serum of HA at 4 week reached a peak and then declined which suggested that hepatic fibrosis at 4 week had generated and then developped into cirrhosis. Compared with normal control group, the IOD value of HMGB1 in liver tissue reached a peak at 2 week then declined.3. The relationship dose of HMGB1 and secretion of PIIIP,CIV in HSC-T6 cells:The secretion of PIIIP,CIV in HSC-T6 cells stimulated by HMGB1 was obviously higher than that in HSC-T6 cells stimulated without HMGB1,and there were significantly dose-dependent relationship in 0.1 mg / L 10 mg / L scope(r₁=0.835,r₂=0.955,P₁=0.0014,P₂=0.0030). The secretion of ECM in HSC-T6 cells reached a peak when the concentration of HMGB1 was 10 mg / L.4. The relationship stimulated time of HMGB1 and the secretion of PIIIP,CIV in HSC-T6 cells: The secretion of PIIIP,CIV in all of the groups were increased at 0 - 24 h and there were significantly time-dependent relationship(r₁=0.970,r₂=0.966,P₁=0.0061,P₂=0.0076).Compared with the control groups, the secretion of ECM in the stimulated groups were higher than that in the control groups at the parallel time points,and there were significantly different at 18h,24h time points(t₁=7.923,t₂=2.138,t₃=6.306,t₄=7.925;P₁=0.0005,P₂ =0.04, P₃=0.0002 P₄=0.0006).Conclusion:(1)The expression of HMGB1 increased in the progress of hepatic fibrosis and there were closely relationship between the expression of HMGB1 and inflammation in the formation of hepatic fibrosis.(2) HMGB1 could promote the secretion of ECM in HSC-T6 cells and showed a dose-dependent and time-dependent relationships. HMGB1 was an important factor of promoting the resistant secretion of ECM in HSC-T6 cells and could play a very important role in the development of hepatic fibrosis.(3) HMGB1 sustained activation to HSC-T6 has great significance to study the mechanisms of occurrence and development of hepatic fibrosis .