The Relevance Of High Mobility Group Protein B1 And Remedial Potential Of Inhibition With Hepatic Fibrosis

A non-histone nucleosomal protein was purified from nuclei approximately 30 years ago and termed high mobility group box 1 (HMGB1).The researches previously reported were exclusively focused on the intranuclear role of HMGB1. Recently we discovered that HMGB1 is released by activated macrophages/monocytes stimulated with LPS, IL-1 and TNF-a, and it was leaked to extracellular spaces from various necrotic cells. The biological functions of secreted HMGB1 could be achieved following binding to RAGE, TLR4 and similar receptors on cell surface. HMGB1 activated TLR4 and the other receptors which were confirmed with significance in the process of hepatic fibrosis. Therefore, we considered the investigation on role of HMGB1 in liver fibrosis and the potential beneficial of HMGB1 antagonist to remedy the hepatic fibrosis in vitro.Objectives: To detect the level of HMGB1 in different degree hepatic fibrosis, explore the relationship between hepatic fibrosis and the level of HMGB1, validate the importance of HMGB1 in the fiber formation process in mouse model of CCl4-induced hepatic fibrosis and consolidate the feasibility of sodium butyrate preventing synthesis and delivery of HMGB1 which leads to delayed hepatic fibrosis progression. We hope a new strategy to prevent hepatic fibrosis could be verified through our investigation on HMGB1 and its antagonist.Methods:1 Enrollment of objects: We randomly selected 73 patients with chronic hepatitis B or cirrhosis aged from 18 to 58. The diagnostic criteria coincided with the Guideline of Chronic Hepatitis B published by Chinese Medical Association, Chinese Association and Chinese Hepatitis Prevention Foundation. Infection of the other hepatitis virus, autoimmune hepatitis, alcoholic hepatitis and tumor were excluded. Among these objects, 54 was male, 19 was female. The average of age was 34.6±10.35 years. The G1-G2 group consisted of 43 objects,G3-G4 of 30,S1-S2 of 34,and the S3-S4 of 39. At the same time 12 healthy volunteers (male:5, female:7) were enrolled as control. The average age was 33.1±9.6 years old. Liver function and the degree of hepatic fibrosis were detected and normalized to the level of HMGB1 to analyze the relationship between HMGB1and hepatic fibrosis.2 Prevention of hepatic fibrosis by HMGB1 antagonist: 50 male BALB/C mice weighting 18-24 g, were divided into 3 groups randomly. First 20 mice was administered with sodium butyrate by water feeding containing 1.5% sodium butyrate in addition to induction of 40% CCl4 2mL/kg (diluted with oleum olivae) by intraperitoneal injection every Tuesday and Friday for 70 days. Another 20 mice was enrolled into fibrosis group, only supplied with 40% CCl4 2mL/kg (diluted with oleum olivae) by intraperitoneal injection every Tuesday and Friday. The remaining 10 mice were included in blank group, supplied with 0.9% isotonic Na chloride 2mL/kg by intraperitoneal injection every Tuesday and Friday. All mice were sacrificed after 70 days to quantify the level of HMGB1 by ELISA and the liver function by immunohistochemistry. The hepatic tissue were fixed in 4% paraformaldehyde to observe the relocalization of HMGB1 after hepatic injury. Western blotting was performed to determine the expression of HMGB1.Results:1 The relevance between HMGB1 and hepatic inflammatory injury: Serum HMGB1 level in blank group was 16.86±3.48 ng/mL, The G1-G2 group was 26.54±5.31 ng/mL, the G3-G4 group was 33.64±8.35ng/mL. It was demonstrated that HMGB1 level in chronic hepatitis B patients was higher than that in healthy volunteers as control and the level in G3-G4 group were higher than that in G1-G2. The levels in three groups had statistically significant differences (P<0.01).2 The relationship between HMGB1 and the degree of hepatic fibrosis in chronic hepatitis patients: Serum HMGB1 level in healthy control group was 16.86±3.48ng/mL, in contrast to the S1-S2 group with 28.83±7.53ng/mL and the S3-S4 group with 30.0±7.61ng/mL.The levels in healthy control group, S1-S2 group and S3-S4 group had statistically significant differences(P<0.01) while the levels in S1-S2 group and S3-S4 group had no significance differences.3 The relationship between HMGB1 and the liver function in chronic hepatitis patients: the HMGB1 level could be positively correlated with serum ALT (r=0.2566, P<0.05) and AST (r=0.4719, P<0.01) level of patients, whislt no correlation with ALB (r=0.114, P>0.05) or TBIL(r=0.141, P>0.05).4 Sodium butyrate could decrease the mice serum HMGB1 level: The mice serum HMGB1 level in control group was obviously higher than that in sodium butyrate treated group. Sodium butyrate administrated simultaneously with CCl4 significantly inhibited HMGB1 expression as shown in the CCl4-only group with HMGB1 level of 30.75±7.43ng/mL and in the CCl4-sodium butyrate group with HMGB1 level of 25.54±3.97ng/mL. There were significant difference between the two groups (P<0.01).5 Sodium butyrate lowered the grade of inflammation and the degree of fibrosis: As for inflammation of the CCl4-only group, inflammation of four mice were measured as G1-G2, 10 mice as G3-G4. In the CCl4-sodium butyrate group, 13 mice were measured as G1-G2, 6 mice as G3-G4. There were significant differences between the two groups(χ2=5.125,P<0.05).As for fibrosis of the CCl4-only group, three mice were graded as S1-S2, 11 mice as S3-S4 in contrast to the CCl4-sodium butyrate group, 12 mice as S1-S2 was 12 and 7 mice as S3-S4. There were significant differences between the two groups(χ2=5.661,P<0.05).6 HE and picrosirius red staining of mice hepatic tissue: Normal lobular architecture of mice in CCl4-only group was destroyed, and liver cell cord was deranged. Necrosis of hepatocytes and pseudlobes could be found. The normal lobular architecture was of mice in CCl4-sodium butyrate group was largely retained. Although fiberic spacer could be spotted, the typical pseudlobes was not observed.7 Immuno staining of HMGB1 in mouse liver: In the blank group, HMGB1 was chiefly expressed inside the nuclei, seldom expressed in the cytoplasm, while undetectable in the extracellular space. In the CCl4-only group,HMGB1 was dispersedly distributed in nuclei, cytoplasma as well as extracellular space which were stained with HMGB1 positive signals especially in the region of serious inflammation or necrosis. Nevertheless, the expression of HMGB1 in the CCl4-sodium butyrate group was obviously downregulated when compared with CCl4-only group.8 Western blotting of HMGB1: As compared with the CCl4-only group, HMGB1 expression was significantly upregulated in the CCl4-only treated mice, with the level much higher than that in but in CCl4-sodium butyrate treated mice.9 Sodium butyrate decreased the mortality of mice undergoing liver fibrosis: During the 70 days of fibrosis induction, 6 mice in the CCl4-only group died with the mortality of 30%. In the CCl4-sodium butyrate group, however, only one mouse died with the mortality of 5%. The difference was considered as statistically significant.Conclusions:1 The HMGB1 level was obviously elevated in the hepatic fibrosis patients compared with healthy volunteers as control, and could be related with grades of hepatic inflammatory injury. HMGB1 might be recognized as a novel marker indicating the initiation of fibrosis following hepatitis.2 In the mice with CCl4-induced fibrosis, HMGB1 level in hepatic tissue and peripheral blood obviously increased and could be tightly regressed to the activity of hepatic inflammation and the degree of hepatic fibrosis.3 In the mice with CCl4-induced fibrosis, the inhibition of HMGB1 expression could ameliorate the activity of hepatic inflammation and detain the progression of fibrosis resulting a lowered mortality of mice undergoing liver fibrosis.4 Sodium butyrate functioning as HMGB1 antagonist, which could obviously decrease the HMGB1 level in hepatic tissue and peripheral blood in mice suffering liver fibrosis, was characterized with the potential of therapeutic application for hepatic fibrosis.