The Role Of RCOR3in Chronic Hepatitis B And Preliminary Study On Its Mechanism

1Backgroud and AimsHepatitis B virus (HBV) infection is a worldwide disease and can result in chronic hepatitis B (CHB), eventually leading to the development of cirrhosis and hepatocellular carcinoma (HCC). Our country has about30million chronic hepatitis B (CHB) patients now, of which5%will develop into severe hepatitis B.Once develop into severe hepatitis B,the survival rate of patients with clinical treatment is less than40%.To prevent the occurrence of severe hepatitis,early detection and early intervention is very importantment. Therefore, there is an urgent need to identify sensitive and reliable biomarkers which can early indicate the occurrence and monitor the development and severity of CHB.Our previuos GeneChip array demonstrated that the differentially expression of RCOR3was significant in the livers of mice in the Con A-induced hepatitis model.As a member of the CoREST family, RCOR3functions as a corepressor for REST and assists REST in its role of transcriptional repression.To date, we haven’t seen any relevant report on REST (RCOR3) and hepatitis B.In the present study, we applied serum from CHB patients to analyze the level of RCOR3in HBV infected patients, and also to evaluate the potential association between the expression of RCOR3and the levels of liver injury. Furthermore, we determined the role of anti-inflammatory agent in the expression of RCOR3and potential mechanism(s) underlying the action. Thus understanding the mechanism(s) of how RCOR3participates in the development of hepatitis B.2Meathods2.1The serum levels of RCOR3in CHB patientsThe study population consisted of211subjects, including25mild chronic hepatitis B (CHB) patients,38moderate CHB patients,24severe CHB patients,60cirrhosis patients,30liver cancer patients and34healthy controls. The levels of RCOR3were measured by ELISA. Meanwhile, the extent of liver injury was also evaluated by determining serum ALT, AST, and Tbusing the standard Reitman-Frankel method.2.2Studying the mechanism(s) of RCOR3in the development of hepatitis B through animal experiments2.2.1Differential expression of RCOR3in the livers of mice in a ConA induced mouse hepatitis modelThe mice were subjected to a tail vein injection of ConA at20mg/kg body weight, and phosphate-buffered saline (PBS) was used as a control. The mice were sacrificed at1.3, or6h separately. Liver samples were collected and fixed in4%phosphate-buffered paraformaldehyde. Paraffin tissue sections were prepared and subjected to hematoxylin-eosin (H&E) staining for standard histological examination. The extent of liver injury was also evaluated by determining serum ALT, AST, and TB using the standard Reitman-Frankel method. Mice liver tissues were homogenized to extract RNA, and then the differential expression of RCOR3was analyzed in the livers ofmice by Q-PCR. 2.2.2The role of anti-inflammatory agent in the expression of RCOR3and potential mechanism(s) underlying the action(1) The efficacy of emodinSix-week old male Balb/c mice were randomly divided into normal control group, emodin group, model group (ConA group), treatment group (12.5mg/kg+ConA group). Treatment group were given emodin by orally at12.5mg/kg body weight, and then the mice were subjected to a tail vein injection of ConA2h later, instead of emodin, CMC-Na (emodin solvent) was used in the ConA group, emodin group used saline instead of ConA, and normal control group without any treatment. The mice were sacrificed at10h after conA injection. Liver samples were collected and fixed in4%phosphate-buffered paraformaldehyde. Paraffin tissue sections were prepared and subjected to hematoxylin-eosin (H&E) staining for standard histological examination. The extent of liver injury was also evaluated by determining serum ALT, AST using the standard Reitman-Frankel method. The serum levels of RCOR3were detected by ELISA.(2) Time-effect relationship of emodinSix-week old male Balb/c mice were randomly divided into normal control group, emodin group, model group (ConA group), treatment group (12.5mg/kg+ConA group). Treatment group were given emodin by orally at12.5mg/kg body weight, and the mice were subjected to a tail vein injection of ConA2h after and5min,10min,30min,60min before emodin administration, instead of emodin,CMC-Na (emodin solvent) was used in the ConA group, emodin group used saline instead of ConA, and normal control group without any treatment. The mice were sacrificed at10h after ConA injection. Mice liver samples were collected and fixed in4%phosphate-buffered paraformaldehyde. Paraffin tissue sections were prepared and subjected to hematoxylin-eosin (H&E) staining for standard histological examination. The extent of liver injury was also evaluated by determining serum ALT. AST using the standard Reitman-Frankel method.(3) Dose-effect relationship of emodinSix-week old male Balb/c mice were randomly divided into normal control group, emodin group, model group (ConA group), treatment group (12.5mg/kg+ConA group). Treatment group were given emodin by orally at1.5625mg/kg,3.125mg/kg,6.25mg/kg,12.5mg/kg,25mg/kg,50mg/kg.100mg/kg body weight, and then the mice were subjected to a tail vein injection of ConA2h later, instead of emodin, CMC-Na (emodin solvent) was used in the ConA group, emodin group used saline instead of ConA, and normal control group without any treatment. The mice were sacrificed at10h after ConA injection. Mouse liver samples were collected and fixed in4%phosphate-buffred paraformaldehyde. Paraffin tissue sections were prepared and subjected to hematoxylin-eosin (H&E) staining for standard histological examination. The extent of liver injury was also evaluat-ed by determining serum ALT. AST using the standard Reitman-Frankel method.(4) Mechanism studyWe homogenized the livers from normal control group, emodin group, model group (conA group) and treatment group (12.5mg/kg+conA group) to extract RNA, and then the differential expression of MIP2and its receptor CXCR2were analyzed in the livers of mice by Q-PCR.2.2.3Statistical analysisAll of the data were processed by SPSS16.0software and presented as means±SE. ANOVA and LSD test were used for comparisons among the groups and between paired data, respectively. When the data were not normally distributed, the Mann-Whitney U test and the one-way non para-metric ANOVA (Kruskal Wallis test) were used to compare quantitative variables between two groups of observation and in more than two groups of data, respectively. Spearman rank correla-tion test was used for correlation analysis. A p value less than0.05was considered significant.3Results3.1Serum RCOR3levels in patients with hepatitis BCompared with the healthy control group (1.30±0.60), the serum RCOR3levels were significantly increased in patients with mild CHB (1.82±0.24), moderate CHB (1.72±0.85), severe CHB (3.96±0.48) and significantly decreased in patients with SHB (0.92±0.09). Moreover, the serum RCOR3levels in severe CHB patients were remarkably increased compared with the mild CHB patients and moderate CHB patients (p<0.01), and there was a significant decline in the serum RCOR3lev-els in patients with SHB compared with the mild CHB, moderate CHB and severe CHB patients (p<0.01). Taken together, the serum RCOR3levels were increased in patients with mild CHB. moderate CHB and severe CHB and were decreased in patients with SHB. Rank correlation analysis demonstrated that there was a significant correlation between serum RCOR3and TB (r=0.466, P＜0.01).3.2Studying the mechanism(s) of RCOR3in the development of hepatitis B through animal experiments3.2.1Differential expression of RCOR3in the livers of mice in a ConA induced mouse hepatitis modelAccording to the results of Q-PCR, RCOR3mRNA was significantly induced at the6h time point in comparison with the0h group (P＜0.01). Meanwhile, there was a significant increase in the mRNA level of RCOR3at the3h and6h time points compared with the1h group (P<0.05and P<0.01, respectively), the overall trend was that the expression of RCOR3was decreased at1h and3h post ConA exposure, but increased at the6h time point. 3.2.2The role of anti-inflammatory agent in the expression of RCOR3and potential mechanism(s) underlying the action(1)The efficacy of emodinCompared with the normal control group (0.003±0.002), the serum RCOR3levels were significantly increased in the conA group (1.66±0.52). However, there was a significant decrease in the seru-m level of RCOR3in the teatment group (0.10±0.08) compared with the model group (P<0.05).(2) Time-effect relationship of emodinEmodin, which was given2h before ConA challenge, can reduce ConA-induced liver injury. Compared with the ConA group (ALT:1647.0±63.24, AST:1139.0±102.79), the serum levels of ALT, AST in treatment group (ALT:165.71±34.49、 AST:290.0±76.28) were significantly decreased. Biopsy also showed there was almost no necrosis and reduced inflammatory cell infiltration in full vision. However, Emodin, which was given after the ConA challenge, can not reduce ConA-induced liver injury.(3) Dose-effect relationship of emodinEmodin, which was given at1.5625mg/kg (ALT:592.0±327.9, AST:596.0±207.2),3.125mg/kg(ALT:574.0±253.9, AST:596.0±69.8),6.25mg/kg(ALT:433.3±244.5, AST:466.7±188.7),12.5mg/kg (ALT:165.7±34.5, AST:290.0±76.28),25mg/kg (ALT:161.4±39.8, AST:248.6±28.9),50mg/kg (ALT:205.4±22.23, AST:289.2±30.31)2h before the ConA challenge, could decline the serum ALT、 AST levels of mice. However, the serum ALT、 AST levels in mice from emodin12.5mg/kg、25mg/kg and50mg/kg group were remarkably declined、 and no significant difference between the three groups.Biopsy of the three groups showed there was almost no necrosis and reduced inflammatory cell infiltration in full vision. However, there were significant increase in the serum levels of ALT, AST in the100mg/kg+conA group (ALT:6496.0±1289.8, AST:4070.0±761.3) compared with the ConA group. And a large area of necrosis was found in this group.(4) The mechanism studyAccording to the results of Q-PCR, the mRNA levels of MIP2and its receptor CXCR2were significantly increased in the ConA group compared with the normal control group, but were significant decreased in treatment group in comparison with the ConA group. Rank correlation analysis demonstrated that there was a significant correlation between serum RCOR3and MIP2, CXCR2(r=0.746, P＜0.01; r=0.764, P＜0.01).4Conclusions(1) The serum RCOR3levels were increased in patients with mild CHB、 moderateCHB and severe CHB and were decreased in patients with SHB in comparison with the health control group. Rank correlation analysis demonstrated that there was a significant positive correlation between serum RCOR3and TB (r=0.466, P<0.01). These results suggest that RCOR3may contribute to the disease progression and pathogenesis of liver injury in CHB patients.(2) Emodin inhibits the expression of RCOR3through MIP2/CXCR2pathway, leading to the reduced inhibitory effect of RCOR3on the anti-inflammtory factors, and thus relieve the degree of liver injury.