| Part1 Study on the correlation of HBV DNA replication and HDAC activity in patients with chronic hepatitis BObjective:The aim of this study was to analyze the correlation of HBV DNA replication and HDAC activity in patients with chronic hepatitis B,and the expression of HDAC2,HDAC6,AH3 and H3 proteins in liver tissues before and after antiviral therapy,as well as the pathological changes of liver tissues.Methods:40 patients with chronic hepatitis B were randomly selected,4 of whom received antiviral therapy.ALT,AST,TBIL and DBIL were detected by ADVIA2400automatic biochemical analyzer.The level of HBV DNA in all patients was determined by HBV DNA quantitative detection kit.According to the level of HBV DNA,40 patients were divided into group A(>10~5IU/m L),group B(10~2IU/m L～10~5IU/m L)and group C(<10~2IU/m L).The activity of HDAC was detected by HDAC kit in 40 patients.The liver tissues of 4 patients with chronic hepatitis B were taken for biopsy before treatment and 24weeks after treatment with entecavir.Pathological and histological diagnostic criteria of viral hepatitis and histological activities index were used to evaluate the grade of hepatitis inflammation and stage of fibrosis.The level of HDAC6,HDAC2,H3 and AH3 protein in liver tissues were detected by Western blot,which are compared with normal liver tissues.Results:(1)There were 20,10 and 10 patients in group A,B and C.The HDAC OD values in serum of group A,B and C were 1.40±0.86,1.34±0.31,0.95±0.71.There were significantly differences between group A and group C,group B and group C.(2)The serum HDAC activity of patients with chronic hepatitis B was positively correlated with HBV DNA concentration(r ~2=0.7153,P<0.01).(3)After 24 weeks of antiviral treatment with entecavir,the expression of HDAC2,HADC6 and AH3 protein in liver tissue of patients with chronic hepatitis B decreased significantly compared with that before treatment(P<0.05).The grade of hepatitis inflammation and stage of fibrosis were also improved compared with that before treatment.There was no significant change in H3protein expression(P>0.05).Conclusion:(1)There is a positive correlation between serum HDAC activity and HBV DNA level in patients with chronic hepatitis B.(2)Antiviral therapy can improve the abnormality of histone acetylation and deacetylation in patients with chronic hepatitis B.Part Ⅱ Changes of related factors in HepG2.2.15 cells after treatment with ETV,IFN-α and TSAObjective: The aim of this study was to investigate the changes of related factors in HepG2.2.15 cells treated with ETV,IFN-α and TSA.Related factors included the level of HBV DNA,pg RNA,cccDNA,HDAC activity,HDAC2,HDAC6,H3 and AH3 protein.Methods: HepG2.2.15 cells transfected with HBV were cultured and divided into TSA group,IFN-alpha group,ETV group and control group.TSA(10 mmol),IFN-α(1000 IU/m L)and ETV(1 mmol)were added to the cells respectively.The control group did not receive any intervention drugs.The supernatants and cells were collected at days 3,6,and 9 after treatments.The content of HBV DNA in the supernatant was determined by the quantitative detection kit of hepatitis B virus nucleic acid.The activity of HDAC in the supernatant was determined by HDAC kit.The level of HBV pg RNA was detected by RT-PCR.Quantitative polymerase chain reaction was used to analyze the level of cccDNA in cells.The level of HDAC6,HDAC2,H3 and AH3 protein were detected by Western blot.Results:(1)Compared with the control group,the expression of HBV DNA was suppressed in the IFN-α and ETV groups at days 3,6 and 9(P < 0.05)and suppressed in the TSA group at day 9(P< 0.05);(2)The HDAC activity was suppressed in the TSA group and IFN-α group at days 3,6 and 9(P < 0.05),in the ETV group at days 6,9(P < 0.05);(3)The expression of pg RNA was enhanced in the TSA group at days 3,6 and 9 and in the ETV group at day 9,but it was suppressed in the IFN-α group at days 3,6 and 9(P < 0.05);(4)cccDNA expression was suppressed in the IFN-α group at days 3,6 and 9(P < 0.05),while TSA and ETV had no direct inhibitory effect on the cccDNA levels;(5)The levels of HDAC2,HDAC6 and AH3 protein were suppressed in the TSA and IFN-α groups compared with the control group at days 3,6 and 9(P < 0.05).ETV also had effects on the HDAC2,HDAC6 and AH3 protein levels at days 6,9(P < 0.05).There was no significant change in H3 protein expression in each group(P >0.05).Conclusion:(1)The changes of HBV replication products during antiviral treatment of chronic hepatitis B are associated with histone deacetylation;(2)Acetylated histone H3 is involved in the process of hepatitis B virus DNA replication. |
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