Study On The Dynamic Changes Of Anti-ubiquitinase USP7 In Serum Of Patients With Hepatitis B And Its Anti-inflammatory Effect

Objectives For the treatment and secondary prevention of viral hepatitis B,we observed the dynamic changes of the USP7(deubiquitinase)in patients with viral hepatitis B(HBV)and verified that the USP7 mediated anti-inflammatory effects at the cellular level.Methods This study includes population description and verification at the cellular level.1 Population study: The clinical data and blood samples of inpatients diagnosed with HBV at the Fifth Hospital of Shijiazhuang from August 2018 to December 2018 were selected.According to the inclusion and exclusion criteria,98 newly diagnosed patients with HBV were screened.Real-time fluorescence quantitative PCR(RT-PCR)and enzyme-linked immunosorbent assay(ELISA)were used to determine the mRNA and protein expression levels of USP7,NF-κB p65,IL-6 and TNF-α in serum.2 Cytology experiment: Hep G2.2.15 cells were divided into 6 groups: blank control group(control group),simple transfection group(siRNA-USP7 group),negative control group(siRNA-NC group),and entecavir intervention group(ETV group),entecavir combined transfection group(ETV + siRNA-USP7 group),and entecavir intervention control group(ETV + siRNA-NC group).First,determine whether Hep G2.2.15 cells can be stably transfected with HBV,and whether this cell strain is established as an in vitro cell model for the functional study of HBV.CCK8 kits were used to determine the optimal drug concentration and treatment time.The USP7 mRNA and protein were detected to determine the siRNA sequence.The content of alanine aminotransferase and aspartate aminotransferase were detected by Microplate reader.The mRNA expression levels of USP7,NF-κB p65,IL-6 and TNF-α were detected by the real-time fluorescence quantitative PCR(RT-PCR),and Western blot and ELISA were used to determine the protein content.The immunoprecipitation method was used to detect the interaction between USP7 and NF-κB p65.3 Statistical analysis: SPSS 22.0 statistical analysis software was used to process the data.All indexes were tested for normality.Non-normal distribution measurement data was described by the median [interquartile range],and the non-parametric test(KW test)was used to calculate the difference between groups.Correlation analysis between the indicators using spearman correlation analysis.The measurement data was expressed as mean ± standard deviation,and the comparison of indicators between multiple groups is by single factor analysis of variance.P <0.05 indicated that the difference was statistically significant.Results 1 Population study: The expression levels of USP7,NF-κB p65,IL-6 and TNF-α in the serum of patients with HBV decreased with the extension of the treatment period(P <0.05,all).USP7 was positively correlated with liver function indexes ALT,AST,inflammation indexes NF-κB p65,IL-6 and TNF-α.2 Cytology experiment: Compared with Hep G2,the expression of HBV-DNA in Hep G2.2.15 was 19,862 times that of Hep G2.Hep G2.2.15 was established as an in vitro functional mechanism research model for hepatitis B virus.According to the CCK8 method,the concentration of entecavir was determined to be 25μg / m L;compared with the control group,the expression levels of ALT and AST in the siRNA-USP7 group decreased,and the expression levels of ALT and AST in the ETV group also decreased.In contrast,the level of ALT and AST expression decreased after combined use of USP7-siRNA,suggesting a reduction in liver injury(P <0.05).Compared with the control group,the USP7 mRNA expression was lower than that of the control group after adding USP7-siRNA(P <0.05),and the corresponding mRNA expression levels of NF-κB p65,IL-6,and TNF-α also decreased(P <0.05),Suggesting that the cellular inflammation damage is reduced after knocking down USP7.Compared with the ETV group,the USP7 mRNA expression was reduced when combined with USP7-siRNA(P <0.05),and the corresponding mRNA expression levels of NF-κB p65,IL-6,and TNF-α also increased The decrease(P <0.05)suggests that inhibition of USP7 on the basis of administration can better reduce liver damage caused by hepatitis B virus;protein results tend to be consistent with mRNA expression.Examination of the interaction between USP7 and NF-κB p65 in the 48 h group found that USP7 and NF-κB p65 did interact.It is suggested that USP7 may increase the degree of inflammatory injury of hepatitis B virus by deubiquitinating NF-κB p65,while inhibiting USP7 can effectively reduce the inflammatory injury of hepatitis B liver.Conclusions 1 Deubiquitinating enzyme USP7 is highly expressed in the serum of hepatitis B patients,and it decreases with the prolongation of the treatment period of hepatitis B patients;2 Deubiquitinating enzyme USP7 and liver function indicators ALT,AST,inflammation regulating factor NF-κB p65 was positively correlated with inflammatory factors TNF-α and IL-6;3 USP7 interacts with NF-κB p65 and exerts a deubiquitinating effect on NF-κB p65 to aggravate the inflammatory damage of HBV hepatitis.After inhibiting USP7,its interaction with NF-κB p65 is reduced,NF-κB p65 is degraded,and the expression level of downstream inflammatory factors also decreases,which reduces the inflammatory damage of hepatitis B.Figure22;Table12;Reference 129.