Ubiquitin-specific peptidase 22 (USP22) regulates the level and function of hepatitis B virus X protein / 中华微生物学和免疫学杂志

Objective:To investigate the effects of the interaction between ubiquitin-specific peptidase 22 (USP22) and hepatitis B virus X protein (HBx) on the protein level and the biological function of HBx.Methods:The interactions between HBx and USP22 were analyzed by GST pull-down, co-immunoprecipitation assay and confocal laser scanning assay. USP22 recombinant plasmids or specific siRNA were transiently co-transfected with HBx plasmids. Western blot were used to detect the protein level of HBx. The half-life and degradation pathway of HBx in the transfected cells treated with cycloheximide (CHX) or proteasome inhibitor MG132 were detected. In vivo ubiquitination assay was used to detect the ubiquitination of HBx with USP22 overexpression. Moreover, dual-luciferase reporter assay and colony formation assay were used to analyze the effects of USP22 on the biological function of HBx. Results:USP22 could interact with HBx in vivo and in vitro. USP22 significantly increased the stability of HBx and inhibited the proteasome-mediated degradation of HBx protein by reducing the ubiquitination of HBx, thereby enhancing the biological function of HBx. Conclusions:USP22 inhibited HBx protein degradation through ubiquitin-dependent proteasome pathway, thus enhancing the stability and biological function of HBx.

Change and significance of HBcAg-specific Th22 cells in patients with hepatitis B virus infection / 临床肝胆病杂志

ObjectiveTo investigate the changes of Th22 cells, interleukin-22 (IL-22), and transcription factor aryl hydrocarbon receptor (AhR) in patients with hepatitis B virus (HBV) infection and their correlation with clinical indices. MethodsA total of 11 patients with acute hepatitis B (AHB) and 38 patients with chronic hepatitis B (CHB) who attended Eighth Hospital of Xi’an from March 2018 to March 2019 were enrolled as AHB group and CHB group, respectively, and 16 healthy controls were enrolled as HC group. The patients with CHB received tenofovir disoproxil fumarate (TDF) antiviral therapy. Peripheral blood samples were collected for AHB patients at baseline and 6 months after discharge, and peripheral blood samples were collected for CHB patients at baseline and at months 6 and 12 of treatment; peripheral blood mononuclear cells (PBMCs) and plasma were isolated, then PBMCs were stimulated with phorbol ester+ionomycin or recombinant HBcAg, and flow cytometry was used to measure nonspecific CD3+CD4+IL-22+ Th22 cells and HBcAg-specific Th22 cells. ELISA was used to measure the plasma level of IL-22, and quantitative real-time PCR was used to measure the mRNA expression of AhR in PBMCs. The t-test and the paired t-test were used for comparison of normally distributed continuous data between two groups; a one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups; the Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups. The chi-square test was used for comparison of categorical data between groups. A Spearman correlation analysis was used to investigate correlation. ResultsThe AHB group had a significantly higher percentage of nonspecific Th22 cells than the CHB group (2.86%±0.45% vs 1.39%±0.33%, t=11.80, P＜0.001) and the HC group (2.86%±0.45% vs 0.80%±0.13%, t=17.30, P＜0.001), and the CHB group also had a significantly higher percentage of nonspecific Th22 cells than the HC group (t=6.825, P＜0.001). The AHB group had a significantly higher percentage of HBcAg-specific Th22 cells than the CHB group (2.97%±0.52% vs 1.22%±0.22%, t=16.58, P＜0.001). The AHB group had a significantly higher plasma level of IL-22 than the CHB group (130.7±39.97 pg/ml vs 66.59±20.83 pg/ml, t=7.176, P＜0.001) and the HC group (130.7±39.97 pg/ml vs 50.63±11.07 pg/ml, t=7.662, P＜0.001), and the CHB group also had a significantly higher plasma level of IL-22 than the HC group (t=2.887, P=0.006). The AHB group had significantly higher mRNA expression of AhR than the CHB group (11.45±3.03 vs 4.81±125, t=10.85, P＜0.0001) and the HC group (11.45±3.03 vs 1.10±0.17, t=13.75, P＜0.001), and the CHB group also had significantly higher mRNA expression of AhR than the HC group (t=11.77, P＜0.001). In both AHB and CHB patients, the percentage of HBcAg-specific Th22 cells was positively correlated with alanine aminotransferase (ALT) level (r=0.638 and 0.830, P=0035 and 0002), and the plasma level of IL-22 was also positively correlated with ALT level (r=0.552 and 0.431, P=0.001 and 0.007). The AHB patients were followed up at 6 months after discharge, and there were significant reductions in the percentage of HBcAg-specific Th22 cells (2.79%±0.56%, t=3.055, P=0.012) and the plasma level of IL-22 (105.8±25.23 pg/ml, t=2.362, P=0.040) from baseline. All CHB patients received TDF antiviral therapy and were followed up at months 6 and 12 of treatment, and there were significant reductions in the percentage of HBcAg-specific Th22 cells (t=4.353 and 3.927, all P＜0.001) and the plasma level of IL-22 (t=4426 and 4.810, both P＜0.0001) from baseline to months 6 and 12 of treatment. ConclusionHBcAg-specific Th22 cells and IL-22 are closely associated with inflammatory response to HBV infection.

HCFC-123-induced toxic hepatitis and death at a Korean fire extinguisher manufacturing facility: a case series

BACKGROUND: Exposure to sustained high concentrations of HCFC-123 is known to be hepatotoxic. We report two simultaneous cases of toxic hepatitis related to exposure to 2,2-dichloro-1,1,1-trifluoroethane (HCFC-123), a common refrigerant, at a Korean fire extinguisher manufacturing facility. CASE PRESENTATION: Patients A and B were men aged 21 and 22 years, respectively, with no notable medical histories. They had recently started working for a manufacturer of fire extinguishers. During the third week of their employment, they visited the emergency center of a general hospital due to fever, lack of appetite, and general weakness. At the time of their visit, they were suspected as having hepatitis due to elevated aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and total bilirubin levels and were hospitalized. However, as their condition did not improve, they were moved to a tertiary general hospital. After conservative treatment, one patient improved but the other died from acute hepatic failure. Assessments of the work environment showed that the short-term exposure levels of HCFC-123 for valve assembly processes were as high as 193.4 ppm. A transjugular liver biopsy was performed in patient A; the results indicated drug/toxin-induced liver injury (DILI). Given the lack of a medical history and the occupational exposure to high levels of HCFC-123, a hepatotoxic agent, the toxic hepatitis of the workers was likely related to HCFC-123 exposure. CONCLUSIONS: Work environment assessments have not included this agent. To the best of our knowledge, we are the first to report a case of death related to HCFC-123-induced liver damage. Our findings suggest that exposure standards and limits for HCFC-123 must be developed in Korea; work environments will have to be improved based on such standards.

miR-215 Enhances HCV Replication by Targeting TRIM22 and Inactivating NF-κB Signaling

PURPOSE: Hepatitis C virus (HCV) infection is a major cause of liver disease. Several miRNAs have been found to be associated with HCV infection. This study aimed to investigate the functional roles and possible molecular mechanisms of miR-215 in HCV replication. MATERIALS AND METHODS: The expression levels of miR-215 and TRIM22 were detected by quantitative real-time PCR (qRT-PCR) and western blot analysis in Con1b subgenomic genotype 1b HCV replicon cells (Con1b cells) and JFH1 full genome infecting Huh7.5.1 cells (Huh7.5.1 cells). HCV RNA levels were measured by qRT-PCR. The protein levels of NS3, NS5A, p65 subunit of NF-κB (p65), and phosphorylated p65 (p-p65) were determined by western blot analysis. The relationship between miR-215 and TRIM22 were explored by target prediction and luciferase reporter analysis. RESULTS: miR-215 overexpression enhanced HCV replication in Con1b cells, while miR-215 knockdown suppressed HCV replication in Huh7.5.1 cells. TRIM22 was confirmed to be a direct target of miR-215. TRIM22 upregulation resulted in a decline in HCV replication, while TRIM22 inhibition led to enhancement of HCV replication. Additionally, exogenous expression of TRIM22 reversed the facilitating effect of miR-215 on HCV replication, while TRIM22 downregulation counteracted the inhibitory effect of miR-215 knockdown on HCV replication. Furthermore, miR-215 targeted TRIM22 to block the NF-κB pathway, and exerted a positively regulatory role on HCV replication. CONCLUSION: miR-215 facilitated HCV replication via inactivation of the NF-κB pathway by inhibiting TRIM22, providing a novel potential target for HCV infection.

Association between interleukin-22 genetic polymorphisms and the prognosis of hepatitis B virus related acute-on-chronic liver failure / 中华传染病杂志

Objective To investigate the association between interleukin-22 (IL-22) single nucleotide polymorphisms (SNPs) and the prognosis of hepatitis B virus related acute-on-chronic liver failure (HBV-ACLF).Methods The patients with HBV-ACLF from the First Affiliated Hospital of Fujian Medical University were retrospectively studied.Seven SNP genotypes of IL-22 gene,including rs2227478,rs2227491,rs1179251,rs1179249,rs2227473,rs2227484,and rs11611206,were detected using imLDRTM multiple SNP typing kit and the distribution features of SNP genotypes were described.The relationship between the distribution of SNP genotypes and alleles and the prognosis of ACLF was analyzed.Comparison of genotypes and allele frequencies between groups were performed by chi-square test of R × C table or Fisher's exact tests.Binary logistic regression analysis was used to analyze whether IL-22 gene polymorphisms was an independent prognostic factor for patients with ACLF.Results A total of 122 patients with HBV-ACLF were included in this study.Ninety-two (75.1％) were male and 30 (24.59 ％) were female.Patients were stratified as survival group (90 cases) and non-survival group (32cases) according to the Results of three months follow-up.The genotype distribution of rs2227484 of IL-22 gene was significantly different between the two groups (x2=6.128,P=0.033).The A allele frequency in the non-survival group (15.6％) was significantly higher than that in the survival group (5.6％) with statistically significance (OR=0.318,95％ CI=0.126-0.804,P=0.012).There was no significant difference in the other six SNP genotypes of IL-22 gene between the two groups (all P＞0.05).However,binary logistic regression showed that rs2227484 of IL-22 gene was not an independent risk factor for the short-term mortality in HBV-ACLF patients (adjusted OR=3.102,95％ CI:0.939-10.250,P=0.063).Conclusions The A allele and AA genotype of rs2227484 of IL-22 gene may be associated with a short-term prognosis in patients with HBV-ACLF.

The dynamic changes and significance of T helper type 17 cells and cell-related cytokines in the peripheral blood of acute hepatitis B patients / 中华传染病杂志

Objective To investigate the expression of T helper type 17 cells (Th17) and cell‐related cytokines ,including interleukin (IL)‐21 ,IL‐22 ,IL‐23 in the peripheral blood of different clinical stages of patients with acute hepatitis B (AHB) .Methods Ten cases of AHB patients were enrolled .The frequency of Th17 cells in the three clinical stages (i .e .acute phase ,convalescent phase and resolved phase) were detected by flow cytometry . IL‐21 , IL‐22 and IL‐23 were measured by enzyme‐linked immunosorbent assay (ELISA) .Control group was composed of ten healthy subjects .The comparison between the two groups was done by t test and the differences among multiple groups were compared by one way ANOVA .Pearson correlation analysis was used for correlation analysis .Results The frequency of Th17 in healthy controls was (0 .68 ± 0 .29)% ,while those in acute phase ,convalescent phase and resolved phase of AHB patients were (18 .22 ± 4 .13)% , (3 .14 ± 1 .90 )% and (3 .31 ± 0 .95 )% , The differences between the two groups were significant (t= 13 .405 ,4 .047 and 8 .342 , respectively ;all P< 0 .01) .The levels of IL‐21 ,IL‐22 and IL‐23 in healthy controls were (42 .00 ± 6 .95) ,(315 .89 ± 96 .16) and (11 .95 ± 6 .95) ng/L ,respectively .Those in acute phase of AHB patients were (575 .39 ± 47 .01) ,(648 .44 ± 47 .12) and (38 .29 ± 4 .68) ng/L ,respectively ,those in convalescent phase were (366 .50 ± 33 .74) ,(405 .04 ± 47 .12) and (25 .10 ± 4 .69) ng/L ,respectively ,while those in resolved phase of AHB patients were (46 .62 ± 8 .28) ,(365 .94 ± 45 .62) and (15 .29 ± 4 .69) ng/L , respectively .Compared with healthy controls ,t values of the levels of IL‐21 in three different phases of AHB patients were 35 .497 ,29 .792 and 1 .354 with P value of <0 .01 ,<0 .01 and 0 .193 ,respectively ;those of IL‐22 were 9 .820 ,2 .632 and 1 .487 with P value of < 0 .01 ,0 .021 and 0 .161 ,respectively ;those of IL‐23 were 9 .944 ,4 .961 and 1 .260 with P values of <0 .01 ,<0 .01 and 0 .226 ,respectively . After comparison of IL‐21 ,IL‐22 and IL‐23 among three different phase of AHB ,F values were 622 .784 , 107 .772 and 60 .743 with all P values less than 0 .01 ,respectively .The levels of IL‐21 ,IL‐22 and IL‐23 were all positively correlated with the serum ALT level in acute phase (r= 0 .655 ,0 .666 and 0 .673 , respectively ;all P<0 .05) .Correlation analysis demonstrated that the frequency of Th17 was positively correlated with the levels of IL‐21 , IL‐22 and IL‐23 in acute phase ( r= 0 .879 ,0 .866 and 0 .879 , respectively ;all P<0 .01) .The frequency of Th17 was positively correlated with the level of IL‐21 in the resolved phase . No correlations between the remaining groups were confirmed . Conclusion The expressions of Th17 and cell‐related cytokines ,including IL‐21 ,IL‐22 and IL‐23 decline with the recovery of A HB .

Detection of Serum miR-122 and miR-22 in Patients with Chronic Hepatitis B by Using of a Quantitative Real-time PCR / 医学研究杂志

Objective To establish a real -time quantitative PCR ( RT-PCR) assay for detecting serum miR -122, miR-22, and evaluate the clinical significance of miR -122 and miR-22 in patients with chronic hepatitis B ( CHB) by using of this assay .Meth-ods The mature miRNAs were reversely transcripted by using of stem -loop primers .SYBR GreenⅠquantitative real-time PCR ( qRT-PCR) was used for quantification of the miRNAs .The sensitivity of this assay was evaluated by using of the 10-fold-diluted miRNA-122 cDNA standards and the specificity was verified by using of melting curve assay .The accuracy was assessed by intra -assay coeffi-cient of variation (CV) of threshold cycle (Ct value), which were calculated from a 20-times-repeat detection of the miR -122 cDNA (2 ×105 , 2 ×106 , 2 ×107 copies/μl) standards.Using the established qRT -PCR assay, we detected the expression of serum miR -122 and miR-22 in the patients with CHB and healthy controls .Results The qRT-PCR assay exhibited good performances in the linear range, sensitivity and reproducibility while detecting miR -122 and miR-22.The relative level of miR -122 and miR-22 was 17.88 vs 5.35 in the CHB patients and 1.80 vs 1.67 in the controls (P=0.000).Conclusion Using of stem-loop primers, we established a qRT-PCR assay for detection of serum miR -122 and miR-22.Serum miR-122 and miR-22 increased significantly in the CHB pa-tients.

Inhibition of HBV replication by small interference RNA in HepG2.2.15 cells / 中华肝胆外科杂志

Objective Virus-specific RNA interference (RNAi) is a powerful inhibitor of gene expression and replication of HBV. It is known to have high efficiency, specificity, and few side effects. We wanted to evaluate the effects of siRNA silencing HBV replication on the growth of hepatocellular carcinomatic(HCC) cells to find out an ideal method for treatment of HCC. Methods We transfected siRNA into HepG2.2. 15 cells (HCC cell inserting HBV gene) and detected the HBsAg and HBV DNA copies for evaluating the inhibitory effects of siRNA. Then we evaluated cell growth and self-renewal ability after transfection of siRNA by MTT. Results The HBsAg level and HBV DNA copies were reduced after the transfection of siRNA, the highest inhibition rate was 83.9%,while the inhibition rate of HBV DNA copies reached 73. 4%. The siRNA group's growth ability and self-renewal rate were lower than the control group in 5 days. Conclusion siRNA can effectively inhibit HBV replication and expression in HepG2.2.15 cells and silencing HBV replication can inhibit HepG2.2.15 cell's growth and self-renewal.

Hepatitis B virus (HBV) S gene-specific antisense locked nucleic acid (LNA) significantly inhibits HBV replication in vitro / 基础医学与临床

Objective To investigate the inhibitory effects of hepatitis B virus (HBV) S gene-specific antisense locked nucleic acid (LNA) on HBV replication and expression in HepG_22.2.15 cells,and to screen the effective short sequence of LNA.Methods Four different lengths of short sequence of antisense locked nucleic acid which were complementary to the initiator of HBV S gene were designed,synthesized and transfected by cationic liposomes into HepG_22.2.15 cells.The HBsAg and HBV DNA of supematant was tested by enzyme linked immunoadsorbent assay(ELISA) and real-time fluorescent quantitative PCR(FQ-PCR) at 24,48 and 72 hours after treatment.LNA's cyto-toxicity on cell was evaluated by MTT method.Results Four different lengths of short sequence of LNA inhibi-ted the expression of HBsAg and the replication of HBV DNA with the inhibition rates of 46.58%,54.38%,72.43% ,69.92% and 27.09% ,28.77% ,34.71% ,32.68% respectively after 72 hours.There's no obvious tox-icity on cell.Conclusion Antisense LNA that targeting at HBV S gene has strong inhibition on HBV in vitro,and the optimal length of LNA sequence might be in the range of 15 base to 25 base.It has a therapeutic potential in the treatment of patients infected with HBV.