Serum levels of microRNAs can specifically predict liver injury of chronic hepatitis B

AIM： To investigate whether circulating microRNAs （miRNAs） can serve as molecular markers to predict liver injury resulted from chronic hepatitis B （CHB）. METHODS： The profiles of serum miRNA expression were first generated with serum samples collected from 10 patients with CHB and 10 healthy donors （Ctrls） by microarray analysis. The levels of several miRNAs were further quantitated by real-time reverse transcription polymerase chain reaction with serum samples from another 24 CHB patients and 24 Ctrls. Serum samples of 20 patients with nonalcohlic steatohepatitis （NASH） were also included for comparison. The comparison in the levels of miRNAs between groups （CHB, NASH and Ctrl） was analyzed with Mann-Whitney U-test. The cor- relation between miRNAs and clinical pathoparameters was analyzed using Spearman correlation analysis or canonical correlation analysis. The receiver-operator characteristic （ROC） curves were also generated to de- termine the specificity and sensitivity of each individual miRNA in distinguishing patients with CriB from Ctrls. RESULTS： miRNA profile analysis showed that 34 miR- NAs were differentially expressed between CriB and Ctrl subjects, in which 12 were up-regulated and 22 down-regulated in CriB subject （fold change 〉 2.0 and P 〈 0.01）. The median levels of miR-122, -572, -575 and -638 were significantly higher （P 〈 1.00 × 10-5） while miR-744 significantly lower （P 〈 1.0× 10-6） in Crib compared with the Ctrl. The levels of miR-122, -572 and -638 were also higher （P 〈 1.00×10-3） while the level of miR-744 lower in CriB （P 〈 0.05） than in NASH, although the difference between them was not as significant as that between CHB and Ctrl. ROC curve analysis revealed that the levels of miR-122, -572, -575, -638 and -744 in serum were sensitive and specific enough to distinguish CriB, NASH and Ctrl. Multivariate analysis further showed that the levels of these miRNAs were correlated with the liver function parameters. Most significantly, it was the scatter plot of principal component with the levels of these miRNAs, but not the parameters of liver function, which clearly distinguished CriB, NASH and Ctrl subjects. CONCLUSION： Serum levels of miR-122, -572, -575, -638 and -744 are deregulated in patients with CHB or NASH. The levels of these miRNAs may serve as po- tential biomarkers for liver injury caused by CHB and NASH.

Development of Quantitative Real-time Polymerase Chain Reaction for the Detection of Vibrio vulnificus Based on Hemolysin (vvhA) Coding System

Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene （vvhA） coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles （Ct value） and fluorescence intensity enhancement （ARn value） were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 103 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.

Ginsenoside Rb1 Pretreatment Attenuates Myocardial Ischemia by Reducing Calcium/Calmodulin-Dependent Protein Kinase Ⅱ-Medicated Calcium Release

Objective:The aim of this study was to investigate the protective effects of ginsenoside Rb1 and assess whether these protective effects are related to calcium/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ).Methods:A myocardial ischemia(IS)rat.model and a myocardial H9 C2 cell hypoxia model were established.MI was induced by occluding the left anterior descending artery for 120 min.Ginsenoside Rb1(10 mg/kg)was administered 30 min before ischemia induction,and the treatment continued for 7 days.Results:In the rat IS injury model,ginsenoside Rb1 reduced myocardial infarct size,mean left ventricular diastolic pressure,incidence of arrhythmia,and levels of serum creatine kinase,lactate dehydrogenase,and malondialdehyde.However,the mean left ventricular systolic pressure,and maximal rising and falling rates of ventricular pressure(±dp/dtmax)increased.In the myocardial H9 C2 cell hypoxia model,ginsenoside Rb1 reduced intracellular calcium concentrations([Ca2+]i)during hypoxia,and markedly reversed the hypoxia-induced decrease in cell survival.Ginsenoside Rb1 was involved in the downregulation of CaMKⅡand the ryanodine receptor,as well as hypoxia-induced H9 C2 cell survival.Conclusion:The findings of the present study suggest that ginsenoside Rb1 attenuates MI injury in rats,partially through the downregulation of CaMKⅡexpression.

DNA Methylation and SNPs in VCX are Correlated with Sex Differences in the Response to Chronic Hepatitis B

The study was conducted to explore the mechanisms of sex differences in the response to chronic hepatitis B(CHB)in terms of DNA methylation,SNP genotype,and gene expression.Genomic DNA was isolated from peripheral blood mononuclear cells(PBMCs)of CHB patients and healthy controls and evaluated using the Human Methylation 450 K Assay.The DNA methylation level at hg37 chromosome(CHR)X:7810800 was further validated using pyrosequencing.SNP genotypes,VCX mRNA expression of PBMCs,and plasma VCX protein concentration were further examined using SNaPshot,RT-qPCR,and Western blot,respectively.Results showed that a total of 5529 CpG loci were differentially methylated between male and female CHB patients.DNA methylation level and CC+CT frequency at CHR X:7810800,VCX mRNA expression of PBMCs,and plasma VCX protein concentration were higher in female than in male CHB patients.The CHR X:7810800 locus was hypermethylated in CHB patients with CC+CT genotypes in comparison with those with the TT genotype.In cases of CC-f CT genotypes,VCX mRNA expression was negatively correlated with the DNA methylation level.CHB patients with higher levels of HBV DNA,AST,and GGT or higher GPRI scores exhibited lower VCX expression.In conclusion,SNPs and DNA methylation at the CHR X:7810800 locus cooperatively regulate VCX expression in CHB.The upregulated VCX expression in female CHB patients might represent a mechanism of protection from more severe liver dysfunction and extensive fibrosis,as observed in male CHB patients.