Anti-fibrosis effects of fenofibrate in mice with hepatic fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the anti-fibrosis effects and mechanisms of fenofibrate on hepatic fibrosis using a mouse model of fibrosis induced by carbon tetrachloride (CCl4).</p><p><b>METHODS</b>Twenty-six male C57BL mice were divided into the following three groups: CCL4-induced untreated model control (n = 10), CCl4-induced fenofibrate-treated model (n = 10), and uninduced/untreated normal control (n = 6). All animals were sacrificed after the 5 weeks of induction and treatment. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA) and procollagen III amino-terminal peptide (PIIINP) were determined by routine biochemistry assays. Liver content of hydroxyproline (HYP) was measured by spectrophotometry. Liver content of malonic aldehyde (MDA) and superoxide dismutase (SOD) was measured by enzymatic assays. mRNA expression levels of liver fibrosis-associated factors were determined by PCR, and included alpha-smooth muscle actin (a-SMA), transforming growth factor-beta1 (TGFbeta1), type I collagen-alpha (Collagen1a), peroxisome proliferator-activated receptor-alpha (PPARa), and the inflammatory cytokines tumor necrosis factor alpha (TNFa) and interleukin-6 (IL-6). Finally, the degree of inflammation and fibrosis were assessed by histological analysis using hematoxylin-eosin and Sirius red staining.</p><p><b>RESULTS</b>Compared to the untreated model group, the fenofibrate-treated model group showed significantly lower levels of serum ALT (55.72+/-1.20 vs. 38.72+/-1.25 IU/L), HA (236.20+/-17.57 vs. 152.9+/-13.06 mug/L) and PIIINP (41.66+/-1.89 vs. 34.32+/-1.53 mug/L) (all P less than 0.05). The fenofibrate-treated group also showed a significantly higher level of hepatic SOD content (untreated model: 67.00+/-4.65 vs. 101.1+/-5.32) but significantly lower level of hepatic MDA content (14.67+/-0.93 vs. 10.17+/-0.60 nmol/mg) and lower level of hepatic HYP content (0.67+/-0.80 vs. 0.41+/-0.50 mg/g) (all, P less than 0.05). In addition, the fenofibrate-treated group showed significantly reduced mRNA expression levels of a-SMA (6.83+/-0.88 vs. untreated model: 11.57+/-1.31), TGFbeta1 (67.83+/-4.65 vs. 112.30+/-4.81), Collagen1a (67.83+/-4.65 vs. 112.30+/-4.81), TNFa (17.43+/-2.32 vs. 37.83+/-4.69), and IL-6 (4.00+/-0.49 vs. 5.62+/-0.54), but significantly increased PPARa (0.30+/-0.03 vs. 0.18+/-0.03) (all, P less than 0.05). Finally, the degree of CCL4-induced hepatic fibrosis was attenuated by the fenofibrate treatment.</p><p><b>CONCLUSION</b>Fenofibrate can reduce the degree of liver fibrosis in mice induced by CCl4. The mechanism may involve up-regulation of PPARa, inhibition of the inflammatory response, and enhancement of SOD antioxidant activity.</p>

Hepatitis B surface antigen affects the expression of lipid metabolism-related genes in HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the influence of hepatitis B virus (HBV)-encoded small surface protein (SHBs) on hepatic cell expression of host genes related to lipid metabolism.</p><p><b>METHODS</b>The full-length SHBs gene was amplified from HBV genotype C by polymerase chain reaction (PCR) and cloned into the pcDNA3.1(+) expression vector for stable transfection into HepG2 cells (selected by G418 screening); cells transfected with empty vector served as control. The SHBs mRNA and protein levels were detected by reverse transcription-PCR and enzyme-linked immunosorbent assay. SHBs effects on expression of genes and proteins related to lipid metabolism were detected by real-time quantitative (q)PCR and western blotting, respectively.</p><p><b>RESULTS</b>The stably transfected cell line HepG2-pn3.1-SHBs was established successfully. qPCR showed that the HepG2-pn3.1-SHBs cells had significantly down-regulated transcription of the ECHS1, APOA1 and LPL genes (0.161+/-0.043 vs. control cells: 0.210+/-0.022, t = 2.479; 0.031+/-0.007 vs. 0.094+/-0.055, t = 2.752; 0.770+/-0.036 vs. 0.982+/-0.031, t = 10.914), but significantly up-regulated ACC and SREBP-1c genes (0.113+/-0.027 vs. 0.059+/-0.022, t = -3.757; 0.019+/-0.002 vs. 0.015+/-0.001, t = -4.330). The CPT1a and PPARa genes' expression was slightly, but not significantly, down-regulated in the HepG2-pn3.1-SHBs cells (0.028+/-0.005 vs. 0.030+/-0.004, t = 1.022; 0.014+/-0.004 vs. 0.015+/-0.002, t = 0.758). Western blotting showed similar expression trends for the corresponding proteins.</p><p><b>CONCLUSION</b>SHBs alters the expression of some host genes with known functions in fatty acid synthesis and decomposition; however, it remains unclear whether the hepatitis B surface antigen can directly contribute to development of hepatic steatosis.</p>

Relationship between the changes in immune cells and HBeAg loss following antiviral treatment in chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the changes in hepatitis B virus (HBV)-specific and non-specific cellular immunity that accompany viral load decline during adefovir dipivoxil (ADV) treatment in patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B, and to explore the antiviral immunity mechanism underlying the treatment response.</p><p><b>METHODS</b>Serial analysis of cellular immunological parameters was performed in HBeAg-positive patients (n = 20) throughout the 48-week course of ADV therapy (10 mg/d). HBV-specific T cell reactivity to HBV core antigen (HBcAg) was assessed by enzyme-linked immunosorbent spot assay and cell proliferation assay at pre-treatment (baseline) and post-treatment weeks 4, 12, 24, 36, and 48. Percentage of regulatory T cells (Tregs), as well as activated peripheral natural killer (NK) cells (expressing the NKG2D receptor), was measured by flow cytometry. Comparisons of means were performed by the two-tailed t-test or the Mann-Whitney rank sum test.</p><p><b>RESULTS</b>After 48 weeks of ADV therapy, HBeAg loss was observed in six of the 20 (30%) patients and 14 patients remained HBeAg-positive. In the patients with HBeAg loss, the viral load reduction was accompanied by a significantly enhanced response rate of HBV-specific interferon (IFN)-gamma-producing CD4+ T cells [measured as (spot forming cells/peripheral blood mononuclear cells); baseline: (661.25+/-281.97) *10(-6) vs. week 48: (280.75+/-104.33) *10(-6), P = 0.045]. In contrast, patients without HBeAg loss showed no significant differences in T cell response rates. The patient groups with and without HBeAg loss showed similar proportions of peripheral blood Tregs during the treatment course, which included a trend of gradual decrease from baseline to week 4 with steady levels thereafter. In addition, both groups showed a similar increase in NKG2D expression that began at week 12 and peaked at week 48.</p><p><b>CONCLUSION</b>HBV-specific T cell reactivity temporally increases in some ADV-treated chronic hepatitis B patients, and this trend is strongly associated with HBeAg loss. Furthermore, recovery of HBV-specific T cell reactivity promotes viral clearance and HBeAg seroconversion.</p>

Prolonged duration of the routine pegylated-interferon alfa-2a therapy produces superior virological response in HBeAg-positive chronic hepatitis B patients: a single-center cohort study / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Hepatitis B e antigen (HBeAg) seroconversion and/or hepatitis B surface antigen (HBsAg) clearance are considered as good prognostic indicators of treatment outcome in HBeAg-positive chronic hepatitis B (CHB) patients. While a sustained virological response (SVR) can be achieved by a finite 48-week course of pegylated-interferon alfa-2a (Peg-IFNalpha-2a), it has been suggested that longer-term treatment can improve the rate of SVR. Therefore, the aim of this study was to compare the effects of prolonged and routine Peg-IFNa-2a therapy in patients with HBeAg-positive CHB.</p><p><b>METHODS</b>Eighty-six consecutive patients diagnosed with HBeAg-positive CHB at our hospital between September 2006 and October 2009 were enrolled in the study. The patients were randomly assigned to receive Peg-IFNa-2a (180 mug once weekly) for either 48 weeks (routine therapy group, n = 53) or 72 weeks (prolonged therapy group, n = 33). Serum samples were collected from each patient every three months until the end of the 24-week follow-up, and standard viral and biochemical tests were carried out. Relapse was defined as HBV DNA concentrations more than 105 copies/mL or an HBeAg-positive test at the end of the 24-week follow-up. Chi-squared test and the t-test were used to determine the significance of intergroup differences. Logistic regression analysis was employed to determine the correlation of outcome parameters to treatment duration, expressed as odds ratio (OR) with 95% confidence interval (CI).</p><p><b>RESULTS</b>The two treatment groups were similar at baseline (pre-treatment) in demographic data, sex ratio, age, alanine aminotransferase (ALT) level, HBV DNA load, and semi-quantitative level of HBeAg (s/co) (all, P more than 0.05). At the end of the 24-week follow-up, there were significant differences between the 48-week treatment group and the 72-week treatment group in patients with HBV DNA negativity (62.3% vs. 97.0%, x2 = 13.273, P = 0.000), HBeAg seroconversion (39.6% vs. 57.6%, x2 = 6.765, P = 0.009), HBsAg clearance (15.1% vs. 36.4%, x2 = 5.155, P = 0.023), and relapse (58.5% vs. 33.3%, x2 = 6.713, P = 0.010). Logistic regression analysis indicated that therapy duration was correlated to HBeAg clearance (OR = 3.702, 95% CI: 1.225 to 11.188) and male sex (OR = 3.005, 95% CI: 1.038 to 8.696) but not to HBeAg level at baseline (OR = 0.999, 95% CI: 0.998 to 1.000) or age (OR = 0.902, 95% CI: 0.839 to 0.970).</p><p><b>CONCLUSION</b>In this single-center cohort study, superior therapeutic benefit was achieved by extending the Peg-IFNa-2a therapy out to 72 weeks for patients with HBeAg-positive CHB. The prolonged duration therapy produced a higher HBsAg loss ratio, HBeAg seroconversion ratio, HBV DNA negativity ratio, and a lower relapse ratio. Furthermore, HBeAg clearance was positively correlated with duration and male sex.</p>

Relation between HBsAg levels during the immune clearance phase of hepatitis B virus infection and liver pathological stages of chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether the level of hepatitis B surface antigen (HBsAg) represents the status of inflammation and stages of fibrosis in livers of patients with chronic hepatitis B (CHB) during the immune clearance phase (IC).</p><p><b>METHODS</b>Liver biopsy samples and sera were collected from 165 consecutive patients (136 males; 29 females) with CHB in IC who were treated in our hospital between March 2009 and June 2011. Routine biochemical tests were carried out to measure indicators of liver function. The relation between HBsAg level and liver pathological stages were determined by Spearman's rank correlation analysis. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of HBsAg level for liver pathological stages. Binary logistic regression was used to analyze potentially relevant indicators, and liver pathology-predicting models were built and analyzed by the ROC method.</p><p><b>RESULTS</b>The mean values of HBsAg (IU/mL) were significantly different at the different liver inflammation stages: G1, 27 716.07+/-32 870.69; G2, 34 478.75+/-40 899.55; G3, 19 408.09+/-24 881.07; G4, 14 286.31+/-28 610.14. Likewise, the mean values of HBsAg (IU/mL) were significantly different at the different liver fibrosis stages: S1, 41 337.23+/-43 236.39; S2, 27 264.32+/-32 517.29; S3, 111 541.77+/-11 538.93; S4, 11 447.37+/-22215.44. Spearman's rank correlation analysis indicated a significant correlation between HBsAg level and liver inflammation stage (rs = -0.244) and fibrosis stage (rs = -0.365). ROC curve analysis of the diagnostic value of HBsAg for inflammation stages S more than or equal to 4 revealed that the area under the curve (AUC) was 0.70. The specificity of diagnosing S more than or equal to 4 was > 95.16% when HBsAg was less than or equal to 32995 IU/mL. Binary logistic regression analysis identified age, serum albumin, cholinesterase, and HBsAg as independent predictors of liver fibrosis.</p><p><b>CONCLUSION</b>HBsAg level is negatively correlated with liver inflammation and fibrosis stages for patients with CHB in the IC phase, and might represent a useful noninvasive marker of the degree of hepatic fibrosis.</p>

Serum HBV DNA level at week 24 as a proper predictor for the effect of 2-year lamivudine treatment / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Lamivudine is the first L-nucleoside analogue approved for the treatment of the patients with chronic hepatitis B (CHB) for over 10 years. The aim of this study was to evaluate the virologic responses at weeks 12 and 24 for the prediction of therapeutic effect and virologic breakthrough after 2 years of lamivudine treatment in the patients with CHB.</p><p><b>METHODS</b>A retrospective study was conducted with 255 hepatitis B e antigen (HBeAg) positive and 122 HBeAg-negative CHB patients treated with lamivudine (100 mg, daily) and duration of treatment was 6 to 72 months. The levels of serum hepatitis B virus (HBV)-DNA at weeks 12 and 24 were evaluated for the predictive value of therapeutic effect and drug resistance after 2 years of lamivudine treatment.</p><p><b>RESULTS</b>HBeAg seroconversion was closely correlated with levels of serum HBV DNA at week 12 (P = 0.000, OR = 0.394) and 24 (P = 0.019, OR = 0.442), while virologic breakthrough was more correlated with baseline levels of serum HBV DNA (P = 0.019, OR = 1.484) and at week 12 (P = 0.049, OR = 1.398) and 24 (P = 0.012, OR = 2.025). At year 2, the virologic response at week 24 was more sensitive compared with week 12 when it was used to predict the efficacy and virologic breakthrough, but was less specific compared with those at week 12. There were no significant differences in terms of predicting positive and negative values of HBV DNA between week 12 and 24 for efficacy and drug resistance at year 2 in both HBeAg positive and HBeAg negative patients.</p><p><b>CONCLUSION</b>Level of serum HBV DNA at 24-week is a proper predictor for the therapeutic effect and virologic breakthrough at year 2 of lamivudine treatment.</p>

Inhibitory effect of combined transfection of p53 and AS genes on K562 cell proliferation / 中国实验血液学杂志

This study was aimed to investigate the inhibitory effect of combined transfection of p53 and angiostatin (AS) genes on K562 cells and to explore its mechanism. pVTRIO2-hp53-hAS was transfected into K562 cells with lipofectamine 2000, RT-PCR was used to determine the expression of gene of interest in transfected cells, MTT growth curve and flow cytometry were used to analyze the cell cycle for observation biological changes of cells, the cellular immunochemistry assay was used to observe the expression differences between VEGF, Bcl-2 and Bax proteins. The results indicated that the genes of interest have been transfected and stably expressed, the increase of K562 with genes of interest was slower than that without genes of interest (p<0.05). And the increase of K562 in double gene group was slower than that in p53 and AS groups (0.264+/-0.011 at last day A290 nm; 0.652+/-0.039 at last day A290 nm; 0.604+/-0.017 at last day A290 nm respectively) (p<0.05). After transfection, the expressions of VEGF and Bcl-2 protein decreased, but the expressions of Bax increased. It is concluded that the combined transfection of p53 and AS genes into K562 cells shows more notable and powerful inhibition on proliferation than those transfected with single one gene. The synergistic mechanism of p53 and AS genes may be commonly influenced the pathway of Bcl-2 and Bax expression.

Anti-apoptosis effect of VEGF on the human chronic myelocytic leukemia cell line K562 / 中国实验血液学杂志

To explore the effects of vascular endothelial growth factor (VEGF) on the mechanisms of CML pathogenesis, the effect of VEGF on K562 cell apoptosis induced by As(2)O(3) was analyzed through morphologic observation, DNA fragmentation agarose gel electrophoresis and DNA ploidy flow cytometry analysis, and the effect of VEGF on the expression of bcl-X(L), Bax and caspase-3 in K562 cells was determined by Western blot, meanwhile the expression difference between bcl-X(L) and Bax mRNA in above conditions was detected by RT-PCR. The results showed that after VEGF added, the apoptosis of K562 cells reduced, however, there was no significant changes in cell cycle distribution (P > 0.05). At the same time, following the increasing of the concentration of VEGF, expression of mRNA and protein of bcl-X(L) was up-regulated and the expression of Bax protein was down-regulated in K562 cells, and the activation of pro-caspase-3 into caspase-3 was inhibited or reduced. It is concluded that VEGF may suppress the apoptosis of K562 cells through its influence on the bcl-X(L)/Bax expression ratio in K562 cells.

Relationship between heat shock protein 70-hom gene polymorphism and ankylosing spondylitis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the association between heat shock protein 70-hom (HSP70-hom) gene polymorphism and ankylosing spondylitis(AS) in Chinese Han patients.</p><p><b>METHODS</b>Genomic DNA from 98 Chinese AS patients and 70 ethnically matched controls were typed for HSP70-hom polymorphism by polymerase chain reaction-restriction fragment length polymorphism.</p><p><b>RESULTS</b>The HSP70-hom genotypes in the AS patients consisted of homozygote AA (60.2%) and BB(4.1%), and heterozygote(35.7%), while the HSP70-hom genotypes in the controls were composed of AA(58.6%), BB(2.9%) and heterozygote(38.6%). No significant difference was found in the distribution of HSP70-hom genotype between these two groups(chi(2) test=0.280, P>0.05). The frequencies of HSP70-hom alleles in AS patients were 77.9%(AA) and 22.1%(BB), while they were 78.1% and 21.9% in the controls. The frequency of HSP70-hom allele in AS patients was not significantly increased, compared with that in controls (chi(2) test=0.002, P>0.05).</p><p><b>CONCLUSION</b>There may be no association between the HSP70-hom gene polymorphism and ankylosing spondylitis in Chinese Han population.</p>

Inhibition of K562 cell proliferation by wild type p16 and p53 genes co-transfection / 中国实验血液学杂志

The tumor suppressor gene p53 and p16, both of which play an important role in inhibition of tumorigenesis, are homozygously deleted in human myeloid leukemia cell line K562. To explore the inhibition of K562 cell proliferation by wild type p16 and p53 genes, both p16 and p53 genes were co-transfected into K562 cells mediated by liposome. The expression of the two genes was measured by immunocytochemical method, the cell cycle was analysed by flow cytometry, and the number of recovered viable cells was assessed after transfection. After co-transfection, the p53 and p16 positive cells were 23% and 28%, respectively. The results showed that co-transfection of p16 and p53 genes significantly inhibits cell proliferation comparing with transfection either by p16 gene or by p53 gene (P < 0.05). Expression of p16 and p53 proteins increased the cell number in G(1) phase but decreased the cell number in S phase. It is concluded that co-transfection of p16 and p53 genes has a stronger growth-inhibitory effect on K562 cell growth than that of transfection only by p16 gene or by p53 gene, may be a pathway for gene therapy in leukemia.