Objective To investigate the effect of microRNA (miRNA )‐548ah targeting histone deacetylase‐4 (HDAC4) on the replication and expression of hepatitis B virus (HBV) .Methods HepG2 , 2 ,15 cells were transfected by mimics and inhibitors . The expressions of miRNA‐548ah and HDAC4 before and after transfection were detected by fluorescent quantitative polymerase chain reaction (PCR) . The expression of HDAC4 protein in HepG2 ,2 ,15 cells was detected by Western blotting .The target gene of miRNA‐548ah was analyzed by bioinformatics methods .3′UTR dual‐luciferase expression vector containing candidate HDAC4 target genes was built to test the luciferase activity .The levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) in the supernatant of cultured HepG2 ,2 ,15 cells were detected by enzyme‐linked immunosorbent assay (ELISA) .The level of HBV DNA in the supernatant of HepG2 ,2 ,15 cells was detected by fluorescent quantitative PCR .The t‐test was used for comparison between two groups ,SNK‐q tests were used for multiple groups comparisons .Results The expressions of miRNA‐548ah in HepG2 ,2 ,15 and HepG2 cells were 5 .74 ± 0 .02 and 2 .96 ± 0 .40 , respectively (t= 11 .89 ,P< 0 .01) ,and the expressions of HDAC4 mRNA were 9 .38 ± 0 .39 and 18 .13 ±0 .34 ,respectively (t = 29 .39 , P < 0 .01) . The expression of miRNA‐548ah in HepG2 ,2 ,15 cells was inhibited by transfection of miRNA‐548ah inhibitors (1 .01 ± 0 .13 ,t= 15 .48 , P< 0 .01) .Compared with control group ,the levels of HBsAg ([6 .45 ± 0 .46 ] IU/mL vs [2 .60 ± 0 .20 ] IU/mL , t = 7 .48 , P <0 .01) ,HBeAg ([5 .49 ± 0 .27] NCU/mL vs [4 .15 ± 0 .34 ] NCU/mL , t = 3 .10 , P < 0 .05 ) and HBV DNA ([3 .93 ± 0 .06] lg copy/mL vs[2 .04 ± 0 .07] lg copy/mL ,t = 18 .89 , P< 0 .01) in the supernatant of cultured HepG2 ,2 ,15 cells significantly decreased in inhibitor group . The expression of HDAC4 in HepG2 ,2 ,15 cells significantly decreased after transfection of miRNA‐548ah mimics (2 .98 ± 0 .94) ,but significantly increased after transfection of miRNA‐548ah inhibitors (23 .77 ± 6 .74 ) , with statistical significance (F= 9 .34 , P< 0 .01) .The expression of HDAC4 protein was also significantly inhibited after transfection of miRNA‐548ah mimics (0 .53 ± 0 .14 vs 0 .23 ± 0 .02 , t = 3 .58 , P = 0 .02) .The activity of luciferase was significantly inhibited by transfection of miRNA‐548ah mimics (7 .62 ± 0 .45 vs 6 .65 ±0 .27 ,t = 3 .18 , P = 0 .03 ) .Conclusion miRNA‐548ah may promote the replication and expression of HBV through the regulation of target gene HDAC4 .

Previous studies have shown that expression of the interferon-sensitive gene (ISG)I5 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by Hi (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by HI (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.

Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononuclear cells (PBMCs) from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8(+) T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8(+) T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8(+) T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immunotherapeutic approaches should be aimed at not only boosting a HBV-specific CD8(+) T response but also improving its function.

Objective To investigate the underlying mechanism of ursolic acid in attenuating diabetic mesangial cells injury induced by high glucose (HG).Methods Rat glomerular mesangial cells were cultured in normal glucose,HG,HG with LY294002 and HG with ursolic acid.The cell proliferation and hypertrophy were detected by MTT and the ratio of total protein content to cell number.miRNA-21 was detected by quantitative real-time PCR.The PI3K-Akt-mTOR pathway,autophagy associated protein and collagen I were detected by Western blotting and quantitative realtime PCR.The autophagosomes were observed by electron microscope.Results Compared with normal control group,the cells exposed to HG showed up-regulating miRNA-21 expression(P ＜ 0.01),down-regulating PTEN protein and mRNA expression(P ＜ 0.01),up-regulating p85PI3K,phospho(p)-Akt,p-mTOR,p62/SQSTMI,collagen I expressions and down-regulating LC3II expression(P ＜ 0.01).Ursolic acid and LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation(P < 0.01),down -regulated the expressions of p85Pl3K,p-Akt,p-mTOR,p62/SQSTMI and collagen I and up-regulated the expression of LC3II(P ＜ 0.01).But LY294002 had no effect on the expression of miRNA-21 and PTEN.Ursolic acid down-regulated miRNA-21 expression(P ＜ 0.01),up-regulated PTEN protein and mRNA expression(P ＜ 0.01).Conclusion Ursolic acid may inhibit high glucose-induced mesangial cell miRNA-21 overexpression,up-regulate PTEN,inhibit the activation of PI3K-Akt-mTOR signaling pathway and the enhanced autophagy to reduce the accumulation of extracellular matrix and ameliorate cell hypertrophy and proliferation.

Objective To study the association between transforming growth factor-β1 (TGF-β1) polymorphism and type 2 diabetes mellitus in Han population of Shanghai. Methods In this case-control study , 1 234 cases of T2DM patients were recruited and 1 272 healthy individuals were selected as control. Five ml of blood sample was collected from each subject ,from which the whole genomic DNA was extracted.The polymorphism was detected by the Taqman technology. Result Significant association was observed in TGF-β1 T896C genotypes and alleles with T2DM (P = 0.0001 and P = 0.004, OR = 1.18 [1.05 ~ 1.33], respectively). Conclusion The polymorphism of T896C in TGF-β1 gene may be associated with T2DM in Han population from Shanghai.

Objective To investigate the expression of Notch signaling molecules, transforming growth factor-β (TGF-β) and fibronectin (FN) in mesangial cell induced by high glucose, and the underlying mechanism of cordyceps sinensis.Methods Rat glomerular mesangial cells were divided into following groups: normal control group (5.5 mmol/L glucose), hypertonic control group (5.5 mmol/L glucose+ 19.5 mmol/L mannitol), high glucose group (25.0 mmol/L glucose), DAPT inhibitor group (25.0 mmol/L glucose + 1 μmol/L DAPT), cordyceps sinensis intervention group (25.0 mmol/L glucose+10 mg/L cordyceps sinensis).Cell proliferation was detected by MTT.The protein and mRNA expression of Notch signaling molecules (Notch1, Jagged1 and Hes1), TGF-β and FN was detected by Western blotting and real time PCR.Results Compared with normal control group, high glucose induced mesangial cell proliferation, as well as the mRNA and protein expression of Notch1, Jagged1, Hes1, TGF-β1 and FN was up-regulated in high glucose group (all P ＜ 0.05).Compared with that in high-glucose group, DAPT and cordyceps sinensis inhibited high glucose-induced mesangial cell proliferation and down-regulated the mRNA and protein expression of Notch pathway, TGF-β1 and FN (all P ＜ 0.05).Conclusion By inhibiting the abnormal activation of Notch signaling pathway and TGF-β signaling pathway, cordyceps sinensis may alleviate high glucose-induced mesangial cell proliferation and reduce extracellular matrix accumulation, thus protecting kidney.

Objective: To investigate the effects of abated microRNA-21 (miRNA-21) on phosphatase and tensin homologue on chromosome ten protein (PTEN) and PI3K/Akt/mTOR pathway, as well as their further influence on the autophagy in high glucose (HG, 25.0 mmol/L) induced rat glomerular mesangial cells. Methods: MiRNA-21 inhibitor and negative control were transfected by liposome 2000 into rat glomerular mesangial cells (HBZY-1). The cells were divided into normal glucose (5.5 mmol/L) group, normal glucose+negative control group, normal glucose+miRNA-21 inhibitor group, HG group, HG+negative control group and HG+miRNA-21 inhibitor group. Cell proliferation and hypertrophy were assayed by MTT and the ratio of total protein to cell number respectively. The miRNA-21 expression was detected using real time PCR. The expressions of PTEN/Akt/mTOR signaling signatures, autophagy-associated protein (p62 and LC3 Ⅱ) and collagen Ⅰ was detected by Western blotting and real time PCR. Autophagosomes were observed using electron microscopy. Results: Compared with those in normal glucose group, in HG group cells had hypertrophy and proliferation, up-regulated miRNA-21 expression, and down-regulated PTEN protein and mRNA expressions (all P<0.01). Also there were and up-regulated p-Akt, p-mTOR, p62 and collagen Ⅰ expression, and lower LC3 Ⅱ expression and autophagosomes (all P<0.01). Further, compared with those in HG group, cells hypertrophy and proliferation in HG+miRNA-21 inhibitor group were reduced, expressions of p-Akt, p-mTOR, p62 and collagen Ⅰ were down-regulated, while expressions of PTEN and LC3 Ⅱ and autophagosomes were up-regulated (all P<0.01). Conclusions MiRNA-21 inhibitor up-regulates PTEN expression, which inhibits the activation of Akt/mTOR signaling pathway, ameliorates cell hypertrophy, proliferation and enhances autophagy to reduce extracellular matrix accumulation.

Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are believed to play a major role in viral clearance and disease pathogenesis during HBV infection. To clarify the differ-ences in host immune responses between self-limited and chronic HBV infections, we constructed three HLA-A*0201/HBV tetramers with immunodominant epitopes of core18-27, polymerase 575-583 and envelope 335-343, and analyzed the HBV-specific CTLs in peripheral blood mononu-clear cells (PBMCs) from patients infected with HBV. The frequencies and expansion ability of HBV-specific CD8+ T cells in most self-limited HBV infected individuals were higher than those in chronic HBV-infected patients. HBV-specific CD8+ T cells could be induced by in vitro peptide stimulation from chronic patients with a low level of serum HBV-DNA but not from those with a high level of serum HBV-DNA. In chronic infection, no significant correlation was found either between the frequencies of HBV-specific CD8+ T cells and the viral load, or between the frequencies and the levels of alanine transaminase. Our results suggested that the frequencies of HBV-specific CTLs are not the main determinant of immune-mediated protection in chronic HBV infection and immuno-therapeutic approaches should be aimed at not only boosting a HBV-specific CD8+T response but also improving its function.

Background: Postherpetic neuralgia (PHN) is the most common complication of acute herpes zoster. The treatment of PHN remains a challenge for clinical pain management. Despite the effectiveness of anticonvulsants, antidepressants, and lidocaine patches in reducing PHN, many patients still face intractable pain disorders.In this randomized controlled study, we evaluated whether hydromorphone through intravenous patient-controlled analgesia (IV PCA) was effective in relieving PHN. Methods: Patients with PHN were randomly divided into two groups, one group received oral pregabalin with IV normal saline, another group received oral pregabalin with additional IV PCA hydromorphone for two weeks. Efficacy was evaluated at 1, 4, and 12 weeks after the end of the treatments. Results: Two hundred and one patients were followed up for 12 weeks. After treatment, numerical rating scale (NRS) score of patients in the hydromorphone group was significantly lower than that of the control group, and the difference of NRS scores between the two groups was statistically significant at 4 and 12 weeks after treatment. The frequency of breakthrough pain in the hydromorphone group was significantly lower than that in the control group 1 and 4 weeks after treatment.After treatment, the quality of sleep in the hydromorphone group was significantly improved compared with the control group. The most common adverse reactions in the hydromorphone group were dizziness and nausea, with no significant respiratory depression. Conclusions IV PCA hydromorphone combined with oral pregabalin provides superior pain relief in patients with PHN, which is worthy of clinical application and promotion.

Objective: To investigate the effect of the Periplaneta Americana polypeptide on the angiogenesis. Method:Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cell scratch assay were used to observe effect of different concentration (6.25,12.5,25,50,100 mg·L^-1) of the Periplaneta Americana polypeptide, CⅡ-3 and skimmed cream on the proliferation and migration of human umbilical vein endothelial cells (HUVECs), and a normal group and a thalidomide group were also established in this study. The tubule formation assay was used to detect the effect of different concentration (25, 50, 100 mg·L^-1) of the Periplaneta Americana extracts on the formation of tubules in HUVECs cells. The adhesion between HepG2 cells and HUVECs cells was observed by cell adhesion assay. The expression of vascular endothelial growth factor (VEGF) proteins in HUVECs was detected by immunocytochemical staining and enzyme linked immunosorbent assay (ELISA). Result:MTT results showed that the Periplaneta Americana polypeptide could inhibit the proliferation of HUVECs in a dose-dependent manner (P<0.05). The effect of different concentrations of PAP-2 was better than that of PAP-1 and PAP-3 at 24, 48 72 h (P<0.05). However, the survival rate of HUVECs was significantly increased after treatment with different concentrations of CⅡ-3 and skimmed cream in a time-dose dependent manner. Cell wound scratch assay indicated that migration of HUVECs could be inhibited by Periplaneta Americana polypeptide in a dose-dependent manner (P<0.05), and the effect of PAP-2 was better than that of PAP-1 and PAP-3 (P<0.05). CⅡ-3 could inhibit migration HUVECs to some extent, but the higher the dose was, the weaker the inhibition effect was. Skimmed cream promoted migration of HUVECs in a dose-dependent manner. Tube formation assay revealed that Periplaneta Americana polypeptide could inhibit tube formation of HUVECs, and the inhibitory effect of PAP-2 was better than that of PAP-1 and PAP-3 (P<0.05). CⅡ-3 and skimmed cream promoted the tube formation of HUVECs to a certain extent. Intercellular adhesion assay stated clearly that Periplaneta Americana polypeptide could block the adhesion between HepG2 and HUVECs (P<0.05), and the effect of PAP-2 was better than that of PAP-1 and PAP-3 (P<0.05). However, CⅡ-3 and skimmed cream could promote the adhesion between HepG2 and HUVECs. Results of ELISA assay and immunocytochemical staining indicated that Periplaneta Americana polypeptide could down-regulate the expression of VEGF of HUVECs in a dose-dependent manner (P<0.05), in which effect of PAP-2 was better than that of PAP-1 and PAP-3 (P<0.05). However, CⅡ-3 and skimmed cream could up-regulate the expression of VEGF in HUVECs. Conclusion:The Periplaneta Americana polypeptide can inhibit the invasion, metastasis and tube formation of HUVECs, and down-regulate the expression of VEGF in HUVECs. The effect of Periplaneta Americana polypeptide is better than CⅡ-3 and skimmed cream, and the among the polypeptide, the effect of PAP-2 is superior to the other two.