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Aim: To assess the efficacy and safety of in vivo electroporation (EP)-mediated dual-plasmid hepatitis B virus (HBV) DNA vaccine vs placebo for sequential combination therapy with lamivudine (LAM) in patients with chronic hepatitis B., Methods: Two hundred and twenty-five patients were randomized to receive either LAM + vaccine (vaccine group, n = 109) or LAM + placebo (control group, n = 116). LAM treatment lasted 72 wk. Patients received the DNA vaccine or placebo by intramuscular injection mediated by EP at weeks 12 (start of treatment with vaccine or placebo, SOT), 16, 24, and 36 (end of treatment with vaccine or placebo, EOT)., Results: In the modified intent-to-treat population, more patients had a decrease in HBV DNA > 2 log 10 IU/mL in the vaccine group at week 12 after EOT compared with the control group. A trend toward a difference in the number of patients with undetectable HBV DNA at week 28 after EOT was obtained. Adverse events were similar. In the dynamic per-protocol set, which excluded adefovir (ADV) add-on cases at each time point instantly after ADV administration due to LAM antiviral failure, more patients had a decrease in HBV DNA > 2 log 10 IU/mL in the vaccine group at week 12 and 28 after EOT compared with the control group. More patients with undetectable HBV DNA at week 28 after EOT in the vaccine group were also observed. Among patients with a viral load < 1000 copies/mL at week 12, more patients achieved HBeAg seroconversion in the vaccine group than among controls at week 36 after EOT, as well as less virological breakthrough and YMDD mutations., Conclusion: The primary endpoint was not achieved using the HBV DNA vaccine. The HBV DNA vaccine could only be beneficial in subjects that have achieved initial virological response under LAM chemotherapy., Competing Interests: Conflict-of-interest statement: Not declared.

Objective: To investigate the effects of LingQiJuanGan Capsule medicated serum on the apoptosis of and the expression of Bcl-2/Bax in hepatic stellate cell-T6 (HSC-T6) activated by platelet-derived growth factor (PDGF)., Methods: Preparation of drug-medicated serum. 30 SD rats were randomly divided into three groups. Serum was obtained from the SD rats administrated intragastricly with saline (10 mL/kg, group A), Fufang Biejia Ruangan Tablet solution (1.5 g/kg, group B) and LingQiJuanGan Capsule solution respectively (4.25 g/kg, group C). Detection of cell apoptosis and the expression of Bcl-2/Bax. HSC-T6 cells were activated by 10 ng/mL PDGF and incubated with drug-medicated serum at 200 mL/L. Cell apoptosis and the expression of Bcl-2/Bax were measured by flow cytometry at 0, 12, 24 and 48 h., Results: Significantly higher apoptotic rates of HSC-T6 in group B and group C were detected at 12, 24, 48 h compared with that of group A (P < 0.05), wheraus no significant difference was found between group B and group C (P > 0.05). With increase of exposure time, the apoptotic rates of HSC-T6 in group B and group C increased (P < 0.05). The levels of Bcl-2 expression in HSC-T6 in group B and C were lower than that of group A (P < 0.05) at 12, 24, 48 h (No significant difference between group B and group C, P > 0.05). The expression level of Bcl-2 decreased with increased exposure time (P > 0.05) in both group B and group C. However, no such trend was observed in group A (P > 0.05). In both group B and group C, the expression level of Bax increased in a time-dependent manner., Conclusion: LingQiJuanGan Capsule can induce apoptosis of PDGF activated HSC-T6 and inhibit hepatic fibrosis in a time-dependent manner by influencing the expression of Bcl-2/Bax.

Objective: To analyze the effects of Ling Qi Juan Gan capsule drug-containing serum at different concentrations on the platelet-derived growth factor (PDGF)-induced proliferative capabilities of and JAK2 and p-STAT3 protein expression in hepatic stellate cells (HSC) using an in vitro system., Methods: Twenty-five Sprague-Dawley rats were randomly divided into five equal groups for intragastric administration of physiological saline (10 ml/kg; group A), Fufang Biejia Ruangan tablet solution (1.5 g/kg; group B), or Ling Qi Juan Gan capsule solution at low dose (2.125 g/kg; group C1), mid dose (4.25 g/kg; group C2), or high dose (8.5 g/kg; group C3). The post-administration serum isolated from each rat (200 ml/L) was used to treat the HSC-T6 cell line following induction by PDGF (10 ng/ml). At 24, 48 and 72 h post-exposure, the cells' proliferation was measured using the Cell Counting Kit-8 (CCK-8) colorimetric assay. In addition, at 24 h post-exposure the expression of JAK2 and p-STAT3 was measured by western blotting (expressed as grey scale intensity). Multiple group comparison of repeated measures data was made by one-way ANOVA with Student-Newman-Keuls post hoc test., Results: Compared to group A, groups C2 and C3 had significantly higher inhibited proliferation at all post-exposure time points examined (24 h: A = 1.550 +/- 0.065, C2 = 1.335 +/- 0.106, C3 = 1.241 +/- 0.205; 48 h: A = 1.311 +/- 0.650, C2 = 1.090 +/- 0.106, C3 = 0.909 +/- 0.191; 72 h: A = 1.039 +/- 0.103, C2 = 0.719 +/- 0.116, C3 = 0.641 +/- 0.110, F = 36.292, all P less than 0.05); in contrast, compared to group A, group C1 showed no inhibition of proliferation at 24 h (1.522 +/- 0.128, P = 0.717) but showed significantly higher inhibition of proliferation at 48 h and 72 h (1.153 +/- 0.183 and 0.753 +/- 0.210, respectively, F = 36.292, P less than 0.05). Compared with group A, all Ling Qi Juan Gan capsule-containing serum-treated groups showed significantly lower expression of both JAK2 (A = 1.605 +/- 0.024 vs. C1 = 1.170 +/- 0.042, C2 = 0.842 +/- 0.036, C3 = 0.555 +/- 0.036, F = 43.091) and p-STAT3 (A = 1.401 +/- 0.030 vs. C1 = 1.229 +/- 0.025, C2 = 0.668 +/- 0.034, C3 = 0.630 +/- 0.026, F = 78.426, all P less than 0.01)., Conclusion: Ling Qi Juan Gan capsule drug-containing serum can inhibit the proliferation of HSC-T6 cells in a dose-dependent manner and cause an overall decrease in the expression of JAK2 and p-STAT3 in activated HSC, thereby leading to a suppression of the JAK/STAT signal transduction pathway.

Objective: To study the influence of diazoxide on mitochondrial apoptosis pathway of human renal proximal tubular cells (HK-2 cells)., Methods: Cultured HK-2 cells were inoculated on 6-well plates, according to stochastic tables law, and they were divided into normal serum-treated group (NSTG) , post-asphyxial serum treatment group (PSTG), and post-asphyxial serum and diazoxide treatment group (PSDTG). The serum from neonates 24 hours after asphyxia in a dilution of 20% (volume fraction) was used for challenge. Diazoxide in a final concentration of 100 mol/L, was used for intervention. The expression of caspase-3 was detected by immunohistochemical method. The translocation rate of Omi/HtrA2 and mitochondria membrane potential were determined by indirect immunofluorescence and confocal microscopy., Results: Compared with that of NSTG, the expression of caspase-3 absorbance (A) value of HK-2 cells in PSTG was significantly increased (25.19 + or - 3.33 vs. 13.63 + or - 1.89, P<0.01), the translocation rate of Omi/HtrA2 of HK-2 cells in PSTG was significantly increased [(56.01 + or - 5.30)% vs.(37.59 + or - 5.60)%, P<0.01], mitochondrial membrane red/green fluorescence intensity ratio was decreased significantly (0.79 + or - 1.42 vs. 1.82 + or - 0.23, P<0.01). Compared with the PSTG, the expression value of caspase-3 of HK-2 cells in PSDTG was significantly decreased (20.17 + or - 2.19), the translocation rate of Omi/HtrA2 of HK-2 cells in PSDTG was significantly decreased [(46.91 + or - 2.70)%], and mitochondrial membrane red/green fluorescence intensity ratio increased significantly (1.47 + or - 0.14), but did not recover to the same degree as that of the NSTG (all P<0.01)., Conclusion: The diazoxide may reduce the expression of caspase-3, intracellular translocation of Omi/HtrA2, and stability of mitochondrial membrane potential, thereby significantly alleviates HK-2 cells injury induced by post-asphyxial-serum of neonate.

Objective: To investigate the mechanism of inducing apoptosis of Omi/HtrA2 in renal tubular cells with post asphyxial serum of neonate., Methods: Human renal proximal tubular cell line HK-2 cell was used as target cell. They were divided into three groups: control group, asphyxia group and Ucf-101 (Omi/HtrA2 special inhibitor) treated group. The challenge concentration of serum obtained from neonates 24 hours after asphyxia was 20%, and the treatment concentration of Ucf-101 was 10 mumol/L. The Omi/HtrA2 translocation in renal tubular cells was observed with confocal microscopy, and the rate of apoptosis was detected with flow cytometer., Results: It was found that Omi/HtrA2 was translocated into cytoplasm in asphyxia group, and the rate of Omi/HtrA2 translocation in HK-2 cells of asphyxia group was significantly increased [(28.1+/-3.6)% vs. (9.4+/-2.1)%, P<0.01]. Compared with the control group, after being treated with post asphyxial serum, the rate of apoptosis of HK-2 cells in asphyxia group was significantly increased [(36.3+/-4.4)% vs.(12.4+/-2.9)%, P<0.01]. Compared with asphyxia group, the rate of apoptosis in HK-2 cells in Ucf-101 treated group was significantly decreased [(27.0+/-3.9)% vs.(36.3+/-4.4)%, P<0.01]., Conclusion: These experimental data demonstrates that post asphyxial serum of neonate can induce apoptosis of HK-2 cells, and translocation of Omi/HtrA2 from mitochondria into cytoplasm may play an important role in its intracellular signal transduction mechanism in induction of apoptosis.

Objective: To study the role of serum from asphyxiated neonates in the inducement of human renal proximal tubular epithelial cells (HK-2) adhesion to neutrophils and possible mechanisms., Methods: HK-2 cells were cultured randomly with 20% serum from neonates (1, 3, and 7 days after asphyxia), pyrrolidine dithiocarbamate (PDTC) or placebo. The activity of myeloperoxidase (MPO), an indicator of adhesion ability of HK-2 cells to neutrophils in suspensions, was detected by the biochemistry assay. Intercellular adhesion molecule-1 (ICAM-1) and nuclear factor-kappaB (NF-kappaB) of HK-2 cells were examined with the immunohistochemical staining., Results: The expression of MPO in the post-asphyxial 1-day serum treatment group were significantly higher than that in the PDTC treatment and the control groups as well as the post-asphyxial 3 and 7-day serum treatment groups (P<0.01). The expression of ICAM-1 and NF-kappaB in the post-asphyxial 1-day serum treatment group was also significantly higher than that in the other groups (P<0.01)., Conclusions: Serum from asphyxiated neonates can induce HK-2 cell adhesion to neutrophils, possibly through activating NF-kappaB and increasing the synthesis and expression of ICAM-1 on the surface of renal tubular epithelial cells.

Objective: Salvia miltorrhiza Bunge (SMB) is a traditional Chinese herb, which is considered to promote blood flow and remove blood stasis. This study examined whether SMB can alleviate injury induced by hypoxia/reoxygenation (H/R) in human kidney proximal tubular cells-2 (HK-2 cells)., Methods: There were 3 experimental groups: control, H/R injury and SMB-treated H/R injury. H/R injury of HK-2 cells was induced by first covering the cells with and then removing liquid paraffin wax. Different concentrations of compound SMB solution (0.05%, 0.10%, 0.15% or 0.20%) were administered to the SMB-treated H/R injury group before the hypoxic injury. After 4, 12 and 24 hrs of hypoxia and 4, 12, 24 and 48 hrs of reoxygenation, morphologic changes of HK-2 cells were observed under an inverted microscope. Cell viability was measured by the MTT method. Lactate dehydrogenase (LDH) activity in the culture supernatants was assayed using biochemical methods; TNF-alpha levels were determined by radioimmunoassay (RIA)., Results: The number of HK-2 cells was significantly reduced in the H/R injury group after hypoxia, and reached a nadir 24 hrs after hypoxia treatment. Various concentrations of SMB-treated groups showed significantly greater number of HK-2 cells than the H/R injury group. SMB solution (0.10%) produced the best effect. The levels of LDH and TNF-alpha in the H/R injury group were significantly increased, and reached a peak between 24 hrs of hypoxia and 4 hrs of reoxygenation when compared to the control group. Pre-treating with 0.10% SMB resulted in significantly lower levels of LDH and TNF-alpha than in the untreated H/R injury group at various time points of H/R., Conclusions: SMB has protective effects against H/R injury of HK-2 cells, possibly through inhibition of inflammatory cytokines.

Aim: To construct chimeric gene vaccine Mtb8.4/hIL-12, express it in COS-7 cells and study its immunogenicity., Methods: Chimeric gene Mtb8.4/hIL-12 was amplified by PCR and cloned into the eukaryotic vector pCI-neo to construct the recombinant plasmid pCI-neo-Mtb8.4/hIL12. After the recombinant plasmid was identified by restriction enzyme digestion analysis, PCR and DNA sequencing, COS-7 cells were transfected with pCI-neo-Mtb8.4/hIL12 through cationic liposome. 48 hours later, the expression of mRNA was detected by RT-PCR and the level of hIL-12 in culture supernatant and cell lysates were detected by Western blot. C57BL/6N mice were vaccinated with chimeric gene vaccine Mtb8.4/hIL-12 three times at the interval of 3 weeks each time. Four weeks after the final inoculation, three mice were sacrificed to assess the cytotoxicity of CTLs and response to cytokine., Results: The recombinant plasmid pCI-neo-Mtb8.4/hIL12 was constructed successfully. After COS-7 cells were transfected with pCI-neo-Mtb8.4/hIL12, chimeric gene Mtb8.4/hIL12 was expressed in COS-7 cells. The chimeric gene vaccine could induce strong antigen-specific immune response. With the increase of IFN-gamma and IL-2 secretion and the decrease of IL-4 secretion, the cytotoxicity of specific CTLs was heightened., Conclusion: Recombinant plasmid pCI-neo-Mtb8.4/hIL12 has been successfully constructed and expressed in COS-7 cells. The constructed chimeric gene vaccine Mtb8.4/hIL12 is of strong immunogenicity and can obviously induce the cytotoxicity of CTLs.

Objective: To construct and express a chimeric Mtb8.4 with signal peptide (MS)/hIL12 eukaryotic expression plasmid, and to study the immunogenicity of the MS/hIL-12 chimeric genetic vaccines., Methods: The MS/hIL-12 chimeric gene was amplified by polymerase chain reaction (PCR) and cloned into the eukaryotic expression vector pCI-neo. The correct pCI-neo-MS/hIL12 (pMSI) recombinant plasmid was identified by PCR, restricted enzyme digestion and DNA sequencing. COS-7 cells were transfected with pMSI constructs by cationic liposome. After 48 hours, mRNA of the target gene was detected by RT-PCR, and hIL-12 protein in culture supernatant and cell lysates was detected by Western blot. C57BL/6N mice were vaccinated with MS/hIL-12 chimeric gene vaccine for three times at 3 week intervals. Four weeks after the final inoculation, three mice were sacrificed for measurement of the cytokine response and cytotoxic T lymphocyte (CTL) induction., Results: The accuracy of plasmid construction was confirmed by a number of molecular biological techniques. Transfection of COS-7 cells with plasmids pMSI lead to transient expression of fusion proteins. The IFN-gamma and IL-2 titers were (1,521 +/- 48) ng/L and (755 +/- 41) ng/L in MS/hIL-12 chimeric gene vaccine group, (820 +/- 50) ng/L and (297 +/- 31) ng/L in MS gene vaccine group, (1,487 +/- 40) ng/L and (767 +/- 50) ng/L in BCG group, (121 +/- 16) ng/L and (62 +/- 10) ng/L in vacant vector group, and (48 +/- 16) ng/L and (32 +/- 17) ng/L in PBS group respectively. The levels of IFN-gamma and IL-2 in MS/hIL-12 chimeric gene vaccine group were higher than those of MS gene vaccine group, vacant vector group and PBS group (P < 0.01) and was similar to the BCG group (P > 0.05). The level of IL-4 in BCG group [(91 +/- 11) ng/L] increased significantly as compared to other groups (P < 0.01). When effector-cell-to-target-cell ratio (E:T ratio) were 100:1, 50:1, and 10:1 respectively, the CTL activity was 77.5%, 51.2%, 30.3% in MS/hIL-12 chimeric gene vaccine group, 56.2%, 37.8%, 11.5% in MS gene vaccine group, 28.9%, 21.4%, 9.8% in BCG group. The cytotoxicity in MS/hIL-12 chimeric gene vaccine group was higher than that of other groups (P < 0.01)., Conclusion: When used to construct the chimeric gene vaccine, hIL-12 could improve the immunogenicity of MS gene vaccine.

Aim: To investigate the relationship between TNF-alpha and renal tubular cell injury caused by anoxia/reoxygenation., Methods: Human renal proximal tubular cell line HK-2 was used as model. Anoxia/reoxygenation were produced by covering/de-covering the cell culture with liquid paraffin wax. The level of TNF-alpha and the activity of lactate dehydrogenase(LDH) in the culture medium was determined by radioimmunoassay(RIA) and biochemical methods, respectively. Trypan blue exclusion was used to measure cell viability., Results: Anoxia/reoxygenation could increase TNF-alpha level and LDH activity, but decrease viability of HK-2 cells. TNF-alpha level was positively correlated with LDH activity (r=0.89, P<0.05) and negatively with the cell viability (r=-0.91, P<0.05)., Conclusion: TNF-alpha induced by anoxia/reoxygenation may participate in the process of renal tubular cell injury.

Objective: To observe the role of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in protecting kidney from injury induced by asphyxia in neonatal rats., Methods: Neonatal rats were used as experimental animals. The changes in intrarenal inflammatory response and renal injury were examined in the control group (n=13), and 2, 24 and 48 hours after asphyxia followed by normal saline treatment in those treated with rhIL-1ra., Results: In normal saline group, the white blood cell count, the blood interleukin-1 (IL-1), IL-8, IL-6, nitric oxide (NO), endothelin (ET-1) levels, and the renal coefficient (LRC), the scores of injured tubules of the left kidney were significantly increased at 2 hours (n=10), 24 hours (n=11), and 48 hours (n=10, P<0.05 or P<0.01). Compared with the normal saline group, the levels of the above parameters, except IL-6, were significantly decreased in rhIL-1ra treatment group at the same time points (P<0.05 or P<0.01). Serum IL-6 at 24 hours and 48 hours was also decreased in rhIL-1ra treatment group significantly (P<0.05 or P<0.01)., Conclusion: The results suggest that rhIL-1ra may protect renal injury after asphyxia via inhibiting intrarenal inflammatory response.

Aim: To describe distribution of the phosphorothioated antisense oligodeoxynucleotides (PS-asODNs) conjugated to galactosylated poly-L-lysine (Gal-PLL) in mice, and to observe their effects on expression of HBV gene in the 2.2.15 cells and transgenic mice., Methods: According to the result of direct sequencing of PCR amplified products, a 16 mer phosphorothioate analogue of the antisense oligodeoxynucleotides (PS-asODNs) directed against the HBV U(5)-like region was conjugated to the hepatotropic Gal-PLL molecules. Its distribution was demonstrated using asODNs labeled with (32)P at the 5' terminus with a T4-polynucleotide Kinase. Its inhibition effect on HBV expression was observed in the transfected 2.2.15 cells and transgenic mice., Results: The Gal-PLL and asODNs could form stable complex at a molar ratio of 2:1. As shown in the HBV-transfected 2.2.15 cells, the inhibition effects of asODNs alone and asODNs conjugated to Gal-PLL, at 10 micromol/L for both, on HBsAg and HBeAg production were different,the former being 70 % and 58 %, respectively, and the latter being 96 % and 82 %, respectively. A more pronounced reduction was also observed in viral DNA load in the culture supernatant for the test with Gal-PLL-asODNs. Among many mouse organs, livers retained more asODNs molecules after administration. The preferential concentration in liver was found to be 52.14 % for Gal-PLL-asODNs, as high as 2.38-fold of that for asODNs (21.9 %). Both elements decreased gradually in liver, with 2.9 % of the former, 5.99 % of the latter retained 24 hours after the administration. The injection interval, therefore, was recommended to be 24 hours. In the transgenic mice, serum HBsAg decreased significantly (P<0.01) at the 12th day after administrating Gal-PLL- asODNs, the serum HBV DNA turned negative in 4 of the 6 mice., Conclusion: Antisense oligodeoxynucleotides conjugated to Gal-PLL can be concentrated in liver and intaked by hepatocytic cells. This may result in specific inhibition of expression and replication of HBV in vitro and in vivo.