Objective To express nonstructural protein 2 transactivated protein (NS2TP) of hepatitis C virus (HCV) in the prokaryotic expression system and prepare polyclonal antibody,and to study the expressions in different liver tissues.Methods NS2TP gene was amplified by polymerase chain reaction (PCR) technique and cloned into the prokaryotic expression vector pET-32a(+),which was transformed into E.coli BL21.The protein was induced by isopropyl thiogalactose (IPTG) and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting assay.The recombinant protein were expressed and purified in a large amount.The rabbit was immunized with the purified protein to prepare polyelonal antibody.The liver tissues of patients with chronic HCV infection and healthy controls were detected by immunohistochemistry method.Results The recombinant NS2TP protein (relative molecular mass:33×103 ) and polyclonal antibody with high titer and specificity were successfully prepared.NS2TP expressions in the liver of patients with chronic HCV infection were higher than those of healthy controls,and were mainly distributed in the nucleus of hepatocytes.Conclusions The NS2TP expression level and intracellular location in liver tissue of patients with chronic HCV infection are understanded,which could bring new clues for further study of the biological function of NS2TP and the pathogenesis of HCV infection.

To investigate the interaction and involvement of sodium hydrosulfide (NaHS), a H(2)S donor, on hippocampus of rats suffering from sepsis-associated encephalopathy, rats were subjected to cecal ligation and puncture (CLP)-induced sepsis. Adult male Sprague-Dawley rats were randomly divided into four groups: Sham group, CLP group, CLP+NaHS group and CLP+aminooxyacetic acid (AOAA, an inhibitor of H(2)S formation) group. The four groups were observed at 3, 6, 9, 12 h after treatment. We examined hippocampal H(2)S synthesis and the expression of cystathionine-β-synthetase (CBS), a major enzyme involved in the H(2)S synthesis in hippocampus. CBS expression was detected by reverse transcription polymerase chain reaction (RT-PCR). The concentrations of inflammatory cytokines (TNF-α, IL-1β) were determined in hippocampus by using enzyme-linked immunosorbent assay (ELISA). Neuronal damage was studied by histological examination of hippocampus. In CLP group, H(2)S synthesis was significantly increased in hippocampus compared with sham group and it peaked 3 h after CLP (P<0.05). Sepsis also resulted in a significantly upregulated CBS mRNA in hippocampus. The levels of TNF-α and IL-1β in the hippocampus were substantially elevated at each time point of measurement (P<0.05), and they also reached a peak value at about 3 h. Administration of NaHS significantly aggravated sepsis-associated hippocampus inflammation, as evidenced by TNF-α and IL-1β activity and histological changes in hippocampus. In septic rats pretreated with AOAA, sepsis-associated hippocampus inflammation was reduced. It is concluded that the rats subjected to sepsis may suffer from brain injury and elevated pro-inflammatory cytokines are responsible for the process. Furthermore, administration of H(2)S can increase injurious effects and treatment with AOAA can protect the brain from injury.

Objective To investigate the changes of the expression of HIF-1α (hypoxia induciblefactor-1a) mRNA in myocardium of rats after cardiopulmonary resuscitation (CPR) and the intervention effects of exogenous hydrogen sulfide on it. Method Forty five male SD rats were randomly divided into three groups: control group ( n = 15 ), cardiopulmonary resuscitation group ( CPR group, n = 15 ) and NaHS + CPR group (n = 15 ) . Rats of control group were anesthetized and intubated without asphyxia and cardiac arrest. The rats of CPR group and NaHS + CPR group were operated to induce cardiac arrest by asphyxiation. In the rats of NaHS + CPR group, NaHS in dose of 50 ug/kg was administrated via the femoral venous line 1 minute before CPR. Hemodynamic was monitored continuously. The expression of HIF - lα mRNA in myocardium of rats in each group was determined by using RT-PCR and the activity of myeloperoxidase (MPO) in the myocardium of rats in each group was assayed by using a patent reagent box 6 h after CPR. The histopathological changes of myocardium were also observed. The t- test was used for statistical analysis. Results There was no statistically significant difference in hemodynamic changes between CPR group and CPR + NaHS group ( P ＞ 0. 05 ) . When compared with the control group, the activity of MPO and the expression of HIF-1α mRNA in CPR group and CPR + NaHS group were both increased, and those increased in CPR + NaHS group was more significant (P ＜ 0. 05) . When compared with CPR group, the expression of HIF-1α mRNA in CPR + NaHS group was higher, however, the activity of MPO in CPR + NaHS group was lower ( P ＜ 0. 05) . There were various histopathological changes of myocardium of rats found in CPR group and CPR + NaHS group, and the damage of myocardium of rats in CPR group was more obvious than that in CPR + NaHS group. Conclusions The expression of HIF-1α mRNA in myocardium of rats was increased after CPR. Exogenous hydrogen sulfide can protect myocardium cells from resuscitation-perfusion injury, and the protection is associated with increase in expression of HIF 1 αmRNA.

Objective To investigate the effects of hydrogen sulfide on sepsis-induced cardiac dysfunction in rats.Methods Male SD rats were randomly ( random number) divided into 4 groups:control group ( Ⅰ group),sepsis group ( Ⅱ group),sepsis + NaHS group (Ⅲ group),sepsis + PAG group (Ⅳ group). Cecal ligation and puncture technique was used in Ⅱ, Ⅲ, Ⅳ group. Hemodynamic was observed,and H2S synthesis,CSE (cystathionine-r-lyase) mRNA,MPO activity and the level of TNF-α,IL-1β were determined.The morphological changes and infiltration of inflammatory cells in myocardium were also observed.Results Compared with Ⅰ group,H2S synthesis,the levels of TNF-α,IL-1 β,the activity of MPO increased ( P ＜ 0.05 or P ＜ 0.01 ),and the expression of CSE-mRNA increased,and blood pressure of rats decreased significantly in Ⅱ group.Compared with Ⅱ group,H2S synthesis,the levels of TNF-α,IL-1β and the activity of MPO increased (P ＜ 0.05),the expression of CSE mRNA did not change noticeably ( P ＞ 0.05),and blood pressure of rats decreased more significantly in Ⅲ group.Compared with Ⅱ group, H2S level decreased,the levels of TNF-α,IL-1β and the activity of MPO decreased significantly,the expression of CSE mRNA decreased and blood pressure of rats decreased in Ⅵ group (P ＜0.05).Histopathological changes of myocardium were aggravated in the following severity order: Ⅰ ＜ Ⅳ ＜＜ Ⅲ.Conclusions CSE/H2S system of the myocardium was upregulated in sepsis rats.Hydrogen sulfide could raise the levels of MPO,TNF-α,IL-1β,aggravating myocardial injury. Contrarily,the inhibitor of H2S could counteract it.

To investigate the interaction and involvement of sodium hydrosulfide (NaHS),a H2S donor,on hippocampus of rats suffering from sepsis-associated encephalopathy,rats were subjected to cecal ligation and puncture (CLP)-induced sepsis.Adult male Sprague-Dawley rats were randomly divided into four groups:Sham group,CLP group,CLP+NaHS group and CLP+aminooxyacetic acid (AOAA,an inhibitor of H2S formation) group.The four groups were observed at 3,6,9,12 h after treatment.We examined hippocampal H2S synthesis and the expression of cystathionine-β-synthetase (CBS),a major enzyme involved in the H2S synthesis in hippocampus.CBS expression was detected by reverse transcription polymerase chain reaction (RT-PCR).The concentrations of inflammatory cytokines (TNF-α,IL-1β) were determined in hippocampus by using enzyme-linked immunosorbent assay (ELISA).Neuronal damage was studied by histological examination of hippocampus.In CLP group,H2S synthesis was significantly increased in hippocampus compared with sham group and it peaked 3 h after CLP (P＜0.05).Sepsis also resulted in a significantly upregulated CBS mRNA in hippocampus.The levels of TNF-α and IL-1β in the hippocampus were substantially elevated at each time point of measurement (P＜0.05),and they also reached a peak value at about 3 h.Administration of NaHS significantly aggravated sepsis-associated hippocampus inflammation,as evidenced by TNF-α and IL-1β activity and histological changes in hippocampus.In septic rats pretreated with AOAA,sepsis-associated hippocampus inflammation was reduced.It is concluded that the rats subjected to sepsis may suffer from brain injury and elevated pro-inflammatory cytokines are responsible for the process.Furthermore,administration of H2S can increase injurious effects and treatment with AOAA can protect the brain from injury.