ABSTRACT
Objective To explore the effect of endogenous sulfur dioxide (SO2)on the apoptosis induced by cobalt chloride (CoCl2)in the human pulmonary arterial endothelial cells (HPAECs).Methods CoCl2was used in the primary HPAECs to mimize hypoxia-induced cell apoptosis.The aspartate aminotransferase 1(AAT1),and the key enzyme generating endogenous SO2 were over -expressed by transfecting HPAECs with lentivirus containing AAT1 cDNA.HPAECs were divided into 4 groups:vehicle group,vehicle + CoCl2 group,AAT1 group and AAT1 + CoCl2 group.The expressions of AAT1,B-cell lymphoma-2 (bcl-2),bcl-associated X protein (bax),Caspase-3 and activated Caspase-3 (cleaved Caspase-3)in the HPAECs were measured by Western blot.The AAT activity was assessed with colorimetry method.The SO2 content in the HPAECs was in situ observed by SO2-specific fluorescent probe.The HPAECs apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)assay.Results There were significant differences in the endogenous SO2 content,the expre-ssions of AAT1 and bcl-2,and the ratio of cleaved Caspase-3/Caspase-3 among 4 groups of HPAECs were (F＝147.364,23.738,6.521,64.884,all P＜0.05).However,there was no difference in the expression of bax among 4 groups of HPAECs (F＝1.620,P＞0.05).Compared with vehicle group,AAT activity [(0.96 ± 0.24)Carmen's unit/μg vs.(2.21 ± 0. 60)Carmen's unit/μg],endogenous SO2 content (40.71 ± 7.72 vs.105.60 ± 16.20)and bcl-2 expression (0.59 ± 0.19 vs.1.02 ± 0.20)in the HPAECs of vehicle +CoCl2 group were significantly de-creased,while the cell apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (1.56 ± 0.25 vs.0.95 ± 0.13)were significantly increased (all P＜0.05).However,there were no differences in the expression of AAT1 (0. 50 ± 0.12 vs.0.53 ± 0.11)in the HPAECs between vehicle group and vehicle+CoCl2 group (P＞0.05). The SO2 content (351.50 ± 42.43 vs.105.60 ± 16.20)and AAT1 expression (1.22 ± 0.33 vs.0.53 ± 0.11)in the HPAECs of AAT1 group were higher than those of vehicle group (all P ＜0. 05 ). Compared with AAT1 group, endogenous SO2content (333.50 ± 46.22 vs.351.50 ± 42.43)and the expression of AAT1 (1.26 ± 0.36 vs.1.22 ± 0.33)and bcl-2 (1.14 ± 0.38 vs.1.03 ± 0.27)in the HPAECs of AAT1 +CoCl2group did not change (all P＞0. 05).Moreover,no difference was observed in the HPAECs apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (0.51 ± 0.17 vs.0.50 ± 0.11)between the two AAT1 -overexpressed groups (all P ＞0. 05).Conclusion Endo-genous SO2inhibited the hypoxic HPAECs apoptosis stimulated by the treatment of CoCl2.
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Functional cure of chronic hepatitis B ( CHB) through antiviral therapy is the goal and main focus of research worldwide.The criteria of functional cure of CHB are undetectable serum HBV DNA, persistent loss of HBsAg, with or without anti-HBs seroconversion, persistence of cccDNA in a transcriptionally inactive status and the absence of spontaneous relapse after treatment cessation .Current antiviral therapy can effectively control viral replication , but is still quite far from achieving functional cure. From the virological point of view , HBV cccDNA reservoir and its transcription activity can hardly be inhibited by the existing antiviral therapy , and the integration of viral genomic DNA leads to the continuous expression of HBsAg, which are the two main causes of the low functional cure rate in CHB patients. Furthermore, the high frequency of viral mutation can also result in a reduction of HBsAg loss .This article reviews the new strategies and advances about functional cure of CHB based on the virology research .
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Objective To investigate the effects of endogenous sulfur dioxide (SO2) on the oxidative stress induced by cobalt chloride (CoCl2) in the rat pulmonary artery smooth muscle cells (PASMCs).Methods Rat PASMCs were treated with 200 μ mol/L CoCl2 to mimic the hypoxia insult.Endogenous SO2 generating enzyme aspartate aminotransferase 1 (AAT1) expression was upregulated or downregulated (AAT1 sh) by transfection with lentivirus.Rat PASMCs were randomly divided into 8 groups:vehicle group,vehicle + CoCl2 group,AAT1 group,AAT1 + CoCl2 group,scramble group,scramble + SO2 group,AAT1 sh group and AAT1 sh + SO2 group.SO2 donor Na2 SO3/NaHSO3 at concentration of 100 μ mol/L were added in scramble + SO2 group and AAT1sh + SO2 group.The expressions of AAT1,superoxide dismutase 1 (SOD1) and SOD2 in PASMCs were detected by Western blot method.In situ SO2 content in PASMCs was detected by fluorescent probe.The superoxide anions in PASMCs were labeled by dihydroethidium (DHE) probe under fluorescent microscope.Results Compared with the vehicle group,the levels of SO2 and the expressions of AAT1 (0.221 ± 0.002 vs.0.446 ± 0.004),SOD1 (0.076 ± 0.028 vs.0.171 ± 0.019) and SOD2 (0.080 ± 0.031 vs.0.196 ± 0.018) significantly decreased (all P ＜ 0.01),and superoxide anion increased in rat PASMCs of vehicle + CoCl2 group.Meanwhile,compared with vehicle + CoCl2 group,the levels of SO2 and the expressions of AAT1 (0.839 ± 0.056 vs.0.221 ± 0.002),SOD1 (0.177 ± 0.020 vs.0.076 ± 0.028) and SOD2 (0.195 ±0.018 vs.0.080-± 0.031) markedly increased (all P ＜ 0.01),and superoxide anion decreased in rat PASMCs of AAT1 + CoCl2 group.On the contrary,compared with the scramble group,the levels of SO2 and the expressions of AAT1 (0.062 ±0.017 vs.0.354 ±0.034),SOD1 (0.054 ±0.029 vs.0.157 ±0.023) and SOD2(0.180 ±0.100 vs.0.586 ± 0.176)significantly decreased (all P ＜ 0.01),and superoxide anion increased in rat PASMCs of AAT1sh group.Furthermore,compared with the AAT1 sh group,the levels of SO2 and the expressions of SOD1 (0.155 ± 0.022vs.0.054 ± 0.029) and SOD2 (0.578 ± 0.200 vs.0.180 ± 0.100) significantly increased (all P ＜ 0.01),and superoxide anion decreased in rats PASMCs of AAT1sh + SO2 group.Conclusion Endogenous SO2/AAT1 inhibits CoCl2-induced oxidative stress in rat PASMCs.
ABSTRACT
BACKGROUND: Tetramethylpyrazine has the protective effect against the central nervous system injury. The structural changes in Nissl bodies were regarded as a marker of neuron injury.OBJECTIVE: To investigate the effect of tetramethylpyrazine on the structure and the quantity of Nissl bodies of cerebral neurons in rat with epilepsy.DESIGN: A comparative study.SETTING: It was conducted at the Physiological Department of Medical School of Xianning College.MATERIALS: From September 2004 to March 2005, it was completed at the Anatomy Department of Tongji Medical College, Huazhong University of Science and Technology. Forty healthy SD rats aging 3-4 months, weighing (250±50) g and regardless of their gender, were selected.METHODS: Rats underwent anesthesia and craniotomy. Then their cerebral cortex were exposed for placing BL-410 Experimental System of Biological Function (TME, China) to record the bilateral EEG of the brain and the seizure in rats with penicillin-induced epilepsy group and the 10 mg/kg tetramethylpyrazine group, 20 mg/kg tetramethylpyrazine group and 40 mg/kg tetramethylpyrazine group. In control groups, the brains of rats were taken out at 1 hour after craniotomy. In penicillin-induced epilepsy group, their brains were taken out at 1 hour after penicillin-induced epilepsy. In 10 mg/kg tetramethylpyrazine group, 20 mg/kg tetramethylpyrazine group and 40 mg/kg tetramethylpyrazine group, after stable penicillin-induced epilepsy, tetramethylpyrazine was injected intraperitoneally at a dose of 10 mg/kg, 20 mg/kg and 40 mg/kg, respectively.When the greatest protective effect of tetramethylpyrazine appeared, the rats' brains were taken out. Brain sections were sliced. Nissl bodies were stained by thionine staining. Under light microscope, structures of Nissl bodies were observed and the images of Nissl bodies were quantitatively analyzed by HPIAS-1000 high acuity color pathologic diagram-writing analyzing system. In each group, the average absorbency of 15 fields was regarded as the average absorbency of Nissl bodies in that group.MAIN OUTCOME MEASURES: In all the groups, the structure and the quantity of Nissl bodies of cortical neurons in rats were studied.of the structure of Nissl bodies in external granular layer cells and external pyramidal layer cells. In control group, multi-layer blue-stained and clumplike or granule-like Nissl bodies could be observed. In penicillin-induced epilepsy group, Nissl bodies were completely resolved and disappeared. In 10 mg/kg tetramethylpyrazine group, Nissl bodies were partly or completely resolved. In 20 mg/kg tetramethylpyrazine group, the quantity of Nissl bodies was significantly increased as compared with those in penicillin-induced epilepsy group and the 10 mg/kg tetramethylpyrazine group. In 40 mg/kg tetramethylpyrazine group, the quantity and the structure of of the average absorbency of stained Nissl bodies in external granular layer cells and external pyramidal layer cells in rat cortex among all the groups.In penicillin-induced epilepsy group, the average absorbency were dramatically lower than that in control group (0.033±0.002, 0.756±0.035, t=4.93,P ＜ 0.01). In 20 mg/kg tetramethylpyrazine group and 40 mg/kg tetramethylpyrazine group, the average absorbency were significantly higher than that in penicillin-induced epilepsy group (0.435±0.011, 0.658±0.029, t=2.98,5.32, P ＜ 0.01). In 40 mg/kg tetramethylpyrazine group, the average absorbency were similar to that in control group (t=1.75, P ＞ 0.05).CONCLUSION: Tetramethylpyrazine can significantly elevated the concentration of Nissl bodies of neurons in rats with epilepsy. Changes in the structure and quantity of Nissl bodies of cerebral neurons may be closely associated with seizure, and tetramethylpyrazine can restore the structure and the quantity of Nissl bodies of neurons, regulate their functions and hereby, inhibit seizures.