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ABSTRACT Background and aim: It is well established that the rate of gastric lesions increases in diabetic rats. Recently, the protective effect of hydrogen sulfide (H2S) in gastric mucosa has been proven. This study aimed to determine the release of H2S and mRNA expression of cystathionine gamma lyase (CSE) in gastric mucosa in alloxan-diabetic rats in response to distention-induced gastric acid secretion. Twenty-four rats were randomly assigned to 4 groups (6 in each). They were the normal-control, distention-control, diabetic-control, and distention-diabetic groups. Under anesthesia, animals underwent a tracheotomy and midline laparotomy. To washout the gastric contents, a catheter was inserted in the stomach through the duodenum. To determine the effect of distention-induced gastric acid secretion on H2S release and mRNA expression of CSE, the stomachs were distended by normal saline. At the end of experiments, animals were sacrificed and the gastric mucosa was collected to determine H2S concentration and to quantify mRNA expression of CSE by quantitative real-time PCR. Mucosal release of H2S and mRNA expression of CSE significantly increased in response to stimulated gastric acid secretion in normal rats (P<0.01), while the increases in diabetic rats were not significant. Basal release of H2S and mRNA expression of CSE in gastric mucosa were significantly in diabetic rats lower than normal rats. On the basis of the results, we conclude that the decreased release of H2S in response to basal and stimulated gastric acid output in alloxan-diabetic rats compared to normal rats is largely due to downregulation of mRNA expression of CSE.
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Animals , Rats , Cystathionine gamma-Lyase , Gastric Acid , Hydrogen Sulfide , AlloxanABSTRACT
Objective To investigate the expressions of endogenous cystathionine-γ-1yase (CSE)/ hydrogen sulfide (H2S) and resolvin E1 (RvE1) in patients with ulcerative colitis (UC),and its effect on the pathogenesis of ulcerative colitis.Methods The distribution and expression of CSE proteins in the rectum mucosa in 60 cases of UC and 30 cases of normal control group were detected by Strept Avidin-Biotin Complex (SABC) immunohistochemistry.The average optical density value of CSE was analyzed with an Image Analyzing systems.The expression of CSE mRNA in the rectum mucosa was detected by real-time polymerase chain reaction (RT-PCR).The levels of H2S and RvE1 in sera were detected by spectrophotometry.Results The expressions of CSE proteins in three groups were detected in the rectum mucosa membrane epithelia.The average optical density value of CSE and the expression of CSE mRNA in patients with active UC were higher than that in normal group and remission of UC.The levels of H2S and RyE1 in patients with active UC were significantly higher than that in normal group and remission of UC.Conclusions The abnormal expressions of CSE/H2S and RyE1 in activity of the UC might play an important role in the pathogenesis of UC.
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Objective:To explore the expressions of endogenous hydrogen sulfide (H2 S)and its synthases cystathionine beta synthase (CBS)and cystathionine gamma lyase (CSE)in the cell lines of normal bladder and bladder cancer,and to clarify their mechanism in the development of bladder cancer.Methods:The bladder cancer cell lines (5637,T24,UM-UC-3,EJ)and human bladder epithelial cell line SV-HUC-1 were selected.The expressions of CBS and CSE in bladder cancer and normal cell lines were analyzed by Western blotting assay and the productivities of H2 S in cell lines were detected by sensitive sulphur electrode assay.The EJ cells were selected based on the previous experimental results and divided into groups as follows:① 10 μmol· L-1 NaHS group, 50 μmol·L-1 NaHS group,100 μmol·L-1 NaHS group and control group.After drug treatment,the cell survival rate was measured by MTT assay at 24 and 48 h.② 5 μg·L-1 cisplatin group,cisplatin (5 μg·L-1 )+ NaHS (100 μmol·L-1 )group and control group.After medicine treatment,the cell survival rate was measured by MTT assay and the cell apoptotic rate was detected by flow cytometry at 48 h. Results:Compared with the normal bladder cells (SV-HUC-1),the expression levels of CBS and CSE and the productivity of H2 S in the bladder cancer cell lines (5637,T24,UM-UC-3 and EJ)were increased obviously (P <0.05 or P <0.01).Compared with control group,exogenous H2 S promoted the cell proliferation of EJ cells.The cell survival rates were increased with the increase of drug dose (P <0.05),which showed a dose-dependent effect.The cell survival rates were increased with the prolongation of time (P <0.05),which showed a time-dependent effect.After medicine treatment,compared with cisplatin group,the cell viability in cisplatin+NaHS group was increased (P <0.05)and the apoptotic rate was decreased (P <0.05).Conclusion:Endogenous H2 S and its synthases CBS and CSE have an increased expression level in bladder cancer cell lines compared with the normal bladder cells.H2 S can enhance the proliferation of bladder cancer cells and decrease the apoptosis induced by cisplatin.
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Objective To explore the inhibitory effects of endogenous hydrogen sulfide, a novel and important gas-eous transmitter generated in mammalian tissues mainly by cystathionine β-synthase (CBS) or cystathionineγ-lyase (CSE) on the apoptosis of the rat hepatic BRL cell line in physiological condition. Methods BRL cells were cultured, and divid-ed randomly into several groups in different phases of the experiment, including negative-siRNA (control) group, CBS siRNA (CBS 1~3) group and CSE siRNA (CSE 1~3) group, which were used to select the most efficient sequences of siRNAs at 48 or 24-hour transfection. Solution group and (CBS+CSE) siRNA group were added to detect the variation of apoptosis. The BRL cell line was observed and evaluated at 0, 4, 8, 12, and 24 hrs after siRNA transfection. When the mechanisms of the apoptosis were detected, CBS/CSE siRNAs were transfected individually or jointly into BRL cells, and compared with nega-tive-siRNA group to examine the variation. The genic and protein expression of CBS/CSE were detected by RT-PCR and Western blot assay. After transfection of CBS/CSE siRNA, the apoptosis of BRL cells was detected by Hoechst stain and flow cytometry (FCM). The mitochondrial membrane potential (MMP) changes were observed by fluorescent staining. Western blot assay was used to examine the protein expression of intracytoplasm cytochrome C (Cyt C) and cleaved-caspase 3. Re-sults CBS and CSE were observed in BRL cells. After transfection of CBS/CSE siRNA, endogenous H2S generation de-creased and the apoptosis of BRL cells increased. Accordingly, the expression of intracytoplasm-Cyt C and cleaved-caspase 3 increased. Conclusion The inhibition of endogenous H2S synthesis induced the apoptosis of BRL cells under physiologi-cal condition, which may be involved in mitochondrial pathway of apoptosis.
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Objective To investigate the protective role of hydrogen sulfide and the expression of cystathionine gamma-lyase/hydrogen sulfide pathway in a mouse model of myocarditis induced by Coxsachie -virus B3(CVB3).Methods A total of 110 five-week-old BALB/c male mice were randomly divided into four groups:the control group, viral myocarditis group, sodium bisulfide (NaHS) group (50 μmol/kg) and DL-propargylglycine (PAG) group (40 mg/kg).The experimental model of viral myocarditis was induced by intraperitoneal injection of CVB 3.Then the four groups were respectively administered with PBS , PBS, NaHS and PAG from day 1 to day 10 after infection.Blood and heart specimens were harvested from 10 mice of each group on day 4 and day 10 for evaluation of myocardial edema .The pathological changes in heart tis-sues were observed through hematoxylin-eosin staining.Levels of H2 S, IL-6 and TNF-αwere measured by ELISA.The expressions of CSE and CVB 3 at mRNA level were determined by quantitative real time PCR ( qRT -PCR ) analysis and the expression of CSE at protein level was detected by Western blot .Results Compared with the control group , the levels of H2 S and the expressions of CSE at mRNA and protein levels were down-regulated in mice with CVB 3-induced myocarditis .With the treatment of NaHS , the levels of H 2 S in serum and tissue were both up-regulated , and the histopathological damage was alleviated .However , PAG as an irreversible CSE inhibitor inhibited the expressions of H 2 S and CSE and aggravated myocardial injury , inflammatory cells infiltration and interstitial edema .Moreover , the RT-PCR analysis also showed that the expression of CVB3 at mRNA level was inhibited by NaHS but enhanced by PAG .Conclusion The expres-sion of CSE/H2 S pathway is down-regulated in mice with CVB 3-induced viral myocarditis .PAG could pro-mote virus propagation and exacerbate the disease through inhibiting the production of endogenous H 2 S, while NaHS as a H2 S donor has a protective effect on infected myocardium by suppressing virus replication at an early stage .
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BACKGROUND/AIMS: Halitosis is a symptom that bothers patients more socially than medically and its pathogenic mechanisms are unclear and treatment armamenterium is limited. Clinicians generally ignored active interventions. Since halitosis is closely associated with volatile sulfur compounds (VSCs), we used a Halimeter and gas chromatography to measure VSCs in patients with Helicobacter-pylori (H. pylori)-associated gastric diseases. METHODS: We categorized 72 patients with H. pylori infection into two groups based on their endoscopic findings: a non-erosive mucosal group (NE, n=24) and an erosive mucosal group (E, n=48). Halitosis was objectively assessed by applying either a Halimeter to breath air or gas chromatography to gastric juice. Simultaneously, the expression of VSC-generating enzyme was measured with reverse-transcriptase PCR using mRNA isolated from biopsy tissues. RESULTS: The levels of VSCs in exhaled breaths or aspirated gastric juices differed significantly between the NE and E groups (p<0.00001), suggesting that VSCs might reflect eroded epithelial damage induced by H. pylori infection. The expressions of cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE) were broadly consistent with the degree of mucosal injury. CONCLUSIONS: Erosive changes in esophagogastroduodenal mucosa were strongly correlated with increased VSC levels, suggesting that halitosis might result from H. pylori-associated erosive lesions.
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Humans , Biopsy , Chromatography, Gas , Cystathionine beta-Synthase , Cystathionine gamma-Lyase , Cytochrome P-450 CYP1A1 , Gastric Juice , Halitosis , Hydrogen Sulfide , Mucous Membrane , Polymerase Chain Reaction , RNA, Messenger , Stomach Diseases , Sulfur , Sulfur CompoundsABSTRACT
Objective:To constract a method of measurement microamount hydrogen sulfide (H_2S) using sensitive sulphur electrode. Methods: According to the physical and chemical characters of H_2S, H_2S, which in the fluids by mean of physical dissolve and chemical shape, is turned to sulphur ion (S 2-) by chemical responses. After the microamount of S 2- was measured by sensitive sulphur electrode, and the concentration of H_2S was converted, a method was constructed to measure the H_2S. It was used to analyze the concentrations of H_2S of plasma in rats and humans, the endogenous concentration of H_2S of cardiovascular tissue in rats, and CSE activity of cardiovascular tissues and cells. Results: The exponential regression of S 2- in the extent including 1 to 80 ?mol/L was found using sensitive sulphur electrod. The H_2S levels of plasma in male and female rats were 40?4 and 41?5 ?mol/L, respectively, and significant difference was not found; those in venous blood plasma of men and women were 33?4 ?mol/L and 35?5 ?mol/L respectively, without significant difference. There were not significant differences in the aortic endogenous levels of H_2S (24?6 and 25?5 nmol/mg pro) and myocardial levels (19?4 and 19?6 nmol/mg protein) between female and male rats. There were no different results of CSE activity in aortal tissue using sensitive sulphur electrode or traditional methods, however, the CSE activity of vascular smooth muscle cells could be accurately measured using sensitive sulphur electrode, which was difficult in using traditional method. Conclusion: The sensitive sulphur electrode assay was fit for the analysis of CSE/H_2S pathway in cardiovascular research.
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Objective: To examine the alteration of pathologic structure and endogenous hydrogen sulfide pathway in rats with pulmonary hypertension induced by high pulmonary blood flow. Methods: Sixteen SD rats were randomly divided into shunting group and control group. An 11 week aortocaval shunting was produced in rats of shunting group, and pulmonary artery mean pressure (mPAP) was evaluated using right cardiac catheterization. The ratios of right ventricular mass to body weight (RV/BW) and right ventricular mass to left ventricular plus septal mass[RV/(LV+S)] were also detected. Pulmonary vascular micro and ultra structures were examined. Meanwhile the concentration of plasma hydrogen sulfide (H 2S) was measured by spectrophotography. The gene expression of cystathionine ? lyase (CSE)was detected by in situ hybridization, and the activity of CSE in lung tissues was measured by H 2S production according to chemical analysis. Results: After 11 weeks of aortocaval shunting, pulmonary artery mean pressure was significantly increased. Muscularization of small pulmonary vessels and relative medial thickness of pulmonary arteries were obviously increased in shunting rats compared with controls. Ultrastructure of intrapulmonary arteries changed obviously in shunting rats. Meanwhile, plasma H 2S concentration was decreased and the activity of CSE (according to H 2S production) in lung tissues decreased in shunting rats. CSEmRNA expression by pulmonary arteries was significantly suppressed. Conclusion: Pulmonary vascular structural remodeling is the important pathologic basis for pulmonary hypertension induced by high pulmonary blood flow. The down regula tion of endogenous H 2S pathway might play an im portant role in the development of high pulmonary blood flow induced pulmonary hypertension.