| Chapter one The expression changes of CSE, NF-κB and IL-8mRNA in gastric ulcer with Helicobacter pylori infectionObjectiveTo study the difference of cystathionine-y-lyase,CSE,[hydrogen sulfide synthesis enzymes,reflect endogenous (hydrogen sulfide, H2S) expression indirectly] in Helicobacter pylori (H.pylori) infected gastric ulcer, and determine expression of inflammatory-related factors NF-κB and IL-8mRNA and explore the role and mechanism of H2S in H.pylori-related gastric ulcer.MethodsWe collected108cases which had underging gastroscopy in the Third Xiangya Hospital from April2011to December2011. Tested H.pylori infection by Rapid Urease Test (RUT),14C-Urea Breath Test(14C-UBT), Pathological examination, and H.pylori culture. Patients were divided into normal control group, H.pylori-negative gastric ulcer and H.pylori-positive gastric ulcer group,36cases in each group. CSE, NF-κB and IL-8mRNA expression was determined by reverse transcriptase PCR, and each gray value was measured by gray level analyzer to calculate the ratio of the gray value of the target gene and the internal reference gene (GAPDH); correlations among genes also involved.Results1.The expression of CSE, NF-κB and IL-8mRNA:1) CSE mRNA expression:Compared with normal group, the CSE mRNA expression in H.pylori-negative gastric ulcer group and H.pylori-positive gastric ulcer group were significantly higher (p<0.05). Compared with H.pylori-negative gastric ulcer group, the CSE mRNA expression in H.pylori positive gastric ulcer group was significantly higher(p<0.05).2) NF-κB mRNA expression:Compared with normal group, the NF-κB mRNA expression in H.pylori-negative gastric ulcer group and H.pylori-positive gastric ulcer group were significantly higher (p<0.05).Compared with H.pylori-negative gastric ulcer group, the NF-κB mRNA expression in H.pylori-positive gastric ulcer group was significantly higher(p<0.05).3) IL-8mRNA expression:Compared with normal group, the IL-8mRNA expression in Hpylori-negative gastric ulcer group and H.pylori-positive gastric ulcer group were significantly higher (p<0.05).Compared with H.pylori-negative gastric ulcer group, the IL-8mRNA expression in H.pylori-positive gastric ulcer group was significantly higher(p<0.05).2. The correlation of CSE, NF-κB and IL-8mRNA expression in different groups1) The control group:the relative expression between CSE and NF-κB mRNA, the relative expression between CSE and IL-8mRNA had no correlation; but the relative expression between NF-κB and IL-8mRNA (r=0.78) showed a positive correlation with statistical significance (p<0.05).2) H.pylori-negative gastric ulcer group:the relative expression between CSE and NF-κB mRNA (r=0.98),the relative expression between CSE and IL-8mRNA (r=0.95),the relative expression between NF-κB and IL-8mRNA (r=0.90) showed a positive correlation with statistical significance (p<0.05).3). H.pylori-positive gastric ulcer group:the relative expression between CSE and NF-κB mRNA (r=0.99),the relative expression between CSE and IL-8mRNA (r=0.85),the relative expression between NF-κB and IL-8mRNA (r=0.90) showed a positive correlation with statistical significance (p<0.05).Conclusion1. CSE mRNA expression in gastric ulcer mucosa is higher, and it has relationship with Helicobacter pylori infection2. There is positive correlation between CSE,NF-κB and IL-8mRNA expression in patients with gastric ulcer, suggesting that H.pylori may stimulate IL-8mRNA generated through NF-κB pathway, leading to the inflammatory response and increas CSE mRNA expression. Chapter Two The effects of Hydrogen sulfide on expressions of CSE,NF-κB, and IL-8mRNA in GES-1cells with Helicobacter pylori infectionObjectiveTo investigate the effects of (hydrogen sulfide, H2S) on expressions of CSE,NF-κB and IL-8mRNA in GES-1cells infected with Helicobacter pylori.MethodsGES-1cells were cultured for24hrs in vitro till80%-90%cell fusion and were divided into Group A (control group, neither H.pylori nor NaHS),Group B(H.Pylori group),Group C (NaHS group,it was further divided into C1,C2,C3,C4group by the concentration of50μM,100μM,200μM,400μM NaHS),Group D (H.Pylori plus NaHS group,it was also further divided into D1,D2,D3,D4group by the concentration of50μM,100μM,200μM,400μM NaHS). The ratio of H.Pylori and cells was100:1, H.Pylori and cells were cultured for3,6and12hours respectively. The expression of CSE,NF-κB and IL-8mRNA were measured by reverse transcriptase PCR, the gray values in each group were measured by gray level analyzer and the ratio of the gray value of the target gene to the internal reference gene (GAPDH) were calculated; then correlations among genes were analyzed.Results1. The expressions of CSE,NF-κB and IL-8mRNA1) the expressions of CSE mRNA:In the different groups,compared with control group,the CSE mRNA expression of H.Pylori group,H.pylori plus50μM NaHS group,H.pylori plus100μM NaHS group was higher(p<0.05),the CSE mRNA expression of200μM NaHS group and400μM NaHS group was lower (p<0.05).Compared with H.pylori group,NaHS group, H.pylori plus200μM NaHS group, H.pylori plus400μM NaHS group was lower(p<0.05). In the different time,compared with3h, the CSE mRNA expression of H.pylori group,200μM NaHS group,400μM NaHS group, H.pylori plus50μM NaHS group, H.pylori plus100μM NaHS group was lower at6h,12h(p<0.05).2) the expressions of NF-κB mRNA:In the different groups,compared with control group, the NF-κB mRNA expression of H.Pylori group,H.pylori plus50μM NaHS group, H.pylori plus100μM NaHS group was higher (p<0.05).The NF-κB mRNA expression of NaHS group had no significant difference (p>0.05). Compared with H.pylori group, the NF-κB mRNA expression of NaHS group H.pylori plus200μM NaHS group, H.pylori plus400μM NaHS group was lower (p<0.05). In the different time,compared with3h, the NF-κB mRNA expression of H.pylori group,H.pylori plus50μM NaHS group,H.pylori plus100μM NaHS group was lower at6h,12h (p<0.05).3) the expressions of IL-8mRNA:In the different groups,compared with control group, the IL-8mRNA expression of H.pylori group,H.pylori plus NaHS group was higher (p<0.05),NaHS group was no difference (p>0.05).Compared with H.pylori group, the IL-8mRNA expression of NaHS group, H. pylori plus200μM NaHS group and H.pylori plus400μM NaHS group was lower(p<0.05). In the different time,compared with3h, the IL-8mRNA expression of H.pylori group, H.pylori plus50μM NaHS group, H.pylori plus100μM NaHS group was higher at6h (p<0.05).Compared with6h, the IL-8mRNA expression of H.pylori group, H.pylori plus50μM NaHS group, H.pylori plus100μM NaHS group was lower at12h(p<0.05)2Correlations analysis of CSE,NF-κB, IL-8mRNA of GES-1cellsH.pylori-treated GES-1and the200μM,400μM NaHS-treated H.pylori-infected GES-1for3h,6h,12h, the relative expression between CSE,NF-κB and IL-8mRNA with positive correlation and significant difference (p<0.05)Conclusion1. H.pylori enhances NF-κB and IL-8mRNA expression and up-regulates CSE mRNA expression。2.200μM,400μM NaHS suppresses H.pylori-induced NF-κB and IL-8mRNA expression and down-regulates CSE mRNA, which may have protective effects on GES-1cells. |
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