Endogenous Hydrogen Sulfide And Its Role In Periodontal Inflammation

Hydrogen sulfide(H2S)was thought of the third air transmitter following nitric oxide(NO)and carbon monoxide(CO)recently.H2S in bodies is mainly produced by cystathionine y-lyase(CSE)and cystathionine ?-synthase(CBS).It has been demonstrated that CBS is located in central nerve system while CSE in cardiovascular system mainly.Endogenous H2S regulates nerve system,cardiovascular system,the endocrine system besides inflammation.Periodontal diseases,including periodontitis and gingivitis,are chronic inflammatory reaction caused by stimulation of periodontal bacteria and plaque.But until now,it is not reported whether there is endogenous H2S in gingival tissue and its role in periodontal disease.Our study detected the two main enzymes of H2S(CSE and CBS)in gingival tissue and measured the change of H2S level when in periodontitis.Then we discussed the possible mechanism of this effect.Part 1 Detection of endogenous H2S in human gingival tissueAim:To detect whether there is endogenous H2S in human gingival tissue.Methods:Collect healthy human gingival tissues.Measure the mRNA and protein level of CBS and CSE through Rt-PCR and western blot respectively.Detect the distribution of the two enzymes in gingival tissue by immunohistochemistry.And further check the H2S levels and producing activity of H2S enzymes with a defined methylene blue test.Results:There were detectable mRNA and protein of CBS and CSE in human gingival tissue.And they were mainly located in cytoplasm of some inflammatory cells,endothelial cells of small blood vessels and gingival fibroblasts.There were also detectable endogenous H2S and it's producing in human gingival tissue.Conclusion:There was endogenous H2S in human gingival tissue.Part 2:The changes of CBS,CSE and H2S level in gingival tissue when in periodontal inflammationAim:To detect the changes of CBS,CSE and H2S level and producing activity in gingival tissue when in periodontal inflammation.Methods:Collect human gingival tissues of gingivitis,moderate periodontitis and severe periodontitis.Measure the mRNA and protein level of CBS and CSE,detect the distribution of the two enzymes and check the H2S levels and producing activity of H2S enzymes in gingival tissues and compare with that in healthy tissue.Results:The mRNA and protein levels of CBS and CSE in moderate periodontitis and severe periodontitis rised up obviously but only CBS not CSE(p>0.05)increased in gingivitis as compared with the healthy(p<0.05).There were lots of inflammatory cells infiltrating in gingivitis and periodontitis with positive CBS and CSE staining increased.And the level of H2S and its producing rate were not changed significantly in gingivitis,moderate periodontitis and severe periodontitis as compared to the control.(p>0.05)Conclusion:When in periodontal inflammation the expression of H2S enzymes(CBS and CSE)increased.That may imply a role of endogenous H2S in periodontitis.Part 3:The effects of H2S on experimental periodontitis in ratsAim:to observe the effect of H2S on experimental periodontitis in SD rats.Method:To build experimental periodontitis in SD rats by ligatures.And rats were divided into four groups randomly.Inject intraperitoneally with NaHS(0.56?mol/L and 0.056?mol/L groups,a donor of H2S),D,L-propargylglycine(PAG,an inhibitor of CSE)and NaCl(as a control)respectively.And the effect of H2S on periodontitis was estimated by inflammation in periodontal tissue.Results:Alvoelar bone resorption and inflammatory cells infiltration were observed in all rats.That means succeed of building experimental periodontitis.But there was no obvious difference among these four groups(p>0.05).Inhabiting CSE increased CBS mRNA in rat gingiva(p<0.05).The other indicators in H2S/CBS-CSE system in four groups had no significant change.(p>0.05)Conclusion:Suppression of CSE could increase gene expression of CBS.But drugs had no effect on alveolar bone resorption,periodontal inflammatory index and other indicators of H2S/CBS-CSE system.Part 4:The mechanism of effect of H2S on experimental periodontitis in ratAim:To explore the mechanism of the effect of H2S on experimental periodontitis in rat.Method:To build experimental periodontitis in SD rats as descreded in part 3.To exam the gene expression of pro-and anti-inflammatory factors in rat gingiva by PCR.To exam the expression of pp38/p38 and ppERKl/2/pERK1/2 by western blot.Results:After giving PAG,gene expression of IL-1?,TNF-?,MMP-8 and MMP-9 increased as compared with NaCl group(p<0.05).And IL-10 increased too(p<0.05).In 0.056NaHS group,gene expression of TNF-? decreased while IL-10 increased(p<0.05).The phosphorylation of p38 and ERK1/2 were not changed in NaHS and PAG groups when compared with NaCl group(p>0.05).The relationship with p38 and ERK1/2 signaling pathway was not found in this study.Concluding:Both pro-inflammatory and anti-inflammatory reactions were stimulated by suppressing of endogenous H2S.And low concentration of H2S would be anti-inflammation.These results further indicated an anti-inflammatory effect of endogenous H2S in periodontitis.