| As a major chemical warfare agent since its introduction on the battlefield,sulfur mustard（2,2’-dichlorodiethyl sulfide,SM）has been applied in a series of conflicts in the past century and has produced a large number of casualties and serious medical problems.SM was produced and stockpiled by many countries and is likely the most widely equipped chemical warfare agent in foreign armies as well as one of biggest chemical terrorism threat that China are facing.Therefore,study on prevention and treatment measures against SM is of strategically importantance for national security and public health.A major target of SM is the lungs,and leading reason for mortality following SM poisoning is due to pulmonary injuries and secondary infections.Oxidative stress is considered as the initial and vital process for the damages.As a thiol/sulfur compound,hydrogen sulfide（H2S）exhibits well-characterized antioxidant properties,which have been extensively investigated and are regarded as the principal mechanism underlying its beneficial effects.H2S can improve pulmonary fibrosis,lung ischemia-reperfusion injury,and acute lung injury by scavenging the reactive oxygen species（ROS）,increasing glutathione,and promoting the translocation of the nuclear transcription factor Nrf2.As reported in the literature,H2S plays an important role in maintaining the normal function of lungs.In our study,we found that H2S participates in regulating SM-induced lung injuries and can reduce the lung oxidative stress level induced by SM in mouse lungs and lung cells.Further experiments showed that H2S can promote S-sulfhydration of Keap1 and enhance dissociation of Nrf2 from Keap1 as well as translocation of Nrf2 to the nucleus.1.The effects and mechanism of H2S in SM-induced lung injuries in miceThe concentrations of H2S were traced using RHP-2.The result revealed a gradual decrease of H2S starting from day 1 and on day 3,there has been a significant drop of H2S compared to the control group（P?0.01）.Western Blot was used to monitor the level of H2S-generating enzymes CBS and CSE.The result showed no significant changes in the protein levels of CBS from day 1 to day 4,but the protein levels of CSE was significantly decreased compared to the control group,especially on day 3（P<0.01）.To evaluate whether or not the changes in endogenous sulfide concentrations were the cause or merely a consequence of SM-induced lung injury,experiments were designed to change endogenous sulfide concentrations in mice lungs.Administration of NaHS targeting endogenous sulfide concentrations resulted in a significant increase in SM-treated mice.In contrast,endogenous sulfide concentrations were reduced significantly in DL-propargylglycine（PPG,a CSE inhibitor）treated mice.Lung tissues of SM-treated mice on day 3 were stained with H&E for a histopathological analysis.The results showed that SM-treated mice exhibited histopathological damage in lungs characterized by diffuse interstitial edema,alveolar thickening,marked decreases in alveolar air space,and lung recruitment of leukocytes.These damages were significantly attenuated by NaHS treatment and aggravated by PPG treatment.Measurement of lung wet/dry weight ratio and bronchoalveolar lavage（BAL）fluid protein demonstrated that treatment with NaHS resulted in a significant protection against SM-induced lung injury as revealed by the dramatically lowered W/D ratio and BAL fluid protein.We also assess the oxidant stress in the lung tissues.There was a marked increase of both H2O2 content and MDA content in SM induced mice lungs.Compared to this,the H2O2 content and MDA content dropped in the group treated by NaHS and SM,but increased significantly in the group treated by SM and PPG.Meanwhile,both the ratio of GSH/GSSG and activity of SOD dropped in SM treated group,but there have been a marked increase in the ratio of GSH/GSSG and activity of SOD among SM and NaSH treated group,but in SM and PPG treated group,both GSH/GSSG ration and SOD activity dropped prominently.We also measured the mRNA levels of five Nrf2 downstream target cytoprotective genes,namely,heme oxygenase-1（HO-1）,NAD（P）H dehydrogenase quinone 1（NQO1）,glutamate cysteine ligase composed of catalytic（GCLC）,glutathione reductase（GR）,and glutamate cysteine ligase composed of modifier（GCLM）.Possible roles of H2S in SM induced lung injuries in mice were discussed.We found that NaHS could enhanceNrf2nuclear translocation,increase the mRNA levels of HO-1,NQO1,GCLC,GR,GCLM expression,alleviate the oxidant stress,while in contrast,PPG could increase the oxidant stress.These results suggested Nrf2 participate in the process of H2S regulating SM induced lung injuries.2.The protective effects and mechanism of H2S against SM-induced oxidative damage in BEAS-2B and MRC-5 cellsFurther,we investigated the protective effects of H2S in cells.CCK-8 results shown that SM does-dependently decreased the survival rate of human lung epithelial cells（BEAS-2B）and human fetal lung fibroblast Cells（MRC-5）.Notably,NaHS（70μM）treatment attenuated SM-induced reduction of BEAS-2B and MRC-5 cell viability.Taken together,these results demonstrated that NaHS inhibits SM-induced cell death.We applied the probe NIR-HS to image endogenous sulfide concentration.ROS analysis was carried out to detect oxidative stress damages.Results showed that cells with the administration of SM exhibited fainter fluorescence emission than that from control cells,suggesting the decreased endogenous levels of H2S in SM exposed cells.SM-treated cells with NaHS pretreatment showed stronger fluorescence and lower ROS than that of the SM group.That indicated that treatment of NaHS could attenuate the decreased endogenous levels of H2S in SM exposed cells.After we constructed the cells with CSE knockdown,NaHS treated cells showed weaker fluorescence than the control cells,their endogenous H2S levels dropped,and ROS levels increased.When CSE was depleted from SM-treated cells and the cells then were incubated with NaHS,almost no fluorescence emission was observed,and there was a significant raise of ROS in the cells at the same time.The results indicated that NaHS could increase the endogenous H2S levels through CSE,and regulate SM-induced oxidative damages in cells.We examined whether Nrf2 was involved in the protective effect of H2S on SM-induced oxidative stress in cells.Among normal cells,compared to SM treated cells,SM+NaHS treated cell nucleus showed a higher expression of Nrf2,in particular,a higher expression of HO-1 and NQO1,at the same time,while ROS was reduced.SM+NaHS treated cells whose Nrf2 protein was redeced by transfection with Nrf2 shRNA showed a marked reduce in Nrf2 expression in cytoplasm and nucleus compared to the previous normal cells,in particular,a significant reduction in HO-1 and NQO1 expression,and increased ROS level.These results indicated that when Nrf2 was silenced,NaHS could no longer function in SM exposed cells.Nrf2 is required in the protective effects of H2S against SM-induced oxidative damage.Further exploration of S-sulfhydrated Keap1 protein expression showed that SM treated cells possess lower S-sulfhydrated Keap1 protein expression;S-sulfhydrated Keap1protein expression was higher in SM+NaHS treated cells than in SM-treated cells.Moreover,after applying DTT（an inhibitor of S-sulfhydration）,S-sulfhydrated Keap1protein expression was remarkably lowered in SM+NaHS+DTT treated cells than in SM+NaHS treated ones,so was Nrf2 expression in nucleus.These findings suggest that DTT could block the protective effects of H2S on SM treated cells,and H2S could promote the translocation of Nrf2 through S-sulfhydration of Keap1 to perform antioxidative stress function.To conclude,the findings in this thesis include:endogenous sulfide concentrations were reduced in SM-induced lung oxidative stress injuries.Treatment of NaHS in SM-treated mice could increase endogenous sulfide concentrations,promote the expression of Nrf2 in nucleus,increase the mRNA expression of HO-1,NQO1,GR and GCLM,as well as the proteins of HO-1 and NQO1,resulting in alleviation of SM-induced lung oxidative stress injuries.Experiments on the cells showed NaHS could increase endogenous sulfide concentrations through CSE and promote translocations of Nrf2 in nucleus through S-sulfhydration of Keap1. |
