| Coronary angioplasty is probably the most important invasive therapeutic tool developed in cardiology in the last 30 years.As we know,it addresses to obtain reperfusion in ischemic heart and especially in acute myocardial infarction.Reperfusion in large coronaries is obtained in almost all cases.But reperfusion in the myocardium is less,which is called a no-reflow phenomenon.It can offset or at least partially offset the beneficial effects of PCI,and is an independent predictor of sudden cardiac death.The mechanism of NR is mainly microcirculation disorder,which is related to the aggregation and adhesion of leukocytes.Endothelial cell damage and functional changes play an important role in it.Pyroptosis is a new type of programmed cell death accompanied by an inflammatory response.It is involved in the pathological process of many cardiovascular diseases including atherosclerosis,diabetic cardiopathy and myocardial infarction.However,it has not been reported whether it is involved in myocardial NR.Hydrogen sulfide?H2S?is the third gas signal molecule after carbon monoxide and nitric oxide being discovered.It has the effects of inhibiting inflammation,relaxing blood vessels and anti-oxidative stress.Our previous studies found that exogenous H2S can limit the area of myocardial infarction after ischemia-reperfusion in rats,and has myocardial protection,but whether it is achieved by inhibiting pyroptosis has not been reported.Therefore,our research aims to investigate whether exogenous H2S can inhibit myocardial NR,and further explore whether its protective effect is related to the inhibition of endothelial cell pyroptosis,then provide new ideas and methods for clinical prevention and treatment of NR.Part 1: Effect and mechanism of H2 on myocardial no-reflow in ratsObjective: To observe the effect of hydrogen sulfide on myocardial no-reflow?NR?in rats and to investigate whether the mechanism is related to pyroptosis.Methods: Based on the successful establishment of a rat myocardial NR model,18 SD male rats were randomly divided into 5 groups:Sham group: LAD was only perforated without ligation;NR group: ligation at LAD caused myocardial ischemia for 1 hour,followed by loosening and reperfusion for 3 hours;H2 group: Na HS?40?mol/L,ip?+NR group;PPG+NR group: PPG?50mg/kg,ip?+NR group;PPG+H2+NR group.We used modified methylene blue method and microplate reader to measure plasma H2 concentration.We used Evan's blue staining to detect myocardial ischemic area,thioflavin S staining to detect myocardial NR area,ECG ST-segment decline rate to determine myocardial NR status,and H/E staining of myocardial tissue sections to detect inflammatory cells infiltration and myocardial damage.We detect the expression level of pyroptosis marker protein in myocardium through western blot.We observe the endothelial cell pyroptosis in myocardial tissue sections of NR group and H2 group via immunofluorescence co-localization.Results: 1.Compared with the NR group,the concentration of plasma H2 in H2+NR group was significantly increased?P<0.01?,while the PPG+NR group is the opposite,and the plasma H2 concentration in the PPG+H2 group were higher than that of PPG+NR group?P<0.05?.2.Compared with the NR group,the NR area of the rat myocardium in the H2+NR group decreased?P<0.05?,the ST segment drop rate was significantly increased?P<0.01?,and the degree of myocardial inflammatory cell infiltration and injury were all reduced,and the PPG+NR group was the opposite.3.Compared with the NR group,the expressions of NLRP3?P<0.05?and pro-caspase-1?P<0.01?,two marker proteins of pyroptosis,in the myocardium of no-reflow area in the H2+NR group were reduced.The expression of pro-caspase-1 in the PPG+NR group was significantly increased?P<0.05?,while the expression of NLRP3 in the PPG+NR group showed a trend of increase,but the difference was not statistically significant?P>0.05?.The expression levels of two pyroptosis marker proteins in rat myocardium of PPG+H2+NR group were higher than that of PPG+NR group?P<0.05?.4.Immunofluoresence showed that compared with that of the control group,the endothelial cell pyroptosis of rats in the H2+NR group was reduced.Conclusion: H2 can reduce myocardial no-reflow in rats,and its protective effect is related to the reduction of pyroptosis.Part 2: Effect of H2 on hypoxia-reoxygenation induced endothelial cell pyrolysisObjective: To observe the effects of H2 on hypoxia-reoxygenation?H/R?induced endothelial cell pyroptosis,and to explore the mechanism of pyroptosis through the classical pathway dependent on caspase-1 activation.Methods: Based on the successful establishment of an endothelial cell H/R model,in order to clarify the effect of H2 on endothelial cell H/R,the endothelial cells were divided into 8 groups: H/R group,12.5 ?mol/L Na HS+H/R group,25?mol/L Na HS+H/R group,50?mol/L Na HS+H/R group,75?mol/L Na HS+H/R group,100?mol/L Na HS+H/R group,150 ?mol/L Na HS+H/R group and 200 ?mol/L Na HS+H/R group.We observe the cell morphology through a microscope,and use a microplate reader to detect the activity of LDH in the cell culture fluid to observe the effects of different concentrations of Na HS on endothelial cell H/R injury;In order to investigate whether H/R can induce endothelial cell pyroptosis,we divided the endothelial cells into 3 groups: Control group,Hypoxia group and H/R group.Western blot was used to detect the protein expression of pro-caspase-1,ASC,NLRP3,GSDMD and IL-18,and ELISA was used to detect the release of IL-1?.Then,endothelial cells are divided into four groups to investigate whether H2 can inhibit pyroptosis of endothelial cells induced by H/R: H/R group,Na HS+H/R group,PPG+H/R group and Na HS+PPG+H/R group.Western blot was used to detect the protein expression of pro-caspase-1,ASC,NLRP3,GSDMD,and IL-18.Finally,we used ELISA to detect the effect of H2 on the secretion of adhesion factor ICAM-1 by endothelial cells?the experimental group is the same as above 8 groups?.Results: 1.Compared with the control group,low concentration of H2 can promote endothelial cells to maintain their normal morphology after H/R injury and reduce the release of LDH?P<0.01?.2.Compared with the hypoxia group,NLRP3?P<0.01?,GSDMD?P<0.05?,pro-caspase-1?P<0.01?,ASC?P<0.05?and IL-18?P<0.05?protein expression levels increased significantly in the H/R group,and the IL-1? release by endothelial cells increased obviously?P<0.01?.It was shown that H/R induced endothelial cell pyroptosis.3.Compared with the H/R group,the protein expression levels of IL-18?P<0.01?,pro-caspase-1?P<0.01?,GSDMD?P<0.05?,NLRP3?P<0.05?,and ASC?P<0.05?in the H2+H/R group were reduced.In contrast,the protein expressions of IL-18?P<0.05?,ASC?P<0.05?and pro-caspase-1?P<0.01?in the PPG group were increased,while GSDMD and NLRP3 showed a trend of increase,but the difference was not statistically significant?P>0.05?.The expression levels of pro-caspase-1?P<0.01?and other pyroptosis marker proteins?P<0.05?of PPG group were significantly higher than those of PPG+H2 group.It was shown that H2 can inhibit H/R-induced endothelial cell pyroptosis.4.Compared with the control group,low concentration of H2 significantly inhibited the release of the adhesion molecule ICAM-1 after H/R injury in endothelial cells?P<0.01?.Conclusion: H2 can inhibit H/R-induced endothelial cell pyroptosis. |
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