| Cardiovascular diseases are the major disease threatening humans’ health,and its incidence is increasing gradually with the development of society andthe change of life style.Moreover, ischemic heart disease has been a majorfocus in cardiovascular diseases, which can lead to arrhythmia,myocardialinfarction.Therefore,how to relieve effectively the injury caused bymyocardial ischemia is a hotspot of medical research at present. The ligationof coronary artery is the most popular method establishing the acutemyocardial ischemia model，which is widely used in studies on mechanism ofmyocardial ischemia. The method of Langendorff isolated heart perfusion isone of the most useful experimental methods in cardiovascular area，withstabilization, reliability and convenient operation.After nitric oxide（NO） and carbon monoxide（CO）, hydrogen sulfide（H2S）was discovered to be the third endogenous signaling gasotransmitter in the1990s. The endogenous hydrogen sulfide is existing widely in the tissues ofmammals and playing a key role in some physiological and pathologicalprocesses.It could be shown that the hydrogen sulfide could antagonizeischemic reperfusion injury and display protective function. However,associations between hydrogen sulfide and myocardial ischemia injury are lesswell established, and its pathogenesis is still unclear. In this experiment, themyocardial ischemia injury model was established using the method ofcoronary artery ligature.The effects of hydrogen sulfide on myocardialischemia injury were observed in isolated hearts in rats and explored thepossible mechanisms from the range of oxidative stress, inflammatory factorand apoptosis.Part1Change of endogenous hydrogen sulfide（H2S）/cystathionine-γ-lyase （CSE） in isolated ischemic rat hearts Objective: To observe the changes of H2S/CSE in isolated ischemichearts in rats at different time points.Methods: The myocardial ischemia injury model was established by theligation of coronary artery.160male SD rats, weighing270±20g,were dividedinto six groups randomly: sham group（n=80,16in each time point）, ischemia30min group（n=16）, ischemia1h group（n=16）,ischemia2h group（n=16）,ischemia3h group（n=16）, ischemia4h group（n=16）.The left anteriordescending coronary artery were ligated in rats of the ischemia group,but therats in the sham group were only threaded without ligation.The hemodynamicparameters,such as the left ventricular developed pressure （LVDP）,±dp/dtmaxand coronary arterial flow（CF）,were respectively recorded to evaluate thecardiac function. The content of H2S and the activity of CSE in cardiac tissuewere respectively detected.Infarct volumes in isolated rat hearts weredetermined by dual staining with Evans-blue and TTC.Results:1There were no statistically significant differences in baselinecardiodynamic data among the experimental groups.Compared with those ofthe sham group,LVDP,±dp/dtmaxand CF were significantly decreased at30min、1h、2h、3h、4h after ischemia（P<0.01）.2There were no statistically significant differences in the content of H2Sand the activity of CSE in cardiac tissue in the sham group from30min to4hafter stabilization. Compared with those of the sham group, there were nostatistically significant differences in the content of H2S and the activity ofCSE in cardiac tissue at30min after ischemia.But during1h to4h afterischemia, the content of H2S and the activity of CSE in cardiac tissue weresignificantly decreased compared with those of the sham control group（P<0.05or P<0.01）.3Compared with those of the sham group, the infarcted volumes weregreatly increased from1h to4h after ischemia （P<0.01）.Conclusion: After ischemia for2h,the content of H2S and the activity ofCSE in cardiac tissue were decreased with larger infarct volumes. It could be suggested that H2S and CSE were involved in myocardial ischemia injury inisolated hearts in rats.Part2Effects of hydrogen sulfide（H2S） on acute myocardial ischemicinjury in isolated hearts in ratsObjective: To observe the effect of hydrogen sulfide （H2S） on cardiacfunction,endogenous hydrogen sulfide/cystathionine-γ-lyase and myocardialinfarct volumes in acute myocardial ischemia injury in isolated hearts in rats.Methods: The myocardial ischemia injury model was established by theligation of coronary artery.Eighty male SD rats, weighing270±20g,weredivided into five groups randomly: sham group（n=16）, model group（n=16）,low dose group of NaHS（n=16）,middle dose group of NaHS（n=16）,high dosegroup of NaHS（n=16）.The left anterior descending coronary artery wereligated in rats of the ischemia group,but the rats in the sham group were onlythreaded without ligation.The normal perfusate was replaced with NaHSperfusate（5μmol/L，10μmol/L，20μmol/L）accordingly in low dose group ofNaHS,middle dose group of NaHS and high dose group of NaHS at2h afterischemia.The hemodynamic parameters,such as the left ventricular developedpressure（LVDP）,±dp/dtmaxand coronary arterial flow（CF）,were respectivelyrecorded to evaluate the cardiac function.The content of H2S and the activityof CSE in cardiac tissue were detected respectively at4h after ischemia.Infarct volumes in isolated rat hearts were determined by dual staining withEvans-blue and TTC.Results:1Compared with those of the sham group, LVDP,±dp/dtmax and CFwere significantly decreased in the model group（P<0.01）.Compared withthose of the model group, LVDP,±dp/dtmaxand CF were significantly increasedin the NaHS low, middle and high dose groups （P<0.05or P<0.01）.2Compared with those of the sham group, the content of H2S and theactivity of CSE in cardiac tissue were significantly decreased in the modelgroup（P<0.01）.Compared with those of the model group, the content of H2Sand the activity of CSE in cardiac tissue were significantly increased in the NaHS low, middle and high dose groups（P<0.05or P<0.01）.3Compared with those of the sham group,the infarct volumes wassignificantly increased in the model group（P<0.01）. Compared with those ofthe model group, the infarct volumes was significantly decreased in NaHSmiddle and high dose groups（P<0.01）.Conclusion: Administration of NaHS could enhance the activity of CSE,increase the content of H2S, and reduce infarct volumes. It could be suggestedthat H2S has cardioprotective effects on acute myocardial ischemic injury.Part3Effects of hydrogen sulfide（H2S） on oxidative stress in acutemyocardial ischemia injury in isolated hearts in ratsObjective: To investigate the effects of H2S on oxidative stress inmyocardial ischemia injury in isolated hearts in rats.Methods: The myocardial ischemia injury model was established by theligation of coronary artery.Forty male SD rats,weighing270±20g,weredivided into five groups randomly: sham group（n=8）, model group（n=8）, lowdose group of NaHS（n=8）,middle dose group of NaHS（n=8）,high dose groupof NaHS（n=8）.The left anterior descending coronary artery were ligated in ratsof the ischemia group,but the rats in the control group were only threadedwithout ligation.The normal perfusate was replaced with NaHS perfusate（5μmol/L,10μmol/L,20μmol/L） accordingly in low dose group of NaHS，middle dose group of NaHS and high dose group of NaHS at2h after ischemia.The content of malondialdehyde（MDA）,the activities of lactate dehydrogenase（LDH）,superoxide dismutase（SOD） and glutathione peroxidase（GSH-PX） wererespectively measured by spectrophotometry. The ultrastructural alterations ofmyocardium were observed by electric microscope.Results:1There were no statistically significant differences in the activity of LDHin perfusate among the experimental groups. Compared with those of the shamgroup, the activity of LDH in perfusate was significantly increased in themodel group（P<0.01）. Compared with those of the model group, the activityof LDH in perfusate was significantly decreased in NaHS low, middle and high dose groups （P<0.01）.2The content of MDA in cardiac tissue was significantly increased,andthe activities of SOD and GSH-PXin cardiac tissue were significantlydecreased in model group compared with those of sham control group（P<0.01）.The content of MDA and the activity of SOD and GSH-PXin cardiactissue were significantly increased in the NaHS low, middle and high dosegroups compared with those of model group（P<0.05or P<0.01）.3The ultrastructure of the myocardial cells exhibited regularmitochondria with uniform size,complete mitochondrial cristae,and intactnuclear membrane in the sham group,but in the model group,the myocardialcells were characterized by mitochondrial swelling,disappearance ordeformation of mitochondrial cristae,disruption of nuclear membrane,andnuclear condensation.Compared with those of the model group,NaHS low,middle and high dose groups had slighter pathological change in myofi-laments, mitochondria and nucleus.Conclusion: Administration of NaHS could improve the injury oforganelles（mitochondria,nucleus,myofilament,etc）,reduce the activity of LDH,decrease the level of MDA and increase the activities of SOD and GSH-PX. Itcould be suggested that H2S has certain protective effect on acute myocardialischemic injury and the mechanism may be related to antioxidation.Part4Effects of hydrogen sulfide（H2S） on inflammatory cytokines inacute myocardial ischemia injury in isolated hearts in ratsObjective: To investigate the effects of H2S on inflammatory cytokinesin acute myocardial ischemia injury in isolated hearts in rats.Methods: The myocardial ischemia injury model was established by theligation of coronary artery.Forty male SD rats,weighing270±20g,weredivided into five groups randomly: sham group（n=8）, model group（n=8）, lowdose group of NaHS（n=8）,middle dose group of NaHS（n=8）,high dose groupof NaHS（n=8）.The left anterior descending coronary artery were ligated in ratsof the model group,but the rats in the sham group were only threaded withoutligation.The normal perfusate was replaced with NaHS perfusate（5μmol/L, 10μmol/L,20μmol/L） accordingly in low dose group of NaHS,middle dosegroup of NaHS and high dose group of NaHS at2h after ischemia.TNF-αmRNA,IL-1βmRNA,IL-6mRNA,IL-10mRNA,ICAM-1mRNA wererespectively detected by RT-PCR.The expression of the nuclear factor-κB（NF-кB） in myocardial tissue was detected by Western blotting.Results:1Compared with those of the sham group,the expression of TNF-α,IL-1β,IL-6and ICAM-1mRNA in cardiac tissue were significantly increasedin the model group（P<0.01）, the expression of IL-10mRNA in cardiac tissuewere significantly decreased in the model group（P<0.01）. Compared withthose of the model group,the expression of TNF-αmRNA,IL-1βmRNA,IL-6mRNA and ICAM-1mRNA in cardiac tissue were significantly decreasedin the NaHS low, middle and high dose groups（P<0.05or P<0.01）, theexpression of IL-10mRNA in cardiac tissue were significantly increased inthe NaHS middle and high dose groups （P<0.01）.2NF-кB hardly express in myocardial tissue in the sham group.But theexpression of NF-кB was significantly increased in nuclei in the model group（P<0.01） compared with that of the sham group. The expression of NF-кB wassignificantly decreased in NaHS middle and high dose groups compared withthat of the model group（P<0.05or P<0.01）.Conclusion: Administration of NaHS could inhibit the activation ofNF-κB,reduce the expression of TNF-α,IL-1β,IL-6and ICAM-1,and increasethe expression of IL-10.It could be suggested that H2S has certain protectiveeffect on acute myocardial ischemic injury and the mechanism may be relatedto inhibiting inflammatory reaction.Part5Effects of hydrogen sulfide（H2S） on apoptosis in acute myocardialischemia injury in isolated hearts in ratsObjective: To investigate the effects of H2S on cardiomyocyte apoptosisin acute myocardial ischemia injury in isolated hearts in rats.Methods: The myocardial ischemia injury model was established by theligation of coronary artery.Forty male SD rats,weighing270±20g were divided into five groups randomly: sham group（n=8）, model group（n=8）, lowdose group of NaHS（n=8）,middle dose group of NaHS（n=8）,high dose groupof NaHS（n=8）.The left anterior descending coronary artery were ligated in ratsof the model group,but the rats in the sham group were only threaded withoutligation.The normal perfusate was replaced with NaHS perfusate（5μmol/L,10μmol/L,20μmol/L）accordingly in low dose group of NaHS,middle dosegroup of NaHS and high dose group of NaHS at2h after ischemia.Histopathological changes of myocardial tissue were observed by hematoxylineosin（HE） staining. The cardiomyocytic apoptosis was assayed by terminaldeoxynucleotidyl transferase nick-end labeling（TUNEL） method. Caspase-3and Cyt-c protein expression were analysised using Western blotting.Results:1The myocardial cells were normal in the sham group,but there wereserious disorder,fracture of myocardial cell,nuclear condensation andinflammatory cell infiltration in the model group in the light microscope.Necrotic degeneration and pathologic changes of myocardiocytes in the NaHSlow, middle and high dose groups were significantly milder than those of themodel group.2According to TUNEL results,compared with that of the sham group,theapoptotic rate of cardiomyocytes was significantly increased in the modelgroup（P<0.01）.Compared with that of the model group, the apoptotic rate ofcardiomyocytes was significantly decreased in NaHS middle and high dosegroups（P<0.01）.3Western blotting showed that the expression of Caspase-3and Cyt-cwere significantly increased in the model group compared with that of thesham group（P<0.01）.The expression of Caspase-3was significantly decreasedin NaHS middle and high dose groups compared with that of the ischemiagroup（P<0.05or P<0.01）.The expression of Cyt-c was significantly decreasedin NaHS low, middle and high dose groups compared with that of the ischemiagroup （P<0.05or P<0.01）.Conclusion: Administration of NaHS could decrease myocardial cells apoptotic,inhibit the expression of Cyt-c and Caspase-3.It could besuggested that H2S has certain protective effect on acute myocardial ischemicinjury and the mechanisms may be related with its antiapoptotic action．... |
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