| Takotsubo cardiomyopathy?TCM?is an acute cardiac syndrome and clinical symptoms which are similar to acute coronary syndromes.About 2% of patients diagnosed coronary syndromes are Takotsubo cardiomyopathy.Mortality of Takotsubo cardiomyopathy is about 1%-4.2%.Variant forms of left ventricular of dysfunction of TCM have been reported,including wall-motion abnormalities,such as mid-ventricular ballooning and apical segments with sparing of the basal segments,such as Tako-tsubo,a Japanese octopus trap.TCM is typically occurs in older women after emotional or physical stress,and symptoms include acute chest pain,dyspnoea,syncope,nausea,vomiting and difficulty breathing.TCM patients have acute cardiac complications such as acute CHF,cardiac shock,ventricular septal defect,acute pulmonary edema,respiratory failure,severe ventricular arrhythmias and cardiac rupture and other serious complications.Takotsubo syndrome is an acute cardiac syndrome characterized by transient left ventricular dysfunction affecting more than one coronary artery territory,in the presence of unobstructed coronary arteries.Due to the gradual understanding and attention of TCM,the number of TCM cases found in clinical continued to be increased,but its pathogenesis,pathological and standardized treatment is still lack of adequate proof.So the further study of the TCM has important clinical significance and social value.Hydrogen sulfide?H2S?has been confirmed to be the third gaseous signal molecule similar to the other gastransmitters,nitric oxide?NO?and carbon monoxide?CO?.As a biologically active mediator,H2 S exhibits potent cardiovascular protective effect.It was reported that endogenous H2 S level is markedly decreased during various ischemia models and excessive stimulation of ?-adrenergic receptor.Exogenous H2 S or increased production of endogenous H2 S reduced myocardial infarction area significantly.Studies have shown that H2 S elicits cardioprotective effects against ischemia in vitro and in vivo via various signaling mechanisms including opening of ATPsensitive potassium?KATP?channels,activation of PKC,elevation of nitric oxide production,inhibiting renin-angiotensin-aldosterone system,and preservation of mitochondrial function.The mechanism of protective effect of H2 S on cardiovascular disease is still unclear,this paper investigate and explore the effect of hydrogen sulfide on Takotsubo cardiomyopathy and the underlying mechanisms via Takotsubo cardiomyopathy animal model and H9c2 cells.This study is composed of three parts as follows.Part one Effects of exogenous hydrogen sulfide on cardiac function in rat models with Takotsubo cardiomyopathyObjective: To investigate the effect of exogenous hydrogen sulfide on cardiac function of TCM modelMethods: 1 TCM models were induced by administering a single i.p.50 mg/kg dose of the ?-adrenergic agonist,isoprenaline?ISO?.2 Power Lab?ML4818?Data Acquisition System?AD Instruments Australia?was used to record the left ventricular end diastolic pressure?LVEDP?,left ventricular systolic pressure?LVSP?,maximum contraction velocity?+dp/dtmax?,maximum relaxation velocity?-dp/dtmax?.The rats were given ISO?50mg/kg,i.p.?or Na HS pre-treatment at different dose?10,50,100,or 200 ?mol/kg,i.p.?.Na HS 100 ?mol/kg was chosen for subsequent experiments.3 Echocardiography was performed using a Visual Sonics 2100 VEVO imaging station to measure the left ventricular ejection fraction?LVEF?and left ventricular fractional shortening?LVFS?.4 The effects of Na HS on myocardial enzymes of TCM rats.5 H&E staining to detect myocardial histomorphological changes in TCM rats.6 Detect the concentration of H2 S in plasma and myocardium and the expression of H2S-producing enzymes in TCM rats with or without Na HS.Results: 1 Compared to the control group,ISO decreased LVSP,+dp/dtmax and –dp/dtmax and increased LVEDP significantly.2 H2 S levels reduced in plasma and myocardial tissue in the ISO group compared to the control group,myocardial CSE and 3-MST were significantly lower than those in the control group.3 Compared to the ISO group,Na HS administration increased LVSP,±dp/dtmax,but decreased LVEDP.4 Echocardiography showed that ISO reduced LVEF,LVFS significantly,caused ventricular wall motion irregular.Exogenous hydrogen sulfide can restore LVEF,LVFS and the irregular wall motion in TCM rats.5 Na HS can decrease CK,CK-MB,LDH and AST activity in TCM rats.6 The myocardial H&E staining showed that the myocardial fibers were intact,arranged neatly,the structure was clearer,and no cell necrosis.7 Compared to the ISO group,Na HS restored H2 S levels in plasma and myocardial tissue,upregulated myocardial CSE and 3-MST expression.Conclusions: 1 TCM rats exhibited left ventricular dysfunction index,irregular ECG and echocardiography.2 Na HS pretreatment can improve left ventricular function in TCM rats,improve LVEF and LVFS,and decrease the irregular wall motion.3 The concentration of H2 S in plasma and myocardial tissue was decreased in TCM rats,which indicated that hydrogen sulfide played an important role in the pathogenesis and pathological damage of Takotsubo cardiomyopathy.Part two Exogenous H2 S can depress myocardial oxidative stress in TCM rats via inhibiting NADPH oxidaseObjective: To explore the mechanism of H2 S to protect the cardiac function of TCM ratsMethods: 1 Myocardial superoxide anion levels was observed by DHE staining.2 H2O2,MDA,SOD and GSH kits were used to detect the oxidative stress level in plasma and myocardial tissues.3 Myocardial NOX2,NOX4,p67 protein expression were detected by Western blot.4 The changes of myocardial apoptosis were observed by TUNEL staining.5 Myocardial expression of Bax and Bcl-x L protein were detected by Western blotResults: 1 DHE staining showed that the levels of superoxide anion in myocardial tissue of TCM rats were significantly increased,and pretreatment with Na HS could reduce the superoxide anion in myocardial tissue of TCM rats.2 H2O2 level in plasma and myocardium in TCM rats were increased.Na HS pretreatment could decrease the level of H2O2.3 MDA level in plasma and myocardium in TCM rats were increased.Na HS pretreatment could decrease the level of MDA.4 GSH level in plasma and myocardium of TCM rats were decreased.Na HS pretreatment did not increase the level of GSH in plasma and myocardium.5 SOD level in plasma and myocardial tissue of the TCM rats had no change.Na HS pretreatment had no effect on SOD level.6 The expression of NOX4 and p67 protein in myocardial tissue were increased in TCM rats,and Na HS pretreatment could reduce the expression of NOX4 and p67 protein.7 TUNEL staining showed that cardiomyocyte apoptosis were significantly increased in TCM rats,and Na HS could reduce the cardiomyocyte apoptosis.8 The expression of Bcl-x L protein was decreased,Bax protein was increased,the Bax/Bcl-x L ratio was increased in TCM rats.Na HS can enhance the expression of Bcl-x L,inhibit the expression of Bax,and decreased the Bax/Bcl-x L ratio.Conclusions: 1 Oxidative stress is involved in the pathological process of Takotsubo cardiomyopathy,Na HS can reduce the level of oxidative stress.2 Na HS reduced the oxidative stress of Takotsubo cardiomyopathy via inhibiting the expression of NOX4,and p67 protein.3 Na HS reduces cardiomyocyte apoptosis in Takotsubo cardiomyopathy.Part three H2 S alleviated ISO injury via p38MAPK/NOX4/ROS pathway in H9c2 cellObjective:To explore protective mechanism of H2 S on H9c2 cellsMethods: 1 Cell culture H9c2 cells were cultured with the DMEM culture medium containing 10% fetal bovine serum?FBS?,penicillin 100 U/m L,streptomycin 100 ?g/m L at 37? in humidified incubator with 5% CO2.2 Lactate dehydrogenase?LDH?release assay Cells were seeded in 96-well cell culture plates with 5000 cells per well,and continually incubated with at various concentrations?10?50?100?200 ?M?for 2h to determine the minimal dose of ISO necessary to induce injury.The dose of ISO 100 ?M was chosen for subsequent experiments.The H9c2 cells pretreated with Na HS at various concentrations?10?50?100?200 ?M?for 30 min,then incubated with ISO 100 ?M for 2 h,to determine the minimal dose of Na HS necessary to protect ISO injury.The dose of Na HS 50 ?M was chosen for subsequent experiments.LDH release assay was used to detect the absorbance at 490 nm wavelength.The percentage of LDH release was calculated according to the instructions of the kits.3 Cell viability measured by CCK8 assay Cells were seeded in 96-well cell culture plates respectively with 5000 cells per well.After adherence,cells were continually incubated with ISO 100 ?M for 2 h,with or without pretreatment of Na HS 50 ?M for 30 min.CCK8 assay was used to detect the optical density?OD?of the cell at 450 nm wavelength and cell growth curves were drawn.4 Superoxide anion levels was observed by DHE staining.5 NOX4,p38,p-p38,JNK,p-JNK,ERK,p-ERK expression in H9c2 were detected by Western blot.Results: 1 LDH release assay showed that different concentration of ISO could increase the cytotoxicity of ISO to H9c2 cells,and the injury of ISO 100 ?M was the most obvious,and ISO 100 ?M was used for the following experiments..2 LDH release assay showed that different concentration of Na HS could reduce the damage of ISO to H9c2 cells,and the protective effect of Na HS50 ?M was the most obvious,and Na HS 50 ?M was used for the following experiments.3 CCK8 experiments showed that Na HS pretreatment could increase the viability of H9c2 treated with ISO.4 ISO increased DHE fluorescence in H9c2 cells,and pretreatment with Na HS could reduce DHE fluorescence in H9c2 cells.5 The expression of NOX4 in H9c2 were increased in ISO group,and Na HS pretreatment could reduce the expression of NOX4.6 Na HS pretreatment could decrease the expression of p-p38/p38 in H9c2 cells induced by ISO.7 Na HS pretreatment could decrease the expression of p-JNK/JNK in H9c2 cells induced by ISO.8 Na HS and ISO had no effect on the expression of p-ERK/ERK in H9c2 cells.9 SB203580 can depress the expression of NOX4 in H9c2 cells induced by ISO.10 SB600125 had no effect on the expression of NOX4 in H9c2 cells induced by ISO.Conclusions: 1 ISO can increase the expression of NOX4 in H9c2 cells and increase the level of superoxide anion.Na HS can depress the expression of NOX4 and reduce the level of superoxide anion induced by ISO.2 ISO can increase the expression of p-p38 MAPK and p-JNK in H9c2 cells,indicating that ISO induced the activation of p38 MAPK pathway and JNK pathway.Na HS can inhibit the activation of p38 MAPK pathway and JNK pathway induced by ISO.3 ISO increased the expression of NOX4 by activating the p38 MAPK pathway,resulting in oxidative stress damage,rather than by activating the JNK pathway 4 Exogenous hydrogen sulfide exerts protective effects on ISO damage by inhibiting the p38MAPK/ NOX4/ROS pathway. |
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