Study On Mechanism And The Protective Effect Of Tanshinone ⅡA And Prohibitin Against Myocardial Ischemia/Reperfusion Injury

OBJECTIVE:It is generally acknowledged that restoring blood perfusion could be the basic measure in prevention of ischemic damage. Recently, thrombolytic therapy, coronary angioplasty and some other new techniques make ischemic tissues and organs restore blood perfusion. Although timely reperfusion of acute ischemic myocardium is essential for myocardial salvage, reperfusion may also result in a unique form of myocardial damage, which called (myocardial ischemia/reperfusion injury, MI/RI). MI/RI result in cardiac disfunction, which bad lead to life quality and prognosis in patients.For this reason, it is of great practical improtance to explore effective measure in prevention of MI/RI and the mechanisms.In our previous proteomics study, we found that Prohibitin protein was in hight expression in the MI/R heart compared with the control hearts, the result was consistent with foreign research. However, the study of PHB function was mostly focused on oncology, the specific cellular function of PHB in myocardial cells has not been clearly elucidated.Salvia miltiorrhiza Bunge has long been used for prevention and treatment of cardiovascular diseases in China. TanshinoneⅡA is one of the major lipid-soluble pharmacologic constituents of of Salvia miltiorrhiza Bunge.The goal of this study was to investigate the protective effect of tanshinoneⅡA in MI/RI. In order to study the machanism of tanshinoneⅡA, we focus our study on antioxidation, antagonizing calcium overload, promoting energy synthesis and the regulation of PHBMETHOD:Animal level:Male Wistar rats were randomly divided into 5 groups, they were Sham operated group (SH group), myocardial ischemia-reperfusion group (MI/R group), high-dose tanshinoneⅡA group, middle-dose tanshinoneⅡA group and low-dose tanshinoneⅡA group. Rats in both SH group and MI/R group were drenched with normal sodium, rats in different doses of tanshinoneⅡA group were drenched with different doses of tanshinoneⅡA physic liquor(20,10,5mg/kg`d), all of administration was performed twice a day. The animal model was conducted by ligating the left anterior descending coronary artery for 15 min and then loosening for 30min.Ⅱlead ECG was continously observed and recorded in order to conduct ventricular arrhythmia score. LVSP, LVEDP,+dp/dtmax,-dp/dtmax and HR was simultaneous recorded by multilead physiological instrument. The size of myocardial infarction was determined by double-staning of evans blue and TTC and weighing. After the operation, took out of the heart, washed it in normal sodium, separated the left ventricle and divided it into two parts. One part was dipped into 10% neutral formaldehyde for fixation, another part was stored in refrigerator with the temperature of -80℃.HE dyeing was used to observe the morphous changes of myocardium in each group. Myocardial cell apoptosis was determined by TUNEL method, and the expression of PHB was determined by immunohistochemical method and western blotting method. Cellular level:Primary cultured neonate rat myocardial cell was cultured in medium with 200μmol/L hydrogen peroxide to simulate oxidative stress.In different doses of TanshinoneⅡA group, the medium was supplemented with different concentrations of TanshinoneⅡA in advangce(1×10-4mol/L,5×10-5mol/L,1×10-5mol/L) for 24 h. The morphological changes of myocardial cell was observed by inverted phase contrast microscope. Cell viability was determined by MTT method.apoptosis rate was determined by Annexin V/PI bivariate dyeing, and it was detected by flow cytometric analysis,so as the level of [Ca2+]i and mitochondrial membrane potential.Besides, Na+-K+-ATPase and Ca2+-Mg2+-ATPase, T-AOC, LDH, MDA, SOD, GSH-PX, CAT were also evaluated in each group.The expression of PHB was determined by immunohistochemical method and western blotting method. siRNA PHB interference was transfected to myocardial cell in order to study the function of PHB.Statistical analysis:The data was analyzed by SPSS13.0. measurement data was presented as the means±SEM. Test of normality and homogeneity of variance was done in each group. If datas accorded with normality and homogeneity of variance, statistical analysis was made by one-way ANOVA followed by LSD test, otherwise statistical analysis was made by rank sum test, and multiple comparison was made by Tamhane's T2.Multiple enumeration data was analyzed by Kruskal-Wallis. Hemodynamics parameters in different time points was analyzed by repetitive measurement and analysis of variance. Level of significanceα=0.05RESULT:(1)Effect of Tanshinone II A on cardiac function in myocardial ischemia/reperfusion injured rats.①Ventricular arrhythmia score. Rats in SH group appeared with premature ventricualr contraction occasionally. Howerer, all rats in MI/R group developed arrhythmia, mainly including premature ventricualr contraction, ventricular tachycardia and ventricular fibrillation,2 rats in the group die of ventricular fibrillation. In different doses of TanshinoneⅡA group, there were occurrence of arrhythmia at different degree. The ventricular arrhythmia score in each group in the sequence from high to low was MI/R group, low-dose of Tanshinone II A, middle-dose of TanshinoneⅡA, high-dose of Tanshinone II A, SH group. The difference of AV score in all the group was significant(x2=29.118, P=0.000).②The area of myocardial infarction. Rats in MI/R group suffered from serious myocardial ischemia and myocardial infarction, so the infarction size was the largest in all the group. TanshinoneⅡA presented decreasing infarction size in a dose dependent manner. The difference of infarction size in all the group was significant(F=95.049,P=0.000).③Hemodynamics parameters.HR, LVSP, and±dp/dtmax was significantly decreased in the sequence of SH group, TanⅡA high dose group, TanⅡA middle dose group, TanⅡA low dose group and MI/R group, while LVEDP was significantl increased in the same sequence. In different doses of TanshinoneⅡA groups, the hemodynamics parameters were improved in a dose dependent manner. The difference of hemodynamics parameters in all the group was significant(each P<0.05).HR, LVSP, and±dp/dtmax was significantly decreased with prolongation of time of ischemia and reperfusion, while LVEDP was significantl increased with prolongation of time of ischemia and reperfusion, the difference of hemodynamics parameters between each time spots was significant(each P<0.05).④Observation of histology.myocardium structure in SH group was characterized with well-arranged myocardial cells, well-distributed HE dyeing and integrated cell membrane. While in MI/R group, the myocardial cells were chaotic, HE dyeing was uneven,cells in some parts of the heart were cloudy swelling, the cardiac muscle fibers was unclear or disappeared.In different doses of TanshinoneⅡA groups, some myocardial cells developed degeneration and necrosis, but it was not severe, the improvement of structure was dose dependent.⑤Cell apoptosis index. There were a large number of apoptotic cells in MI/R group. The apoptotic cells characterized with srtong TUNEL positive staining. Cell apoptosis index in different TanshinoneⅡA groups was significantly increased dose dependently. The difference of cell apoptosis index in all the group was was significant(x2=186.968,P=0.000).(2) Antioxidation of TanshinoneⅡA and the mechanism.①The optimum concentration of H2O2 and TanshinoneⅡA. Myocardial cells were exposed to various concentrations of H2O2 (50-800μmol/L)for 2 h. Cell cytotoxicity was determined by MTT assay, the result was that H2O2 significantly inhibited the viability of both cell in a dose-dependent manner. When myocardial cells were exposed to TanshinoneⅡA at the concentration of 1×10-5mol/L,5×10-5mol/L,1×10-4mol/L for 24 h, there was no statistical significance in cell viability compared with normal control group(each P>0.05).But when the contration was 5×10-4mol/L, cell viability was significantly decreased(P=0.003).②orphological changes of cardiocytes. Normal cardiocyte were in fusiform or rhomubus shapes, with spurious leg interlaced like a net, cell beat simultaneously, cardiocytes treated with H2O2 for 2 h beat weakly, with spurious leg shortened or dispeared, endochylema condensed. The appearance of cardiocytes in TanshinoneⅡA groups had been improved.③Cell apoptosis related indexes.Apoptosis rate, intracellular concentration of calcium was significantly increased, and mitochondrial membrane potential levels was significantly down-regulated by treatment with H2O2 (200μmol/L) for 2h. Pretreatment with TanshinoneⅡA for 24 h provided significant protection against down-regulation of apoptosis rate, intracellular concentration of calcium caused by H2O2, while the mitochondrial membrane potential levels was significantly up-regulated.④The effect of TanshinoneⅡA on oxidation balance system. T-AOC,SOD activity, GSH-PXA activity, CAT activity was significantly decreased in model group, while LDH activity and MDA content was significanltly increased. T-AOC, SOD activity, GSH-PXA activity and CAT activity was significantly increased in TanshinoneⅡA groups in a dose dependent manner, and LDH activity and MDA content was significanltly decreased in the manner. The difference in all groups was significant(each P=0.000).⑤Na+-K+-ATPase, and Ca2+-Mg2+-ATPase was significantly decreased in model group, while pretreatment with TanshinoneⅡA for 24 h provided significant increasement of Na+-K+-ATPase, and Ca2+-Mg2+-ATPase in a dose dependent manner. The difference in all group was significant(each P=0.000).(3)The function of PHB.①siRNA transfection efficiency. The transfection efficiency of siRNA PHB was significantly increased depended on siRNA PHB concentration(F=295.323,P=0.000).The expression of prohibitin reduced by 74.11±3.23% in condition of siRNA PHB.Besides, apoptosis rate, [Ca2+]i, LDH activity and MDA content was significantly increased, and MMP levels(P<0.05),T-AOC, SOD activity, GSH-PXA activity, CAT activity, Na+-K+-ATPase and Ca2+-Mg2+-ATPasewas significantly decreased(each P=0.000).(4)Effect of MI/R, oxidative stress and tanshinoneⅡA on PHB protein expression in myocardial cells.At animal level, the expression of PHB was remarkably increased in MI/R group, and tanshinoneⅡA attenuated PHB expression in a dose dependent manner, the difference in all groups was significant(P=0.000).At cell level, the expression of prohibitin increased remarkably under oxidative stress conditions, the increase was attenuated by tanshinoneⅡA too, the difference in all groups was significant(P=0.000).CONCLUSION(1)Myocardial ischemia reperfusion injury could result in ventricular arrhythmia, damage on myocardial structure, cardiac dysfunction. TanshinoneⅡA show satisfactory protection against the above damage by MI/RI.(2) Hydrogen peroxide mediated myocardial injury through oxidative imbalance, energy metabolic disturbance, intracellular calcium overload, apoptosis and so on. TanshinoneⅡA protect myocardial cell against injury probably by strengthen the activity of antioxidant enzyme and energy synthesis, and inhibiting lipid peroxidation and intracellular calcium overload.(3)PHB showed multiple effect on myocardial cells, such as remarkable antioxidation, enhancing ATPase activity, and inhibition of intracellular calcium overload and apoptosis.(4) PHB protein was in high expression in condition of MI/RI and oxidative stress.TanshinoneⅡA decreased the expression of PHB probably through the inhibition of oxidative damage, so that compensatory increase by oxidative stress may be inhibited.