| Objective:The purpose of this study is to establish an rat ischemia/reperfusion (I/R) heart and cardiomyocyte model, and to observe the effects of Salvianolic acid A (SAA) pretreatment on the I/R myocardium in vitro and cardiomyocyte contraction function and apoptosis with these indexes for myocardium infarction area, cardiac hemodynamics, myocardium necrosis markers, single cardiomyocytes contractile function, apoptosis related proteins, dual specificity protein phosphatase (DUSP)2/4/16and ERK1/2/JNK pathways related protein expression. Meanwhile, the cardioprotection against I/R injury and underlying molecular mechanisms are able to be discussed, thus a kind of new drugs and therapeutic targets can be provides for clinical treatment of myocardium I/R.Methods:1. Clean grade adult male Wistar rats, weight220-260g, were divided into normal control group (CON group, n=6), I/R group (I/R, n=6), SAA pretreatment group (SAA+I/R, n=6), ERK1/2inhibitors PD098059+I/R group (PD+I/R), ERK1/2inhibitors PD098059+SAA+I/R group (PD+SAA+I/R), JNK inhibitors SP600125+I/R group (SP+I/R). The cardioprotection of SAA (20μM) on I/R heart in vitro was observed by application of Langendorff heart connection device in vitro and I/R process was simulated by stopping perfusion following reperfusion. The heart in vitro in CON group was continuously perfused K-H solution perfusion for160min; the heart in vitro in I/R group was perfused for10min, then ischemia for30min following reperfusion for120min; In SAA+I/R group, the heart in vitro was perfused for10min, then SAA pretreatment for20min, ischemia for30min following reperfusion for120min; In PD+I/R group, the heart in vitro was perfused for10min, then PD pretreatment for20min, the following procedure was the same as I/R group; In PD+SAA+I/R group, PD was pretreated for30min, the following process was the same as SAA+I/R group; In SP+SP group, SP was pretreated for20min, the following procedure was the same as I/R group; During the course of reperfusion, A filling water sac connecting pressure sensor was inserted into left ventricle via pulmonary vein orifice, left atrium, and mitral valve, the filling water sac was located in LVEDP for5-15mmHg. heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and maximum rate of left ventricular pressure (+dp/dtmax) were recorded for30min prior to I/R and120min after I/R. At the same time, lactate dehydrogenase (LDH) from coronary effluent for reperfusion15min was assayed. After reperfusion, heart was frozen, myocardial infarction areas were measured by TTC staining method.Adult rat cardiomyocytes were isolated in conventional enzymolysis approach, then I/R process was simulated by lacking of oxygen, sugar following recovering sugar, oxygen to observe cardioprotection of SAA for I/R cardiomyocytes in vitro.In cells level, there were five groups.(1) CON group:cardiomyocytes were cultivated for18h;(2) I/R group:cardiomyocytes were cultivated for13h, then were placed in three gas incubator for3h, next, cardiomyocyt es were cultured in high glucose DMEM medium and CO2incubator to simulate reperfusion.(3) SAA+I/R group:cardiomyocytes were cultivated for1h, then I/R was performed after different concentrations of SAA (1,5,10,15^M) pretreatment for12h. The optimal concentration of SAA was determined according to cell survival rate by observing the rod rate of cardiomyocytes and then was performed in following experiments.(4) PD+SAA+I/R group:PD was added for1h prior to SAA pretreatment, then the following process was as the same as SAA+I/R group;(5) SP+I/R group:pretreatment with SP for1h, I/R was performed. The contraction amplitude of cardiomyocytes was determined with a single detection technology by isopropyl adrenaline stimulating; Cardiomyocytes apoptosis was detected by TUNEL and DAPI; The protein expression levels for Bcl-2, Bax, JNK, phosphorylation-JNK (p-JNK), ERK1/2, phosphorylation-ERK1/2(p-ERK1/2), DUSP2/4/16were assayed by Western blot.Results:1. In cardiac I/R level in vitro, compared with CON group, various parameters of heart function were reduced significantly(P<0.01) and LDH release, area of myocardial infarction, cell apoptosis were increased significantly following I/R (P<0.01); Compared with I/R group, various parameters of heart function were improved significantly ((P<0.05)) and LDH release, area of myocardial infarction, cell apoptosis were reduced significantly among SAA+I/R group, SP+I/R group, PD+SAA+I/R group (P＜0.01); The results, PD+I/R compared with different groups, were as the same as I/R group; In PD+SAA+I/R group, heart function parameters were reduced and LDH release, area of myocardial infarction, cell apoptosis rate were increased (P<0.05) by comparsion with SAA+I/R group; However, SP+I/R group compared with SAA+I/R group, the indexes mentioned above were not significant different (P>0.05); Compared with PD+SAA+I/R group, various parameters of heart function were elevated significantly (P＜0.05)) and LDH release, area of myocardial infarction, cell apoptosis were reduced significantly in SP+I/R group (P＜0.01). With regard to the values of hemodynamic indicators, LDH and apoptosis rate between PD+I/R group and I/R group were not statistically different (P>0.05), so the related test in PD+I/R group was not performed at cell level.2. At cellular level, compared with CON group, contraction amplitude of single cardiomyocyte, protein expression level for p-ERK1/2, Bcl-2and DUSP4/16were decreased significantly(P<0.01), however, p-JNK, Bax and DUSP2protein expressions were increased significantly (P＜0.05-0.01) following I/R; With SAA pretreatment, contraction amplitude of single cardiomyocyte. protein expression level for p-ERK1/2, Bcl-2, DUSP4/16were increased and p-JNK, Bax, DUSP2protein levels were reduced significantly (P＜0.05); Compared with I/R group, contraction amplitude of single cardiomyocyte, protein expression level for p-ERK1/2, Bcl-2, DUSP4/16were increased significantly and p-JNK, Bax, DUSP2protein expressions were reduced significantly (P＜0.05) among SAA+I/R group, SP+I/R group, PD+SAA+I/R group (P<0.01); In PD+SAA+I/R group, contraction amplitude of single cardiomyocyte, protein expression level for p-ERK1/2, Bcl-2, DUSP4/16were reduced significantly and p-JNK, Bax, DUSP2protein expressions were activated significantly (P<0.05) by comparsion with SAA+I/R group; However, SP+I/R group compared with SAA+I/R group, the indexes mentioned above were not significant different (P>0.05); Compared with PD+SAA+I/R group, the protein expression levels for p-ERK1/2, Bcl-2, DUSP4/16and single cardiomyocyte contraction amplitude were increased significantly and p-JNK, Bax, DUSP2protein levels were lowered in SP+I/R group (P<0.05).Conclusion:During the course of I/R, JNK pathway can inhibit the ERK1/2activation via DUSP2and ERK1/2inhibit ERK1/2activation via DUSP4/16.SAA can inhibit DUSP2-mediated JNK pathway for activation of ERK1/2and activate DUSP4/16-mediated ERK1/2by JNK dephosphorylation to play anti-apoptosis role in I/R cardiomyocytes. |
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