| Objective:Overtraining-induced acute myocardial injury(OTIAMI) is one of the most important causes resulted in sudden death. Up to the present, there is no effective drug therapy of OTIAMI in clinical. Therefore, it is very important to clarify the pathogenesis and to find the methods to prevent and treat OTIAMI. However, the previous studies on overtraining-induced acute myocardial injury limited mostly in the ordinary morphological and functional aspects, the cellular and molecular pathogenesis responsible for OTIAMI is unclear. Accumulating evidences indicate that myocardial apoptosis is involved in the development of OTIAMI . However, the molecular mechanism of myocardial apoptosis in OTIAMI is unknown. It has been reported that Anisodamine attenuates TNF-α-induced myocardial apoptosis and protects the heart from ischemia-reperfusion injury, and that Inula Britannica regulates the cell apoptosis by anti-inflammation and Ixeris Sonchifolia Hance has the action of anti-oxidative stress. But, little is known about the effect of Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance on OTIAMI and its molecular mechanism is not yet reported. In the current study, we investigated the pathological feature and pathogenesis of OTIAMI, and the role of myocardial apoptosis in development of OTIAKI, and caspase-related signal pathway mediating myocardial apoptosis in process of OTIAMI, by detecting the changes of expression of the key signaling kinases and cytokine in the myocardial tissue of OTIAMI rats. In addition, the protective effect and molecular mechanism of Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance on the development of OTIAMI were studied in the OTIAMI rats.Methods: Eighty male Wistar rats were randomly divided into five groups: control group(CN, n=8), exhaustive swimming(ES, n=24), Anisodamine(AD, n=16), Inula Britannica(IB, n=16) and Ixeris Sonchifolia( IS, n=16). The rats of CN were quiet and no swimming. The rats of ES swimed to exhaustive state and sacrificed at immediately(ESI), 6 hour(ES 6 h) and 24 hour(ES 24 h) after exhaustive swimming. The rats of AD received a single intraperitoneal injection of Anisodamine at a dose of 10mg/kg body weight at 20 minutes before swimming and then swimed to exhaustive condition. The rats of IB took orally Inula Britannica at the dose of 25 ml/kg body weight at 24 h before swimming and then swimed to exhaustive condition. The rats of IS received a single intraperitoneal injection of Ixeris Sonchifolia Hance at a dose of 20g/kg body weight at 20 minutes before swimming and then swimed to exhaustive condition. The rats of AD, IB and IS group were sacrificed at 6 hour ( AD 6 h, IB 6 h, IS 6 h) and 24 hour (AD 24 h, IB 24 h, IS 24 h) after exhaustive swimming. The animal model of overtraining-induced acute myocardial injury was developed by exhaustive swimming. The CK and CK -MB levels in serum were measured by biochemistry analyzer. The myocardial tissue morphological changes were observed by Olympus microscopy and transmission electron microscopy. The myocardial cell apoptosis was measured by the method of terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling(TUNEL) and flow cytometry. The changes of NF-КB, Fas-L, caspase-8, caspase-9 in myocardial tissue were measured by the way of flow cytometry. The expression of TNF-α, NF-КB, Bax, Bcl-2, Bid, Fas-L, caspase-8 and caspase-9 were observed by immunohistochemistry. The changes of SOD and MDA in serum and myocardial tissue were measured. All data were expressed asˉx±s. F and T test was separately used to analyse the difference between the two groups and within groups, which was finished with SPSS 12.0 software.Results:1. The changes of overtraining-induced myocardial apoptosis and effect of Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance on OTIAKITo deterimine the changes of overtraining-induced myocardial injury and effect of Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance on OTIAKI, the myocardial morphorological, functional, metablic changes were investigated in this section. The results were as follows:The level of serum CK and CK-MB increased markedly in ESI rats( p<0.05),and in ES 6 h rats( p <0.05), and decreased to CN levels in ES 24 h rats. The level of serum CK and CK-MB at 6 h in AD, IB and IS rats was decreased compared with ES rats. HE staining showed that the morphological slight changes were observed in ESI, ES 6 h and ES 24 h rats. Transmission electron microscopy showed that myocardial cell and the chromatin of nuclear were swollen, the organelles decreased, mitochondrion was partly swollen, the protruding of mitochondrion was not in order, the protruding and membrane of mitochondrion were partly blended and disappeared. There were not obvious morphological changes in myocardial tissue and the changes of myocardial tissue ultrastructure were slight in 6 h and 24 h of AD, IB and IS rats. It was revealed by the way of TUNEL and flow cytometry that the number of apoptostic cell in myocardial tissue was increased markedly in ESI rats( p<0.05), and in ES 6 h rats( p <0.05), and decreased in ES 24 h rats, but higher than that in CN rats. TUNEL and Flow Cytometry assay showed that myocardial cell apoptostic ratios were lower than that in ES rats but higher than that in CN rats at 6 h and 24 h of AD, IB and IS rats. The SOD in serum and myocardial tissue decreased temporarily after exhaustive swimming, and to the lowest level at ES 6 h. There was a opposite changes in MDA. The SOD and MDA reversed to CN level at ES 24 h. Compared with ES, the SOD in serum and myocardial tissue increased markedly in AD 6 h, IB 6 h and IS 6 h rats, the MDA decreased significantly in AD 6 h, IB 6 h and IS 6 h rats( p <0.05). These results suggest that the overtraining could induced acute myocardial injury involving the functional, morphological changes and obvious myocardial cell apoptosis by increasing oxidative stress2. Expression of apoptosis-related gene and anti-apoptpsis mechanism of Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance in cardiomyocyte of overtraining ratsTo understand the pathogenesis of cardiomyocyte apoptosis induced by overtraining, in this part, the expression of apoptosis-related genes and influences of Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance were studied. The results were as follows:The expression of Bax was stronger, the expression of Bcl-2 was weaker, the Bax/Bcl-2 ratio increased remarkably at immediately in exhausted rat. The changes were up to peak at 6 h and recovered slightly at 24 h in exhausted rats. The Bax/Bcl-2 ratio had significantly positive correlation with cardiomyocyte apoptosis ratio (r=0.727, p <0.05). After treatment of Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance, compared with that of control rats, the expression of Bax decreased remarkably, the expression of Bcl-2 increased significantly, the Bax/Bcl-2 ratio decreased remarkably. Those changes were more obvious in Anisodamine group and Inula Britannica group. These results suggest that the imbalance of Bax and Bcl-2 is one of the important pathogenesis in development of overtraining-induced myocardial cell apoptosis, Anisodamine, Inula Britannica and Lxeris Sonchifolia Hance have the obvious anti- apoptosis effect of myocardial cell by regulating the expression of Bax, Bcl-2 and the Bax/Bcl-2 ratio.3. Expression of caspase-related apoptosis signal molecules in overtraining-induced acute myocardial injuryTo understant cell apoptosis signal transduction pathway, the expressions of a group of caspase-related cell apoptosis signal molecular were observed by immunohistochemistry and flow cytometry in this part. The results showed that positive stainings for TNF-αwere observed in myocardial tissue of ESI. The expressions of TNF-αwas increased markedly in ESI rats( p <0.05), and in ES 6 h rats( p <0.05), and decreased in ES 24 h rats, but higher than that in CN rats. The expressions of NF-КB were higher progressively at 0h, 6 h and 24 h after exhaustive swimming( p <0.05), and the level of NF-КB was significantly increased in ESI, ES 6 h and ES 24 h rats (p <0.05). The expressions of Fas-L, Caspase-8, Caspase-9 and Bid were increased markedly in ESI rats( p <0.05), and in ES6h rats( p <0.05), and decreased in ES 24 h rats, but higher than CN rats. These results suggest that caspase-related cell apoptosis signal transduction pathway plays an important role in the development of overtraining-induced myocardial cell apoptosis.4. Effect of Anisodamine,Inula Britannica and Ixeris Sonchifolia on expression of caspase-related myocardial apoptosis signal molecules in OTIAMI ratsTo understand the molecular mechanism of protective effect of Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance on OTIAMI in rats, the influences of above three drugs on the expression of apoptosis-related signal molecular were observed. The results were as follows:①Compared with ES, the expressions of TNF-αin myocardial tissue decreased markedly but higher than that in CN rats at 6 h and 24 h of AD, IB and IS rats (p<0.05).②The expressions of NF-КB in myocardial tissue decreased markedly but higher than that in CN rats at 6 and 24 hour of AD, IB and IS rats.③Immunohistochemistry staining and flow cytometry showed that the expressions of Fas-L in myocardial tissue decreased markedly but higher than that in CN rats at 6 h of AD, IB, IS rats and in AD 24 h, IB 24 h and IS 24 h rats.④Immunohistochemistry staining and flow cytometry showed that the expressions of Caspase-8 in myocardial tissue were decreased markedly in AD 6 h, IB 6 h and IS 6 h rats but higher than that in CN rats and returned to basic level in 24 h of AD, IB, IS rats.⑤Immunohistochemistry staining and flow cytometry showed that the expressions of Caspase-9 in myocardial tissue decreased markedly but higher than CN rats at 6 h in AD, IB, IS rats, and decreased to CN level at 24 h in AD, IB, IS rats.⑥Immunohistochemistry staining showed that the expressions of Bid of myocardial tissue decreased markedly but higher than that in CN rats at 6 h and 24 h in AD, IB, IS rats, the expressions of Bid in myocardial tissue among three drug groups was no significant difference. These results suggest that above three drugs can decrease the apoptosis signal transduction in caspase-related signal pathway.Conclusions:1. The exhaustive swimming could induced acute myocardial injury includling the functional and morphological changes, via inducing myocardial cell apoptosis.2. The caspase-related signal transduction pathway play a key role in the development of myocardial apoptosis induced by OTIAMI .3. Anisodamine, Inula Britannica and Ixeris Sonchifolia Hance have cardioprotective effects in OTIAMI rats by decreasing the overtrining-induced myocardial cell apoptosis. |
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