| ObjectiveIn recent years, with the rapid development of competitive sports, excessive movement has gradually become the focus of sports medicine and clinical medicine.Overtraining-induced acute myocardial injury is one of the important reasons for the athletes sudden death, At present it is lack of effective prevention and control measures in clinically.Therefore, further understanding the pathogenesis of OTIAMI, explore the prevention and control measures is a major task faced by the sports medicine, military medicine and clinical medicine at present. This study intents to observe the myocardial histopathological changes and the dynamic changes of p38 MAPK, p-p38 MAPK, COX-2, HSP70 in rats' serum and myocardial tissue at different time points of convalescence after acute exhausted exercise(at0h, 3h, 6h, 12 h and 24 h after exercise); at the same time study the effects of 0.90g/kg and0.45g/kg Nuo Di Kang capsule to the myocardial histopathological changes and the dynamic changes of p38 MAPK, p-p38 MAPK, COX-2, HSP70 in rats' serum and myocardial tissue at3 h, 6h and 24 h after acute exhausted exercise, in order to investigate the role of p38 MAPK,COX-2 and HSP70 in the acute myocardial injury and the intervention effect of Nuo Di Kang capsule, in order to provide important experimental theroetical basis for the occurrence and rehabilitation of acute myocardial injury.Methods1. Established model and grouping sample collectionThe overtraining-induced acute myocardial injury animal model was established by exhausted swimming, with 2% of body weight attached to the tail. 160 healthy male Wistar rats were randomly divided into a sedentary control group(n=20), a exhausted exercise group(n=80), high-dose Nuo Di Kang capsule intervention group(0.90g/kg, n=30) and low-dose Nuo Di Kang capsule intervention group(0.45g/kg, n=30). Sedentary control group rats were taken materials without any exercise; exhausted exercise model group rats were forced to take exhausted exercise, According to different recovery times after exercise,exhausted exercise groups were divided into 0h, 3h, 6h, 12 h and 24 h after exercise(n=10),then taken materials at the different time points; 0.90g/kg and 0.45g/kg Nuo Di Kang capsule intervention groups were divided into drug treatment 3h, 6h and 24 h groups respectively in each group(n=10), Intragastric administration of corresonding drugs were given before exhausted exercise, once a day for ten days.2. The target detectionThe exhaustion swimming time of each group was recorded and Rats from each group were taken 2ml blood from abdominal venous respectively, prepared serum, stored at-20?until analysis. The left ventricular myocardial tissue was removed quickly, taken the cardiac apex to conventionally dehydrated and fixed for three days in neutral formalin,embedded in paraffin, cutted into slices, reserved for use. Diveded the rest of the myocardial tissue into three groups: the first part was used to prepared 10% myocardial tissue homogenate, centrifuged and the supernatant was got, then stored at-20? until use, detected the protein expression of p38 MAPK, COX-2 and HSP70 in rats' serum and myocardial tissue;the second part was used to prepare myocardial frozen specimen in liquid nirtrogen quickly,stored at-80?until use, detected the protein expression of p-p38 MAPK, COX-2 and HSP70 in rats' myocardial tissue; The last part was stored at-80?until use, detected the m RNA expression of p38 MAPK, COX-2 and HSP70 in rats' myocardial tissue.HE staining was used to observe myocardial pathological changes; ELISA was used to detect the p38 MAPK, COX-2 and HSP70 content in rats' serum and myocardial tissue; The protein expression of p-p38 MAPK, COX-2 and HSP70 in rats' myocardial tissue were detected by Western blotting and immunohistochemical method; The m RNA expression of p38 MAPK, COX-2 and HSP70 in rats' myocardial tissue were detected by RT-PCR(Real-time fluorescent quantitative PCR) method.Results1. The myocardial morphological changes at different time points after exhausted in rats and the intervention effects of Nuo Di Kang capsuleHE results showed that myocardial tissue ultrastructure in rats of sedentary control group were normal. Myocardial tissue ultrastructure in rats of different time points after exhausted exercise all had damage with different degree, the damage of 24 h after exercise groups was most serious among all the model groups. The rats' myocardial structures of Nuo Di Kang capsule 0.90g/kg dose 6h, 24 h and 0.45g/kg dose 24 h groups were all improved.2. Dynamic changes of the content of p38 MAPK, COX-2 and HSP70 at different time points after exhausted exercises in rats' serum and the intervention effects of Nuo Di Kang capsuleELISA experiment results showed that compared with sedentary control group, the rats' serum p38 MAPK and COX-2 content of 3h, 6h, 12 h and 24 h after exhausted exercise were increased obviously(P<0.01 and P<0.05), the peak of p38 MAPK content appeared at 3h and24 h. the rats' serum HSP70 content of 0h, 3h, 6h, 12 h and 24 h after exhausted exercise increased obviously(P<0.01), peaked at 24 h. Compared with 3h, 6h, 24 h after exhausted exercise respectively, the rats' serum p38 MAPK and COX-2 content of high-dose and low-dose Nuo Di Kang capsule all reduced obviously(P<0.05 and P<0.01), while the HSP70 content of Nuo Di Kang capsule was increased obviously(P<0.05).3. Dynamic changes of the content of p38 MAPK, COX-2 and HSP70 at different time points after exhausted exercises in rats' myocardial tissue and the intervention effects of Nuo Di Kang capsuleELISA experiment results showed that compared with sedentary control group, the rats' p38 MAPK, COX-2 and HSP70 content in the myocardial tissue of 3h, 6h, 12 h and 24 h after exhausted exercise were increased obviously(P<0.01 and P<0.05), Compared with 3h, 6h and24 h after exhausted exercise groups, the rats' p38 MAPK content of low-dose and high-dose Nuo Di Kang capsule 3h, 24 h groups were all reduced obviously(P<0.01), high-dose Nuo Di Kang capsule 6h group was also reduced obviously(P<0.01); the rats' COX-2 content of low-dose and high-dose Nuo Di Kang capsule 3h, 6h, 24 h groups were all reduced obviously(P<0.01); while the HSP70 content of low-dose and high-dose Nuo Di Kang capsule3 h, 24 h groups were increased obviously(P<0.01), high-dose Nuo Di Kang capsule 6h group was increased obviously(P<0.01).4. Dynamic changes of the expression of p-p38 MAPK, COX-2 and HSP70 proteins at different time points after exhausted exercises in rats' myocardial tissue and the intervention effects of Nuo Di Kang capsuleThe western-blotting results showed that compared with sedentary control group, the rats' p-p38 MAPK content of 3h, 6h, 12 h and 24 h in the myocardial tissue after acute exhausted exercise were increased obviously(P<0.05), peaked at 3h; the rats' COX-2 and HSP70 content were also increased obviously(P<0.05 and P<0.01). Compared with 3h, 6h and 24 h after exhausted exercise, the rats' p-p38 MAPK and COX-2 content in the myocardial tissue of low-dose and high-dose Nuo Di Kang capsule groups were all reduced obviously(P<0.01), while the HSP70 content of 0.90g/kg dose Nuo Di Kang capsule was increased obviously(P<0.05).The results of immunohistochemical showed that p-p38 MAPK, COX-2 and HSP70 hardly expressed in the sedentary control group, obviously increased in the lesion area in the model groups. the rats' p-p38 MAPK and COX-2 content were reduced obviously, while the HSP70 content was increased obviously in the low-dose and high-dose Nuo Di Kang capsule groups.5. Dynamic changes of the expression of p38 MAPKm RNA, COX-2m RNA and HSP70 m RNA at different time points after exhausted exercises in rats' myocardial tissue and the intervention effects of Nuo Di Kang capsuleThe results of RT-PCR showed that compared with sedentary control group, the rats' p38 MAPKm RNA, COX-2m RNA and HSP70 m RNA content of 3h, 6h and 24 h in the myocardial tissue after exhausted exercise were increased obviously(P<0.05).Compared with3 h, 6h and 24 h after exhausted exercise, the rats' p38 MAPKm RNA and COX-2 m RNA content of Nuo Di Kang capsule 0.90g/kg dose and 0.45g/kg dose were all reduced obviously(P<0.01); HSP70 m RNA was increased obviously in all the Nuo Di Kang capsule groups, peaked at 6h followed by a decline.Conclusion1. The acute myocardial injury model was established successfully by exhausted exercise.2. It was confirmed that exhausted exercise could cause rats' myocardial tissue damage in different degrees at different time points. 0.90g/kg and 0.45g/kg Nuo Di Kang capsule intervention groups could improve myocardial morphological changes.3. ELISA, immunohistochemical and Western-blotting methods all confirmed that exhausted exercise could cause the rats' p38 MAPK, p-p38 MAPK, COX-2 and HSP70 protein expression in the serum and myocardial tissue increased in different degrees at different time points. Nuo Di Kang capsule could reduced the p38 MAPK, p-p38 MAPK and COX-2 protein expression level and increased the HSP70 protein expression level.4. q RT-PCR method confirmed that the p38 MAPKm RNA, COX-2m RNA and HSP70 m RNA expression in the myocardial tissue at different time points after exhausted exercise all increased in different degrees. 0.90g/kg and 0.45g/kg Nuo Di Kang capsule could reduced the p38 MAPKm RNA and COX-2m RNA protein expression level and increased the HSP70 m RNA protein expression level.5. Different methods further confirmed that p38 MAPK, COX-2 and HSP70 all played an important role in the development of myocardial injury induced by acute exhaustion exercise.6. Nuo Di Kang capsule had protect effects on myocardial injury induced by exhaustion exercise, the mechanism may be related to reduced the p38 MAPK, COX-2 and increased the over expression of HSP70 in the myocardial tissue. |
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