| Objective : Overtraining is the pathological states characterized by multiorgan dysfunction induced by overload of exercise and beyond the body ability. In recent years, the studies on overtraining-induced organ injury is paid great attention. It has been reported that Anisodamine relieves angiospasm,improves microcirculation and has the protective effects on ischemic acute renal injury.In the current report ,the authors observed the expression of mitochondrial protein Smac / DIABLO, the X-linked inhibitor of apoptosis protein (XIAP) and Caspase-9 in the renal tissue at different time after exhaustive swimming in rats to study the mechanism of overtraining-induced acute kidney injury(OTIAKI).In addition, we also observed the effect of Anisodamine to understand the protective mechanism of Anisodamine on overtraining-induced acute kidney injury of rats.Methods: 1. Thirty six male Wistar rats were randomly divided into three groups:control group(CN, n=6), exhaustive swimming(ES, n=18), Anisodamine(AD,n=12). 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 intraperitoneal injection of Anisodamine at the dose of 10 mg/kg body weight at 20 minutes before swimming and then swimed to exhaustive condition. 2. A animal model of OTIAKI was induced by exhaustive swimming : Rats of each group were given adapt swimming 30 min at one day before the experiment day, then put into the water till exhausted. 3. Sample: 6 Rats of each group were sacrificed after exhausted at different time. One part renal tissue of rats were collected in order to observe immunohistochemical staining changes.The other part tissue of rats were saved in refrigerator of -80℃for Western blot analysis. 4. The expression of Smac/DIABLO and XIAP in renal tissue were detected by immunohistochemical staining. 5. The expression of Smac/DIABLO , XIAP and Caspase-9 in renal tissue were detected by Western blot analysis. 6.The results of immunohistochemical staining were analyzed with the pathological image analysis system. 7. Statistical analysis was performed using SPSS 12.0. All data were expressed as X_±S, F and T test was separately used to analyze the difference between the two groups and among groups . p<0.05 was considered significant difference.Results:1. The expression of Smac/DIABLO in renal tissue of rats in every group:There was only little expression of mitochondrial protein Smac/DIABLO in the renal tissue of the CN group rats,but the expression of Smac/DIABLO was increased in ESI group(p<0.05),and the expressions of Smac/DIABLO in renal tissue increased progressively in ES 6h and ES 24h group(p<0.05). Compared with ES , the expression of Smac/DIABLO in renal tissue decreased markedly in AD 6h and AD 24h group(p<0.05).2. The expression of XIAP in renal tissue of rats in every group:The X-linked inhibitor of apoptosis protein was slightly expressed in the renal tissue of the CN group rats.The expression of XIAP was decreased in ESI group(p<0.05).And the expressions of XIAP in renal tissue decreased progressively in ES 6h and ES 24h group(p<0.05). Compared with the ES group, the expression of XIAP in renal tissue increased markedly in the AD 6h and the AD 24h group(p<0.05).3. The expression of Caspase-9 in renal tissue of rats in every group:Compared with CN, the expression of Caspase-9 in renal tissue was increased remarkably in ESI rats (p<0.05), and these changes were more prominent in ES 6 h rats and reached the higher level in ES 24h rats. Compared with ES, the expression of Caspase-9 in renal tissue decreased remarkably at 6 h and 24h of AD rats ( p<0.05).Conclusions:1.The expression of Smac/DIABLO and Caspase-9 in the renal tissue was increased and the expression of XIAP was decreased in ES rats,which suggested that the changes of expression of Smac/DIABLO,XIAP and Caspase-9 could play a key roll in the cell apoptosis of kidney induced by overtraining through caspase-dependent pathway.2.Anisodamine could down-regulate the expression of Smac/DIABLO and Caspase-9,up-regulate the expression of XIAP, which could be one of the very important protective mechanism of Anisodamine on OTIAKI in rats. |
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