过度训练可通过破坏Bax/Bcl-2平衡激活caspase依赖的凋亡通路诱导大鼠肾小管上皮细胞凋亡

目的 观察过度训练大鼠肾组织Bax、Bcl-2及 caspase-3的表达及其与肾小管上皮细胞凋亡的关系，探讨caspase依赖的凋亡信号途径在其中的作用及其机制。 方法 将48只雄性Wistar大鼠按随机数字表法分为对照组（CN）、力竭运动组（ES）、旋覆花素干预组（IB）。CN组为安静对照；ES组又根据力竭后恢复时间分为力竭后即刻（ESI）、力竭后6 h（ES 6 h）和力竭后24 h（ES 24 h）组；IB组于力竭运动前24 h 给予旋覆花素25 ml/kg分3次灌胃后进行力竭运动，分为IB 6 h和IB 24 h组。采用大鼠游泳至力竭建立过度训练模型。TUNEL法检测肾小管上皮细胞凋亡。免疫组织化学法检测肾组织Bax、Bcl-2及 caspase-3的表达。Western印迹法测定肾组织caspase-3蛋白的表达。用图像分析系统测定肾小管凋亡细胞、Bax、Bcl-2及 caspase-3表达的平均吸光度并计算Bax和Bcl-2的比值。用Pearson法分析Bax和Bcl-2的比值与caspase-3之间的相关性。用非参Spearman法分析Bax/Bcl-2的比值和caspase-3与细胞凋亡之间的相关性。 结果 TUNEL法显示，过度训练大鼠肾组织凋亡细胞主要分布在肾小管上皮细胞，力竭后即刻、6 h及24 h肾小管上皮细胞凋亡数呈进行性增多（P < 0.01）；免疫组化显示，过度训练大鼠肾组织caspase-3的表达及分布与Bax/Bcl-2比值变化及凋亡细胞的分布一致。图像分析显示，大鼠肾小管上皮细胞Bax/Bcl-2比值与caspase-3的表达于力竭后即刻、力竭后6 h、力竭后24 h逐渐增高（均P < 0.05），均显著高于对照组（均P < 0.05）。Western 印迹测定结果也显示caspase-3蛋白表达的变化趋势。过度训练大鼠肾小管上皮细胞Bax/Bcl-2比值与caspase-3的表达呈正相关（r = 0.865，P < 0.05）；Bax/Bcl-2比值和caspase-3的表达与肾小管上皮细胞凋亡之间均呈正相关（r = 0.674，r = 0.837，均P < 0.05）。用旋覆花素干预后，过度训练引起的肾小管上皮细胞Bax/Bcl-2比值增高和caspase-3的过度表达及肾小管上皮细胞的过度凋亡均被显著抑制（均P < 0.05）。 结论 过度训练可通过破坏肾小管上皮细胞Bax/Bcl-2的平衡，激活caspase依赖的凋亡信号通路，进而诱导大鼠肾小管上皮细胞凋亡。这可能是过度训练引起肾小管上皮细胞凋亡的分子机制之一。

Overtraining induces renal tubular cells apoptosis through activating caspase-related signal pathway by impairing the balance of Bax and Bcl-2 in exhaustive swimming rats

Objective To observe the expression changes of renal tissue Bax, Bcl-2 and caspase-3, to wxamine the correlation between the ratio of Bax to Bcl-2, caspase-3 and renal tubular cells apoptosis, and to investigate the role of caspase-related signal pathway. Methods Forty-eight male Wistar rats were randomly divided into three groups: control (CN, n=8), exhaustive swimming (ES, n=24) and inula britannica (IB, n=16) group. The rats of CN were quiet without swimming. The rats of ES swam to exhaustive state and were sacrificed at immediately(ESI), 6 hour(ES 6 h) and 24 hour(ES 24 h) after exhaustive swimming respectively. The rats of IB took orally inula britannica at the dose of 25 ml/kg body weight at 24 h before swimming and then swam to exhaustive state. The rats of IB group were sacrificed at 6 hour (IB 6 h) and 24 hour (IB 24 h) after exhaustive swimming. The animal model of overtraining-induced acute kidney injury was developed by exhaustive swimming. The renal cell apoptosis was measured by the method of TUNEL. The expressions of Bax, Bcl-2, caspase-3 in renal tissue were observed by immunohistochemistry. The expression of caspase-3 protein was examined by Western blotting. The correlation between the ratio of Bax to Bcl-2 and caspase-3 was analysed by Pearson method, and the correlation between the ratio of Bax to Bcl-2, caspase-3 and renal tubular cell apoptosis was analysed by Spearman method. Results The number of renal tubular apoptotic cells was increased progressively in ESI to ES 24 h rats by TUNEL (P<0.05). Immunohistochemistry staining showed that the ratio of Bax to Bcl-2 and caspase-3 in renal tubular cells were increased progressively at 0 h, 6 h and 24 h after exhaustive swimming compared with control group (P<0.05). The change of renal tissue caspase-3 was also revealved by Western blotting analysis. The ratio of Bax to Bcl-2 and caspase-3 in renal tubular cell was correlated positively (r=0.865, P<0.05), The ratio of Bax to Bcl-2, and caspase-3 was also correlated positively to renal tubular cell apoptosis (r=0.674, r=0.837, P<0.05) in ES rats. Pretreatment with inula britannica inhibited the up-regulation of the ratio of Bax to Bcl-2, caspase-3 and cell apoptosis in renal tubular cell induced by exhaustive swimming. Conclusion Overtraining can induce renal tubular cells apoptosis through activating caspase-related signal pathway by impairing the balance of Bax and Bcl-2, which may be one of the important molecular mechanisms of overtraining-induceed renal tubular cells apoptosis.