过度训练部分通过炎性信号途径诱导肾小管上皮细胞凋亡

Overtraining induces renal cell apoptosis partly through inflammatory signal pathway in exhaustive swimming rats

目的观察过度训练大鼠肾组织肿瘤坏死因子α（TNF—α）、核因子-（B（NF—KB）的表达及其与’肾组织细胞凋亡的关系以及旋覆花素对其的影响，探讨炎性信号途径在其中的作用。方法采用大鼠游泳至力竭建立过度训练模型。将48只雄性Wistar大鼠按随机数字表法分为对照组（CN，n=8）、力竭运动组（ES，n=24）、旋覆花素干预组（IB，n=16）。CN组为安静对照。ES组又根据力竭后恢复时间分为力竭即刻（ESI，n=8）、力竭后6h（ES6h，n=8）和力竭后24h（ES24h，n=8）。IB组于力竭运动前24h给予旋覆花素25ml／kg，分3次灌胃后进行力竭运动，分为IB6h（n=8）和IB24h（n=8）组。TUNEL法检测。肾组织细胞凋亡。免疫组织化学法检测肾组织TNF-α和NF—KB的表达。流式细胞术检测肾组织细胞NF．KB的变化。用图像分析系统测定肾组织凋亡细胞、TNF-α和NF-KB表达的平均吸光度。采用Pearson法分析TNF-α与NF—KB之间的相关性；采用非参数Spearman法分析TNF-α和NF—KB与细胞凋亡之间的相关性。结果TUNEL法显示，过度训练大鼠于力竭后即刻、6h及24h肾组织细胞凋亡数呈进行性增多（P〈0．01）。免疫组化显示，对照组大鼠肾组织有TNF-α轻微表达，主要分布于肾小管上皮细胞；力竭后即刻（0．136±0．009）、力竭后6h（0．171±0．011）、力竭后24h（0．229±0．008）大鼠肾组织TNF-α表达逐渐升高，均显著高于对照组（0．109±0．010）（均P〈0．05）。对照组大鼠肾组织NF—KB有轻微表达；力竭后即刻其表达即显著增高（0．129±0．α1）（P〈0．05）；力竭后6h（0．166±0．009）显著高于对照组（0．095±0．α0）及力竭后即刻组（均P〈0．05）；力竭后24h（0．218±0．019）则进一步增高，显著高于力竭后即刻和6h组（均P〈0．05）。流式细胞术检测结果也证实了力竭后大鼠。肾组织NF—KB有同样变化趋势。过度训练大鼠。肾组织TNF-α与NF—KB的表达呈正相关（r=0．955，P〈0．01）；肾组织TNF-α和NF-KB表达与肾组织细胞凋亡之间均呈正相关（r=0．953，r=0．939，均P〈0．01）。用旋覆花素干预后，与同时间点力竭组比较，过度训练引起的肾组织TNF-α（6h：0．142±0．012，24h：0．130±0．010）和NF-KB（6h：0．138±0．010，24h：0．136±0．011）的过度表达均被显著抑制（均P〈0．05）。结论过度训练可导致肾组织炎性反应增强，旋覆花素能部分逆转这种炎性反应。这可能是过度训练引起肾组织细胞凋亡及旋覆花素抗凋亡的分子机制之一。

Objective To observe the changes in the expression of renal tissue TNF-α , NF-KB and the interrelation to renal cell apoptosis, and their influences of Inula Britannica（an inhibitor of inflammatory signal pathway） in exhausted swimming rats, and to investigate the role of inflammatol7 signal pathway. Methods Forty-eight male Wistar rats were randomly divided into three groups： control group （CN, n=8）, exhaustive swimming group （ES, n=24） and lnula Britannica group （IB, n=16）. The rats of CN were quiet without swimming. The rats of ES swam to exhaustion and were sacrificed at immediately （ESI, n=8）, 6 hour （ES 6 h, n=8） and 24 hour （ES 24 h, n=8） after exhaustiing swimming. The rats of IB group took orally lnula 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, n=8） and 24 hour （IB 24 h, n=8） after exhaustiing swimming. The renal cell apoptosis was measured by the method of terminal- deoxynueleotidyl transferase mediated d-UTP nick end labeling （TUNEL）. The expression of TNF-α in renal tissue was examined by imnmnohistochemistry. The changes of NF-KB in renal tissue were measured by flow cytometry and immunohistochemistry. The interrelation between TNF-α and NF-KB was analyzed by Pearson method, and the interrelation between TNF-α, NF-KB and renal tissue cell apoptosis was analyzed by Spearman method. Results The number of renal tissue apoptotic cells was increased progressively from ESI to ES 24 h rats （P 〈0.05）. Immunohistochemistry staining showed that the positive expressions of renal tissue TNF-α and NF-kB were increased progressively at 0 h （0.136±0.009, 0.129±0.011）, 6 h （0.171±0.011, 0.166± 0.009） and 24 h （0.229±0.008, 0.218±0.019） after exhaustiing swimming in ES compared with control group （0.109±0.010, 0.095±0.010） （ all P〈0.05）. The similar changes of renal tissue NF-KB was also revealved by flow cytometry. The expression of TNF-α was positively correldted with NF-KB （r=0.955, P〈O.01 ）, and renal cell apoptosis was also positively correlated with TNF-α and NF-KB （r=0.953, r=0.939, P〈O.01） in ES rats. Pretreatment with Inula Britcmnica, inhibited the up-regulation of expressions of renal tissue TNF-α （6 h：0.142±0.012, 24 h：0.130±0.010） and NF-KB （6 h：0.138±0.010, 24 h：0.136±0.011 ） induced by exhausting swimming. Conclusion Overtraining can induce the up-regulating expressions of renal tissue TNF-α and NF-KB, and Inula Britannica can partly counter the above changes in exhaustied swimming rats, which may be one important mechanisms of overtraining-indueed renal tissue cell apoptosis and the anti- apoptosis effect of lnula Britannica.