过度训练可部分通过上调TLR4的表达激活死亡受体凋亡通路诱导大鼠肾小管上皮细胞凋亡

Overtraining Induces Renal Tubular Cells Apoptosis Through Activating Death Receptor Signal Pathway by Impairing TLR4 in Exhaustive Swimming Rats

目的:观察过度训练大鼠肾组织TLR4、Fas及Caspase-8表达的变化,探讨TLR4在死亡受体凋亡通路中的作用。方法:将48只雄性SD大鼠按随机数字表法分为对照组(CN)、力竭运动组(ES)、山莨菪碱干预组(AD)。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);AD组于力竭运动前20 min腹腔注射山莨菪碱10 mg/kg后进行力竭运动,分为AD 6 h和AD 24 h组。采用大鼠游泳至力竭建立过度训练模型。采用Western印记测定肾组织TLR4和Fas的表达,免疫组织化学法检测肾组织Caspase-8的表达,并分别对TLR4与Fas、Caspase-8、肾组织细胞凋亡率进行相关性分析。结果:Western印记测定结果显示,过度训练大鼠肾组织TLR4及Fas的表达于力竭后即刻、6 h及24 h逐渐增高,均显著高于对照组(P<0.05)。免疫组化显示,过度训练大鼠肾组织Caspase-8的表达于力竭后即刻、6 h及24 h逐渐增强,均显著高于对照组(P<0.05)。过度训练大鼠肾组织TLR4与Fas、Caspase-8和细胞凋亡率的表达均呈正相关性(r=0.848,P<0.05;r=0.916,P<0.05;r=0.95,P<0.05)。用山莨菪碱干预后,过度训练引起的肾组织TLR4、Fas及Caspase-8的过度表达均被显著抑制(均P<0.05)。结论:过度训练可通过上调TLR4强化死亡受体凋亡通路,进而诱导大鼠肾小管上皮细胞凋亡。

Objective：To investigate the changes of the expression of renal tissue TLR4,Fas,Caspase-8 and the influences of TLR4 in death receptor signal pathway.Methods：Forty-eight male SD rats were randomly divided into three groups： control（CN,n=8）,exhaustive swimming（ES,n=24） and Anisodamine（AD,n=16）.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 AD received intraperitoneal injection of Anisodamine at the dose of 10 mg/kg body weight at 20 minutes before swimming and then swam to exhaustive condition.The rats of AD group were sacrificed at 6 hour（AD 6 h） and 24 hour（AD 24 h） after exhaustive swimming.The expression of TLR4 and Fas protein in renal tissue were examined by Western Blotting.The expression of Caspase-8 in renal tissue was observed by immunohistochemistry.Results：The expression of TLR4 and Fas and Caspase-8 were significantly increased in group ES as compared with control group.Respectively,TLR4 had significantly positive correlation with the value of Fas and Caspase-8 and renal tubular cell apoptosis（r=0.848,P0.05;r=0.916,P0.05;r=0.95,P0.05）in ES rats.Pretreatment with Anisodamine inhibited the up-regulation of TLR4,Fas,Caspase-8 and cell apoptosis in renal tublar cells induced by exhaustive swimming.Conclusion：Overtraining can induce renal tubular cells apoptosis through activating Fas-Caspase-8 signal pathway by impairing TLR4.