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目的目的探讨体外缺血后处理对肾小管上皮细胞缺血再灌注损伤后TLR4/NF-k B表达的影响。方法将人肾小管上皮细胞HK-2同步化24 h后,细胞模拟缺血3 h,然后行3个10 min再灌注/10 min缺血循环,最后正常培养至24 h建立肾缺血后处理的体外模型。流式细胞仪和Hoechst检测细胞凋亡,免疫细胞化学检测BAX表达情况,蛋白印迹法检测TLR4和NF-k B蛋白水平。结果与正常对照组相比,模拟缺血再灌注损伤组细胞凋亡比率增加,差异有统计学意义(P<0.05),而缺血后处理组凋亡比率明显下降。Hoechst染色结果显示,模拟缺血再灌注组细胞凋亡明显,而缺血后处理组细胞凋亡减轻。免疫细胞化学染色结果显示,Bax在模拟缺血再灌注组中表达明显增加,而在缺血后处理组中显著减轻。Western blot结果显示,TLR4和NF-k B蛋白表达在模拟缺血再灌注组中明显升高,而在缺血后处理组TLR4和NF-k B蛋白表达显著减轻。结论体外缺血后的处理能显著减弱缺血再灌注后的损伤,机制可能与抑制TLR4/NF-k B通路有关。
Objective To investigate the effect of ischemic postconditioning on TLR4 / NF-κB expression in renal tubular epithelial cells after ischemia-reperfusion injury. Methods Human renal tubular epithelial cells (HK-2) were synchronized for 24 h. After 3 h of ischemia, the cells were subjected to ischemia-reperfusion for 10 min followed by 10 min reperfusion and 10 min reperfusion. In vitro model. Flow cytometry and Hoechst were used to detect apoptosis. Immunocytochemistry was used to detect the expression of BAX. Western blotting was used to detect the protein levels of TLR4 and NF-κB. Results Compared with the normal control group, the apoptotic rate in simulated ischemia-reperfusion injury group increased significantly (P <0.05), while the apoptotic rate in ischemic postconditioning group decreased significantly. The results of Hoechst staining showed that the apoptosis of mock-ischemia-reperfusion group was obvious, while the apoptosis of ischemic postconditioning group was alleviated. Immunocytochemical staining showed that Bax was significantly increased in the simulated ischemia-reperfusion group and significantly decreased in the ischemic postconditioning group. Western blot results showed that TLR4 and NF-|ÊB protein expression in simulated ischemia-reperfusion group was significantly increased, while in ischemic postconditioning group TLR4 and NF-k B protein expression was significantly reduced. Conclusion The post-ischemic treatment can significantly reduce the injury after ischemia-reperfusion, which may be related to the inhibition of TLR4 / NF-κB pathway.