体外模拟缺血后处理对人HK-2细胞缺血再灌注损伤的保护作用研究

Research on protective effect of simulated ischemic postconditioning on ischemia reperfusion injury use human HK-2 cell in vitro

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 effects of ischemic postconditioning（IPO）on the expression of TLR4/NF-κB,using the human kidney cortex proximal tubule epithelial HK-2 cells in vitro ischemic postconditioning model.[Methods]Vitro model of renal ischemic postconditioning were established by making the cells exposed to three cycles of reperfusion condition for 10 minutes and ischemic condition for 10 minutes after placing the cells in ischemic condition for 3 hours,and then to the time required.Flowcytometry and Hoechst were used to accessing apoptosis.Immunocytochemistry was performed to detect the expression of Bax.The expression spot and protein levels of TLR4 and NF-κB were also analyzed.[Results]Compared with the control group,apoptosis rate was aggravated in ischemia reperfusion injury（IRI）group,and the difference was statistically significant（P〈0.05）,while this rate was significantly decreased in IPO group.The result of Hoechst staining indicated that IPO could reduce apoptosis induced by IRI.Immunocytochemistry found that the expression of Bax was up-regulated in IRI group,and IPO could down regulate its expression.Western blot showed that TLR4 and NF-κB expression increased significantly in IRI group,and their expression decreased significantly in IPO group.[Conclusion]In vitro ischemic postconditioning significantly weakens the injury by ischemia-reperfusion and the mechanism may be through inhibiting TLR4/NF-κB pathway to exert protective effects.