缺血再灌注导致急性肾损伤中的细胞死亡形式初探

Forms of cell death in acute renal injury induced by ischemia reperfusion

目的探究在肾缺血再灌注损伤(IRI)模型中存在的细胞死亡形式。方法将24只Wistar大鼠随机分为4组:正常组(NC组)、假手术组(Sham组)、肾缺血组(RI组)、肾缺血再灌注组(RIR组)。RI组及RIR组通过钳夹双肾动脉行30min双肾动脉阻断,诱导肾缺血,RIR组阻断30min后松开钳夹恢复血流180min制成肾缺血再灌注模型。NC组不处理,Sham组开腹但不阻断双侧肾动脉。再灌注180min结束后收集血清和肾组织。免疫组化、免疫印迹法对肾组织中混合系列蛋白激酶样结构域蛋白(MLKL)和谷胱甘肽过氧化物酶4(GPX4)进行定位及表达水平检测,TUNEL法分析肾小管细胞凋亡情况。定量分析超氧化物歧化酶(SOD)、丙二醛(MDA)的变化。结果TUNEL法显示肾脏细胞凋亡明显增加。Westem blot检测显示RIR组与NC组相比,MLKL表达上升,GPX4表达降低(P<0.05),免疫组化病理切片结果与其一致。SOD活力显著下降(P<0.05),MDA水平明显上升(P<0.05)。结论RIR模型中肾小管细胞死亡数增加,抗氧化能力下降,RIR中有坏死性凋亡(necroptosis)和铁坏死(ferroptosis)两种细胞死亡形式的参与。

Objective To explore the forms of cell death in the model of renal ischemia reperfusion injury(IRI).Methods Twenty-four Wistar rats were randomly divided into four groups:the normal group(NC group),the Sham group(Sham group),the renal ischemia group(RI group)and renal ischemia reperfusion group(RIR group).Renal ischemia was induced in RI group and RIR group by clamping bilateral renal artery for 30min.In the RIR group,the renal ischemia-reperfusion model was established after loosening the clamp for 180min to restore the blood flow.The NC group received no treatment,while the Sham group underwent laparotomy without clamping bilateral renal arteries.The serum and renal tissues were collected after 180min of reperfusion.Immunohistochemistry and western blotting were employed to detect the localization and expressions of the mixed lineage kinase domain-like protein(MLKL)and glutathione peroxidase 4(GPX4)in kidney tissues.TUNEL assay was adopted to analyze the apoptosis of renal tubular cells.The changes of superoxide dismutase(SOD)and malondialdehyde(MDA)were quantitatively analyzed.Results TUNEL assay showed that the apoptosis of renal cells increased significantly.Western blot analysis showed that,compared with the NC group,the RIR group had increased expression of MLKL and decreased expression of GPX4(P<0.05).The results of immunohistochemical pathological section were consistent between two groups.The SOD activity decreased significantly(P<0.05)and the level of MDA increased significantly(P<0.05).Conclusion In the renal RIR model,there is an increase in the death number of renal tubule cells and an decrease in the antioxidant capacity.Necroptosis and ferroptosis are two forms of cell death involved in RIR.