动力相关蛋白-1在缺氧/复氧诱导人肾小管上皮细胞焦亡机制中的作用

Role of dynamin-related protein 1 in hypoxia / reoxygenation-induced human renal tubular epithelial cell pyroptosis

目的观察人肾小管上皮细胞(HK-2细胞)缺氧/复氧(hypoxia/reoxygenation,H/R)损伤时线粒体动力相关蛋白1(dynamin-related protein 1,Drp1)、Nod样受体热蛋白结构域相关蛋白3(nucleotide binding oligomerization domain-like receptor family pyrin domain containing 3,Nlrp3)以及炎性因子的表达改变和细胞焦亡情况,探讨Drp1在H/R诱导肾小管上皮细胞焦亡中的作用。方法 HK-2细胞用含10%胎牛血清的DMEM/F12液培养于37℃、5%CO2和95%O2的恒温培养箱。将细胞分为正常对照组、H/R组、H/R+Drp1抑制剂(H/R+Mdivi-1)组、H/R+si Nlrp3腺病毒(H/R+si Nlrp3)组、H/R+空腺病毒(H/R+Scra)组。检测细胞培养液上清乳酸脱氢酶LDH活性,Western blot检测Drp1、Nlrp3、凋亡相关蛋白-1(caspase-1)、IL-1β表达,2',7'-二氯荧光黄双乙酸盐(DCFH-DA)检测细胞活性氧(ROS)水平,流式分析仪检测细胞焦亡情况。结果与正常对照组比较,H/R组培养液上清LDH和细胞内ROS水平明显升高,Drp1、Nlrp3、caspase-1、IL-1β表达增高,细胞焦亡明显增加,差异有统计学意义(P<0.05);与H/R组比较,H/R+Mdivi-1组培养液上清LDH和细胞内ROS水平明显降低,Nlrp3、caspase-1、IL-1β表达降低,细胞焦亡明显减少,差异有统计学意义(P<0.05)。结论在H/R条件下,Drp1通过激活Nlrp3炎性体诱导人肾小管上皮细胞焦亡。

Objective To study the expressions of dynamin-related protein 1（ Drp1）,nucleotide binding oligomerization domain-like receptor family pyrin domain containing 3（ Nlrp3） and inflammatory cytokines and the pyroptosis during hypoxia / reoxygenation（ H / R） injury in human renal tubular epithelial cell line（ HK-2）,and discuss the relationship between Drp1 and H / R-induced pyroptosis of HK-2 cells.Methods HK-2 cells were cultured in DMEM / F12 containing 10% fetal bovine serum in a thermostatic incubator（ 37 ℃,5% CO2）. Then the cells were divided into normal control group,H/R group,H/R ＋Mdivi-1（ Drp1 inhibitor） group,H / R ＋ si Nlrp3 group and H / R ＋ Scra group. LDH activity in the supernatant of cell culture was tested with an automatic biochemical analyzer. The expressions levels of Drp1,Nlrp3,caspase-1 and IL-1β were detected by Western blotting. Reactive oxygen species was measured by 2＇,7＇-dichlorodihydrofluorescein diacetate（ DCFH-DA） assay. Pyroptosis was analyzed with a flow cytometer.Results The H / R group exhibited higher celluar ROS level and higher expressions of Drp1,Nlrp3,caspase-1 and IL-1β proteins than the normal control group（ P〈 0. 05）,and the pyroptosis index were increased in the H / R group（ P 〈0. 05）. Mdivi-1 could dramatically decrease the level of ROS and pyroptosis and relieve the dysfunction of HK-2 cells（ P〈 0. 05）. Conclusion Drp1 can induce pyroptosis of human renal tubular epithelial cells through activating Nlrp3 in H / R injury.