微小RNA-137对人肾小管上皮细胞缺氧/复氧损伤的影响

Effects of microRNA-137 on hypoxia/reoxygenation injury of human renal tubular epithelial cells

目的探究微小RNA-137(miR-137)通过磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/AKT)信号通路对人肾小管上皮细胞缺氧/复氧(H/R)损伤的影响。方法取人肾小管上皮细胞HK-2,将其随机分为对照组和模型组。对照组细胞常规培养,模型组细胞缺氧4 h、复氧2 h以构建H/R模型。将HK-2细胞转染miR-137模拟物或阴性对照寡核苷酸,随后构建H/R损伤模型,分别命名为miR-137 mi组和miR-NC组。用原位末端标记(TUNEL)法检测各组细胞的凋亡率;用丙二醛(MDA)检测试剂盒、谷胱甘肽过氧化物酶(GSH-PX)测定试剂盒及超氧化物歧化酶(SOD)试剂盒检测各组细胞MDA、GSH-PX和SOD的含量;用酶联免疫吸附(ELISA)法检测各组细胞单核细胞趋化蛋白-1(MCP-1)、细胞间黏附分子-1(ICAM-1)、白细胞介素-1β(IL-1β)的含量;用蛋白质印迹(Western blot)法检测磷酸化PI3K(p-PI3K)蛋白及磷酸化AKT(p-AKT)蛋白的表达水平。结果对照组、模型组、miR-NC组和miR-137 mi组的细胞凋亡率分别为(5.06±0.10)%,(13.74±1.39)%,(11.75±1.20)%和(8.32±0.76)%;MDA的含量分别为(38.37±2.33),(45.38±3.02),(45.95±3.27),(33.49±1.78)nmol·mg^(-1);GSH-PX的含量分别为(573.17±5.15),(204.46±2.77),(205.39±1.72),(415.80±4.48)U·mg^(-1);SOD的活性分别为(11.42±1.84),(4.92±0.20),(5.01±0.34),(7.93±0.57)U·mg^(-1);MCP-1的含量分别为(1.57±0.13),(4.78±0.38),(4.30±0.14),(2.95±0.62)ng·mL^(-1);ICAM-1的含量分别为(0.18±0.05),(0.56±0.09),(0.53±0.04),(0.25±0.09)ng·mL^(-1);IL-1β的含量分别为(23.62±2.85),(43.83±1.70),(44.07±2.33),(31.86±1.67)pg·mL^(-1),以上指标,对照组和模型组相比,miR-NC组和miR-137 mi组相比,差异均有统计学意义(均P<0.05)。结论miR-137可减少人肾小管上皮细胞H/R损伤模型细胞凋亡、降低氧化应激以及炎症反应,其作用机制与PI3K/AKT信号通路有关。

Objective To investigate the effect of microRNA-137(miR-137)mediating hypoxia/reoxygenation(H/R)injury in renal tubular epithelial cells through regulating phosphatidylinositol-3 kinase/protein kinase B(PI3 K/AKT)signaling pathway.Methods Human renal tubular epithelial cells HK-2 were randomly divided into control group and model group.Cells in control group were cultured routinely.Cells in model group were subjected to hypoxia for 4 h and reoxygenation for 2 h to construct the H/R model.HK-2 cells were transfected with miR-137 mimics or negative control oligonucleotides,and then the H/R injury was modeled,which were named as miR-137 mi group or miR-NC group,respectively.The apoptosis rate of each group was detected by terminal transferase-mediated d UTP nick end-labeling(TUNEL)assay;the contents of malondialdehyde(MDA),glutathione peroxidase(GSH-PX)and superoxide dismutase(SOD)in each group were measured by MDA assay kit,GSH-PX assay kit and SOD assay kit;the contents of monocyte chemoattractant protein-1(MCP-1),intercellular adhesion molecule-1(ICAM-1)and interleukin-1β(IL-1β)in each group were detected by enzyme-linked immunosorbent assay(ELISA).Western blot assay was used to detect the expression of the phosphorylated PI3 K(p-PI3 K)protein and phosphorylated AKT(p-AKT)protein.Results The apoptosis rates of control group,model group,miR-NC group and miR-137 mi group were(5.06±0.10)%,(13.74±1.39)%,(11.75±1.20)%and(8.32±0.76)%,respectively;the contents of MDA were(38.37±2.33),(45.38±3.02),(45.95±3.27),(33.49±1.78)nmol·mg^(-1);the contents of GSH-PX were(573.17±5.15),(204.46±2.77),(205.39±1.72)and(415.80±4.48)U·mg^(-1);the activities of SOD were(11.42±1.84),(4.92±0.20),(5.01±0.34)and(7.93±0.57)U·mg^(-1);the contents of MCP-1 were(1.57±0.13),(4.78±0.38),(4.30±0.14)and(2.95±0.62)ng·mL^(-1);the contents of ICAM-1 were(0.18±0.05),(0.56±0.09),(0.53±0.04)and(0.25±0.09)ng·mL^(-1);and the contents of IL-1βwere(23.62±2.85),(43.83±1.70),(44.07±2.33)and(31.86±1.67)pg·mL^(-1).There were statistically significant differences in the above indicators between control group and model group,and between miR-NC group and miR-137 mi group(all P<0.05).Conclusion miR-137 can inhibit the apoptosis,and alleviate the oxidative stress and inflammatory response in cells with H/R injury by regulating PI3 K/AKT signaling pathway.