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LRP-1抑制缺氧/复氧诱导的肾细胞损伤
作者:段斌1  职瑾2  杜鹏1  高妍婷1  李振江1 
单位:1. 陕西省人民医院 肾病血透中心, 陕西 西安 710068;
2. 西安市第一医院, 陕西 西安 710002
关键词:低密度脂蛋白受体相关蛋白1 缺氧/复氧 肾细胞 JNK 
分类号:R33-33
出版年·卷·期(页码):2017·36·第十二期(1697-1704)
摘要:

目的:探讨低密度脂蛋白受体相关蛋白1(LRP-1)在肾小管上皮细胞(HK2)缺氧/复氧损伤中的作用及其可能机制。方法:建立HK-2缺氧/复氧模型;实时荧光定量PCR (qRT-PCR)检测LRP-1 mRNA表达量;Western blotting检测LRP-1、JNK蛋白表达量和JNK磷酸化水平;构建重组载体pcDNA.3.1-LRP-1并转染细胞过表达LRP-1;转染LRP-1 siRNA抑制LRP-1表达;试剂盒方法检测caspase-3活性;Annexin-Ⅴ fluorescein isothiocyanate conjugate and propidium iodide (Annexin-Ⅴ FITC/PI)方法检测细胞凋亡;MTT法检测细胞生长活力;试剂盒方法检测乳酸脱氢酶(LDH)漏出量;JNK抑制剂SP600125孵育HK-2。结果:HK-2缺氧/复氧诱导LRP-1表达量上调;过表达LRP-1显著下调JNK磷酸化水平,降低缺氧/复氧诱导的HK-2凋亡,降低caspase-3活性和LDH漏出量,且提高细胞生长活力。抑制LRP-1则作用相反。另外,SP600125抑制JNK磷酸化水平,且提高细胞生长活力,降低caspase-3活性。结论:LRP-1可通过调控JNK降低肾细胞的缺氧/复氧损伤,为肾脏缺血再灌注的防治提供新的靶点。

Objective: To explore the effects of low density lipoprotein receptor-related protein 1(LRP-1) on hypoxia/reoxygenation-induced renal tubular epithelial cell (HK-2) injury and the related mechanism.Methods: The model of HK-2 with hypoxia/reoxygenation injury was established. The mRNA expression of LRP-1 was measured by qRT-PCR. The protein expressions of LRP-1 and JNK and JNK phosphorylation level were detected using Western blotting. Recombinant vector pcDNA.3.1-LRP-1 was constructed and transfected into cells to overexpress LRP-1. LRP-1 siRNA was transfected into cells to inhibit the expression of LRP-1. Caspase-3 activity was detected by using the kit. Cell apoptosis was tested by Annexin-Ⅴ fluorescein isothiocyanate conjugate and propidium iodide (Annexin-Ⅴ FITC/PI) assay. Cell viability was detected by MTT. LDH leakage was quantified by using the kit. HK2 was incubated with JNK inhibitor SP600125.Results: LRP-1 was induced by the hypoxia/reoxygenation injury in HK-2. LRP-1 overexpression obviously down-regulated JNK phosphorylation level, hypoxia/reoxygenation-induced cells apoptosis, caspase-3 activity and LDH leakage, whereas promoted cell viability. LRP-1 inhibition had the reversed effect. What's more, SP600125 decreasd the JNK phosphorylation level and LDH leakage, whereas increased cell viability.Conclusions: LRP-1 can constrain the kidney cells injury caused by hypoxia/reoxygenation via regulating JNK, providing the theoretical basis for the treatment of renal ischemia reperfusion.

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