Necrostatin-1 Attenuates Renal Ischemia and Reperfusion Injury via Meditation of HIF-1α/mir-26a/TRPC6/PARP1 Signaling
Necrostatin-1 Attenuates Renal Ischemia and Reperfusion Injury via Meditation of HIF-1α/mir-26a/TRPC6/PARP1 Signaling
复制标题
Necrostatin-1 通过调节 HIF-1 alpha/mir-26a/TRPC6/PARP1 信号传导减轻肾缺血和再灌注损伤
DOI：
10.1016/j.omtn.2019.06.025
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发表时间：
2019-09-06
影响因子：
8.8
通讯作者：
Zhao, Hongwen
中科院分区：
文献类型：
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作者：
Shen, Bingbing;Mei, Mei;Zhao, Hongwen
坏死性下垂、氧化应激和炎症是缺血性急性肾损伤的主要致病因素。肿瘤抑制素-1(NEC-1)是受体相互作用蛋白激酶-1(RIP1)激动域的抑制剂，已有报道可调节肾缺血再灌注(I/R)损伤，但其潜在作用机制尚不清楚。用HK-2细胞建立体外I/R模型，缺氧后复氧2、6、12h。为进行体内研究，建立了大鼠肾I/R模型，再灌注后采集大鼠血清和肾组织标本，以评估肾功能和检测组织学变化。分别用细胞计数试剂盒(CCK)-8法和流式细胞术分析细胞存活率和坏死性下垂。通过实时定量聚合酶链式反应、免疫印迹、免疫荧光染色和酶联免疫吸附试验检测与坏死性下垂、氧化应激和炎症相关的分子的表达水平。荧光素酶和染色质免疫沉淀(ChIP)检测以确定相关的下游信号通路。我们发现，NEC-1预处理显著降低了I/R损伤细胞和大鼠模型中缺氧诱导因子-1α(HIF-1α)和miR-26α的表达，以及与坏死性下垂相关的因子(RIP1、RIP3和Sirtuin-2)、氧化应激(丙二醛[MDA]、NADP(+)/NADPH比率)和炎症(白介素[IL]-1β、IL-10和肿瘤坏死因子α[TNF-α])的水平。然而，这些效应可以被miR-26α的过度表达或TRPC6基因敲除而逆转。机制研究表明，HIF-1α与miR-26a的启动子区域直接结合，TRPC6是miR-26a的潜在靶基因。我们的研究结果表明，NEC-1通过抑制坏死性下垂、氧化应激和炎症反应而有效地保护肾I/R损伤，并可能通过HIF-1α/miR-26a/TRPC6/PARP1信号通路发挥作用。
Necroptosis, oxidative stress, and inflammation are major contributors to the pathogenesis of ischemic acute kidney injury. Necrostatin-1 (Nec-1), an inhibitor of the kinase domain of receptor-interacting protein kinase-1 (RIP1), has been reported to regulate renal ischemia and reperfusion (I/R) injury; however, its underlying mechanism of action remains unclear. HK-2 cells were used to create an in vitro I/R model, in which the cells were subjected to hypoxia, followed by 2, 6, and 12 h of reoxygenation. For the in vivo study, a rat model of renal I/R was established in which samples of rat blood serum and kidney tissue were harvested after reperfusion to assess renal function and detect histological changes. Cell viability and necroptosis were analyzed using the Cell Counting Kit (CCK)-8 assay and flow cytometry, respectively. The expression levels of molecules associated with necroptosis, oxidative stress, and inflammation were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to confirm the relevant downstream signaling pathway. We found that pretreatment with Nec-1 significantly decreased hypoxia-inducible factor-1 alpha (HIF-1 alpha) and miR-26 alpha expression, as well as the levels of factors associated with necroptosis (RIP1, RIP3, and Sirtuin-2), oxidative stress (malondialdehyde [MDA], NADP(+)/NADPH ratio), and inflammation (interleukin [IL]-1 beta, IL-10, and tumor necrosis factor alpha [TNF-alpha]) in I/R injury cells and the rat model. However, these effects could be reversed by miR-26 alpha overexpression or TRPC6 knockdown. Mechanistic studies demonstrated that HIF-1 alpha directly binds to the promoter region of miR-26a, and that TRPC6 is a potential target gene for miR-26a. Our findings indicate that Nec-1 can effectively protect against renal I/R injury by inhibiting necroptosis, oxidative stress, and inflammation, and may exert its effects through mediation of the HIF-1 alpha/miR-26a/TRPC6/PARP1 signaling pathway.