基于Keap1-Nrf2/HO-1信号通路探讨miR-141对大鼠脑缺血再灌注后的炎症反应及保护神经元功能的研究

miR-141 targeting Keap1-Nrf2/HO-1 signaling pathway alleviates the inflammation and protects neurons in rats with cerebral ischemia reperfusion injury

目的探究miR-141靶向KELCH样ECH关联蛋白-核因子E2相关因子2/血红素加氧酶-1(Keap1-Nrf2/HO-1)信号通路调控脑缺血再灌注(IR)后的炎症反应及保护神经元功能的作用机制。方法60只大鼠随机分为sham组、IR组、agomirNC组、miR-141组;agomir-NC组、miR-141组大鼠侧脑室注射agomir NC、miR-141 agomir,sham组、IR组注射等体积生理盐水。用大脑中动脉栓塞(MCAO)构建IR模型,sham组只插线不结扎。72 h后行神经功能学评分;TTC染色、TUNEL染色检测脑梗死体积和细胞凋亡;ELISA法、qRT-PCR、Western blot检测IL-1β、IL-6、TNF-α、miR-141、Keap1、Nrf2、HO-1、NLRP3水平。同时将体外培养并转染的小胶质细胞BV-2分为control组、OGD组、agomir-NC组、miR-141组、miR-141+pcDNA3.1-NC组、miR-141+Keap1组;糖氧剥夺(OGD)法建立IR细胞模型。TargetScanHuman预测及双荧光素酶报告验证miR-141对Keap1的靶向调控关系;MTT法、AnnexinV-FITC/PI双染法测定BV-2细胞增殖、凋亡能力;再次应用ELISA法、qRT-PCR、Western blot检测白介素-1β(IL-1β)、白介素-6(IL-6)、TNF-α、miR-141、Keap1、Nrf2、HO-1、NLRP3水平。结果过表达miR-141可促进IR大鼠神经功能恢复,减少脑梗死体积和细胞凋亡,降低IL-1β、IL-6、TNF-α、NLRP3水平,升高Nrf2、HO-1水平。BV-2细胞中miR-141能够负向调控Keap1表达,过表达miR-141抑制Keap1-Nrf2/HO-1信号通路,促进细胞增殖,抑制细胞凋亡,并下调IL-1β、IL-6、TNF-α、NLRP3的表达。结论miR-141能够靶向调控Keap1-Nrf2/HO-1信号通路,减轻IR的炎症反应,限制小胶质细胞激活,保护神经元。

The purpose of this study was to explore the mechanism of miR-141 targeting KELCH-like ECHrelated protein-nuclear factor E2-related factor 2/heme oxygenase-1(Keap1-Nrf2/HO-1)signal pathway in regulating inflammatory response and protecting neuronal function after ischemia reperfusion(IR).Total of 60 rats were recruited and randomly divided into sham group,IR group,agomir-NC group and miR-141 group.The rats in the agomir-NC and miR-141 groups were injected with agomir NC and miR-141 agomir in the lateral ventricle,while sham group and IR group were injected with the same volume of normal saline.IR model was constructed by middle cerebral artery occlusion(MCAO),while in the sham group,the thread was inserted without ligation.72 hours later,neurological score was calculated;TTC staining and TUNEL staining were used to detect cerebral infarction volume and cell apoptosis;ELISA,qRT-PCR,and Western blot were used to detect the levels of IL-1β,IL-6,TNF-α,miR-141,Keap1,Nrf2,HO-1 and NLRP3.At the same time,the microglia BV-2 cultured and transfected in vitro were divided into control group,OGD group,agomir-NC group,miR-141 group,miR-141+pcDNA3.1-NC group and miR-141+Keap1 group.IR cell model was constructed by oxygen and glucose deprivation(OGD).TargetScanHuman prediction and dual luciferase report verify the targeted regulation of miR-141 on Keap1;MTT and AnnexinV-FITC/PI double staining were used to determine the proliferation and apoptosis ability of BV-2 cells;ELISA,qRT-PCR and Western blot were used to detect the expression levels of interleukin-1β(IL-1β),interleukin-6(IL-6),TNF-α,miR-141,Keap1,Nrf2,HO-1 and NLRP3.Data showed that miR-141 overexpression could promote the recovery of neurological function in IR rats,reduce the cerebral infarction volume and apoptosis,decrease the levels of IL-1β,IL-6,TNF-αand NLRP3,and increase the levels of Nrf2 and HO-1.In BV-2 cells,miR-141 could negatively regulate the expression of Keap1.Furthermore,overexpression of miR-141 inhibited Keap1-Nrf2/HO-1 signaling pathway,promoted cell proliferation,inhibited apoptosis,and downregulated the expression of IL-1β,IL-6,TNF-αand NLRP3.In conclusion,miR-141 can target and regulate Keap1-Nrf2/HO-1 signal pathway,inhibit the inflammatory response of IR,limit the activation of microglia and protect neurons.