Dex基于CREB/PGC-1α信号通路调控星形胶质细胞线粒体功能在大鼠脑缺血再灌注损伤中的作用

The role of dexmedetomidine based on CREB/PGC-1α signaling pathway to regulate d astrocyte mitochondrial function in rats with cerebral ischemia-reperfusion injury

目的 探究右美托咪定(Dex)基于CREB/PGC-1 α信号通路调控星形胶质细胞线粒体功能在大鼠脑缺血再灌注损伤中的作用。方法 采用改良的Zen-land法制作大鼠脑缺血再灌注损伤(middle cerebral artery occlusion,MCAO)模型,分别在造模后评估假手术(Sham)组、脑缺血再灌注模型(MCAO)组,脑缺血再灌注加药(MCAO+Dex)组大鼠神经功能损伤,脑梗死面积,实时荧光定量PCR(qPCR)和蛋白印迹检测环磷腺苷效应元件结合蛋白(CREB)、过氧化物酶体增殖物激活受体-γ共激活因子-1α(PGC-1α)的mRNA及蛋白表达;透射电子显微镜检测线粒体形态,免疫荧光法检测星形胶质细胞活性氧物质(ROS)及细胞凋亡。制备氧糖剥夺/复氧(OGD/R)星形胶质细胞模型,分为对照组(Control)、溶剂组(OGD/R-Dex0)、低剂量组(OGD/R-Dex-L)、高剂量组(OGD/R-Dex-H),qPCR检测CREB、PGC-lα的mRNA水平,免疫荧光和WB检测CREB、PGC-1α的蛋白表达。在OGD/R星形胶质细胞模型Dex处理的基础上过表达CREB或PGC-1 α,分为过表达对照组(OE-NC)、过表达CREB组(OE-CREB)、过表达PGC-1 α组(OE-PGC-1 α),流式细胞术检测细胞凋亡,探针法检测线粒体ROS水平。结果Dex预处理改善MCAO大鼠神经功能评分,减少脑梗死面积,抑制CREB、PGC-1 α表达,改善线粒体形态,抑制细胞凋亡和星形胶质细胞ROS水平。Dex可抑制OGD/R星形胶质细胞中CREB、PGC-1α的mRNA及蛋白表达水平及线粒体ROS水平。过表达CREB和PGC-1α逆转由Dex诱发的星形胶质细胞ROS水平和细胞凋亡抑制。结论 Dex可下调CREB和PGC-1 α表达,进而抑制星形胶质细胞ROS水平和细胞凋亡,从而在大鼠脑缺血再灌注损伤中发挥保护功能。

Objective To explore the role of dexmedetomidine(Dex)based on CREB/PGC-1αsignaling pathway in regulating astrocyte mitochondrial function in rats with cerebral ischemia-reperfusion injury.Methods The modified Zen-land method was used to make a rat cerebral ischemia-reperfusion injury(middle cerebral artery occlusion,MCAO)model.The neurological damage,cerebral infarction area were evaluated in the sham-operated(Sham)group,the cerebral ischemia-reperfusion model(MCAO)group,and the cerebral ischemia-reperfusion plus drug(MCAO+Dex)group.cerebral infarction area,Quantitative real-time PCR(qPCR)and Western blotting(WB)detection of cyclic adenosine monophosphate response element binding protein(CREB),peroxisome proliferator-activated receptor-coactivator-1α(PGC-1α)mRNA and protein expression;Mitochondrial morphology was detected by transmission electron microscopy,and reactive oxygen species(ROS)and apoptosis of astrocytes were detected by immunofluorescence.Oxygen-glucose deprivation/reoxygenation(OGD/R)astrocyte models were prepared and divided into control group(Control),solvent group(OGD/R-DexO),low-dose group(OGD/R-Dex-L),High-dose group(OGD/R-Dex-H).The mRNA levels of CREB and PCC-1αwere detected by qPCR,and the protein expressions of CREB and PGC-1αwere detected by immunofluorescence and WB.The OGD/R astrocyte model was overexpressed CREB or PGC-1αon the basis of Dex treatment,and divided into overexpression control group(OE-NC),overexpression CREB group(OE-CREB),overexpression PGC-1αgroup(OE-PGC-1α),Cell apoptosis was detected by flow cytometry,and mitochondrial ROS levels were detected by probe method.Results Dex pretreatment improved neurological function scores,reduced cerebral infarct size,inhibited the expression of CREB and PGC-1α,improved mitochondrial morphology,and inhibited apoptosis and astrocyte ROS levels in MCAO rats.Dex can inhibit the mRNA and protein expression levels of CREB and PCC-1αand the level of mitochondrial ROS in OGD/R astrocytes.Overexpression of CREB and PGC-1αreversed the Dex-induced inhibition of ROS levels and apoptosis in astrocytes.Conclusion Dex can down-regulate the expression of CREB and PGC-1α,thereby inhibiting the level of ROS and apoptosis in astrocytes,thereby exerting a protective function in cerebral ischemia-reperfusion injury in rats.