PKA-PINK1/Parkin信号通路在氧糖剥夺/复氧复糖后大鼠皮质星形胶质细胞凋亡和焦亡中的作用  

Effects of PKA-PINK1/Parkin signal pathway on apoptosis and pyroptosis of rat cortical astrocytes exposed to oxygen-glucose deprivation/resuscitation

作　　者：于红[1] 马美娜[1] 周真真 郭庆夺[1] 于红美[1] YU Hong;MA Mei-na;ZHOU Zhen-zhen;GUO Qing-duo;YU Hong-mei(Department of Anesthesiology, Cangzhou Central Hospital, Hebei Province, Cangzhou 061000, China)

机构地区：[1]河北省沧州市中心医院麻醉科,河北沧州061000

出　　处：《河北医科大学学报》2021年第2期129-133,171,共6页Journal of Hebei Medical University

摘　　要：目的评价蛋白激酶A(protein kinase A,PKA)-PTEN诱导性激酶蛋白1(PTEN induced putative kinase 1,PINK1)/E3泛素连接酶(E3 ubiquitin ligases,Parkin)信号通路在氧糖剥夺/复氧复糖后大鼠皮质星形胶质细胞凋亡和焦亡中的作用。方法体外培养SD新生大鼠原代皮质星形胶质细胞,将细胞接种于6孔板或96孔板,根据随机数字表法将星形胶质细胞分为4组(n=42):对照组(C组)、氧糖剥夺组(oxygen-glucose deprivation,OGD/R组),H89+氧糖剥夺(H89+OGD/R,HO组),H89对照组(H组),H89为PKA特异性抑制剂。采用无糖培养基在无氧条件下(95%N2和5%CO2的混合气体内)孵育6 h,再放置在含有5%CO2的正常环境下培养24 h的方法制备氧糖剥夺/复氧复糖损伤模型,HO组和H组在氧糖剥夺前1 h向培养基内加入H8910μmol/L,C组和H组PBS洗涤后置于含有5%CO2的正常培养箱中培养。根据上述各组处理后,采用膜联蛋白(Annexin V)-FITC/碘化丙啶(Propidium Iodide,PI)免疫荧光双染流式细胞学法测定细胞凋亡率,采用半胱天冬酶-1裂解片段(cleaved caspase-1)/PI免疫荧光双染流式细胞学法测定细胞焦亡率,采用四甲基偶氮唑法测定细胞存活率,采用Fluo-3/AM免疫荧光法测定细胞内钙离子浓度,二氯荧光黄双乙酸盐(2′,7′-dichlorodihydrofluorescein diacetate,DCFH-DA)法测定活性氧(reactive oxygen species,ROS)含量,酶联免疫吸附法检测PKA活性,蛋白免疫印迹法法检测PINK1和Parkin的表达。结果与C组比较,OGD/R组和HO组皮质星形胶质细胞的凋亡率和焦亡率、细胞内游离钙离子浓度、ROS含量、PKA活性、PINK1和Parkin表达升高,细胞存活率下降(P<0.05);与OGD/R组比较,HO组皮质星形胶质细胞的凋亡率和焦亡率、细胞内游离钙离子浓度、ROS含量、PKA活性PINK1和Parkin表达下降,细胞存活率升高(P<0.05);C组与H组以上各项指标差异无统计学意义(P>0.05)。结论抑制PKA-PINK/Parkin信号通路过度活化能够减轻氧糖剥夺/复氧复糖Objective To investigate the effects of Protein Kinase A(PKA)-PTEN induced putative kinase 1(PINK1)/E3 ubiquitin ligases(Parkin)signal pathway on apoptosis and pyroptosis of rat cortical astrocytes exposed to oxygen-glucose deprivation/resuscitation(OGD/R).Methods Primary cortical astrocytes were cultured in vitro from SD neonatal ratsand seededon 6 or 96-well plates.They were divided into four groups(n=42):control group(C group),oxygen-glucosede privation group/resuscitation group(OGD/R group),and H89+OGD/R group(HO group),and H89 control group(H)group according random number table method.H89 is a specific inhibitor of PKA.OGD/Rmodel was established by 95%N2-5%CO2 and sugar-free culture-medium for 6 h and subsequently at normal culture-medium containing 5%CO2 for 24 h.At 1 h before OGD/R exposure,the cultures in HO and H group were treated with H8910μmol/L.C and H groups were rinsed with PBS and cultured in a normal incubator containing 5%CO2.Subsequently,the apoptosis,pyroptosis and viability of cortical astrocytes were assessed by Annexin V-FITC/(Propidium Iodide)PI,cleaved caspase-1/PI and methyl thiazolyl tetrazolium(MTT)assays,respectively.Intracellular calcium ion concentration and reactive oxygen species(ROS)content were assessed by Fluo-3/AM fluorescence and 2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA)assays respectively.The activity of PKA was measured by enzyme-linked immunosorbent assay.The expressions in PINK1 and Parkin were measured by Western blot assay.Results Compared with C group,there were significant increases of astrocytes apoptosis,pyroptosis,intracellular free calcium ion concentration,ROS content,PKA activity,and expressions of PINK1 and Parkin,but decreases of viability in OGD/R and HO group(P<0.05).Compared with OGD/R group,there were significant decreases of astrocytes apoptosis,pyroptosis,intracellular free calcium ion concentration,ROS content,PKA activity,and expressions of PINK1 and Parkin,but increases of viability in HO group(P<0.05).There were no significant differen

关 键 词：凋亡 焦亡 蛋白激酶类 

分 类 号：R743[医药卫生—神经病学与精神病学]