摘要
目的:应用缺血再灌注高分化大鼠嗜铬神经瘤细胞(PC12)体外细胞模型研究细胞缺血再灌注损伤的分子机制。方法:PC12细胞经氧糖剥夺/复氧(OGD/R)处理体外模拟缺血再灌注,分为为对照组(Control组)、OGD 4 h后分别复氧不同时间(R0、R6、R12、R18和R24 h)作为实验组,观察各组细胞自噬和凋亡水平;以细胞自噬和凋亡水平同时最高作为药物干预的模型组,OGD/R+3-MA组细胞先进行3-甲基腺嘌呤(3-MA,2.5 Mm/L)预处理2 h后再行OGD4/R24处理作为药物干预处理组;流式细胞术检测各组(Control组、OGD4 h后复氧不同时间段组)PC12细胞的凋亡率,DCFH-DA荧光探针法检测各组(Control、OGD4 h后复氧不同时间段组)PC12细胞内活性氧(ROS)表达水平,蛋白免疫印迹法(Western blot)分析各组PC12细胞自噬标志分子微管相关蛋白轻链-1A/1B(LC3-Ⅰ/LC3-Ⅱ)、泛素结合蛋白(P62)和自噬相关基因BECN1(Beclin-1),以及凋亡分子裂解型胱天蛋白水解酶3(Cleaved-caspase3)的表达变化。结果:与Control组比较,OGD4 h后复氧不同时间(R0、R6、R12、R18和R24 h)诱导了细胞凋亡和ROS水平的升高,凋亡和ROS伴随复氧时间的延长而增加(P<0.05),其中OGD4+R24组凋亡和ROS的升高最明显(P<0.05);与Control组比较,OGD4 h后复氧不同时间段(R0、R6、R12、R18和R24 h)组的LC3-Ⅱ/LC3-Ⅰ的比率、P62、Beclin-1和C-caspase3的表达均增加(P<0.05),其中OGD4+R24组上述自噬和凋亡相关分子的表达升高最明显(P<0.05);与OGD4+R24组比较,OGD/R+3-MA组细胞可以明显降低OGD/R诱导的LC3-Ⅱ/LC3-Ⅰ比率以及Beclin-1、P62和Cleaved-caspase3的表达(P<0.05)。结论:OGD/R诱导了PC12自噬性死亡,其机制可能是OGD/R导致的ROS生成过量导致自噬功能障碍,最终导致细胞自噬性死亡,加剧缺血再灌注损伤。
Objective:To investigate the molecular mechanism of cellular injury in highly differentiated rat pheochromocytoma cell line(PC12)induced by ischemia-reperfusion.Methods:PC12 cells were treated with oxygen-sugar deprivation/reoxygenation(OGD/R)to establish ischemia-reperfusion,and divided into control group and OGD 4 h then reoxygenation at different times(R0,R6,R12,R18 and R24 h)as experimental groups.The levels of autophagy and apoptosis were observed.The group with the highest level of both autophagy and apoptosis was selected for drug intervention group,pretreated with 3-methyladenine(3-MA,2.5 Mm/L)for 2 h and then treated for OGD4/R24.Cellular apoptosis was detected by flow cytometry,and ROS was measured using DCFH-DA probe.Western blot was used to detect the expression levels of LC3-Ⅰ/LC3-Ⅱ,ubiquitin binding protein(P62),BECN1(Beclin-1)and Cleaved-caspase3.Results:Compared with the control group,reoxygenation at 4 h after OGD induced an increase in apoptosis and ROS levels.The longer the reoxygenation time,the more the apoptosis and ROS(P<0.05).The highest levels of apoptosis and ROS was observed at OGD4/R24 group when compared with the control group(P<0.05).Compared with the control group,reoxygenation at 4 h after OGD,the ratio of LC3-Ⅱ/LC3-Ⅰ,the expression of P62,Beclin-1 and C-caspase3 were all increased(P<0.05).The increases in autophagy-and apoptosis-related genes were the most obvious in OGD4/R24 group when compared with control group(P<0.05).Compared with OGD4/R24 group,the ratio of LC3-Ⅱ/LC3-Ⅰ,the expression of P62,Beclin-1 and C-caspase3 were all decreased in OGD/R+3-MA(P<0.05).Conclusion:OGD/R induces autophagic death of PC12 cells.The mechanism may be the excessive ROS induced by OGD/R,leading to autophagy dysfunction,which eventually leads to autophagic death and exacerbates ischemia-reperfusion injury.
作者
谢鹏
任真奎
吕菊
胡玉梅
吴昌学
禹文峰
XIE Peng;REN Zhenkui;LV Jv;HU Yumei;WU Changxue;YU Wenfeng(Key Laboratory of Endemic and Ethnic Diseases,Ministry of Education,Guizhou Medical University,Guiyang 550004,China;Key Laboratory of Medical Molecular Biology,Guizhou Medical University,Guiyang 550004,China;Department of Clinical laboratory,People's Hospital of Southwest Guizhou Autonomous Prefecture,Xingyi 562400,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2020年第4期379-386,共8页
Journal of Guizhou Medical University
基金
国家自然科学基金(81360199)
黔科中引地[(2019)4008号]
贵州省卫生健康委科学技术基金(gzwjkj2019-1-039)
黔西南州科技局基金(2019-1-10)。
关键词
再灌注损伤
自噬
细胞损伤
活性氧
凋亡
3-甲基腺嘌呤
嗜铬神经瘤细胞
reperfusion injury
autophagy
cellular injury
ROS
apoptosis
3-methyladenine(3-MA)
adrenal pheochromocytoma cell
