α-硫辛酸对缺血再灌注诱导的PC12细胞应激性凋亡抑制作用的研究  

Study of Inhibitory Effect ofα-Lipoic Acid on Apoptosis of PC12 Cells Induced by Ischemia-Reperfusion

作　　者：谢鹏 任真奎 吕菊 胡玉梅 吴昌学[1,2] 禹文峰[1,2] XIE Peng;REN Zhenkui;LV Jv;HU Yumei;WU Changxue;YU Wenfeng(Key Laboratory of Molecular Biology of Guizhou Medical University,Guiyang 550004,Guizhou,China;Education Ministry Key Laboratory of Endemic and Minority Diseases of Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Laboratory,People's Hospital of Southwest Guizhou Autonomous Prefecture,Xingyi 562400,Guizhou,China)

机构地区：[1]贵州医科大学分子生物学重点实验室,贵州贵阳550004 [2]贵州医科大学地方病与少数民族疾病教育部重点实验室,贵州贵阳550004 [3]黔西南州人民医院检验科,贵州兴义562400

出　　处：《贵州医科大学学报》2020年第4期373-378,386,共7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)。

摘　　要：目的:探讨α-硫辛酸(α-LA)抑制缺血再灌注诱导的大鼠肾上腺嗜铬神经瘤细胞(PC12)内质网应激性凋亡的分子机制。方法:传代培养PC12细胞分为正常组(Control组)、氧糖剥夺/再灌注(OGD/R)4 h组(OGD4组)、OGD 4 h后复氧不同时间段组(OGD4+R6、OGD4+R12、OGD4+R18以及OGD4+R24组)、在OGD4 h后复氧阶段给予不同浓度(0.01、0.05、0.1、0.25、0.5、1、2、5和10 mmol/L)α-LA共同复氧培养24 h作为α-LA处理组(OGD/R+α-LA组);Control组细胞在完全培养基、正常氧培养箱中培养,OGD/R组和OGD/R+α-LA组细胞按相应方法进行培养处理;蛋白免疫印迹法检测各组PC12细胞中内质网应激[葡萄糖调节蛋白78(GRP78)、环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)]和凋亡[B淋巴细胞瘤-2相关X(Bax)、裂解型胱天蛋白水解酶3(Cleaved-caspase3)]相关分子的表达;细胞活性检测试剂盒-8(CCK-8试剂盒)检测各组PC12细胞的存活率,分析α-LA作用的有效工作浓度。结果:与Control组比较,OGD4 h后复氧不同时间段均诱导了PC12细胞发生内质网应激性的凋亡,表现为内质网应激相关分子GRP78、CHOP和凋亡蛋白Bax、Cleaved-caspase3的表达伴随复氧时间段的延长而逐渐升高(P<0.05),在OGD4+R24组升高最明显(P<0.05);与Control组比较,OGD4+R24组细胞的存活率明显下降(P<0.05);与OGD4+R24组比较,OGD/R+α-LA组α-LA在0.05~5 mmol/L浓度范围内增加了细胞的存活率(P<0.05),0.5 mmol/L时细胞存活率升高最明显(P<0.05),10 mmol/L时细胞存活率明显降低(P<0.05);与OGD4+R24组比较,OGD/R+α-LA(0.5 mmol/L)组凋亡分子Bax、Cleaved-caspase3以及内质网应激相关分子GRP78、CHOP表达明显降低(P<0.05)。结论:缺血再灌注诱导了PC12细胞发生内质网应激性的凋亡,α-LA能够降低缺血再灌注诱导的PC12损伤,其机制可能与降低了缺血再灌注诱导的内质网应激性凋亡有关。Objective:To explore the molecular mechanism by whichα-lipoic acid(α-LA)inhibits endoplasmic reticulum(ER)stress-mediated apoptosis in rat adrenal pheochromocytoma cells(PC12)induced by ischemia-reperfusion.Methods:PC12 cells were cultured under normal condition(control group),oxygen-sugar deprivation/reperfusion(OGD/R)4 h group(OGD4 group),and OGD4+reoxygenation(R)at different time periods(OGD4+R6,OGD4+R12,OGD4+R18,and OGD4+R24 groups).During rexoygenation,OGD4/R were then givenα-LA at different concentrations(0.01,0.05,0.1,0.25,0.5,1,2,5 and 10 mmol/L)for 24 h as OGD/R+α-LA group.Western blot was used to detect glucose-regulated protein 78(GRP78),cyclic adenosine phosphate response element binding transcription factor homolog protein(CHOP),Bax and Cleaved-caspase3.Cell viability was detected using CCK-8 kit.Results:Compared with the control group,reoxygenation induced PC12 cells to undergo ER-mediated apoptosis in OGD4 group,and the expression levels of GRP78,CHOP,Bax and Cleaved-caspase3 gradually increased over the prolongation of reoxygenation period(P<0.05),and reached the highest levels in OGD4+R24 group(P<0.05).When compared with the control group,the cell survival rate was remarkably decreased in the OGD4+R24 group(P<0.05).α-LA increased the cell survival rates at the concentration range of 0.05~5 mmol/L in OGD/R+α-LA group(P<0.05).The survival rate was the highest level at 0.5 mmol/L ofα-LA,but reduced at 10 mmol/L ofα-LA(P<0.05).When compared with the OGD4+R24 group,the expression levels of Bax,Cleaved-caspase3,GRP78 and CHOP were significantly reduced in OGD/R+α-LA(0.5 mmol/L)(P<0.05).Conclusion:Ischemia-reperfusion induces endoplasmic reticulum stress-mediated apoptosis in PC12 cells.α-LA can reduce ischemia-reperfusion-induced cell injury by downregulating GRP78 and CHOP.

关 键 词：再灌注损伤 大鼠 Α-硫辛酸 肾上腺嗜铬神经瘤细胞 细胞损伤 氧糖剥夺/再灌注 内质网压力介导的凋亡 

分 类 号：R965[医药卫生—药理学]