外源性一氧化碳释放分子对体外培养PC12细胞的谷氨酸兴奋性神经毒性  

Excitatory neurotoxicity of CO-releasing molecules-2 on PC12 cells

作　　者：罗欣 高怡 杨金翰 金亚平[1] 赵凤红[1] 王高阳[1] LUO Xin;GAO Yi;YANG Jinhan;JIN Yaping;ZHAO Fenghong;WANG Gaoyang(Department of Occupational Health,School of Public Health,China Medical University,Shenyang,Liaoning 110122,China)

机构地区：[1]中国医科大学公共卫生学院劳动卫生教研室,辽宁沈阳110122

出　　处：《职业卫生与应急救援》2022年第2期140-144,共5页Occupational Health and Emergency Rescue

基　　金：国家自然科学基金(81803203);辽宁省博士科研启动基金(20170520450)。

摘　　要：目的探讨一氧化碳释放分子2(CORM-2)对PC12细胞谷氨酸信号通路的影响及作用机理,揭示急性一氧化碳中毒迟发性脑病(delayed encephalopathy after acute carbon monoxide poisoning,DEACMP)发生的可能机制,为CO的中毒及其后遗症的防治提供实验依据。方法以高分化的PC12细胞为研究对象,经不同浓度的CORM-2暴露后,用MTS法检测细胞活力,确定合适的染毒浓度及染毒时间,收集细胞后用蛋白免疫印迹法检测N-甲基-D-天冬氨酸(NMDA)受体主亚基(NR1)及NMDA受体2B亚基(NR2B)蛋白的表达,检测细胞内钙离子浓度及细胞释放的谷氨酸含量,用流式细胞术检测细胞的凋亡情况。结果随着CORM-2浓度的增加,PC12细胞的活力呈剂量依赖性降低(P<0.05);各剂量与对照组相比,NMDA受体的NR1亚基的表达未发生显著的变化(P>0.05),250μmol/L和300μmol/L剂量组NR2B蛋白的相对表达水平以及Ca^(2+)浓度、谷氨酸水平均高于对照组和200μmol/L剂量组(P<0.05);随着CORM-2浓度的增加,PC12细胞的凋亡率也呈剂量依赖性增加(P<0.05)。结论随着COMR-2浓度的增加,PC12细胞的活力呈剂量依赖性降低。CORM-2可使PC12细胞谷氨酸信号通路激活。CORM-2可能通过激活谷氨酸信号通路引起Ca^(2+)内流,进而导致细胞凋亡。这些结果对认识CO中毒机制具有重要意义。Objective The effect and mechanism of CO-releasing molecules-2(CORM-2)on glutamate signal pathway in PC12 cells was explored,in order to reveal the possible mechanism of DEACMP caused by acute carbon monoxide poisoning and furthermore improve the treatment of CO poisoning in the future.Methods Highly differentiated PC12 cells were exposed to different concentrations of CO-releasing molecules-2.The cell viability was detected by MTS method,and the appropriate concentration and time of exposure were determined.After the cells were collected,the expression of NMDA receptor major subunit(NR1)and NMDA receptor 2B subunit(NR2B)protein,intracellular calcium concentration and glutamate release were detected by Western blotting,and the apoptosis of cells was detected by flow cytometry.Results With the increase of CO-releasing molecules-2 concentration,the viability of PC12 cells decreased in a dose-dependent manner(P<0.05).Compared with the control group,the expression of NR1 subunit of NMDA receptor had no significant change(P>0.05).The relative expression level of NR2B protein,Ca^(2+)concentration and glutamate level in 250μmol/L and 300μmol/L CO-releasing molecules-2 treated groups were higher than those in the control and 200μmol/L CO-releasing molecules-2 treated groups(P<0.05).With the increase of CO-releasing molecules-2 concentration,the apoptosis rate of PC12 cells also increased in a dose-dependent manner(P<0.05).Conclusions With the increase of CO-releasing molecules-2 concentration,the viability of PC12 cells decreased in a dose-dependent manner.CO-releasing molecules-2 can activate glutamate signal pathway in PC12 cells and then induce Ca^(2+)influx,finally cause cell apoptosis.These results have important implications for understanding the mechanism of CO poisoning.

关 键 词：PC12细胞 一氧化碳释放分子2 谷氨酸 信号通路 急性一氧化碳中毒 迟发性脑病 

分 类 号：R135.1[医药卫生—劳动卫生]