摘要
目的探讨普伐他汀预处理对香烟提取物(CSE)诱导的小鼠血管平滑肌细胞(VSMCs)凋亡的影响及可能机制。方法 2017年6月—2018年1月,采用负压吸引装置抽吸香烟烟雾并经过滤除菌制备CSE原液,取10代以内生长状态良好的对数期VSMCs进行实验。(1)将实验细胞分为不同浓度CSE诱导组,其中空白组使用不含CSE的DMEM培养基培养12 h,低浓度组、中浓度组、高浓度组分别于含1%、5%、10%CSE的DMEM培养基培养12 h。(2)将实验细胞分为不同剂量普伐他汀预处理组,其中对照组使用不含普伐他汀的DMEM培养基培养12 h;低剂量组、中剂量组、高剂量组分别给予1μM、10μM、100μM普伐他汀预处理2 h,然后使用含5%CSE的DMEM培养基培养12 h;部分实验设置阳性对照组,阳性对照组给予4-苯丁酸(4-PBA)2 m M预处理2 h,然后使用含5%CSE的DMEM培养基培养12 h。采用流式细胞术检测细胞凋亡率,采用实时荧光定量聚合酶链反应检测Bcl-2、Bax、GRP78、Chop m RNA相对表达量,采用Western Blot检测Bcl-2、Bax、GRP78、Chop蛋白相对表达量。结果 (1)低浓度组、中浓度组和高浓度组细胞凋亡率高于空白组,中浓度组和高浓度组细胞凋亡率高于低浓度组,高浓度组细胞凋亡率高于中浓度组(P<0.05)。(2)低浓度组、中浓度组和高浓度组Bcl-2 m RNA和蛋白相对表达量低于空白组,中浓度组和高浓度组Bcl-2 m RNA和蛋白相对表达量低于低浓度组,高浓度组Bcl-2蛋白相对表达量低于中浓度组(P<0.05);低浓度组、中浓度组和高浓度组Bax m RNA和蛋白相对表达量高于空白组,中浓度组和高浓度组Bax m RNA和蛋白相对表达量高于低浓度组,高浓度组Bax m RNA相对表达量高于中浓度组(P<0.05)。(3)低浓度组、中浓度组、高浓度组GRP78、Chop m RNA和蛋白相对表达量高于空白组,中浓度组、高浓度组GRP78、Chop m RNA和蛋白相对表达量高于低浓度组,高浓度组GRP78 m RNA、Chop蛋白相对表达量高于中浓度组(P<0.05)。(4)低剂量组、中剂量组、高剂量组细胞凋亡率低于对照组,中剂量组细胞凋亡率低于低剂量组和高剂量组(P<0.05)。(5)低剂量组、中剂量组、高剂量组Bcl-2蛋白相对表达量高于对照组,Bax蛋白相对表达量低于对照组(P<0.05);中剂量组Bax蛋白相对表达量低于低剂量组和高剂量组(P<0.05)。(6)阳性对照组、低剂量组、中剂量组、高剂量组GRP78和Chop蛋白相对表达量低于对照组,低剂量组、中剂量组、高剂量组GRP78蛋白相对表达量低于阳性对照组,中剂量组GRP78蛋白相对表达量低于低剂量组、Chop蛋白相对表达量低于阳性对照组(P<0.05)。结论 CSE可诱导小鼠VSMCs凋亡,且呈浓度依赖性;普伐他汀预处理对CSE诱导的小鼠VSMCs凋亡具有一定保护作用,其中10μM普伐他汀预处理的抗细胞凋亡作用最佳,其机制可能与调控内质网应激信号途径有关。
Objective To investigate the impact of pravastatin pretreatment on cigarette smoke extract（CSE）-induced apoptosis of vascular smooth muscle cells（VSMCs） in mice and its mechanism. Methods This experiment was carried out from June 2017 to January 2018. Negative pressure suction device was used to collect cigarette smoke, and CSE stoste was prepared by filtration sterilization, log phase VSMCs（within 10 cell propagations） with good growing status were selected for the experiment.（1） VSMCs were divided into blank group（cultivated without CSE）, low-concentration group（cultivated with 1% CSE）, moderate-concentration group（cultivated with 5% CSE） and high-concentration group（cultivated with 10% CSE） according to the concentration of CSE, and then were cultivated for 12 hours on DMEM medium.（2） VSMCs were divided into control group（without pravastatin pretreatment）, low-dose group（with 1 μM pravastatin pretreatment for 2 hours）, moderate-dose group（with 10 μM pravastatin pretreatment for 2 hours） and high-dose group（with 100 μM pravastatin pretreatment for 2 hours） according to the concentration of CSE, and then were cultivated for 12 hours on DMEM medium with 5% CSE. Meanwhile some experiments set positive control group（with 2 m M 4-PBA pretreatment for 2 hours and then were cultivated for 12 hours on DMEM medium with 5% CSE）. Flow cytometry was used to detect the apoptosis rate, real-time fluorescence quantitative PCR was used to detect the relative expression quantity of m RNA of Bcl-2, Bax, GRP78 and Chop, and Western Blot was used to detect the relative expression quantity of protein of Bcl-2, Bax, GRP78 and Chop. Results（1） Apoptosis rate in low-concentration group, moderate-concentration group and high-concentration group was statistically significantly higher than that in blank group, respectively, apoptosis rate in moderate-concentration group and high-concentration group was statistically significantly higher than that in low-concentration group, respectively, meanwhile apoptosis rate in high-concentration group was statistically significantly higher than that in moderate-concentration group（P〈0.05）.（2） Relative expression quantity of m RNA and protein of Bcl-2 in low-concentration group, moderate-concentration group and high-concentration group was statistically significantly lower than that in blank group, respectively, relative expression quantity of m RNA and protein of Bcl-2 in moderate-concentration group and high-concentration group was statistically significantly lower than that in low-concentration group, respectively, meanwhile relative expression quantity of m RNA and protein of Bcl-2 in high-concentration group was statistically significantly lower than that in moderate-concentration group, respectively（P〈0.05）; relative expression quantity of m RNA and protein of Bax in low-concentration group, moderate-concentration group and high-concentration group was statistically significantly higher than that in blank group, respectively, relative expression quantity of m RNA and protein of Bax in moderate-concentration group and high-concentration group was statistically significantly higher than that in low-concentration group, respectively, meanwhile relative expression quantity of m RNA of Bax in high-concentration group was statistically significantly higher than that in moderate-concentration group（P〈0.05）.（3） Relative expression quantity of m RNA and protein of GRP78 and Chop in low-concentration group, moderate-concentration group and high-concentration group was statistically significantly higher than that in blank group, respectively, relative expression quantity of m RNA and protein of GRP78 and Chop in moderate-concentration group and high-concentration group was statistically significantly higher than that in low-concentration group, respectively, meanwhile relative expression quantity of m RNA of GRP78 and relative expression quantity of protein of Chop in high-concentration group was statistically significantly higher than that in moderate-concentration group, respectively（P〈0.05）.（4） Apoptosis rate in low-dose group, moderate-dose group and high-dose group was statistically significantly lower than that in control group, respectively, meanwhile apoptosis rate in moderate-dose group was statistically significantly lower than that in low-dose group and high-dose group, respectively（P〈0.05）.（5） Relative expression quantity of protein of Bcl-2 in low-dose group, moderate-dose group and high-dose group was statistically significantly higher than that in control group, respectively, while relative expression quantity of protein of Bax in low-dose group, moderate-dose group and high-dose group was statistically significantly lower than that in control group, respectively（P〈0.05）; relative expression quantity of protein of Bax in moderate-dose group was statistically significantly lower than that in low-dose group and high-dose group, respectively（P〈0.05）.（6） Relative expression quantity of protein of GRP78 and Chop in positive control group, low-dose group, moderate-dose group and high-dose group was statistically significantly lower than that in control group, respectively, relative expression quantity of protein of GRP78 in low-dose group, moderate-dose group and high-dose group was statistically significantly lower than that in positive control group, relative expression quantity of protein of GRP78 in moderate-dose group was statistically significantly lower than that in low-dose group, meanwhile relative expression quantity of protein of Chop in moderate-dose group was statistically significantly lower than that in positive control group（P〈0.05）. Conclusion CSE may induce apoptosis of VSMCs with concentration dependence in mice; pravastatin pretreatment has certain protective effect on CSE-induced apoptosis of VSMCs in mice（10 μM pravastatin pretreatment has the best anti-apoptosis effect）, the mechanism may correlated with regulate and control of endoplasmic reticulum stress signal pathway.
作者
廖思聪
祁春雷
王大新
LIAO Si-cong;QI Chun-lei;WANG Da-xin(Department of Cardiology, the Second Xiangya Hospital of Central South University, Changsha 410011, China;Department of Cardiology, the People＇ s Hospital Northern Jiangsu, Yangzhou 225001, China)
出处
《实用心脑肺血管病杂志》
2018年第3期44-50,共7页
Practical Journal of Cardiac Cerebral Pneumal and Vascular Disease
基金
国家重大基础研究项目"973"计划项目(2007CB936104)
江苏省"六大人才高峰"项目(2014-SWYY-052)
