橙皮素通过减轻氧化应激和线粒体损伤抑制细颗粒物诱导的大鼠H9c2细胞凋亡  

Hesperetin inhibits PM2.5-induced apoptosis in H9c2 cells by attenuating oxidative stress and mitochondrial damage

作　　者：曹静[1] 吕吉元[2] 张明升[3] 师锐赞[3] 冯巧爱 Cao Jing;Lyu Jiyuan;Zhang Mingsheng;Shi Ruizan;Feng Qiao＇ai(Department of lntensive Care Unit, First Hospital of Shanxi Medical University, Taiyuan 030001, China)

机构地区：[1]山西医科大学第一医院重症医学科,太原030001 [2]山西医科大学第一医院心血管内科,太原030001 [3]山西医科大学药理教研室 [4]山西博爱医院心血管内科

出　　处：《中华心血管病杂志》2018年第5期382-389,共8页Chinese Journal of Cardiology

基　　金：山西省国际科技合作项目（2012081046）

摘　　要：目的 探讨橙皮素对细颗粒物PM2.5诱导的心肌样细胞损伤是否具有保护作用及其机制.方法 将培养的大鼠心肌样细胞H9c2分为4组,即对照组、PM2.5组、橙皮素组和橙皮素＋PM2.5组.对照组细胞在普通培养基中正常培养.PM2.5组,以含PM2.5800μg/ml的培养基进行培养.橙皮素组,先用含橙皮素40 μμmol/L的培养基预处理1h（37℃）后换用普通培养基正常培养.橙皮素＋PM2.5组,先用含橙皮素40 μmol/L的培养基预处理1 h（37℃）后再以含PM2.5800 μg/ml的培养基进行培养.处理相应的时间后,采用Annexin V-FITC/PI双染流式细胞技术和Hoechst 33258染色检测H9c2细胞凋亡情况,采用DCFH-DA法检测H9c2细胞活性氧（ROS）的生成量,采用JC-1染色法检测H9c2细胞线粒体膜电位（MMP）,采用Western blot法检测H9c2细胞凋亡相关蛋白及MAPK信号通路蛋白的表达水平.结果 （1）各组H9c2细胞的凋亡情况：流式细胞术检测结果显示,PM2.5组H9c2细胞凋亡率明显高于对照组[（48.94±3.20）％比（8.13±1.40）％,P〈0.01],橙皮素＋PM2.5组H9c2细胞凋亡率[（34.80±2.21）％]虽亦明显高于对照组（P〈0.01）,却明显低于PM25组（P=0.003 2）.Hoechst 33258染色结果显示,橙皮素＋PM2.5组H9c2细胞凋亡数目明显少于PM2.5组.（2）各组H9c2细胞内ROS生成量：PM2.5组H9c2细胞内ROS荧光强度明显高于对照组[（49.69±5.05）％比（10.57±1.33）％,P〈0.01],橙皮素＋PM2.5组[（35.08±3.90）％]虽亦高于对照组（P〈0.01）,却明显低于PM25组（P=0.0002）.（3）各组H9c2细胞MMP：PM2.5组H9c2细胞绿色荧光强度明显高于对照组[（20.28±4.69）％比（10.50±2.72）％,P〈0.01],橙皮素＋PM2.5组[（13.41±2.89）％]虽亦高于对照组（P=0.029 4）,却明显低于PM25组（P〈0.01）.（4）各组H9c2细胞凋亡相关蛋白B淋巴细胞瘤-2（Bcl-2）和活化的半胱氨酸天冬氨酸蛋白酶3（caspase-3）的表达水平：PM2.5组H9c2细胞Bcl-2蛋白�Objective To investigate the effects of hesperetin on fine particulate matter （PM2.5） induced apoptosis in H9c2 cells and related mechanisms.Methods H9c2 cells were divided into 4 groups：control group （cells were cultured without intervention）,PM2.5 group （cells were treated with 800 μg/ml PM2.5）,hesperetin group （H group,cells were treated by 40 μmol/L hesperetin for 1 h at 37 ℃）,and hesperetin＋PM2.5 group （H＋PM2.5 group,cells were pretreated with hesperetin before PM2.5 treatment）.Cells were cultured for corresponding interval.Apoptotic cells were detected by Annexin V-FITC/PI apoptosis detection kit and Hoechst staining.The intracellular reactive oxygen species （ROS） levels were measured by DCFH-DA Fluorescence Probe and mitochondrial membrane potential （MMP） was detected with JC-1 staining,respectively in these groups.Apoptotic related protein and phosphorylated MAPK expression levels were determined by Western blot.Results （1） Flow cytometry results showed that the apoptosis rate of PM2.5 group （（48.94± 3.20）%） was significantly higher than that of control group （（8.13 ± 1.40）%,P〈0.01）,which was significantly reduced in H＋PM2.5 group （（34.80±2.21）%） （P=0.003 2 vs.PM2.5 group,P〈0.01 vs.control group）.The number of Hoechst 33258 positive apoptotic cells was distinctly less in H＋PM2.5 group than in PM2.5 group.（2） The ROS levels was significantly higher in PM2.5 group （（49.69 ± 5.05）%） than in control group （10.57± 1.33）%,P〈0.01）,which was significantly reduced in H＋PM2.5 group （（35.08±3.90）%）（P=0.000 2 vs.PM2.5 group,P〈0.01 vs.control group）.（3） Green fluorescence indicating the JC-1 monomer form,which represented MMP loss of H9c2 cells,was significantly higher in PM2.5 group （（20.28±4.69）%）than in control group （（10.50±2.72）%,P〈0.01）,which was significantly decreased in H＋PM2.5 group （（13.41 ±2.89）%） （P〈0.01 vs.PM2.5 group,P=0.029 4 vs.control group）.

关 键 词：心肌 细胞凋亡 橙皮素 细颗粒物 线粒体 活性氧 

分 类 号：R285.5[医药卫生—中药学]