内质网应激在PM2.5诱导大鼠肺泡巨噬细胞凋亡中的作用

目的 本研究通过用PM2.5干预传代培养的大鼠肺泡巨噬细胞,检测细胞存活率、凋亡率,及内质网应激信号分子C/EBP同源蛋白(C/EBP honologus protein,CHOP)、葡萄糖调节蛋白78 (glucoser regulated ptotein 78,GRP78)的mRNA相对表达量,探讨内质网应激在PM2.5诱导肺泡巨噬细胞凋亡过程中的作用.方法 用不同浓度的PM2.5干预传代培养的肺泡巨噬细胞(NR8383细胞),分6个浓度组(分别为H0:0 mg/L,空白对照组,加等体积的PBS液;H1:40 mg/L;H2:80 mg/L;H3:160 mg/L;H4:320 mg/L;H5:640 mg/L),经12h、24 h、48h3个时间后,用MTT法检测NR8383细胞的存活率,选择最佳的干预时间.将传代的NR8383细胞接种于6孔板,浓度组设置同MTT,每组设3个复孔(n=3),经最佳干预时间后,采用流式细胞术检测各浓度组细胞的早期凋亡率及总凋亡率,用RT-PCR法检测各浓度组CHOP mRNA、GRP78 mRNA的相对表达量.结果 PM2.5干预12 h后,H1组与H0组、H2组与H1组细胞存活率间的差异无统计学意义(P＞0.05),余组间差异有统计学意义(P＜0.05),且随干预浓度的增加,细胞存活率下降.干预24 h后,除H5组与H4组差异无统计学意义(P＞0.05),余各组细胞存活率的组间差异有统计学意义(P ＜0.05).干预48 h后,H4组与H3组及H5组与H4组差异无统计学意义(P＞0.05),余各组细胞存活率的组间差异有统计学意义(P ＜0.05).各浓度组在PM2.5环境中分别暴露12 h、24 h、48 h后,以暴露24 h后各浓度组间差异较12h、48 h明显,确定24h为最佳的干预时间.经不同浓度的PM2.5干预24 h后,H1组较H0组、H5组较H4组的细胞早期凋亡率无明显增加,差异无统计学意义(P＞0.05),余各组与H0组比较,细胞早期凋亡率明显增加,差异有统计学意义(P＜0.05).干预24 h后,各浓度组的细胞总凋亡率较H0组均增加明显,且随干预浓度的增加,细胞凋亡率相应增加,组间差异有统计学意义(P ＜0.05).不同浓度PM2.5干预24 h后,H1组较H0组、H4组较H3组的GRP78 mRNA的相对表达量无明显增加,差异无统计学意义(P＞0.05),余各组与H0组比较,GRP78 mRNA的相对表达量均增加,组间差异有统计学意义(P＜0.05).干预24 h后,各浓度组的细胞CHOP mRNA的相对表达量较H0组增加明显,随干预浓度的增加,其相对表达量相应增加,组间差异有统计学意义(P ＜0.05).结论 PM2.5诱导NR8383细胞凋亡,内质网应激参与PM2.5诱导NR8383细胞凋亡的过程.

Effect of endoplasmic reticulum stress in PM2.5 induced apoptosis in alve olar macrophages of rats

Objective In this study,the subcultured rat alveolar macrophages were intervened by PM2.5,then tested the survival rate,the apoptosis rate,the relative expression of CHOP mRNA and GRP78 mRNA in the endoplasmic reticulum stress,to discuss the role of endoplasmic reticulum stress in the process of PM2.5 induced alveolar macrophages to apoptosis.Methods The subcultured alveolar macrophages (NR8383 cells) were exposed to different concentrations of PM2.5,according to the concentration gradient,divided into 6 groups (respectively for H0:0 mg/L,blank control group,with an equal volume of PBS;H1:40 mg/L;H2:80 mg/L;H3:160 mg/L;H4:320 mg/L;H5:640 mg/L),after the different intervention time (12 hours,24 hours,48 hours),the survival rate of cells was detected by MTT method,and selected the best intervention time.Seeded the subcultured NR8383 cells into the 6 hole plate,setted 6 concentration groups same with the MTT method,each group had 3 multiple holes (n =3),after the optimal intervention time,detected the early apoptosis rate and the total apoptosis rate of each concentration group by the flow cytometry,and detected the relative expression of CHOP mRNA and GRP78 mRNA by RT-PCR method.Results Be intervented with PM2.5 after 12 hours,compared with the H0 group,the decline of cell survival rate of the H1 group was not obvious,there was not statistically significant in the difference(P ＞0.05).Compared with the H1 group,the decline of cell survival rate of the H2 group was not obvious,there was not statistically significant in the difference(P ＞0.05).Then,the difference of the cell survival rate between the other groups there was statistically significant (P ＜ 0.05),and the cell survival rate decreased with the increase of the concentration of intervention.After 24 hours,compared with the H4 group,the decline of cell survival rate of the H5 group was not obvious,there was not statistically significant in the difference(P ＞0.05),then,the difference of the cell survival rate between the other groups there was statistically significant (P ＜0.05).After 48 hours,compared with the H3 group,the decline of cell survival rate of the H4 group was not obvious,there was not statistically significant in the difference (P ＞ 0.05),compared with the H4 group,the decline of cell survival rate of the H5 group was not obvious,there was not statistically significant in the difference(P ＞ 0.05),and the difference of the cell survival rate between the other groups there was statistically significant (P ＜0.05).All the groups were intervented with PM2.5 after 12 hours,24 hours and 48 hours,after exposured for 24 hours,the difference between the concentration groups was more obvious than that of 12hours and 48 hours,determined the 24 hours as the optimal intervention time.Intervented with PM2.5 after 24 hours,the difference of the early apoptosis rate between H1 group and H0 group,H5 group and H4 group there was not statistically significant in the difference(P ＞ 0.05),then the other groups,early apoptosis rate rather than the H0 group was more higher,the differencethere was statistically significant (P ＜0.05).After 24 hours,compared with the H0 group,the total apoptosis rate of all the concentration groups increased,and the apoptosis rate increased with the increase of the concentration of intervention,the difference between the groups was statistically significant (P ＜0.05).After 24 hours,the difference of the relative expression of GRP78 mRNA between H1 group and H0 group,H4 group and H3 group there was not statistically significant in the difference(P ＞0.05),then compared with the H0 group,the relative expression of GRP78 mRNA of the other groups was increased,the difference between the groups was statistically significant (P ＜0.05).After 24 hours,compared with the H0 group,the relative expression of CHOP mRNA of the other groups was increased,the difference between the groups was statistically significant (P ＜0.05),and the relative expression of CHOP mRNA increased with the increase of the concentration of intervention,the difference between the groups was statistically significant (P ＜0.05).Conclusions PM2.5 induced the apoptosis of NR8383 cells,and the endoplasmic reticulum stress participated in the process of the apoptosis.