摘要
目的探讨丹参多糖(SMP)调控脂多糖(LPS)诱导的心肌细胞凋亡的作用机制。方法不同浓度(0、0.5、1、2mg/mL)SMP预处理SD大鼠心肌细胞12h后,1μg/mL LPS刺激大鼠心肌细胞24h,以LPS单独处理构建心肌细胞损伤模型,对照组加入等量生理盐水,四唑盐比色法(MTT)观察心肌细胞存活率,流式细胞仪检测心肌细胞凋亡率,免疫印迹(Western blot)检测各组心肌细胞自噬相关蛋白LC3、Beclin1及凋亡相关蛋白Bax、Caspase3表达量;SMP联合自噬抑制剂氯喹(CQ)或自噬激活剂雷帕霉素靶蛋白抑制剂(Torin1)处理LPS处理的大鼠心肌细胞24h,MTT观察细胞存活率,流式细胞仪(FACS)检测细胞凋亡率,Western blot检测各组细胞自噬相关蛋白LC3、Beclin1及凋亡相关蛋白Bax、Caspase3表达量。结果与对照组比较,LPS处理24h后,心肌细胞活力显著下降[(43.57±1.07)%比(100.03±2.12)%,P<0.01],凋亡细胞率升高[(30.34±3.13)%比(4.02±0.13)%,P<0.01],凋亡相关蛋白Bax、cleaved-Caspase3表达量增多,自噬相关蛋白Beclin1和LC3II表达量增多。与LPS组比较,1、2mg/mL SMP预处理组心肌细胞存活率显著增加[(69.72±2.15)%、(89.65±3.23)%比(43.57±1.07)%,P<0.05,P<0.01],凋亡率显著降低[(19.18±1.34)%、(12.28±1.05)%比(30.34±3.13)%,P<0.05,P<0.01],凋亡相关蛋白Bax和Cleaved-Caspase3表达量减少,自噬相关蛋白Beclin1和LC3II蛋白表达增加。与SMP处理组比较,CQ抑制细胞活力[(60.01±1.99)%比(79.42±4.23)%,P<0.05],增加细胞凋亡率[(35.63±2.45)%比(20.13±1.65)%,P<0.05],促进Bax、Cleaved-Caspase3和LC3II蛋白表达,抑制Beclin1蛋白表达;Torin1上调细胞活力[(85.63±1.08)%比(79.42±4.23)%,P<0.05],减少细胞凋亡率[(9.78±0.74)%比(20.13±1.65)%,P<0.05],促进LC3-II、Beclin1蛋白表达,抑制Bax、cleaved-Caspase3蛋白表达。结论SMP能促进自噬抑制LPS诱导的心肌细胞凋亡。
Objective To explore the mechanism of Salviae Miltiorrhizae(danshen root)polysaccharide(SMP)regulating lipopolysaccharide(LPS)-induced myocardial cell apoptosis.Methods After pretreatment of SD rat cardiomyocytes with different concentrations(0,0.5,1,2 mg/mL)of SMP for 12 h,1μg/mL of LPS was used to stimulate the rat cardiomyocytes for 24 h,and the model of myocardial cell injury was constructed by LPS alone.The control group was treated with equal amount of normal saline.The survival rate of cardiomyocytes was observed by MTT,apoptosis rate of cardiomyocytes was detected by flow cytometry,expression of autophagy-related protein LC3 and Beclin 1,and apoptosis-related protein Bax and caspase 3 were detected by Western blot.Then,SMP com-bined with chloroquine(CQ),an autophagy inhibitor,or Torin 1,an autophagy activator,was used to treat LPStreated cardiomyocytes for 24 h,and the same detections were performed.Results Compared with the cell viability[(100.06±1.02)%]and the proportion of apoptotic cells[(4.02±0.13)%]of control group,cell viability of cardiomyocytes treated with LPS[(43.57±1.07)%]decreased significantly,the proportion of apoptotic cells[(30.34±3.13)%],P<0.01,respectively.Expression of Bax,cleaved-Caspase 3,Beclin 1 and LC3 II increased.Compared to the LPS group,in 1 and 2 mg/mL SMP-pretreatment group,cell viability increased:[(69.72±2.15)%,(89.65±3.23)%vs(43.57±1.07)%,P<0.05,P<0.01],apoptosis rate decreased:[(19.18±1.34)%,(12.28±1.05)%vs(30.34±3.13)%,P<0.05,P<0.01],expression of Bax and cleaved-Caspase 3 decreased,Beclin 1 and LC3 II increased.Compared to SMP treatment group,CQ inhibited cell viability[(60.01±1.99)%vs(79.42 vs 4.23)%,P<0.05],increased cell apoptosis[(35.63±2.45)%vs(20.13±1.65)%,P<0.05],promoted the expression of Bax,Cleaved-Caspase3 and LC3 II proteins,inhibited the expression of Beclin 1 protein.Torin 1 increased cell viability[(85.63±1.08)%vs(79.42±4.23)%,P<0.05],reduced apoptosis rate[(9.78±0.74)%vs(20.13±1.65)%,P<0.05],promoted the expression of LC3-II and Beclin 1,and inhibited the expression of Bax and cleaved-Caspase3.Conclusion SMP could promote autophagy and inhibit LPS-induced cardiomyocyte apoptosis.
作者
张建成
林淑琴
胡鹏飞
ZHANG Jiancheng;LIN Shuqin;HU Pengfei(Department of General Practice,Zhenhai District People's Hospital,Ningbo,Zhejiang province,315202,China;Department of Neurology,Ningbo Medical Center Li Huili Eastern Hospital,Ningbo,Zhejiang province,315040,China;Department of Cardiology,The Second Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou,Zhejiang province,310005,China)
出处
《浙江中西医结合杂志》
2020年第4期281-285,共5页
Zhejiang Journal of Integrated Traditional Chinese and Western Medicine
关键词
大鼠
丹参多糖
心肌细胞
凋亡
自噬
Rat
Salviae Miltiorrhizae(Danshen Root)Polysaccharide
Cardiomyocyte
Apoptosis
Autophagy
