[关键词]
[摘要]
目的 研究注射用丹参多酚酸(Salvianolate Lyophilized Injection,SLI)对氧糖剥夺/再灌注(oxygen-glucose deprivation/reperfusion,OGD/R)损伤小鼠脑神经瘤细胞株Neuro-2a的保护作用及机制。方法 体外培养Neuro-2a细胞,建立OGD/R损伤模型,给予SLI(10、25、50 μg/mL)以及自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)干预,检测SLI对OGD/R损伤Neuro-2a细胞存活率、乳酸脱氢酶(lactate dehydrogenase,LDH)漏出量及细胞色素C(cytochrome C,Cyt-C)的释放;采用流式细胞仪检测细胞凋亡率;采用Western blotting法检测凋亡及自噬相关蛋白的表达情况。结果 与OGD/R组比较,SLI显著提高OGD 4 h/R 24 h Neuro-2a细胞存活率(P<0.05、0.01),降低LDH漏出量(P<0.05、0.01),抑制细胞凋亡和细胞内Cyt-C释放(P<0.05、0.01),调节活化的半胱氨酸天冬氨酸蛋白酶-3(cleaved cystein-asparate protease,cleaved Caspase-3)、B淋巴细胞瘤2(B cell lymphoma-2,Bcl-2)蛋白表达(P<0.05、0.01)。SLI还可以增加OGD 4 h/R 6 h细胞自噬相关蛋白微管相关蛋白1轻链3Ⅱ(microtubule-associated protein 1 light chain 3II,LC3Ⅱ)、自噬效应蛋白(Beclin-1)表达(P<0.05、0.01),减少自噬选择性底物p62蓄积(P<0.05、0.01)。加入自噬抑制剂,可以抵消SLI对OGD/R损伤Neuro-2a细胞的保护及凋亡抑制作用。此外,SLI可以降低磷酸化蛋白激酶B(phosphorylated protein kinase B,p-Akt)水平以及哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)表达(P<0.05、0.01),介导细胞自噬的发生。结论 SLI对OGD/R损伤后的Neuro-2a细胞具有保护作用,其机制可能是通过调节Akt/mTOR信号通路介导的细胞自噬,进而发挥抑制细胞凋亡的作用。
[Key word]
[Abstract]
Objective To study the protective effect and mechanism of Salvianolate Lyophilized Injection (注射用丹参多酚酸, SLI) on oxygen-glucose deprivation/reperfusion (OGD/R)-injured mouse brain neuroma cell line Neuro-2a. Methods Neuro-2a cells were cultured in vitro, OGD/R injury model was established, SLI (10, 25, 50 μg/mL) and autophagy inhibitor 3-methyladenine (3-MA) were administered for intervention. Effects of SLI on survival rate, lactate dehydrogenase (LDH) leakage and cytochrome C (Cyt-C) release of OGD/R-injured Neuro-2a cells were detected; Apoptosis was detected by flow cytometry; Expressions of apoptosis and autophagy-related proteins were detected by Western blotting. Results Compared with OGD/R group, SLI significantly increased the survival rate of Neuro-2a cells induced by OGD 4 h/R 24 h (P < 0.05, 0.01), decreased the leakage of LDH (P < 0.05, 0.01), inhibited cell apoptosis and Cyt-C release (P < 0.05, 0.01), regulated cleaved cystein-asparate protease (cleaved Caspase-3) and B lymphoma-2 (Bcl-2) protein expressions (P < 0.05, 0.01). SLI increased the expressions of autophagy-associated protein microtubule-associated protein 1 light chain 3II (LC3II) and autophagy effector protein (Beclin-1) in cells induced by OGD 4 h/R 6 h (P < 0.05, 0.01), reduced the accumulation of autophagy-selective substrate p62 (P < 0.05, 0.01). The addition of an autophagy inhibitor could counteract the protective and apoptosis inhibitory effects of SLI on OGD/R-injured Neuro-2a cells. In addition, SLI could reduce the level of phosphorylated protein kinase B (p-Akt) and expression of mammalian target of rapamycin (mTOR) (P < 0.05, 0.01) to mediate autophagy. Conclusion SLI has a protective effect on Neuro-2a cells after OGD/R injury, its mechanism may be regulating the autophagy mediated by Akt/mTOR signaling pathway, thereby inhibiting apoptosis.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金资助项目(81573644);国家重点科技攻关项目(2012ZX09101202)