玉米黄素对衣霉素诱导的SH-SY5Y细胞周期、自噬、凋亡的保护作用

Protective Effect of Zeaxanthin on Tunicamycin-Induced Cell Cycle Arrest,Autophagy and Apoptosis of SH-SY5Y Cells

目的:探讨玉米黄素对衣霉素(tunicamycin,TM)诱导的SH-SY5Y细胞活性、细胞周期、自噬及凋亡的影响。方法:分别设置空白对照组(不作处理)、玉米黄素组(5μmol/L玉米黄素)、TM损伤组(5μg/mL TM)和损伤加保护组(5μmol/L玉米黄素预处理+5μg/mL TM进一步共处理),采用噻唑蓝法检测各处理对SH-SY5Y细胞存活率的影响,从而筛选药物共处理时间;采用流式细胞术测定细胞周期的变化;通过Western blot法测定自噬相关蛋白Beclin1、Beclin1-C和微管结合蛋白1轻链3(microtubule-associated protein1 light chain 3,LC3)B以及凋亡相关蛋白B淋巴细胞瘤-2(B-cell lymphoma 2,BCL2)的表达量。结果:玉米黄素(5μmol/L)能减轻TM对SHSY5Y细胞存活率的抑制作用,与TM损伤组相比,损伤加保护组在处理时间为36 h时差异极显著(P<0.01),48 h时差异显著(P<0.05);而TM损伤组相较于空白对照组在处理24、36、48 h时均表现出极显著差异(P<0.01)。与空白对照组相比,TM处理可以极显著增加细胞周期中G0/G1期细胞数量(P<0.01),显著或极显著增加LC3B(P<0.01)、Beclin1(P<0.05)及Beclin1-C(P<0.05)表达水平,极显著降低G2/M期细胞数量(P<0.01)、抗凋亡蛋白BCL2表达水平(P<0.01)。而玉米黄素联合TM处理与TM损伤组比较,均能减轻TM引起的改变,其中BCL2蛋白水平显著提高(P<0.05)。结论:玉米黄素可减轻TM处理引起的SH-SY5Y细胞周期G1期阻滞、促细胞自噬与促凋亡作用,其作用机制与玉米黄素对细胞存活率、细胞周期分布和自噬、凋亡相关蛋白表达的调节有关。

Objective:To investigate the effect of co-treatment with zeaxanthin(Zea)and tunicamycin(TM)on the viability,cell cycle,autophagy and apoptosis of SH-SY5Y cells.Methods:The cells were divided into four groups:blank control(without drug treatment),Zea(5μmol/L),TM-induced injury(5μg/mL)and Zea(5μmol/L)+TM(5μg/mL).The methylthiazol tetrazolium(MTT)method was used to evaluate the survival rate of SH-SY5Y cells to determine the drug co-treatment time for further experiments;flow cytometry was used to determine the change of cell cycle;Western blot was used to determine the expression of autophagy-related proteins such as Beclin1,Beclin1-C and microtubule-associated protein 1 light chain 3B(LC3B),and apoptosis-related protein B-cell lymphoma 2(BCL2).Results:Zea treatment(5μmol/L)reduced the inhibitory effect of TM on the survival rate of SH-SY5Y cells.Compared to the injury group,a significant difference in the survival rate of SH-SY5Y cells was observed for the combined treatment group at 36(P<0.01)and 48 h(P<0.05).In addition,compared to the blank control group,TM treatment displayed a significant difference in the survival rate of SH-SY5Y cells at 24,36,and 48 h(P<0.01).Compared to the blank group,TM treatment significantly increased the number of G0/G1 phase cells in the cell cycle(P<0.01)and the expression of LC3B(P<0.01),Beclin1(P<0.05)and Beclin1-C(P<0.05),and significantly reduced the number of G2/M phase cells(P<0.01)and the expression level of anti-apoptotic protein BCL2(P<0.01).The combined treatment alleviated the changes caused by TM,and significantly increased BCL2 protein expression compared to the injury group(P<0.05).Conclusion:Zea can significantly extenuate TM-induced cell cycle G1 arrest,autophagy and apoptosis of SH-SY5Y cells,and its underlying mechanism is related to the modulatory of Zea on cell survival,cell cycle distribution,and the expression of autophagy and apoptosis-related proteins.