辅酶Q10对高糖引起的人冠状动脉内皮细胞凋亡的抑制作用及其机制

Inhibitory effect of coenzyme Q10on apoptosis of human coronary endothelial cells induced by high glucose and its mechanism

目的:探讨辅酶Q10(Co-Q10)对高糖诱导的人冠状动脉内皮细胞(HCAECs)凋亡的抑制作用,并阐明其可能的作用机制。方法:HCAECs分为对照组,高糖组,高糖联合5、10和20μmol·L^-1Co-Q10处理组。对照组HCAECs采用常规培养方法培养24h;高糖组采用30mmol·L^-1葡萄糖处理细胞24h;高糖联合5、10和20μmol·L^-1Co-Q10处理组分别采用5、10和20μmol·L^-1Co-Q10联合30mmol·L^-1葡萄糖处理细胞24h。采用CCK-8法检测各组细胞活性,Hoechst-PI双染色法检测高糖组和高糖联合10μmol·L^-1Co-Q10处理组细胞凋亡率,Mito-tracker染色检测高糖组和高糖联合10μmol·L^-1Co-Q10处理组细胞线粒体膜电位,MitoSOX染色检测高糖组和高糖联合10μmol·L^-1Co-Q10处理组细胞线粒体活性氧(mtROS)水平,Westernblotting法检测高糖组和高糖联合10μmol·L^-1Co-Q10处理组细胞中B细胞淋巴瘤/白血病2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)、Bcl-2相关死亡启动子(Bad)和X连锁凋亡抑制蛋白(x-IAP)表达水平。结果:与对照组比较,高糖组细胞活性明显降低(P<0.01);与高糖组比较,高糖联合5、10和20μmol·L^-1Co-Q10处理组细胞活性均明显升高(P<0.05或P<0.01),高糖联合10μmol·L^-1Co-Q10处理组升高最为明显(P<0.01)。与高糖组比较,高糖联合10μmol·L^-1Co-Q10处理组细胞凋亡率、线粒体膜电位和mtROS水平均明显降低(P<0.01)。Westernblotting法检测,与高糖组比较,高糖联合10μmol·L^-1Co-Q10处理组HCAECs中Bax和Bad表达水平明显降低(P<0.01),Bcl-2和x-IAP表达水平均明显升高(P<0.01)。结论:Co-Q10可能通过抑制线粒体凋亡相关通路减少高糖引起的HCAECs凋亡,对细胞起保护作用。

Objective : To investigate the inhibitory effect of coenzyme Q10 (Co-Q10) on the apoptosis of human coronary endothelial cells (HCAECs) induced by high glucose, and to elucidate its possibl e mechanism. Methods : The HCAECs were divided into control group, high glucose group and high glucose combined with 5, 10,and 20 μmol·L^-1 Co-Q10 treatment groups;the HCAECs in control group were cultured for 24 h using a routine culture method. The cells in high glucose group were treated with 30 mmol·L^-1 glucose for 24 h;the cells in high glucose combined with 5, 10,and 20 μmol·L^-1 Co-Q10 treatment groups were treated with 5, 10,and 20 μmol·L^-1 Co-Q10 combined with 30 mmol·L^-1 glucose for 24 h, respectively. The cell viabilities of HCAECs in various groups were measured by CCK-8 assay. The apoptotic rates of HCAECs in high glucose group and high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were detected by Hoechst-PI double staining. The cell mitochondrial membrane potentials of HCAECs in high glucose group and high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were determined by Mito-tracker staining.The mitochondrial reative oxygen species (mtROS) levels in the HCAECs in high glucose group and high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were measured by MitoSox staining. The protein expression levels of B cell lymphoma/leukemia 2 protein (Bcl-2), Bcl-2 assaciated X protein (Bax), Bcl-2 assaciated death promoter (Bad) and X-linked inhibitor of apoptosis protein (x-IAP) in the HCAECs in high glucose group and high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were detected by Western blotting method. Results : Compared with control group, the cell viability of HCAECs in high glucose group was significantly reduced ( P <0.01);compared with high glucose group, the cell viabilities of HCAECs in high glucose combined with 5, 10,and 20 μmol·L^-1 Co-Q10 treatment groups were significantly increased( P <0.05 or P <0.01), especially in high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group ( P <0.01). Compared with high glucose group, the apoptotic rate, the mitochondrial membrane potential and the mtROS level of HCAECs in high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were significantly decreased ( P <0.01).The Western blotting results showed that compared with high glucose group, the expression levels of Bax and Bad proteins in the HCAECs in high glucose combined with 10 μmol·L^-1 Co-Q10 treatment group were decreased significantly( P <0.01), and the expression levels of Bcl-2 and x-IAP proteins were increased si gnificantly ( P <0.01). Conclusion : Co-Q10 may reduce the apoptosis of HCAECs induced by high glucose through inhibiting the mitochondrial apoptosis-related pathway to ptotect the cells.