目的 探讨丹酚酸B通过线粒体凋亡途径干预动脉粥样硬化(Atherosclerosis，AS)的作用机制。方法 将小鼠单核巨噬细胞RAW264。7 培养后分为空白组，细胞色素C(Cytochrome C，cytC)组，丹酚酸B低、中、高浓度组。各组干预时间均为24h。用噻唑蓝(MTT)实验检测细胞存活率，并用流式细胞仪检测细胞凋亡率。采用蛋白免疫印迹(Western Blotting)法分别检测cytC的释放和B淋巴细胞瘤-2基因(B-cell lymphoma-2，Bcl-2)、半胱天冬氨酸蛋白酶-3(Cysteinyl aspartate specific proteinase-3，Caspase-3)、半胱天冬氨酸蛋白酶-9(Cysteinyl aspartate specific proteinase-9，Caspase-9)和Bcl-2相关X蛋白质(Bcl-2-associated X protein，Bax)蛋白表达水平。结果 与空白组比较，cytC组细胞活性降低(P<0。05)，细胞平均凋亡率较高(P<0。05);cytC组中促凋亡蛋白cytC、Bax、Caspase-3 及Caspase-9 的表达均升高(P<0。05)，抑凋亡蛋白Bcl-2 的表达下降(P<0。05)。与cytC组比较，丹酚酸B低、中浓度组细胞活性均上升(P<0。01，P<0。05)，各组促凋亡蛋白cytC、Bax、Caspase-3 及Caspase-9 表达均下降(P<0。01，P<0。05)，丹酚酸B中、低浓度组细胞中抑凋亡蛋白Bcl-2 的表达升高(P<0。01)。丹酚酸B中、高浓度组与空白组细胞平均凋亡率比较，差异有显著统计学意义(P<0。01)。结论 丹酚酸B通过降低cytC、Bax、Caspase-3、Caspase-9 蛋白表达抑制cytC诱导的RAW264。7 细胞凋亡，发挥在AS发展进程中改善粥样斑块沉积的作用，可能与抑制线粒体凋亡途径相关。
Study on the effect of salvianolic acid B in intervening atherosclerosis through the mitochondrial apoptosis pathway
Objective To investigate the mechanism of salvianolic acid B intervening atherosclerosis through the mitochondrial apoptosis pathway.Methods Mouse monocyte macrophages RAW264.7 were randomly divided into blank group,cytochrome C(cytC)group,and salvianolic acid B group with low,medium and high concentration.The intervention time of each group was 24h.The cell survival rate was measured by MTT assay and the apoptosis rate was measured by flow cytometry.The release of cytC and B-cell lymphoma-2(Bcl-2),protein expression levels of cysteinyl aspartate specific proteinase-3(Caspase-3),cysteinyl aspartate specific proteinase-9(Caspase-9)and Bcl-2-associated X protein(Bax)were detected by Western Blotting,respectively.Results Compared with the blank group,the cell activity of the cytC group decreased(P<0.05),the apoptosis rate of the cytC group increased,the expressions of pro-apoptotic proteins cytC,Bax,Caspase-3 and Caspase-9 in the cytC group increased(P<0.05),and the expression of anti-apoptotic protein Bcl-2 decreased(P<0.05).Compared with the cytC group,the cell activity of the low and medium salvianolic acid B concentration groups increased(P<0.01,P<0.05),the expressions of pro-apoptotic proteins cytC,Bax,Caspase-3 and Caspase-9 decreased(P<0.01,P<0.05),and the expression of anti-apoptotic protein Bcl-2 in the cells of the medium and the low concentration of salvianolic acid B increased(P<0.01).There was significance of the apoptosis rate between the blank group and the medium and high salvianolic acid B concentration group(P<0.01).Conclusion Salvianolic acid B inhibits cytochrome C-induced apoptosis of RAW264.7 cells by reducing the expression of cytC,Bax,Caspase-3 and Caspase-9 proteins,and has the effect of improving atheroplaque deposition in the development of AS,which may be related to the inhibition of mitochondrial apoptosis pathway.
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