三黄泻心汤活血化瘀优势方抗动脉粥样硬化的作用机制

目的:研究三黄泻心汤活血化瘀优势方对氧化低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVECs)损伤的影响并探讨其抗动脉粥样硬化的作用机制。方法:采用体外培养的HUVECs,分为空白组,ox-LDL损伤组(模型组),三黄泻心汤活血化瘀优势方15.63,31.25,62.50 mg·L-1组;用200 mg·L-1ox-LDL孵育24 h,造成HUVECs损伤模型;细胞存活率采用噻唑蓝(MTT)比色法测定,放射免疫法检测细胞上清液中内皮素(ET-1),6-酮-前列腺素F1α(6-keto-PGF1α),血栓烷素B2(TXB2)含量,半自动生化分析仪检测抗氧化应激指标超氧化物歧化酶(SOD),丙二醛(MDA),一氧化氮(NO)水平。采用试剂盒荧光定量法检测活性氧(ROS)含量,检测细胞间黏附分子-1(ICAM-1),血管细胞黏附分子-1(VCAM-1)及单核细胞趋化因子-1(MCP-1)水平酶联免疫吸附测定。细胞凋亡率及线粒体膜电位变化采用流式细胞术,采用蛋白免疫印迹法(Western blot)检测凋亡相关蛋白B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2),Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax),半胱氨酸蛋白酶-3(cysteine aspartate-specific protease-3,Caspase-3),Caspase-9表达。结果:与空白组比较,模型组HUVECs存活率降低;MDA,MCP-1,ICAM-1,VCAM-1的水平升高,SOD和NO含量降低,ROS的分泌增加,细胞凋亡率升高,Bax,Caspase-3和Caspase-9的表达量增加,Bcl-2的表达量减少(P0.05,P0.01)。与模型组比较,不同质量浓度的三黄泻心汤活血化瘀优势方使HUVECs存活率明显上升,显著降低MDA,MCP-1,ICAM-1,VCAM-1的水平;提高SOD和NO含量,且减少ROS的分泌,降低细胞凋亡率,降低Bax,Caspase-3和Caspase-9的表达量,增加Bcl-2的表达量(P0.05,P0.01)。结论:在ox-LDL诱导损伤的HUVECs模型上,三黄泻心汤活血化瘀优势方具有抑制细胞凋亡,减轻炎性反应和抗氧化的作用,可能与抗动脉粥样硬化有密切关系。

Anti-atherosclerosis Mechanism of Sanhuang Xiexintang in Activating Blood and Resolving Stasis Formula in Vitro

Objective:To investigate the anti-atherosclerosis mechanism of Sanhuang Xiexintang in activating blood and resolving stasis (SXTAR) in vitro. Method:The cell injury model was established with oxidized law-density lipoprotein(ox-LDL) in cultured human umbilical vein endothelial cells live (HUVECs) in vitro, and divided into control group, model group, and SXTAR 15.63,31.25,62.50 mg·L^-1 groups. The HUVECs livability and the concerning markers in cell supernatant, like malondialdehyde(MDA), monocyte chemoattractant protein-1(MCP-1), intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1(VCAM-1), superoxide dismutase(SOD) and nitric oxide(NO), were detected; reactive oxygen species(ROS) was measured by fluorescence quantitation. Apoptosis rate and mitochondrial membrane potential were determined by flow cytometry. Western blot was employed for testing expressions of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cysteine aspartate-specific protease-3(Caspase-3). Result:The survival rate of HUVECs in model group was significantly lower than that of control group. In the model group, MDA, MCP-1, ICAM-1, VCAM-1 were significant increases, SOD and NO content were decreased, apoptosis rate, ROS and expressions of Bax, Caspase-3, Caspase-9 were up-regulated, and expression of Bcl-2 was down(P<0.05,P<0.01)-regulated. SXTAR significantly increased the survival rate of HUVECs. Compared with the model group, SXTAR significantly decreased contents of MDA, MCP-1, ICAM-1 and VCAM-1.SOD and NO content were increased, apoptosis rate, ROS and expressions of Bax, Caspase-3, Caspase-9 was down-regulated, and expression of Bcl-2 was up-regulated(P<0.05,P<0.01). Conclusion:SXTAR can protect HUVECs injured by ox-LDL, which may be related with inhibiting the endothelial cell apoptosis, reducing inflammation and resisting oxidation.