软脂酸对人脐静脉内皮细胞凋亡影响及其机制的研究

Effect and its mechanism of palmitic acid on the apoptosis of human umbilical vein endothelial cells

目的探讨软脂酸(PA)对人脐静脉内皮细胞凋亡的影响及其可能机制。方法 (1)将人脐静脉内皮细胞分为:①正常对照(NC)组;②PA组(浓度分别为0.2、0.4、0.8mmol/L),培养24h,采用MTT法检测细胞增殖情况,流式细胞术及Hoechst 33258核染色检测各组细胞凋亡情况。(2)人脐静脉内皮细胞分为:①NC组;②PA组(0.4mmol/L);③PA+雷帕霉素(PA+Rapamycin)组。采用West-ern blot法分析各组马铃薯球蛋白(Tuberin)、P-Tuberin、核糖体蛋白S6激酶(S6K)、P-S6K、Bcl-2、Bax,以及Caspase3蛋白表达水平。结果经MTT检测显示,与NC组比较,PA组细胞增殖(OD值)下降(P<0.05),且呈剂量依赖性。经Western blot分析显示,与NC组比较,PA组细胞凋亡率增加(P<0.05),且呈剂量依赖性。与NC组比较,PA组P-Tuberin、P-Tuberin/Tuberin、S6K、P-S6K/S6K、Bax、Caspase3表达水平增高,Bcl-2表达降低(P<0.05);PA+Rapamycin组S6K、P-S6K/S6K、Bax、Caspase3表达水平低于PA组,Bcl-2表达高于PA组(P<0.05)。结论 PA能够诱导人脐静脉内皮细胞凋亡,其机制可能是通过上调tuberin/mTOR活性,而降低Bcl-2表达、增加Bax及Caspase3表达,最终导致血管内皮细胞凋亡。

Objective To explore the effect of palmitic acid （PA） on the apoptosis of human umbilical vein endothelial cells and its mechanism. Methods （1）. Human umbilical vein endothelial cells （HUVECs） were divided into two groups： The normal control group （0.4% Bovine Serum Albumin） and the palmitic acid （PA） group （PA at the concentrations 0.2mmol/L, 0.4mmol/L, and 0.8mmol/L）. All the cells were cultured for 24 hours, then the cell proliferation was determined by MTT assay, and flow cytometry and Hoechst 33258 staining were used to assess the apoptosis of cells. （2）. HUVECs were divided into three groups： the normal control group, PA group （at the concentration 0.4mmol/L）, and PA＋Rapamycin group （the cells were pretreated with Rapamycin and then PA）. The expression levels of Tuberin, P-Tuberin, S6K, P-S6K, Bcl-2, Bax and Caspase 3 protein were measured by western blot analysis using specific antibodies. Results Compared with the normal control group, the cell proliferation （OD value） of HUVECs was decreased in the PA group （P〈0.05）, and in a PA dose dependent manner. Compared with the normal control group, the apoptosis rate of HUVECs in the PA group was increased （P〈0.05）, and in a PA dose dependent manner. Compared with the normal control group, the expression levels of Tuberin, P-Tuberin/Tuberin, S6K, P-S6K/S6K, Bax, and Caspase 3 were increased （P〈0.05）, while the level of Bcl-2 was decreased in the PA group （P〈0.05）. Compared with the PA group, the expression levels of Tuberin, P-Tuberin/Tuberin, S6K, P-S6K/S6K, Bax, and Caspase 3 were lower （P〈0.05）, and the expression level of Bcl-2 was higher in the PA＋Rapamycin group （P〈0.05）. Conclusion Palmitic acid is inducing the apoptosis of HUVECs. Its mechanism may be associated with upregulation of tuberin/mTOR activity, decreasing the expression of Bcl-2 and increasing the expression of Bax and Caspase 3, thus leading eventually to the apoptosis of HUVECs.