巨噬细胞对内皮细胞凋亡的影响及通心络的干预作用

Effect of Macrophages on Apoptosis of Endothelial Cells and Interventing Effect of Tongxinluo

摘要:

目的：研究不同亚型巨噬细胞对内皮细胞凋亡的影响及通心络的干预作用，并探讨其可能的作用机制。方法：以THP-1细胞为研究对象，实验分为5组，分别为空白组，佛波酯（PMA）组，M2组，M1组及通心络组。采用PMA（5 μg·L^-1）分别联合白细胞介素（IL）-4（100 μg·L^-1），脂多糖（LPS，200 μg·L^-1）诱导THP-1细胞分别向M2和M1型巨噬细胞极化并给以通心络（300 mg·L^-1）进行干预，采用酶联免疫吸附方法（ELISA）检测其标志分子IL-10，肿瘤坏死因子-α（TNF-α），IL-6的表达。取巨噬细胞培养上清作为条件培养基培养内皮细胞，采用流式细胞仪检测内皮细胞凋亡率、蛋白免疫印迹方法检测凋亡相关蛋白半胱氨酸天冬氨酸蛋白酶-3（Caspase-3），B细胞淋巴瘤/白血病-2（Bcl-2），Bcl-2相关X蛋白（Bax）蛋白的表达。结果：与空白组比较，M2组细胞培养上清中IL-10水平显著升高（P<0.01）；M1组细胞培养上清中IL-10水平显著降低，IL-6及TNF-α水平显著升高（P<0.01）；与M1组比较，通心络组细胞培养上清中IL-10水平升高，IL-6及TNF-α水平下降（P<0.01）。与M2组内皮细胞比较，M1型巨噬细胞细胞培养的内皮细胞凋亡率显著升高（P<0.01），促凋亡蛋白Caspase-3，Bax蛋白的表达明显升高（P<0.05，P<0.01），抗凋亡蛋白Bcl-2蛋白的表达显著降低（P<0.01）；与M1组比较，通心络组内皮细胞的凋亡率显著降低（P<0.01），促凋亡蛋白Caspase-3，Bax蛋白的表达明显降低（P<0.05，P<0.01），抗凋亡蛋白Bcl-2蛋白的表达显著升高（P<0.01）。结论：通心络可以抑制巨噬细胞向M1型极化。M2型巨噬细胞培养上清对内皮细胞的凋亡无明显影响，M1型巨噬细胞培养上清可以促进内皮细胞凋亡；通心络预培养的M1型巨噬细胞培养上清培养的内皮细胞凋亡率显著下降。

Abstract:

Objective: To study the effect of different subtypes of macrophages on the apoptosis of endothelial cells and the interventing effect of Tongxinluo (TXL). Method: THP-1 cells were taken as the study object. Cells were divided into five groups: control group, phorbol myristate acetate(PMA) group, M2 group, M1 group and TXL group. THP-1 cells were respectively induced by PMA (5 μg·L^-1) combined with interleukin(IL) -4 (100 μg·L^-1) or lipopolysaccharide(LPS) (200 μg·L^-1) to M2 and M1 type polarized macrophages, and treated by TXL with 300 mg·L^-1. The expressions of marker molecules IL-10, tumor necrosis factor-α(TNF-α), IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). The culture supernatant was taken as conditioned medium to culture endothelial cells, and the apoptosis rate and the expression of apoptosis related proteins were detected by flow cytometry and Western blot, respectively. Result: Compared with control group, the level of IL-10 increased in M2 group (P<0.01), and the level of IL-10 decreased, and IL-6, TNF-α increased in M1 group (P<0.01). Compared with M1 group, the level of IL-10 increased, and IL-6, TNF-α decreased in TXL group (P<0.01). Compared with M2 group, the apoptosis rate and the expressiona of Caspase-3, Bax increased (P<0.05, P<0.01), the expression of Bcl-2 decreased in M1 group (P<0.01). And compared with M1 group, the apoptosis rate and the expressiona of Caspase-3, Bax decreased (P<0.05, P<0.01), and the expression of Bcl-2 increased in TXL group (P<0.01). Conclusion: The polarization of macrophages to M1 type can be inhibited by TXL. The supernatant of M2 macrophages has not a significant effect on the apoptosis of endothelial cells. The apoptosis of endothelial cells increases in the cultured supernatant of M1 macrophages, and TXL can reduce the apoptosis of endothelial cells.