同型半胱氨酸诱导小鼠胶质细胞表达白介素-1β和肿瘤坏死因子-α的研究

Study on the mechanism of homocysteine-induced microglia expression of interleukin -1β and tumor necrosis factor-α

目的探讨同型半胱氨酸诱导小胶质细胞表达白介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）的机制。方法将体外培养的小鼠小胶质瘤细胞（BV-2细胞）分成空白对照组、半胱氨酸（Cys）组、同型半胱氨酸（Hcy）组和同型半胱氨酸＋还原型谷胱甘肽（Hcy＋GSH）组，培养72h。应用反转录实时定量聚合酶链式反应（RT-qPCR）方法检测IL-1β和TNF-αmRNA表达，酶联免疫吸附法（ELISA）测定培养上清中细胞因子的蛋白浓度，蛋白质印迹法（Westernblot）检测NF-κB／p65的含量。结果空白对照组、Cys组、Hcy组和Hcy＋GSH组细胞内IL-1β 和TNF-αmRNA（F=48．63、130．76，均P〈0．05）和蛋白表达（F=702．91、293．69，均P〈0．05）以及NF-κB／p65含量（F=212．06，P〈0．05）差异均有统计学意义；与空白对照组比较，Hcy组细胞内IL-1β 和TNF-αmRNA和蛋白表达明显增加，Hcy＋GSH组较Hcy组表达水平下降，Hcy组NF-κB／p65含量高于其他组，差异均具有统计学意义（均P〈0．05）。结论Hcy可诱导小胶质细胞表达IL-1β和TNF-α，NF-κB信号通路可能参与了此过程。

Objective To investigate the mechanism of homocysteine induced microglia （BV-2 cells） expression of interleukin-1β（IL-1β） and tumor necrosis factov-α （TNF-α）. Methods The BV- 2 cells were divided into blank control group, eysteine （Cys） group, homocysteine （Hey） group and homocysteine and glutathione （Hcy＋GSH） group, and the BV-2 cells in these groups were incubated with cysteine or homocysteine or homocysteine and glutathione together for 72 h. The mRNA expressions of IL-1β and TNF-α were assessed by RT-qPCR. The protein expressions of IL-1βand TNF a in supernatant were detected by enzyme linked immunosorbent assay （ELISA）. Western blot was used to observe the changes of NF-κB/p65 expression. Results There were significant differences in mRNA and protein expressions of IL-1β and TNF-α and NF-κB/p65 protein expression between groups （F=48.63, 130.76, 702.91, 293.69, 212.06, respectively, all P=0.000）. The secretions of IL-1β and TNF-α were significantly improved by homocysteine （P〈0.05）, and were reversed by the treatment with glutathione （P〈0.05）. Western blot assay result showed that NF-κB/ p65 was elevated after treatment with homocysteine （P〈0.05）. Conclusions Homocysteine can induce microglia expression of interleukin --1β and tumor necrosis factor-α, and NF-κB signaling pathway may be involved in this process.