同型半胱氨酸通过上调Rap1a诱导小鼠小胶质细胞炎性极化

Homocysteine induces inflammatory polarization in mouse microglia by up-regulating Rap1a

目的:研究不同浓度同型半胱氨酸(homocysteine,Hcy)对BV2小鼠小胶质细胞介导的炎症反应的影响,探讨Ras相关蛋白1a(Ras-related protein 1a,Rap1a)在Hcy诱导的BV2细胞炎症反应中的作用机制。方法:体外培养小鼠小胶质细胞系BV2细胞,用Hcy干预,建立高同型半胱氨酸血症细胞模型,将细胞分成空白对照组、50μmol/L Hcy组、100μmol/L Hcy组和150μmol/L Hcy组,采用RT-qPCR法检测BV2细胞M1型极化标志物、炎症因子(IL-6、IL-1β和TNF-α)及Rap1a的mRNA表达水平,ELISA法检测BV2细胞炎症因子IL-6、IL-1β和TNF-α的含量;Western blot法检测细胞内Rap1a的蛋白表达水平。进一步利用慢病毒转染过表达和敲减Rap1a对其功能加以验证。结果:100μmol/L以上浓度Hcy干预下,BV2细胞发生炎性极化,M1型极化标志物CD80和CD86 mRNA表达升高(P<0.05);IL-6、IL-1β和TNF-α等炎症因子mRNA及蛋白表达显著上调(P<0.05);Rap1a蛋白及mRNA表达水平均显著升高(P<0.05);Rap1a mRNA水平与CD80 mRNA及IL-1β和TNF-α含量均呈明显正相关(P<0.05)。过表达Rap1a和敲减Rap1a慢病毒的感染复数均为80,荧光显微镜观察可达有效转染率,过表达Rap1a加剧了Hcy诱导的BV2细胞炎性极化(P<0.05);敲减Rap1a可抑制Hcy诱导的BV2细胞炎性极化(P<0.05)。结论:Hcy可促进BV2小鼠小胶质细胞M1型极化、介导炎症反应。Rap1a可能是Hcy诱导BV2细胞炎症反应的重要调节因子。

AIM:To investigate the impact of homocysteine(Hcy)on the inflammatory response mediated by BV2 mouse microglia,and to explore the mechanism of Ras-related protein 1a(Rap1a)in the Hcy-induced inflammatory response in BV2 cells.METHODS:Mouse microglial cell line BV2 was cultured in vitro,and Hcy intervention was used to establish a hyperhomocysteinemia cellular model.The cells were divided into 4 groups:blank control group,and 50μmol/L,100μmol/L and 150μmol/L Hcy groups.The mRNA expression levels of M1 polarization markers,inflammatory factors IL-6,IL-1βand TNF-α,and Rap1a in BV2 cells were detected by RT-qPCR.Additionally,the levels of inflamma-tory factors IL-6,IL-1βand TNF-αin BV2 cells were measured using an ELISA kit.The protein expression level of Rap1a was detected by Western blot assay.To verify the function of Rap1a,viral transfection was employed for both over-expression and knockdown experiments.RESULTS:Under the intervention of Hcy concentration above 100μmol/L,BV2 cells exhibited inflammatory polarization,as indicated by the increased mRNA expression of M1 polarization markers CD80 and CD86(P<0.05).The mRNA and protein expression levels of inflammatory factors IL-6,IL-1βand TNF-αwere significantly up-regulated(P<0.05).Additionally,the mRNA and protein expression levels of Rap1a also showed a significant increase(P<0.05).Moreover,Rap1a mRNA level was positively correlated with CD80 mRNA,IL-1βcontent and TNF-αcontent(P<0.05).The multiplicities of infection of the viruses with Rap1a overexpression and Rap1a knock-down were both 80,and the effective transfection was observed through fluorescence microscopy.Overexpression of Rap1a exacerbated the inflammatory polarization of BV2 cells induced by Hcy(P<0.05),while knockdown of Rap1a attenuated this polarization(P<0.05).CONCLUSION:Hcy can promote M1 polarization of BV2 mouse microglia,leading to in-flammatory response,which indicates that Rap1a could potentially serves as a critical regulatory factor in the Hcy-induced inflammatory response of BV2 cells.