Sesn2通过激活mTOR信号通路调节LPS诱导的巨噬细胞M1极化和炎症反应

Sesn2 modulates LPS-induced macrophage M1 polarization and inflammatory response by activating mTOR signaling pathway

目的:探讨sestrin 2(Sesn2)在巨噬细胞中的功能及其通过哺乳动物雷帕霉素靶蛋白(mTOR)通路对巨噬细胞极化和炎症的影响。方法:用小干扰RNA构建Sesn2沉默(si-Sesn2)的RAW264.7小鼠巨噬细胞胞株,通过脂多糖(LPS)处理RAW264.7细胞建立体外炎症模型。RT-qPCR评估白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)、CD86和诱导型一氧化氮合酶(iNOS)的mRNA水平;ELISA检测培养液中IL-1β、IL-6和TNF-α水平;流式细胞术确定CD86标记的M1型巨噬细胞百分比;Western blot分析mTOR、p70核糖体蛋白S6激酶(p70S6K)和真核翻译启动因子4E结合蛋白1(4EBP1)的蛋白表达水平。结果:在LPS诱导的体外炎症模型中,Sesn2在RAW264.7细胞中的表达上调,IL-1β、IL-6、TNF-α以及M1型巨噬细胞标志物CD86和iNOS的表达显著增加,伴随mTOR、p70S6K和4EBP1蛋白表达水平的升高。而si-Sesn2进一步促进了上述效应,但这些变化可通过雷帕霉素预处理减轻。结论:Sesn2可通过mTOR/p70S6K/4EBP1信号通路调控巨噬细胞M1型极化,并影响促炎因子的表达。

AIM:To investigate the function of sestrin 2(Sesn2)in macrophages and its impact on macrophage polarization and inflammatory response.METHODS:Knockdown of Sesn2 in RAW264.7 mouse macrophages was established using siRNA,and an in vitro inflammation model was created by treating RAW264.7 cells with lipopolysaccharide(LPS).The mRNA levels of interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α),CD86 and inducible nitric oxide synthase(iNOS)in RAW264.7 cells were detected by RT-qPCR.The concentrations of IL-1β,IL-6 and TNF-α in the culture medium were measured by ELISA.The percentage of M1-type macrophages marked by CD86 was determined by flow cytometry.The protein levels of mammalian target of rapamycin(mTOR),p70 ribosomal protein S6 kinase(p70S6K)and eukaryotic translation initiation factor 4E binding protein 1(4EBP1)were analyzed by Western blot.RESULTS:In the LPS-induced in vitro inflammation model,the expression levels of Sesn2,inflammatory factors(IL-1β,IL-6 and TNF-α),and M1 macrophage markers(CD86 and iNOS)in RAW264.7 cells were significantly increased,along with elevated protein expression of mTOR,p70S6K and 4EBP1.Silencing of Sesn2 with siRNA further enhanced these effects.However,if rapamycin was added before LPS in the experiment,these changes could be mitigated.CONCLUSION:Sesn2 can regulate M1 macrophage polarization through mTOR/p70S6K/4EBP1 signaling pathway and affect the expression of pro-inflammatory factors.