目的 探讨高盐刺激下巨噬细胞表型偏移的可能机制,为相关心血管疾病的防治提供新的实验依据。方法 选取Raw264.7小鼠巨噬细胞为研究对象,构建TonEBP慢病毒干扰稳定细胞株,利用Real-time PCR技术检测TonEBP干扰效率,选取干扰效率最高的感染组细胞株用于后续实验。给予各组相应干预后同步培养24 h,利用Real-time PCR检测各组细胞TonEBP、NF-κB及其M1、M2型巨噬细胞表型标志物mRNA的表达水平;Western blot法检测各组TonEBP、NF-κB、pNF-κB、pIKKα/β的蛋白表达水平。结果 成功构建TonEBP -shRNA稳定干扰细胞株,与Control组相比其TonEBP干扰效率达70％;Real-time PCR结果显示单纯高盐干预下Raw264.7细胞TonEBP、NF-κB和M1型巨噬细胞表型标志物的mRNA表达水平显著上调(P＜0.05),M2型巨噬细胞表型标志物mRNA表达水平显著下调(P＜0.05);高盐刺激下分别干扰TonEBP、NF-κB的表达后,M1型巨噬细胞表型标志物的mRNA表达水平显著下调,M2型巨噬细胞表型标志物mRNA表达水平显著上调。Western blot结果显示单纯高盐刺激下Raw264.7细胞TonEBP、p-IKKα/β、NF-κB、p-NF-κB的蛋白表达水平均明显上调,NF-κB,p-NF-κB,p-NF-κB与NF-κB比值增加;高盐刺激下分别干扰TonEBP、NF-κB的表达后,p-IKKα/β、p-NF-κB蛋白表达水平均显著下调。结论 TonEBP/NF-κB信号通路的激活是高盐刺激下Raw264.7细胞向M1型巨噬细胞发生偏移的可能机制;调节TonEBP/NF-κB信号通路,可改变高盐诱导下Raw264.7细胞表型偏移方向。
Abstract
Objective To investigate the possible mechanism of macrophage phenotypic polarization under hypersaline stimulation so as to provide data for future research into the prevention and treatment of salt-sensitive hypertension and other macrophage related cardiovascular diseases.Methods Using Raw264.7 mouse macrophages as the subjects, a TonEBP lentivirus interference stable cell line was constructed. The interference efficiency of TonEBP gene was detected by real-time PCR , and the cell lines of the infected group with the highest interference efficiency were selected for subsequent experiments.The mRNA expression levels of TonEBP、NF-κB 、M1-type and M2-type macrophage phenotypic markers in each group were detected by real-time PCR after twenty hours of corresponding intervention.Western blot was used to detect the protein expression levels of TonEBP、NF-κB、p-NF-κB and p-IKKα/β.Results Stable interfering cell lines of TonEBP-shRNA were constructed, and the TonEBP interfering efficiency was over 70% compared with the control group (P＜0.05).The mRNA expression levels of TonEBP、NF-κB andM1 macrophage phenotypic markers in the HS group were significantly up-regulated,(P＜0.05), while the mRNA expression levels of M2 type macrophage phenotypic markers were significantly down-regulated(P＜0.05).mRNA expression of M1-type macrophage phenotypic markers was significantly down-regulated,(P＜0.05) while mRNA expression of M2-type macrophage phenotypic markers was significantly up-regulated by interfering with the expressions of TonEBP and NF-κB respectively under high salt stimulation(P＜0.05).Compared with the control group, the protein expression levels of TonEBP、p-IKKα/β、NF-κB and p-NF-κB in the HS group were significantly up-regulated(P＜0.05), and the ratio of p-NF-κB to NF-κB was increased(P＜0.05).Compared with the HS group, protein expression levels of p-IKKα/β、NF-κB and NF-κB were significantly decreased after interference with the expression of TonEBP and NF-κB respectively under high salt stimulation(P＜0.05),but difference in the ratio of p-NF-κB to NF-κB was of no statistical significance(P＞0.05).Conclusions Activation of TonEBP/NF-κB signaling pathway is a possible mechanism of Raw264.7 cell shifting to M1 phenotype under high salt stimulation.The regulation of TonEBP/ NF-κB signaling pathway can change the direction in which the phenotypes of Raw264.7 cells are polarized under high salt induction.
关键词
高盐 /
心血管疾病 /
巨噬细胞 /
表型 /
信号通路
Key words
high salt /
cardiovascular disease /
macrophages /
phenotype /
signaling pathway
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基金
国家自然科学基金项目(81600328);天津市自然科学基金项目(16JCQNJC11800);武警后勤学院中心实验室开放基金项目 (2015ZXKF11);国家重点研发计划资助(项目号:2017YFC1307600,课题号: 2017YFC1307602)
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