C型凝集素Dectin-2对动脉粥样硬化M2型巨噬细胞活化和凋亡的作用及机制研究

Effect and Mechanism of C-Type Lectin Dectin-2 on Activation and Apoptosis of M2 Macrophages in Atherosclerosis

目的探究树突状细胞相关凝集素-2(Dectin-2)对动脉粥样硬化进程中巨噬细胞M2型活化和凋亡的作用及机制研究。方法利用白介素4(IL-4)刺激巨噬细胞M2型活化,荧光定量PCR(Q-PCR)检测Dectin-2表达;巨噬细胞敲降Dectin-2,Q-PCR检测巨噬细胞M2活化标志物精氨酸酶1(Arg1)、抵抗素样分子α(FIZZ1)、几丁质酶3(Ym1)表达;氧化修饰低密度脂蛋白(ox-LDL)刺激巨噬细胞,流式细胞法检测巨噬细胞凋亡水平;IL-4刺激巨噬细胞0、5、30、60min,蛋白免疫印迹(WB)法检测信号传导及转录激活蛋白6(STAT6)磷酸化变化。结果Q-PCR结果显示,与对照组比较,IL-4(10ng/mL)、IL-4(20ng/mL)组巨噬细胞Dectin-2表达明显升高[(1.78±0.11)、(2.47±0.12)比(1.00±0.07),P<0.05];敲降Dectin-2后,加入IL-4刺激巨噬细胞,与IL-4+siNC组比较,IL-4+siDectin-2组中Arg1、FIZZ1、Ym1 mRNA相对表达量均明显降低[Arg1:(175.36±12.36)比(278.23±23.12),P<0.05;FIZZ1:(1625.13±13.15)比(2151.12±45.12),P<0.05;Ym1:(25.15±1.36)比(54.16±1.36),P<0.05];流式结果显示,与ox-LDL+siNC组相比,ox-LDL+siDectin-2组细胞凋亡率明显降低[(12.36±1.07)%比(30.04±0.62)%,P<0.05];WB结果显示,敲降Dectin-2明显抑制IL-4诱导的STAT6磷酸化水平升高。结论IL-4刺激巨噬细胞Dectin-2表达上调,抑制Dectin-2表达可以明显抑制巨噬细胞M2型活化以及ox-LDL诱导的凋亡,其作用机制可能是通过调控STAT6磷酸化水平。

Objective To investigate the effect and mechanism of dendritic cell-related lectin 2(Dectin-2)on activation and apoptosis of M2 macrophages in the process of atherosclerosis.Methods Interleukin-4(IL-4)was used to stimulate activation of M2 macrophages,and the expression of Dectin-2 was detected by quantitative fluorescence PCR(Q-PCR).Dectin-2 was knocked down in macrophages,and expressions of M2 macrophage activation markers,argininase 1(Arg1),resistin-like molecular cut(FIZZ1)and chitinase 3(Ym1),were examined by Q-PCR.Oxidation-modified low-density lipoprotein(ox-LDL)stimulated macrophages and flow cytometry was used to test the apoptotic level of macrophages.IL-4 stimulated macrophages for 0,5,30,and 60min,and the changes of signal transduction and transcriptional activation protein 6(STAT6)phosphorylation were checked by Western blot(WB).Results Compared to the control group,RT-PCR results showed that the expression of Dectin-2 in macrophages of IL-4(10ng/mL)and IL-4(20ng/mL)treatment groups increased significantly[(1.78±0.11)and(2.47±0.12)vs(1.00±0.07),P<0.05].Compared to IL-4+siNC group,after Dectin-2 knockdown and IL-4 was added to stimulate macrophages,the relative mRNA expression of Arg1,FIZZ1 and Ym1 in IL-4+siDectin-2 group decreased significantly,i.e.,Arg1:[(175.36±12.36)vs(278.23±23.12)],FIZZ1:[(1625.13±13.15)vs(2151.12±45.12)],Ym1:[(25.15±1.36)vs(54.16±1.36)],P<0.05,respectively.Compared to IL-4+siNC group,flow cytometry results indicated that the cell death rate of the ox-LDL+siDectin-2 group reduced significantly[(30.04±0.62)%vs(12.36±1.07)%,P<0.05].WB results showed that IL-4-induced STAT6 phosphorylation level was inhibited significantly after Dectin-2 knockdown.Conclusion IL-4 can stimulate the up-regulation of Dectin-2 expression in macrophages,and inhibit the expression of Dectin-2,which can significantly inhibit activation and ox-LDL-induced apoptosis of M2 macrophages,possibly through the regulation of STAT6 phosphorylation level.