牛磺酸促进线粒体自噬抑制M2型巨噬细胞极化

Taurine inhibits M2 polarization of macrophages by promoting mitophagy

目的研究牛磺酸对M2型巨噬细胞抑炎极化的调控作用,初步探索线粒体自噬在其中的作用机制。方法人外周血的单核细胞系THP-1细胞经100 nmol/L佛波酯(PMA)处理48 h后诱导分化为M0型巨噬细胞;M0型巨噬细胞经20 ng/mL白细胞介素4(IL-4)刺激48 h,诱导极化为M2型巨噬细胞;加入(40、80)mmol/L牛磺酸与IL-4共处理M2巨噬细胞48 h。实时荧光定量PCR检测M2型巨噬细胞极化相关标志物C型甘露糖受体1(MRC-1)、CC趋化因子配体22(CCL22)、树突状细胞特异性细胞间黏附分子结合非整合素(CD209)的mRNA表达;多功能酶标仪及激光共聚焦显微镜检测和观察线粒体、溶酶体数量变化,JC-1线粒体膜电位检测试剂盒检测线粒体膜电位水平;Western blot法检测线粒体自噬相关蛋白PTEN诱导激酶1(PINK1)、微管相关蛋白1轻链3(LC3)的表达。结果M0极化为M2时,MRC-1、CCL22、CD209 mRNA表达水平明显上升;线粒体数量和线粒体膜电位增加;溶酶体数量减少;PINK1蛋白表达水平增加,LC3Ⅱ/LC3Ⅰ比值下降。40 mmol/L、80 mmol/L牛磺酸分别处理后,MRC-1、CCL22、CD209的mRNA水平明显下降;线粒体数量和线粒体膜电位下降;溶酶体数量增加;PINK1水平及LC3Ⅱ/LC3Ⅰ比值增加,与牛磺酸浓度呈剂量依赖性。结论牛磺酸通过降低线粒体膜电位,促进线粒体自噬,减少细胞的线粒体数量,抑制M2型巨噬细胞极化相关标志物的表达,负调控M2型巨噬细胞极化,避免过度抑炎极化。

Objective To investigate the molecular mechanism of taurine regulating the polarization of M2 macrophages by mitophagy.Methods THP-1 cells were divided into four groups:MO group(THP-1 cells were treated by 100 nmol/L phorbol myristate ester for 48 hours to polarize into MO),M2 group(THP-1 cells were induced to polarize into M2 macrophages by 20 ng/mL interferon-4(IL-4)for 48 hours),M2 combined with taurine groups(added with 40 or 80 mmol/L taurine on the basis of M2 macrophages).The mRNA expression of mannose receptor C type 1(MRC-1),C-C motif chemokine ligand 22(CCL22)and dendritic cell-specific ICAM-3 grabbing non-integrin(CD209)in M2 macrophages were detected by quantitative real-time PCR.Mitochondrial and lysosome probes were used to detect the number of mitochondria and lysosomes by multifunction microplate reader and confocal laser scanning microscope.The level of mitochondrial membrane potential(MMP)was detected by JC-1 MMP assay kit.The expression of mitophagy-related proteins PTEN-induced putative kinase 1(PINK1)and microtubule-associated protein 1 light chain 3(LC3)were detected by Western blot analysis.Results Compared with MO group,the expression of MRC-1,CCL22,CD209 and PINK1,the number of mitochondria and the level of MMP in M2 group were significantly increased,whereas the number of lysosomes and LC3 II/LC3 I ratio were decreased.Compared with M2 group,the expressions of MRC-1,CCL22 and CD209,the number of mitochondria and the level of MMP in M2 combined with taurine group dropped significantly while the number of lysosomes was found increased,and the protein expression of PINK1 and LC3 II/LC3 I ratio were also increased.Conclusions Thepolarizationof M2macrophages is regulated by taurine to prevent excessive polarization via reducing the level of MMP,improving the level of mitophagy,reducing the number of mitochondria,and inhibiting the mRNA expression of polarization markers in M2 macrophages.