4-辛基衣康酸抑制糖酵解调控M2型巨噬细胞极化

4-octylitaconic acid inhibits glycolysis and regulates the polarization of M2 macrophages

目的:研究4-辛基衣康酸(4OI)对M2型巨噬细胞(Mφ)极化的干预作用,初步探讨糖酵解及脂肪酸氧化在其中的作用机制。方法:将Mφ分为M0组(100 nmol/L佛波酯刺激48 h诱导为M0型)、M2组[M0组基础上用20 ng/mL白细胞介素4(IL-4)刺激48 h诱导为M2型]、M2+4OI-L组(M2组基础上用100μmol/L 4OI处理12 h)、M2+4OI-H组(M2组基础上用200μmol/L 4OI处理12 h)。实时荧光定量PCR检测M2型Mφ极化相关标志物白细胞介素10(IL-10)、甘露糖受体(MRC-1)、细胞趋化因子(CCL-22)、DC特异性细胞间黏附分子3结合非整合素(CD209)、细胞因子信号抑制物(SOCS1)、过氧化物酶体增殖物激活受体γ(PPAR-γ)、脂肪酸氧化限速酶碱棕榈酰基转移酶-1α(CPT-1α)的mRNA表达水平;微量法酶活性试剂盒检测糖酵解限速酶己糖激酶(HK)和丙酮酸激酶(PK)活性,刃天青法检测细胞内呼吸链代谢酶活性。结果:M0极化为M2时,与M0组比较,伪足伸出细胞和梭形细胞占比和M2型极化相关标志物CCL-22、IL-10、MRC-1、CD209、SOCS1和PPAR-γmRNA表达水平上升(P<0.01);HK和PK活性升高(P<0.05);CPT-1αmRNA水平和线粒体呼吸链代谢酶活性升高(P<0.01)。4OI干预后,与M2组比较,伪足伸出细胞和梭形细胞占比和M2型极化相关标志物CCL-22、IL-10、MRC-1、CD209、SOCS1和PPAR-γmRNA表达水平下降(P<0.05);HK和PK活性降低(P<0.05);CPT-1αmRNA水平进一步升高(P<0.01);线粒体呼吸链代谢酶活性无明显变化。结论:4OI可能通过抑制M2型Mφ糖酵解降低M2型Mφ极化相关标志物的表达,通过进一步促进脂肪酸氧化水平,维持M2细胞内线粒体呼吸链代谢酶活性和细胞氧化磷酸化稳定。

Objective:To study the effect of 4-octylitaconic acid(4OI)on the polarization of M2 macrophages and to explore the mechanism of glycolysis and fatty acid oxidation in 4OI.Methods:Mφwas divided into M0 group(induced by 100 nmol/L phorbol ester for 48 h),M2 group(based on M0 group,induced by 20 ng/mL interleukin 4(IL-4)for 48 h),M2+4OI-L group(based on M2 group,treated with 100μmol/L 4OI for 12 h)and M2+4OI-H group(based on M2 group,treated with 200μmol/L 4OI for 12 h).Real-time fluorescence quantitative PCR was used to detect the polarization-related markers of M2 macrophages interleukin-10(IL-10),mannose receptor(MRC-1),chemotactic factor(CCL-22),DC-specific intercellular adhesion molecule-3 binding non-integrin(CD209),cytokine signal inhibitor(SOCS1),peroxisome proliferator-activated receptor-gamma(PPAR-γ),mRNA expression level of Alkaline palmitoyltransferase-1α(CPT-1α)in fatty acid oxidation rate-limiting enzyme.The enzyme activity kit was used to detect the glycolysis rate-limiting enzymes hexokinase(HK)and pyruvate kinase(PK)activity,and the intracellular respiratory chain metabolic enzyme activity was detected by resazurin disc test.Results:Compared with M0 group,when M0 polarized into M2,the percentage of pseudopod protruding cells and spindle cells,the mRNA expression of CCL-22,IL-10,MRC-1,CD209,SOCS1 and PPAR-γof M2 increased(P<0.01),HK and PK activity increased(P<0.05).CPT-1αmRNA level and mitochondrial respiratory chain metabolic enzyme activity increased(P<0.01).After 4OI intervention,compared with M2 group,the percentage of pseudopod protruding cells and spindle cells and the expression of M2 po-larization related markers CCL-22,IL-10,MRC-1,CD209,SOCS1 and PPAR-γmRNA decreased(P<0.05).HK and PK activity decreased(P<0.05),CPT-1αmRNA level further increased(P<0.01),while there was no significant change in mitochondrial respiratory chain metabolic enzyme activity.Conclusion:4OI may reduce the expression of polarization-related markers in M2 macrophages by inhibiting glycolysis of M2 macrophages,and further promote the level of fatty acid oxidation to maintain the metabolic activity of mitochondrial respiratory chain and oxidative phosphorylation in M2 cells.