游离脂肪酸抑制AMPK信号通路促进小鼠巨噬细胞葡萄糖酵解和M1极化  

Free fatty acid promotes glycolysis and M1 polarization via inhibiting AMPK pathway in mouse macrophages

作　　者：左惠演 黄秀清[1] 沈涛[1] 唐蔚青[1] 马佳睿 徐芳芷 黎健[1] 窦琳[1] ZUO Huiyan;HUANG Xiuqing;SHEN Tao;TANG Weiqing;MA Jiarui;XU Fangzhi;LI Jian;DOU Lin(Beijing Hospital&National Geriatrics Center&Beijing Institute of Geriatrics of the National Health Commission&Beijing Key Laboratory of Geriatrics of the National Health Commission&Institute of Geriatrics of the Chinese Academy of Medical Sciences,Beijing 100730,China)

机构地区：[1]北京医院国家老年医学中心、国家卫生健康委北京老年医学研究所、国家卫生健康委北京老年医学重点实验室、中国医学科学院老年医学研究院,北京市100730

出　　处：《中国动脉硬化杂志》2023年第3期205-211,共7页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金项目(81600618、81770858和81770228);北京市自然科学基金项目(7182144)。

摘　　要：[目的]探讨游离脂肪酸对小鼠巨噬细胞葡萄糖酵解和M1极化的影响,从巨噬细胞代谢重编程和M1极化阐明非酒精性脂肪性肝炎(NASH)的发病机制,为NASH的防治提供新的思路。[方法]以油酸/棕榈酸(O/P)处理RAW264.7细胞建立游离脂肪酸处理巨噬细胞模型;转染AMP活化蛋白激酶(AMPK)特异siRNA抑制AMPK表达;使用阿卡地新(AICAR)激活AMPK活性。采用qPCR检测巨噬细胞M1型分子标志物(TNF-α、IL-6和MCP-1)、M2型分子标志物(CD206和IL-10)以及葡萄糖酵解相关基因(PGM1、PGK1、GPI1、LDHA、ALDOA、GLUT1和HK2)mRNA水平,采用Western blot检测p-mTOR/mTOR、p-Raptor/Raptor、p-Akt/Akt、p-AMPK和AMPK蛋白水平。[结果] O/P处理RAW264.7细胞后,p-mTOR/mTOR和p-Akt/Akt蛋白水平升高(P<0.05),p-Raptor/Raptor、p-AMPK和AMPK蛋白水平降低(P<0.05),葡萄糖酵解相关基因PGM1、PGK1、GPI1、LDHA、ALDOA、GLUT1和HK2 mRNA水平升高(P<0.05),促炎因子TNF-α、IL-6和MCP-1 mRNA水平升高(P<0.05),抑炎因子CD206和IL-10 mRNA水平降低(P<0.05)。抑制RAW264.7细胞中AMPK表达后,p-mTOR/mTOR和p-Akt/Akt蛋白水平升高(P<0.05),p-Raptor/Raptor、p-AMPK和AMPK蛋白水平降低(P<0.05),葡萄糖酵解相关基因PGM1、PGK1、GPI1、LDHA、ALDOA、GLUT1和HK2 mRNA水平升高,促炎因子TNF-α、IL-6和MCP-1 mRNA水平升高(P<0.05),抑炎因子CD206和IL-10 mRNA水平降低(P<0.05)。激活AMPK活性可逆转O/P对巨噬细胞葡萄糖酵解和M1极化的影响。[结论]在RAW264.7细胞中游离脂肪酸通过抑制AMPK信号通路促进葡萄糖酵解和M1型极化。Aim To explore the effect of free fatty acid on the M1 polarization and glycolysis in mouse macrophages. The mechanism of non-alcoholic steatohepatitis(NASH) pathogenesis is clarified from the perspective of metabolism reprogramming and polarization in macrophages, which will provide a new way for prevention and treatment of NASH.Methods Oleic acid/palmitate acid(O/P) was used to treat RAW264.7 cells to establish cell model. AMPK-specific siRNA was transfected into RAW264.7 cells to knockdown AMPK protein expression. Acadesine(AICAR) was used to stimulate the activation of AMPK. The mRNA levels of M1 type molecular markers(TNF-α, IL-6 and MCP-1), M2 type molecular markers(CD206 and IL-10) and glycolysis related genes(PGM1, PGK1, GPI1, LDHA, ALDOA, GLUT1 and HK2) were detected by qPCR, and the protein levels of p-mTOR/mTOR, p-Raptor/Raptor, p-Akt/Akt, p-AMPK and AMPK were detected by Western blot. Results After treatment of RAW264.7 cells with O/P, the protein levels of p-mTOR/mTOR and p-Akt/Akt increased(P<0.05), the protein levels of p-Raptor/Raptor, p-AMPK and AMPK decreased(P<0.05), the mRNA levels of glucose glycolysis related genes PGM1, PGK1, GPI1, LDHA, ALDOA, GLUT1 and HK2 increased(P<0.05), the mRNA levels of pro-inflammatory factors TNF-α, IL-6 and MCP-1 increased(P<0.05), while the mRNA levels of anti-inflammatory factors CD206 and IL-10 decreased(P<0.05). After inhibiting AMPK expression in RAW264.7 cells, the protein levels of p-mTOR/mTOR and p-Akt/Akt increased(P<0.05), the protein levels of p-Raptor/Raptor, p-AMPK and AMPK decreased(P<0.05), the mRNA levels of glucose glycolysis related genes PGM1, PGK1, GPI1, LDHA, ALDOA, GLUT1 and HK2 increased(P<0.05), the mRNA levels of pro-inflammatory factors TNF-α, IL-6 and MCP-1 increased(P<0.05), while the mRNA levels of anti-inflammatory factors CD206 and IL-10 decreased(P<0.05). Activation of AMPK activity could reverse the effect of O/P on macrophage polarization and glycolysis. Conclusion Free fatty acid promotes M1 polarization and glycolysis via i

关 键 词：AMP活化蛋白激酶 巨噬细胞极化 糖酵解 非酒精性脂肪性肝炎 游离脂肪酸 

分 类 号：R363[医药卫生—病理学] R5[医药卫生—基础医学]