大黄酚调节NF-κB/HMGB1-PI3K/Akt/mTOR轴抑制ox-LDL诱导的巨噬细胞泡沫化机制研究  

Mechanism of chrysophanol in inhibiting ox-LDL-induced macrophage foaminess through NF-κB/HMGB1-PI3K/Akt/mTOR pathway

作　　者：吴春林 胡亚男 刘毅强 黎晖[1] 温泉 WU Chun-lin;HU Ya-nan;LIU Yi-qiang;LI Hui;WEN Quan(School of Basic Medical Sciences,Guangzhou University of Chinese Medicine,Guangzhou 511400,China;Ziwei Community Health Service Center of Longgang District People's Hospital,Shenzhen 518100,China;Guangdong Hospital of Traditional Chinese Medicine,Guangzhou 511400,China)

机构地区：[1]广州中医药大学基础医学院,广东广州511400 [2]深圳市龙岗区人民医院紫薇社区卫生服务中心,广东深圳518100 [3]广东省中医院,广东广州511400

出　　处：《中国中药杂志》2024年第23期6439-6449,共11页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82104615)。

摘　　要：旨在探讨大黄酚(chrysophanol, Chr)减轻氧化型低密度脂蛋白(oxidized low-density lipoprotein, ox-LDL)所致炎症和泡沫细胞形成的潜在机制,探索大黄酚与冠状动脉粥样硬化的相关作用靶点及通路,为临床新药研发提供理论依据。采用体外培养RAW264.7巨噬细胞,通过细胞计数试剂盒(cell counting kit-8,CCK-8)确定大黄酚和ox-LDL对RAW264.7巨噬细胞适当给药浓度后,用不同浓度的大黄酚(10、15μmol·L^(-1))、不同浓度的ox-LDL(含或不含80 mg·mL^(-1))处理RAW264.7巨噬细胞24 h,即将RAW264.7巨噬细胞分为4组,对照组、模型组(80 mg·mL^(-1)ox-LDL)、大黄酚低剂量组(80 mg·mL^(-1)ox-LDL+大黄酚10μmol·L^(-1))、大黄酚高剂量组(80 mg·mL^(-1)ox-LDL+大黄酚15μmol·L^(-1)),并用油红O染色检测各组的脂质累积情况、流式细胞术分析各组中白细胞分化抗原36(CD36)的表达情况、蛋白免疫印迹(Western blot)法检测A1类清道夫受体(SR-A1)、B族Ⅰ型清道夫受体(SR-B1)、自噬相关蛋白5(Atg5)、苄氯素1(Beclin1)、自噬衔接蛋白p62(P62)、微管相关蛋白轻链3(LC3)Ⅱ与LC3Ⅰ比值(LC3Ⅱ/LC3Ⅰ)和核转录因子κB P65(NF-κB P65)、抑制性κB激酶β(IKKβ)、核因子κB激酶亚基β抑制因子(IκB)、高迁移率族蛋白B1(HMGB1)、磷脂酰肌醇3-激酶(PI3K)、蛋白激酶B (Akt)、哺乳动物雷帕霉素靶蛋白(mTOR)磷酸化的蛋白表达,实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction, RT-qPCR)检测三磷酸腺苷结合盒转运子A1(ABCA1)、三磷酸腺苷结合盒转运体G1 (ABCG1)、白细胞介素-1β(IL^(-1)β)、肿瘤坏死因子-α(TNF-α)、HMGB1、诱导型一氧化氮合酶(iNOS)、精氨酸酶1(Arg1)、巨噬细胞半乳糖型凝集素-1(Mgl-1)和NF-κB P65的mRNA表达水平,免疫荧光分析确定HMGB1在各组RAW264.7细胞中的定位,后增加自噬抑制剂3-甲基腺嘌呤(3-methyladenine, 3-MA)作为抑制剂对照进行反向验证,即重新将RAW264.7巨噬细胞�The aim of this study was to investigate the underlying mechanism of chrysophanol(Chr)in reducing inflammation and foam cell formation induced by oxidized low-density lipoprotein(ox-LDL)and to investigate the targets and pathways related to effects of Chr on coronary atherosclerosis,providing a theoretical basis for the development of new clinical drugs.RAW264.7 macrophages were cultured in vitro,and after determining the appropriate concentrations of Chr and ox-LDL for treating RAW264.7 macrophages using a cell counting kit-8(CCK-8),the macrophages were treated with different concentrations of Chr(10,15μmol·L^(-1))and ox-LDL(with or without 80 mg·mL^(-1))for 24 h.RAW264.7 macrophages were divided into four groups:control group,model group(80 mg·mL^(-1) ox-LDL),treatment group(80 mg·mL^(-1) ox-LDL+10μmol·L^(-1) Chr),and treatment group(80 mg·mL^(-1) ox-LDL+15μmol·L^(-1) Chr).Lipid accumulation in each group was detected by oil red O staining.CD36 expression was analyzed by flow cytomet ry.Western blot was used to detect the expression of scavenger receptor class A1(SR-A1),scavenger receptor class B type I(SR-B1),autophagy-related protein 5(Atg5),Beclin-1,autophagy adaptor protein p62(P62),the ratio of microtubule-associated protein light chain 3Ⅱto LC3Ⅰ(LC3Ⅱ/LC3Ⅰ),nuclear factor kappa-light-chain-enhancer of activated B cells RelA(NF-κB P65),inhibitor ofκB kinaseβ(IKKβ),nuclear factor ofκB inhibitor(IκB),high mobility group box protein 1(HMGB1),phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),and phosphorylated mammalian target of rapamycin(mTOR).Real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the mRNA expression levels of ATP-binding cassette transporter A1(ABCA1),ATP-binding cassette transporter G1(ABCG1),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),HMGB1,inducible nitric oxide synthase(iNOS),arginase 1(Arg1),macrophage galactose-type lectin-1(Mgl-1),and NF-κB P65.Immunofluorescence analysis was performed to determine the localization of

关 键 词：动脉粥样硬化 大黄酚 炎症 自噬 泡沫细胞形成 NF-κB/HMGB1 PI3K/AKT/MTOR 

分 类 号：R285[医药卫生—中药学]