芹菜素对小鼠RAW264.7巨噬细胞极化和炎症反应的作用及其机制  

Effect of apigenin on polarization and inflammation of mouse RAW264.7 macrophages and its mechanism

作　　者：李海涛[1] 李沁[1] 蔡飞[1] 胡国富[1] 滕云飞[1] LI Haitao;LI Qin;CAI Fei;HU Guofu;TENG Yunfei(Department of Vascular Surgery,Affiliated Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430000,China)

机构地区：[1]华中科技大学同济医学院附属协和医院血管外科,湖北武汉430000

出　　处：《吉林大学学报（医学版）》2023年第3期549-556,共8页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金项目(81900432)。

摘　　要：目的:探讨不同浓度芹菜素(API)对氧化低密度脂蛋白(ox-LDL)诱导的小鼠单核巨噬细胞RAW264.7炎症反应和极化的作用,并阐明其可能机制。方法:将RAW264.7细胞分为RAW264.7组(不做任何处理)和RAW264.7+API组(2、4、8、16和32μmol·L^(-1)API)。药物处理细胞24 h后,CCK-8法检测各组细胞增殖率,选取API实验浓度。将RAW264.7细胞分为RAW264.7组(正常RAW264.7细胞)、RAW264.7+ox-LDL组(0.08 g·L^(-1)ox-LDL诱导24 h)、RAW264.7+ox-LDL+低剂量API组(2μmol·L^(-1)API和0.08 g·L^(-1)ox-LDL诱导24 h)和RAW264.7+ox-LDL+高剂量API组(8μmol·L^(-1)API和0.08 g·L^(-1)ox-LDL诱导24 h),药物处理细胞24 h,采用油红O染色观察各组RAW264.7细胞中泡沫细胞形态表现,酶联免疫吸附测定(ELISA)法检测各组细胞培养上清液中肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素4(IL-4)和白细胞介素10(IL-10)水平,Western blotting法检测各组细胞中核转录因子κB(NF-κB)p65、磷酸化NF-κB(p-NF-κB)p65、诱导型一氧化氮合成酶(iNOS)、信号转导与转录激活因子6(STAT6)、磷酸化STAT6(p-STAT6)和精氨酸酶1(Arg-1)蛋白表达水平。结果:CCK-8法检测,随着API的浓度增加,RAW264.7细胞增殖率降低(P<0.05),选择无毒的低浓度(2μmol·L^(-1))和高浓度(16μmol·L^(-1))API作为后续实验API浓度。油红O染色,RAW264.7组极少数RAW264.7细胞被油红O染色;RAW264.7+ox-LDL组较多细胞被染成暗红色,胞内脂质明显增加,表明成功建立了RAW264.7源性泡沫细胞;RAW264.7+ox-LDL+低剂量API组和RAW264.7+oxLDL+高剂量API组少量细胞被油红O染色。ELISA法检测,与RAW264.7组比较,RAW264.7+ox-LDL组、RAW264.7+ox-LDL+低剂量API组和RAW264.7+ox-LDL+高剂量API组细胞培养上清液中TNF-α和IL-1β水平升高(P<0.05),IL-4和IL-10水平降低(P<0.05);与RAW264.7+oxLDL组比较,RAW264.7+ox-LDL+低剂量API组和RAW264.7+ox-LDL+高剂量API组RAW264.7细胞培养上清液中TNF-α和IL-1β水平降低(P<0.05),IL-4和ILObjective:To discuss the effects of different concentrations of apigenin(API)on the inflammatory and polarization response of the mouse mononuclear macrophage cells RAW264.7 induced by oxidized low-density lipoprotein(ox-LDL),and to clarify their possible mechanisms.Methods:The RAW264.7 cells were divided into RAW264.7 group(without any treatment)and RAW264.7+API group(2,4,8,16 and 32μmol·L^(-1) API).After the cells were treated for 24 h.CCK-8 assay was used to detect the proliferation rates of the cells in various groups.The experimental concentration of API was selected.The RAW264.7 cells were divided into RAW264.7 group(normal RAW264.7 cells),RAW264.7+ox-LDL group(induced with 0.08 g·L^(-1) ox-LDL for 24 h),RAW264.7+ox-LDL+low dose of API group(induced with 2μmol·L^(-1) API and 0.08 g·L^(-1) ox-LDL for 24 h)and RAW264.7+ox-LDL+high dose of API group.Oil red O staining was used to observe the morphology of the foam cells in various groups.The levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-4(IL-4),and interleukin-10(IL-10)in the cell culture supernant were detected by enzyme-linked immunosorbent assay(ELISA)method.The expression levels of neclear factor-κB(NF-κB)p65,phosphorylated NF-κB(p-NF-κB)p65,inducible nitric oxide synthase(iNOS),signal transducer and activator of transcription 6(STAT6),phosphorylated STAT-6(p-STAT6),and arginase-1(Arg-1)in the cells in various groups were detected by Western blotting method.Results:The results of CCK-8 assay showed that the proliferation rates of the cells were significantly decreased with the increasing of the concentrations of API(P<0.05).The non-toxic low concentration(2μmol·L^(-1))and high concentration(8μmol·L^(-1))of API were selected to use in the follow-up experiment.The Oil red O staining results showed that few RAW264.7 cells were stained with Oil red O;a lot of RAW264.7 cells in RAW264.7+ox-LDL group were stained as dark red,and the lipids in the cells were increased,indicating the foam cell model was successfully e

关 键 词：芹菜素 动脉粥样硬化 核因子ΚB 信号转导与转录激活因子6 巨噬细胞极化 

分 类 号：R543.5[医药卫生—心血管疾病]