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泻心汤含药血清对RAW264.7源性泡沫细胞TLR9/MyD88/NF-κB p65信号通路的影响 被引量:6

Effect of Xiexintang Containing Serum on Expression of TLR9/MyD88/NF-κB p65 Signal Pathway in Foam Cell
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摘要 目的:通过体外细胞实验研究泻心汤对巨噬细胞源性泡沫细胞Toll样受体9(TLR9)信号通路的影响,探讨泻心汤抗动脉粥样硬化的作用机制。方法:50只SPF级雄性SD大鼠随机分为泻心汤低、中、高剂量组(1. 4,4. 2,12. 6 g·kg-1·d-1)及正常组,除正常组20只外,其余每组10只,正常组给予等体积的纯水灌胃,各组连续灌胃7 d,末次给药1 h后,分离血清,制备泻心汤低、中、高剂量组含药血清及正常组血清。用氧化低密度脂蛋白(ox-LDL)干预RAW264. 7巨噬细胞分化为泡沫细胞。采用油红O染色法鉴定细胞泡沫化;噻唑蓝(MTT)比色法观察含药血清对巨噬细胞源性泡沫细胞增殖的影响后,选择各剂量浓度为20%的含药血清作用于泡沫细胞模型,采用酶联免疫吸附测定(ELISA)检测白细胞介素-1β(IL-1β),γ-干扰素(INF-γ)的含量;实时荧光定量PCR(Real-time PCR)及蛋白免疫印迹法(Western blot)分别测定TLR9,髓样分化因子88(MyD88),核转录因子-κB(NF-κB) mRNA及蛋白表达。结果:油红O染色显示出ox-LDL干预后细胞内红色颗粒明显,泡沫细胞模型制备成功;MTT比色法结果显示,与正常组血清比较,泻心汤高剂量组含药血清在10%~30%浓度范围内,其细胞增殖差异无显著性。后续选择各剂量浓度为20%的含药血清干预经ox-LDL诱导的泡沫细胞,与正常组比较,模型组用ox-LDL干预后诱导了TLR9,MyD88,NF-κB p65,IL-1β,INF-γ的高表达(P <0. 05,P <0. 01);与模型组比较,不同剂量组泻心汤含药血清干预后降低了TLR9,MyD88,NF-κB p65,IL-1β,INF-γ的表达(P <0. 05,P <0. 01),且部分干预作用呈现出剂量依赖性。结论:泻心汤含药血清可抑制泡沫细胞TLR9/MyD88/NF-κB p65通路以及促炎因子IL-1β,INF-γ的转录和过表达,这可能是泻心汤抗动脉粥样硬化重要作用机制之一。 Objective:The effect of Xiexintang on Toll like receptor 9(TLR9)signaling pathway in macrophage derived foam cells was studied by in vitro cell experiments.Method:The fifty SPF male SD rats were randomly divided into low,medium and high dose groups(1.4,4.2,12.6 g·kg-1·d-1)and normal groups.Except 20 rats in the normal group,10 rats in each group were given equal volume of pure water gavage in the blank group.After the last Administration for 7 days,serum was separated,and the serum containing drugs in the low,medium and high dose groups of Xiexintang was prepared.Oxidized low density lipoprotein(ox-LDL)was used to intervene the differentiation of RAW264.7 macrophages into foam cells.The cell foam was identified by oil red O staining.After observing the effect of drug containing serum on the proliferation of macrophage derived foam cells by methye thiazolye telrazlium(MTT)method,the serum containing 20%concentration of each drug was selected to act on the foam cell model.The expression of interleukin(IL)-1βand interferon(INF)-γwas determined by enzyme-linked immunosorbent assay(ELISA)and real-time fluorescence quantitative PCR(Realtime PCR).TLR9,myeloid differentiation factor 88(MyD88)and nuclear factor(NF)-κB were detected by Western blot.Result:Oil red O staining showed that the red particles were obvious after ox-LDL intervention.The foam cell model was successfully prepared.MTT results showed that there was no significant difference in cell proliferation between the high dose group of Xiexintang in the 10%~30%concentration range and the normal group serum.Follow up selection of the serum containing 20%concentration of each dose intervened the foam cells induced by ox-LDL.Compared with the normal group,the model group after ox-LDL intervention induced the high expression of TLR9,My D88,NF-κB p65,IL-1β,INF-γ(P<0.05,P<0.01).Compared with model group,the serum of Xiexintang containing different dosage groups reduced the expression of TLR9,MyD88,NF-κB p65,IL-1β,INF-γ(P<0.05,P<0.01),and part of the interv
作者 于红红 俞琦 蔡琨 陈茜 盛蒙 许丽婷 田维毅 YU Hong-hong;YU Qi;CAI Kun;CHEN Xi;SHENG Meng;XU Li-ting;TIAN Wei-yi(Guizhou University of Traditional Chinese Medicine,Guiyang 550002,China)
机构地区 贵州中医药大学
出处 《中国实验方剂学杂志》 CAS CSCD 北大核心 2019年第18期24-29,共6页 Chinese Journal of Experimental Traditional Medical Formulae
基金 国家自然科学基金项目(81860779,81860776) 贵州省国内一流建设学科项目(GNYL[2017]008号) 贵州省普通高等学校工程研究中心项目(黔教合KY字[2015]337号)
关键词 泻心汤 泡沫细胞 TOLL样受体9 动脉粥样硬化 氧化低密度脂蛋白 Xiexintang foam cells Toll like receptor 9 atherosclerosis oxidized low density lipoprotein
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