摘要
考察清热解毒药对白花蛇舌草-半枝莲的组分(YDW11)对巨噬细胞表型M1/M2的调控及其机制。利用脂多糖(lipopolysaceharides,LPS)/干扰素-γ(interferon-γ,IFN-γ)和白介素4(interleukin 4,IL-4)/白介素13(interleukin 13,IL-13)分别诱导鼠源巨噬细胞RAW264.7为M1和M2表型,MTT法检测YDW11对细胞活力的影响;Griess反应法检测细胞上清液中亚硝酸盐含量的变化;Trans-well小室迁移实验考察M2型巨噬细胞对乳腺癌细胞4T1的体外迁移及YDW11干预的影响;qRT-PCR法检测YDW11对细胞内诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS)、白介素1β(interleukin1β,IL-1β)、精氨酸酶1(arginase-1,Arg-1)、甘露糖受体(mannose receptor,MR)基因表达的影响;Western blot法检测YDW11对i NOS和Arg-1蛋白表达的影响;Taqman microRNA RT-PCR法检测YDW11对miR155表达的影响。质谱(MS)联用超高效液相色谱(UPLC)检测YDW11中的化学成分。结果显示,等比水提取白花蛇舌草-半枝莲药对的乙酸乙酯组分(YDW11)呈剂量依赖性抑制巨噬细胞M1表型中亚硝酸盐含量,同时抑制M2型巨噬细胞引起的乳腺癌细胞4T1体外迁移加剧,且对未刺激的巨噬细胞M0无细胞毒性。YDW11呈剂量依赖性抑制M1表型标记物i NOS和M2表型标记物Arg-1基因及蛋白的表达,抑制M1表型中IL-1β和M2表型中MR的基因表达,调控M1表型上升而M2表型下调的miR155的表达。MS-UPLC联用结果显示,YDW11中含有对羟基苯乙酮、野黄芩苷、木犀草素和芹菜素4种化学成分。结果表明,YDW11部分通过调控miR155的表达,抑制巨噬细胞M1和M2表型标志性产物i NOS和Arg-1的基因和蛋白表达,而调控巨噬细胞M1/M2表型的极化过程。
To explore the regulatory effect and relevant mechanisms of the fraction of Hedyotis diffusa and Scutellaria barbata herb couple( YDW11) on polarization of macrophage between M1/M2 phenotypes. RAW264. 7 cells were induced with LPS/IFN-γ or IL-4/IL-13 to establish M1 or M2 macrophage cell model. MTT assay was used to measure the cell cytotoxicity of YDW11. Griess reaction was used to detect the changes of nitrite accumulation in the cell supernatant. Trans-well assay was used to measure the migration capability. QRT-PCR was used to assay mRNA expressions of i NOS,IL-1β,Arg-1 and MR. Western blot was used to detect the effect of YDW11 on i NOS and Arg-1 protein expressions. Taqman MicroRNA RT-PCR was used to detect the effect of YDW11 on miR155 expression under M1 and M2 phenotype conditions. In addition,MS-UPLC assay was carried out to identify the constituents in YDW11.The results showed that the ethyl acetate of H. diffusa and S. barbata extracted in 1∶ 1 ratio with water( YDW11) showed the activity in suppressing the nitrite content in M1 macrophages without cytotoxicity. YDW11 also inhibited the migration of breast cancer cells with the help of M2 macrophages by blocking their polarization towards M2. YDW11 decreased i NOS,IL-1β,Arg-1 and MR mRNA expressions and i NOS and Arg-1 protein expressions. YDW11 down-regulated miR155 expression in M1 phenotype,and up-regulated miR155 expression in M2 phenotype. Based on MS-UPLC,four compounds were identified in YDW11,including 4’-hydroxyacetophenone,scutellarin,luteolin and apigenin. YDW11 inhibited M1/M2 phenotypes of macrophages by regulating the expression of miR155.
作者
徐元
陈龙
蒋义鑫
杨月
章丹丹
XU Yuan;CHEN Long;JIANG Yi-xin;YANG Yue;ZHANG Dan-dan(Institute of Interdisciplinary Integrative Medicine Research,Shanghai University of Traditional Chinese Medicine, Shanghai 201203,China;Laboratory of Analysis and Test,Experiment Center of Science and Technology, Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2018年第18期3722-3728,共7页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81773946,81573673,81001666)
上海卫计委青年项目(20144Y0143)
上海教委创新项目(13YZ048)
上海教委优青项目(SZY07029)
