祛痰方和祛痰化瘀方调控单核-巨噬细胞分化抗动脉粥样硬化的作用研究

Effects of Eliminating Phlegm Prescription and Eliminating Phlegm and Removing Blood Stasis Prescription on Regulating the Monocyte Macrophage Differentiation And Anti Atherosclerosis

目的:观察和比较祛痰方和祛痰化瘀方调控Apo E-/-小鼠外周血单核细胞亚型分化,祛痰方和祛痰化瘀方含药血清体外调控巨噬细胞和泡沫细胞亚型分化,探讨其抗动脉粥样硬化的作用。方法:采用12周龄Apo E-/-雄性小鼠,模型组给予高脂饮食,治疗组同时给予祛痰方和祛痰化瘀方干预,4周后流式细胞仪检测外周血单核细胞亚型。采用祛痰方和祛痰化瘀方含药血清干预原代培养的巨噬细胞和泡沫细胞分化,流式细胞仪检测巨噬细胞和泡沫细胞表面受体CD206表达(CD206+M2型)比例;实时定量PCR检测巨噬细胞和泡沫细胞M1型因子Nos2和M2型因子Arg1的表达。结果:12周龄Apo E-/-小鼠高脂喂养4周后,外周血炎症型单核细胞(Ly6Chi)的比例增高,定居型单核细胞(Ly6Clo)的比例降低;祛痰方具有降低Ly6Chi和增高Ly6Clo比例的趋势;祛痰化瘀方干预显著降低Ly6Chi的比例,提高Ly6Clo的比例。祛痰方含药血清增加巨噬细胞Arg1的表达,但仅轻度提高M2型巨噬细胞比例;祛痰化瘀方含药血清可提高M2型巨噬细胞比例和Arg1的表达,抑制Nos2的表达,促进单核细胞向M2型巨噬细胞分化。ox-LDL促进M2型泡沫细胞分化;祛痰方含药血清仅轻度提高M2型泡沫细胞比例;祛痰化瘀方含药血清进一步提高M2型泡沫细胞比例,但没有明显改善Arg1和Nos2的表达。结论:祛痰方调节单核-巨噬-泡沫细胞分化作用不明显,而祛痰化瘀方通过调节单核、巨噬、泡沫细胞的功能性分化,可能是祛痰化瘀方减缓、抑制As发生发展的重要作用环节之一。

Objective： To observe and compare the effects of Qutan-Fang and Qutan-Huayu-Fang on regulating the differentiation of monocytes in an apolipoprotein E knockout（Apo E-/-） mouse model,and Qutan-Fang-containing serum and Qutan-Huayu-Fang-containing serum on differentiation of macrophages and foam cells in vitro. Methods： In the present study,12 weeks old male homozygous Apo E-/-mice were fed with a normal chow diet,a high cholesterol Western diet（WD） and WD supplemented with Qutan-Fang and Qutan-Huayu-Fang. After 4 weeks,the subtypes of monocytes in peripheral blood were detected by FACS. Bone marrow cells were differentiated to bone marrow-derived macrophages（BMDM） with Qutan-Fang-containing serum or Qutan-Huayu-Fang-containing serum for 7 days; BMDM were stimulated by ox-LDL to form foam cells with or without Qutan-Fang-containing serum and Qutan-Huayu-Fang-containing serum,the M2 subtype surface receptor CD206 of macrophages and foam cells were detected by FACS,the genes Nos2 and Arg1 were assayed by Real-time PCR. Results： Hypercholesterolaemia increased the ratio of inflammatory subset of monocytes（Ly6hi）and decreased the ratio of resident subset of monocytes（Ly6Clo） in peripheral blood in 12 weeks apo E-/-mice with Western diet for another 4 weeks. Qutan-Huayu-Fang decreased the ratio of Ly6 Chiand increased the ratio of Ly6 Clo. In vitro,Qutan-Huayu-Fang-containing serum increased the differentiation of M2 macrophages from bone marrow cells. QutanHuayu-Fang-containing serum induced mRNA expression of Arg1 and inhibited mRNA expression of Nos2 of these cells.Qutan-Fang-containing serum induced mRNA expression of Arg1,but it had no significant effect on the differentiation of M2 subtype of BMDM. Ox-LDL induced M2 subtype of foam cells from BMDM,and Qutan-Huayu-Fang-containing serum increased differentiation of M2 subtype of foam cells from BMDM. Qutan-Fang-containing serum had no significant effect on the differentiation of M2 subtype of foam cells. Conclusion： Hypercholesterolaemia and atherosclerosis are associated with abnormal differentiation of monocytes,macrophages and foam cells. Qutan-Huayu-Fang can inhibit atherosclerosis by regulating the differentiation of monocytes,macrophages and foam cells. Qutan-Fang has significant effect on regulating the differentiation of monocytes,macrophages and foam cells.