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目的观察黄连解毒汤体内调控载脂蛋白E基因敲除(ApoE~(-/-))小鼠外周血单核细胞亚型分化,黄连解毒汤含药血清体外调控巨噬细胞和泡沫细胞亚型分化的作用。方法将15只ApoE~(-/-)小鼠随机分为ApoE~(-/-)普食组、ApoE~(-/-)高脂组及ApoE~(-/-)高脂加中药治疗组,每组5只,分别给予普食及高脂饮食4周。5只C57BL/6野生型小鼠作为野生普食对照组,ApoE~(-/-)高脂加中药组给予黄连解毒汤5 g/(kg·d)灌胃,其他组给予等量纯净水灌胃。4周后流式细胞仪检测外周血单核细胞亚型。另选30只SD大鼠给予黄连解毒汤5 g/(kg·d)灌胃,每12 h 1次,共5次,制备黄连解毒汤含药血清。采用5%黄连解毒汤含药血清对体外原代骨髓衍生的巨噬细胞(bone marroW-derived macrophage,BMDM)和泡沫细胞分化进行干预,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 ApoE~(-/-)小鼠高脂饮食喂养4周后,外周血炎症型单核细胞(Ly6C~(high))比例增高;黄连解毒汤干预可显著降低炎症型单核细胞比例(P<0.05)。与对照血清比较,5%黄连解毒汤含药血清可显著增加CD206+M2型BMDM的比例(P=0.034)。实时定量PCR结果 显示,含药血清可上调巨噬细胞M2亚型基因Arg1的表达水平(P<0.05),抑制巨噬细胞M1亚型基因Nos2的表达水平(P=0.017)。在ox-LDL促进M2型泡沫细胞分化的基础上,黄连解毒汤含药血清可进一步提高CD206+M2型泡沫细胞比例及Arg1的表达(P<0.01)。黄连解毒汤含药血清抑制Th1因子作用下的M2型泡沫细胞向M1型逆转,显著提高Arg1表达水平,降低Nos2表达水平(P<0.01)。结论黄连解毒汤能降低高脂导致的ApoE~(-/-)小鼠外周血炎症性单核细胞亚群比例;黄连解毒汤含药血清体外促进M2型巨噬、泡沫细胞分化。黄连解毒汤可能通过调节单核、巨噬、泡沫细胞的功能性分化,从而减缓、抑制高脂血症引发的AS的发生、发展。
Objective To observe the differentiation of peripheral blood mononuclear cells (PBMCs) of Peripheral blood mononuclear cells (PBMCs) treated with Huanglian Jiedu Decoction in vivo and the serum containing Huanglian Jiedu Decoction The role of differentiation. Methods Fifteen ApoE ~ (- / -) mice were randomly divided into ApoE ~ (- / -) diet group, ApoE ~ (- / -) high fat diet group and ApoE ~ (- Group, with 5 rats in each group, which were fed with normal diet and high-fat diet for 4 weeks respectively. Five C57BL / 6 wild-type mice were used as wild food control group. ApoE ~ (- / -) high-fat plus Chinese herb group was given Huanglian Jiedu Decoction 5g / (kg · d) Gavage. After 4 weeks, peripheral blood mononuclear cells were detected by flow cytometry. 30 SD rats were given Huanglian Jiedu Decoction 5 g / (kg · d) intragastrically, once every 12 h, a total of 5 times, preparation Huanglian Jiedu Decoction serum. Intervention was made with 5% Huanglian Jiedu Decoction-containing serum on differentiation of primary bone marrow-derived macrophage (BMDM) and foam cells in vitro. Flow cytometry was used to detect macrophage and foam cell surface receptor CD206 (Type of CD206 + M2). The expression of Nos2 and Arg1 in macrophages and foam cells were detected by real-time quantitative PCR. Results The ratio of Ly6C ~ (high) in peripheral blood mononuclear cells increased after 4 weeks of high-fat diet in ApoE ~ (- / -) mice. The treatment of Huanglian Jiedu Decoction could significantly reduce the proportion of inflammatory monocytes <0.05). Compared with the control serum, 5% Huanglian Jiedu Decoction serum could significantly increase the proportion of CD206 + M2 type BMDM (P = 0.034). The results of real-time PCR showed that the drug-containing serum could up-regulate the expression of Arg1 (P <0.05) and inhibit the expression of Nos2 in macrophage M1 subtype (P = 0.017). On the basis of ox-LDL promoted the differentiation of M2 foam cells, Huanglian Jiedu decoction containing serum could further increase the proportion of CD206 + M2 foam cells and the expression of Arg1 (P <0.01). The Huanglian Jiedu Decoction-containing serum inhibited the M2-type foam cells under the action of Th1 factor to reverse M1, significantly increasing Arg1 expression and decreasing Nos2 expression (P <0.01). Conclusions Huanglian Jiedu Decoction can reduce the proportion of inflammatory monocyte subsets in peripheral blood of ApoE ~ (- / -) mice induced by high fat. Huanglian Jiedu Decoction serum can promote the differentiation of M2 macrophages and foam cells in vitro. Huanglian Jiedu Decoction may regulate mononuclear, macrophage, foam cell functional differentiation, thereby slowing and inhibiting hyperlipidemia-induced AS occurrence and development.