黄连解毒汤调控单核、巨噬细胞及泡沫细胞分化的实验研究

Huanglian Jiedu Decoction Regulated and Controlled Differentiation of Monocytes,Macrophages,and Foam Cells:an Experimental Study

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 effect of Huanglian Jiedu Decoction（HLJDD） in in vivo regulating differentiation of monocytes in an apolipoprotein E knockout（ApoE^-/-） mouse model,and to observe the effect of HLJDD-containing serum in in vitro regulating differentiation of macrophages and foam cells.Methods Fifteen apoE^-/-mice were randomly divided into the common diet group,the hyperlipidemia group,and the hyperlipidemia ＋ HLJDD treatment group,5 in each group.Mice in the common diet group were fed with a chow diet.Mice in the hyperlipidemia group were fed with high cholesterol wild diet（WD）.Those in the hyperlipidemia ＋HLJDD treatment group were fed with high cholesterol WD supplemented with HLJDD.All mice were fed for 4 weeks.Five C57BLJ5 wild types were recruited as the wild common diet control group.HLJDD was administered to mice in the hyperlipidemia ＋ HLJDD treatment group by gastrogavage at the daily dose of 5 g/kg.Equal volume of purified water was given by gastrogavage to mice in the rest 3 groups.Four weeks later,subtypes of monocytes in the peripheral blood were detected by FACS.HLJDD administered to another 30 SD rats by gastrogavage at the daily dose of 5 g/kg,once for every 12 h for 5 times in total,thereby preparing 5%HLJDD containing serum to intervene the differentiation of in vitro primary bone marrow-derived macrophage（BMDM） and foam cells.The M2 subtype surface receptor CD206 of macrophages and foam cells were detected by FACS.The expression of Nos2 and Arg1 genes were assayed by Real-time PCR.Results The ratio of inflammatory subset of monocytes（Ly6C^high） increased in the peripheral blood after ApoE^-/-mice were fed with high fat diet for 4weeks.HLJDD significantly decreased the ratio of inflammatory subset of monocytes（P〈0.05）.Compared with the vehicle serum,5%HLJDD containing serum significantly increased differentiation of CD206^＋ M2 BMDM（P =0.034）.Results of real-time quantitative PCR showed that the expression level of Arg1 mRNA could be up-regulated by HLJDD containing serum（P〈0.05）,and that of Nos2 mRNA down-regulated（P = 0.017）.ox-LDL induced the differentiation of M2 subtype foam cells from BMDM,and HLJDD containing serum could further elevate the ratio of CD206~＋ M2 foam cells and increase the Arg1 mRNA expression level（both P〈0.01）.HLJDD containing serum could inhibit the inversion of M2 subtype of foam cells to M1 subtype induced by Th1 factors,significantly elevate the Arg1 mRNA expression level,and decrease the Nos2 mRNA expression level（all P〈0.01）.Conclusions HLJDD could lower hyperlipidemia induced inflammatory monocyte subtype ratios in the peripheral blood of ApoE^-/-mice.HLJDD containing serum promoted in vitro differentiation of M2 macrophages and foam cells.HLJDD attenuated and inhibited the occurrence and development of atherosclerosis induced by hyperlipidemia possibly through regulating the functional differentiation of monocytes,macrophages,and foam cells.