祛痰、化瘀和祛痰化瘀方对apoE^(-/-)小鼠全身和主动脉血管局部免疫反应影响的对比研究

Comparative Study on the Effects of Eliminating Phlegm Recipe,Removing Blood Stasis Recipe,Removing Phlegm and Blood Stasis Recipe on the Systemic and Local Immune Response in Aortic Blood Vessel of ApoE-/- Mice

目的:观察祛痰方、化瘀方和祛痰化瘀方对高脂饮食导致的apo E^(-/-)小鼠全身和主动脉血管局部免疫反应的影响。方法:12周龄雄性apo E^(-/-)小鼠给予高脂饮食,治疗组同时给予基础方、祛痰方、化瘀方或祛痰化瘀方干预。4周后,检测血脂水平、外周血粒、单核细胞比例及其表面受体TLR4的表达;检测主动脉病理变化和细胞因子表达水平;进一步采用LPS刺激检测血浆细胞因子的水平。结果:4周高脂饮食导致apo E^(-/-)小鼠外周血TC和LDL水平显著增高;粒和单核细胞比例及其TLR4的表达显著升高;LPS刺激后,IL-12、TNF-α和MCP-1水平显著升高;主动脉组织不仅MCP-1、IL-1β和IL-6表达显著升高,IL-10也显著升高,Arg-1显著降低。4周的中药干预并未引起血脂明显改善,但祛痰化瘀方降低外周血粒细胞比例;对于LPS刺激高脂饲喂apo E-/-小鼠,基础方和活血化瘀方降低IL-12表达水平;对于主动脉血管局部,基础方降低主动脉血管IL-1β和IL-6表达,提高Arg-1表达;祛痰方显著抑制MCP-1、IL-1β和IL-6表达,而对抑炎因子无明显作用;活血化瘀方降低MCP-1、IL-1β和IL-6表达,提高IL-10和Arg-1表达;祛痰化瘀方仅抑制IL-1β表达,但提高IL-10、TGF-β和Arg-1表达。结论:高脂饮食引发apo E^(-/-)小鼠全身性炎症反应和血管局部炎症反应。中药祛痰方、化瘀方及祛痰化瘀方通过不同免疫调节机制,以独立于降脂作用的方式调节高脂血症引发的血管局部免疫反应。

Objective： To observe and compare the effect of Qutang-Fang,Huayu-Fang and Qutang-Huayu-Fang on apo E-deficient mice with Western diet（ WD） by modulating systemic and aortic immune responses. Methods： In the present study,12 week-old homozygous male apo E-（-/-）mice were fed with a normal chow diet,a high cholesterol WD and WD supplemented with Basic-Fang,Qutang-Fang,Huayu-Fang and Qutang-Huayu-Fang. After 4 weeks,the proportions of granulocyte and monocyte,and their surface receptor TLR4 of peripheral blood were detected by flow cytometry. Serum cytokines stimulated by LPS for 3 hours were detected by cytometric bead array（ CBA） mouse inflammation kit. The pathogenesis of aorta was detected by HE staining,and cytokines in aorta were detected by real-time PCR. Results： 4-week WD increased the levels of TC and LDL in apo E-（-/-）mice. Hyperlipidemia increased the proportions of granulocyte and monocyte,and expression of their surface receptor TLR4 in apo E-（-/-）mice; and induced serum IL-12,TNF-α and MCP-1levels after LPS stimulation for 3 hours. In aorta,4-week WD induced atherosclerosis lesions of aorta in apo E-（-/-）mice,increased expressions of cytokines MCP-1, IL-1β, IL-6, IL-10, and decreased Arg-1 expression. After 4 weeks admistration of Basic-Fang,Qutang-Fang,Huayu-Fang or Qutang-Huayu-Fang on Apo E-（-/-）mice with WD,these medicines did not change the serum lipid levels significantly. Qutang-Huayu-Fang decreased the proportions of granulocyte. BasicFang and Huayu-Fang lowered serum IL-12 level in respond to LPS challenge. In aorta,basic-Fang decreased expressions of cytokines IL-1β and IL-6,increased Arg-1; Qutang-Fang dramatically lowered the expressions of inflammatory cytokines MCP-1,IL-1β and IL-6,but did not affect the expressions of anti-inflammatory cytokines; Huayu-Fang lowered the expressions of inflammatory cytokines MCP-1,IL-1β and IL-6,and a ugmented anti-inflammatory cytokines IL-10 and Arg-1; Qutang-Huayu-Fang just decreased inflammatory cytokine IL-1β,but significantly increased the expressions of antiinflammatory cytokines IL-10、TGF-β and Arg-1. Conclusion： Hyperlipidemia induced systemic and aortic inflammatory disproportions in apo E-（-/-）mice. Qutang-Fang,Huayu-Fang and Qutang-Huayu-Fang can modulate these inflammatory disproportions by different mechanisms and in a lipids-lower-independent manner.