黄连解毒汤对高脂饮食apoE^(-/-)小鼠全身和主动脉血管局部免疫反应影响的研究

Protection of Huanglian Jiedu Decoction on Systemic and Vascular Immune Responses of High Fat Induced apoE^(- /-) Mice

目的观察黄连解毒汤对高脂饮食喂养的apoE基因敲除(apoE-/-)小鼠全身和主动脉血管局部免疫反应的影响。方法以8只野生型C57BL6小鼠为野生普食组,24只apoE-/-小鼠随机分为apoE-/-普食组、apoE-/-高脂组、apoE-/-高脂加中药组,每组8只。普食组与高脂组分别给予普食和高脂饮食4周。ApoE-/-高脂加中药组同时给予黄连解毒汤5 g/(kg·d)灌胃,其他小鼠采用等量纯净水灌胃。4周后,检测血脂水平、外周血单核细胞比例及其表面Toll样受体4(Toll-like receptor 4,TLR4)和清道夫受体CD36的表达;检测主动脉病理变化和主动脉血管局部细胞因子表达水平;进一步采用脂多糖(Lipopolysaccharide,LPS)刺激检测血浆细胞因子的水平。结果 (1)与野生普食组比较,apoE-/-普食组TC、TG和LDL-C水平显著升高(P<0.05,P<0.01);与apoE-/-普食组比较,apoE-/-高脂组TC和LDL-C水平显著增高(P<0.05);apoE-/-高脂组与apoE-/-高脂加中药组血脂各项指标比较,差异无统计学意义(P>0.05)。(2)与野生普食组比较,apoE-/-普食组外周血单核细胞比例无明显变化,TLR4表达水平显著增多(P<0.05);与apoE-/-普食组比较,apoE-/-高脂组小鼠外周血单核细胞比例及TLR4表达水平显著增多(P<0.05);与apoE-/-高脂组比较,apoE-/-高脂加中药组外周血单核细胞比例及其表面TLR4表达显著降低,清道夫受体CD36表达水平亦显著降低(P<0.05)。(3)LPS刺激3 h后,与野生普食组比较,apoE-/-普食组血浆TNF-α和IL-10表达显著增加(P<0.05);与apoE-/-普食组比较,apoE-/-高脂组小鼠血浆IL-12、TNF-α、MCP-1和IL-10表达增加,但差异无统计学意义(P>0.05);黄连解毒汤干预4周后,与apoE-/-高脂组比较,apoE-/-高脂加中药组MCP-1表达显著下调,而IL-10水平进一步显著上升,差异有统计学意义(P<0.05)。(4)与野生普食组比较,apoE-/-普食组主动脉血管壁炎症因子IFN-γ、MCP-1、TNF-α和IL-1βmRNA水平显著升高,抑炎因子IL-10亦显著升高(P<0.05,P<0.01);与apoE-/-普食组比较,apoE-/-高脂组主动脉血管壁不仅IFN-γ、MCP-1、TNF-α和IL-1β水平进一步显著升高,且抑炎因子IL-12、IL-10和TGF-β亦显著升高(P<0.05,P<0.01);黄连解毒汤干预4周后,与apoE-/-高脂组比较,apoE-/-高脂加中药组主动脉血管壁炎症因子MCP-1、TNF-α、IL-1β和IL-12显著降低(P<0.05,P<0.01)。结论高脂饮食引发apoE-/-小鼠全身性炎症反应和血管局部炎症反应,血管局部炎症反应程度超过全身性炎症反应。黄连解毒汤干预后能够减轻炎症反应,并且对血管局部的作用更为显著,同时增强全身抗炎反应。

Objective To observe the effect of Huanglian Jiedu Decoction （HJD） on systemic and vascular immune responses of high fat diet fed apoE deficient （apoE-/-） mice. Methods Eight wild type C57BL6 mice were recruited as the wild type common food group. Totally 24 apoE-/- mice were ran- domly divided into the ApoE-/-common food group, the ApoE-/- hyperlipidemia group, and the ApoE-/- hyperlipidemia plus HJD group, 8 in each group. In the present study, the common food mice and high fat fed mice were fed with a chow diet or a high cholesterol diet for 4 weeks. HJD was given to mice in the ApoE-/- hyperlipidemia plus HJD group at the daily dose of 5 g/kg by gastrogavage, while equal volume of pure water was given to mice in the rest groups by gastrogavage. Four weeks later, the plasma levels of blood lipids, the ratio of peripheral blood mononuclear cells, and expressions of Toll-like receptor 4 （TLR-4） and CD36 on the monocytes were detected. The pathological changes and expressions of cyto- kines in local aorta were detected. The plasma cytokine levels in response to lipopolysaccharide （LPS） were analyzed. Results （1） Compared with the wild type common food group, TC, TG, and LDL-C sig- nificantly increased in the ApoE-/-common food group （P 〈0.05,P 〈0.01 ）. Compared with the ApoE-/- common food group, TC and LDL-C significantly increased in the hyperlipidemia group （P 〈0.05）. There was no statistical difference in each index between the ApoE-/- hyperlipidemia group and the ApoE-/- hy- perlipidemia plus HJD group （P〉0.05）. （2） Compared with the wild type common food group, no obvi- ous change of the ratio of peripheral blood mononuclear cells happened, the TLR4 expression level signif- icantly increased in the ApoE-/-common food group （P 〈0.05）. Compared with the ApoE-/-common food group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly in- creased in the ApoE-/- hyperlipidemia group （P 〈 0.05）. Compared with the ApoE-/- hyperlipidemia group, the ratio of peripheral blood mononuclear cells and the TLR4 expression level significantly de- creased. Besides, the CD36 expression level also significantly decreased （P 〈0.05）. （3） After stimula- ted by LPS for 3 h, compared with the wild type common food group, plasma TNF-α and IL-10 expres- sions significantly increased in the ApoE-/-common food group （P 〈 0.05 ）. Compared with the ApoE-/- common food group, plasma expressions of IL-12, TNF-α, MCP-1, and IL-10 increased, but with no sta- tistical difference in the ApoE-/- hyperlipidemia group （P 〉0.05）. After 4-week intervention of HJD, com- pared with the ApoE-/- hyperlipidemia group, the MCP-1 expression was significantly down-regulated, while the IL-10 expression significantly increased, showing statistical difference （P 〈0.05）. Compared with the wild type common food group, mRNA expression levels of IFN-α,MCP-1, TNF-α, IL-10, and IL-1β significantly increased （P 〈0.05,P 〈0.01 ）. Compared with the ApoE-/- common food group, not only mRNA expression levels of IFN-α, MCP-1, TNF-α, and IL-1β, further significantly increased, but also IL- 12, IL-10, and TGF-β significantly increased （P 〈0.05 ,P 〈0.01 ）. After 4-week intervention of HJD, com- pared with the ApoE-/- hyperlipidemia group, mRNA expression levels of MCP-1, TNF-α, IL-1 β, and IL-12 significantly decreased in the ApoE-/- hyperlipidemia plus HJD group （P 〈0.05 ,P 〈0.01 ）. Conclusions High fat diet induced systemic reaction and inflammatory reactions of local vessels. The local inflammato- ry response of vessels exceeded systemic inflammatory response. Intervention of HJD could attenuate inflammatory response, especially in local arteries. Meanwhile, it enhanced systemic anti-inflammatory reactions.