抑制Toll样受体4对apoE^(-/-)小鼠动脉粥样硬化病变的影响

Effect of Inhibition of Toll-like Receptor 4 on The Development of Atherosclerosis in Apolipoprotein E Knockout (apoE^(-/-)) Mice

通过Toll样受体4(TLR4)抑制剂表没食子儿茶素没食子酸酯(EGCG)对TLR4途径的抑制,研究apoE-/-小鼠TLR4及多种炎症因子的表达和动脉粥样硬化病变程度的改变,以探讨TLR4途径在动脉粥样硬化病变发生中的作用.5周龄雄性apoE-/-小鼠50只,随机分成4组:基础饮食组对照组(n=12)、高脂饮食组对照组(n=12)、基础饮食+EGCG组(n=13)、高脂饮食+EGCG组(n=13).给药14周后处死动物,从主动脉根部连续冰冻切片,油红O染色观察主动脉窦处动脉粥样硬化(As)斑块面积,定量分析主动脉粥样硬化斑块大小及占管腔的面积百分比,采用Realtime-PCR检测主动脉TLR4mRNA和CD14mRNA的表达,蛋白质印迹检测TLR4和CD14蛋白表达,ELISA检测小鼠血清中单核细胞趋化蛋白-1(MCP-1),肿瘤坏死因子-α(TNF-α)浓度.研究结果提示:EGCG显著减轻apoE-/-主动脉窦部的动脉粥样硬化病变,高脂对照组的主动脉窦AS斑块面积为(2.37±0.08)mm2,高脂饲料+EGCG组的主动脉窦AS斑块的面积为(1.05±0.13)mm2,EGCG组小鼠主动脉窦粥样斑块面积比相应对照组明显减少(P<0.05),高脂饮食+EGCG组小鼠TLR4蛋白表达显著降低(P<0.05),MCP-1,TNF-α的含量减少,与高脂饮食对照组相比差异有显著性(P<0.05).TLR4信号转导途径在高脂所致的AS发生当中有着重要作用,该信号途径的激活至少是AS发生当中的一个重要环节.

It is known that （-）-Epigallocatechin-3-gallate （EGCG） is the inhibitor of TLR4 signaling pathway activation. To investigate a possible role of TLR4 signaling pathway in the development of atherosclerosis, the effects of EGCG on the development of atherosclerosis, the expression of TLR4 and inflammatory cytokine production in apoE^-/- mice was investigated. Fifty male apoE^-/- mice （5wk old） were divided into four groups： basic diet group （control group）, high-fat diet group （control group）, EGCG ＋ basic diet group （EGCG group）, and high-fat diet ＋ EGCG group （EGCG group）. EGCG （10 mg/kg） was injected intraperitoneally every day. Areas of aortic plaque areas were measured by oil red O staining. Western blot was used to detect the expression of TLR4 and CD14 in mouse aorta. The expression of TLR4 mRNA and CD14 mRNA were detected by Real-time PCR. Serum concentrations of MCP-1 and TNF-α were determined with ELISA. Compared with high-fat diet group, EGCG groups showed marked decreases in aortic atherosclerosis （P 〈 0.05）, concomitantly with significant decreases in levels of the expression of TLR4, TNF-α and MCP-1. The mean lesion area was （2.37 ±0.08） mm^2 in the high-fat diet ＋ EGCG group, whereas the atherosclerotic lesion was only （1.05 ± 0.13） mm^2 in the high-fat diet ＋ EGCG group. The TLR4 expression was obviously higher in high-fat diet group than that in other groups （P 〈 0.05）. Compared with high-diet group, the serum concentrations of MCP-1 and TNF-α were significantly decreased in EGCG groups （P 〈 0.05）. These results suggest that TLR4 signaling pathway may play an important role in the development of atherosclerosis in apoE^-/- mice induced by high-fat diet.