青心酮对载脂蛋白E缺陷小鼠主动脉粥样硬化形成及斑块中主要细胞的TLR4表达的影响

Effect of 3,4-dihydroxyacetophenone on Atherogenesis in ApoE-Deficient Mice and the Expression of TLR4 in Three Main Cells of Atherosclerosis Plaque

关键词:  青心酮  Toll样受体4  动脉粥样硬化

Key words:  3,4-dihydroxyacetophenone  Toll-like receptor 4  atherosclerosis

摘要:

目的 观察青心酮对小鼠动脉粥样硬化(AS)斑块中平滑肌细胞、内皮细胞、巨噬细胞Toll样受体4表达(Toll-like receptor 4，TLR4)的影响，及对载脂蛋白E基因敲除小鼠主动脉粥样硬化病变的影响。方法 取小鼠胸主动脉，进行平滑肌细胞、内皮细胞的原代培养，RAW 264.7(小鼠巨噬细胞)细胞株传代培养。实验分4组：空白对照组，脂多糖(LPS)组，青心酮组，辛伐他汀组。收集各组细胞总mRNA及蛋白，应用RT-PCR及Western-blot方法检测TLR4 mRNA及蛋白。取30只8周龄雄性ApoE(-/-)小鼠，随机分成3组：模型组(n=10)，每天生理盐水灌胃；青心酮治疗组(n=10)经胃管灌注青心酮10 mg·kg^-1·d^-1，辛伐他汀治疗组(n=10)经胃管灌注辛伐他汀10 mg·kg^-1·d^-1。所有实验小鼠均饲以"西方类型膳食"饲料12周。剪取主动脉根部切片及HE染色观察主动脉粥样硬化病变情况。结果 1×10^-7 mol·L^-1青心酮组血管平滑肌细胞、内皮细胞、巨噬细胞TLR4 mRNA和蛋白含量明显低于未处理组(P<0.01)；与辛伐他汀组比较无显著性差异；青心酮组AS病灶形成减少。结论 青心酮可下调LPS活化的小鼠平滑肌细胞、内皮细胞、巨噬细胞TLR4 mRNA和蛋白的表达，在一定程度上能减少AS病灶的形成，可能通过TLR4途径发挥其作用。

Abstract:

OBJECTIVE To explore the effect of 3,4-dihydroxyacetophenone(DHAP) on the expression of Toll-like receptor 4(TLR4) mRNA and protein in activated mouse vascular smooth muscle cells (VSMC), endothelial cells (EC) and macrophage (MC) to observe the change of aortic atherosclerosis at apolipoprotein E knockout (ApoE-/-) mouse treated with DHAP. METHODS Normal thoracic aortas were obtained from 4 adult mice, and the VSMC, EC were cultured. The MC (RAW264.7 cell lines) was obtained from ATCC. The cells were divided into four groups: control group; LPS group; DHAP group; simvastatin group. Changes of TLR4 mRNA and protein were analyzed by RT-PCR and Western blot. In addition, 30 same age male mice of ApoE(-/-) were fed with a Western diet (21% fat, 0.15% cholesterol) for 12 weeks, and these mice were divided into three groups at random including: model group; DHAP treated group receiving DHAP administered via gastric tube at 10 mg·kg^-1·d^-1 for consecutive 12 weeks; simvastatin treated group receiving simvastatin administered via gastric tube at 10 mg·kg^-1·d^-1 for consecutive 12 weeks. The changes of aortic atherosclerosis were analyzed. RESULTS The VSMC, EC and MC pretreated by 1×10^-7 mol·L^-1 DHAP expressed TLR4 mRNA and protein lower than unpretreated ones (P<0.01) in activated. The lesions of aortic atherosclerosis were inhibited in two treated groups. CONCLUSION It suggested that DHAP could be effective for the prevention and treatment of AS, and TLR4 path might be the mechanism of DHAP to treat AS.