化瘀祛痰方对ApoE^(-/-)小鼠主动脉MAPK信号通路相关蛋白表达的影响

Effect of Huayu Qutan Recipe in Regulating Aortic MAPK Pathway-related Gene Expression in Atherosclerosis Mice

目的:探讨载脂蛋白敲除(Apoe-/-)小鼠主动脉丝裂原活化蛋白激酶(MAPK)信号通路相关mRNA的表达变化及化瘀祛痰方药对其的干预作用,探讨化瘀祛痰方药抗AS作用及可能的机制。方法:50只Apo E-/-小鼠随机分为模型组,丹参酮ⅡA组(30 mg·kg^(-1)·d^(-1)),化瘀祛痰高剂量组(20 g·kg^(-1)·d^(-1)),化瘀祛痰中剂量组(10 g·kg^(-1)·d^(-1)),化瘀祛痰低剂量组(5 g·kg^(-1)·d^(-1)),10只C57BL/6/J小鼠作为正常组。全自动生化仪检测血清甘油三酯(triglyceride,TG),胆固醇(cholesterol,TC),高密度脂蛋白胆固醇(high-density lipoprotein cholesterol,HDL-C),低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C)的含量;苏木素-伊红(HE)检测各组小鼠主动脉斑块情况;油红O检测各组小鼠肝脏脂质沉积情况;酶联免疫吸附测定(ELISA)测定各组小鼠血清中血管细胞黏附分子-1(vascular cell adhesion protein 1,VCAM-1),细胞间黏附分子-1(ICAM-1),白细胞介素-6(interleukin-6,IL-6),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)含量;蛋白免疫印迹法(Western blot)检测各组小鼠主动脉c-Jun氨基末端激酶(c-Jun n-terminal kinase,JNK),磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase,p-JNK),p38 MAPK,p-p38 MAPK,细胞外调节激酶(extracellular regulated protein kinases,ERK),磷酸化细胞外调节激酶(extracellular regulated protein kinases,p-ERK)蛋白的表达变化。结果:与正常组比较,模型组小鼠血清TC,TG,LDL-C水平明显升高,HDL-C水平明显降低;主动脉管腔见较大粥样斑块,肝细胞中见大量脂质沉积;血清内相关炎性因子TNF-α,IL-6,VCAM-1,ICAM-1含量增加;血管内相关MAPK信号通路p-p38,p-JNK蛋白表达明显增加(P<0.05)。与模型组比较,丹参酮ⅡA组与化瘀祛痰各组小鼠TC,TG,LDL-C明显下降,HDL-C明显升高,主动脉管腔中粥样斑块面积和肝脏脂质沉积量明显减少,血清内相关炎性因子TNF-α,IL-6,VCAM-1,ICAM-1含量降低(P<0.05);血管内相关MAPK信号通路p-p38,p-JNK蛋白表达明显降低(P<0.05),p-ERK表达趋势不明显。结论:化瘀祛痰方药可抑制Apo E-/-小鼠主动脉斑块形成,其机制可能与调控血管MAPK信号通路基因表达有关。

Objective: To investigate the expression changes of mitogen activated protein kinase ( MAPK) signaling pathway-related genes in ApoE - / - mice and the intervention effect of Huayu Qutan recipe on them,in order to explore the anti-atherosclerotic ( AS) effect and possible mechanism of Huayu Qutan recipe. Method: Fifty ApoE - / - mice were randomly divided into model group,tanshinone IIA group ( 30 mg·kg - 1·d - 1 ) , and high-dose phlegm group ( 20 g·kg - 1·d - 1 ) ,the middle-dose group ( 10 g·kg - 1·d - 1 ) ,and the low-dose group ( 5 g·kg - 1·d - 1 ) ,and 10 C57BL /6 /J mice were included in the blank controls group. Automated biochemical analyzer was used to detect serum triglyceride ( TG) ,cholesterol ( TC) ,high-density lipoprotein cholesterol ( HDL-C) ,low-density lipoprotein cholesterol ( LDL-C) content;hematoxylin-eosin ( HE) was used to detect aortic plaque in each group;oil red O was used to detect liver lipid deposition in each group;enzyme-linked immuno sorbent assay ( ELISA) was used to determine vascular cell adhesion protein-1 ( VCAM-1) ,intercellular adhesion molecule-1 ( ICAM-1 ) ,interleukin-6 ( IL-6 ) ,tumor necrosis factor-α ( TNF-α) in serum of each group;Western blot was performed to detect c-Jun N-terminal kinase ( JNK) ,phosphorylated c-Jun N-terminal kinase ( p-JNK) ,c-Jun N-terminal kinase ( JNK) ,p38 MAPK,p-p38 MAPK,extracellular regulated protein kinases ( ERK) ,and expression of p-ERK. Result: Compared with the blank control group,serum TC,TG, LDL-C levels in the model group were significantly increased,while HDL-C levels were significantly decreased;aortic plaques were observed in the aortic lumen,and lots of lipid deposition were observed in the liver cells. Serum inflammatory factors TNF-α,IL-6,VCAM-1,ICAM-1 increased ( P < 0. 05);and intravascular associated MAPK signaling pathway p-p38 MAPK,p-JNK gene expressions increased significantly ( P < 0. 05) . Compared with the model group,TC,TG,LDL-C in the tanshinone IIA group and the phlegm group were significantly decreased,HDL-C was significantly increased,and atherosclerotic plaque area and liver lipid were remarkably reduced in the aortic lumen. The deposition amount was significantly reduced,and the levels of inflammatory factors such as TNF-α,IL-6,VCAM-1 and ICAM-1 were decreased. The expressions of p-p38 MAPK and p-JNK genes in intravascular associated MAPK signaling pathway were significantly decreased. p-ERK expression trend was not obvious ( P < 0. 05) . Conclusion: Huayu Recipe can inhibit the formation of aortic plaque in ApoE - / - mice,and its mechanism may be related to the regulation of vascular MAPK signaling pathway gene expression.