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
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摘要
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 C57 BL/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.
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 C57 BL/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.
引文
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[11]朱旑,徐标,高玲,等.糖尿病载脂蛋白E基因缺陷小鼠动脉粥样硬化斑块炎症反应增强[J].中国动脉硬化杂志,2008,16(5):349-352.
[12]Sen U,Moshal K S,Singh M,et al.Homocysteine induced bio-chemical stress predisposes to cytoskeletal remodeling in stretched endothelial cells[J].Mol Cell Bioehem,2007,302(1/2):133-143.
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[14]Lecleire S,Hassan A,Marrion-Letellier R,et al.Combined glutamine and arginine decrease proinflammatory cyto-kine production by biopsies from Crohn's patients in association with changes in nuclear factor-kappa B and p38 mitogen-acti-vated protein kinase pathways[J].J Nutr,2008,138(12):2481-2486.
[15]刘倩,范颖,李新,等.黄芪葛根配伍调节糖尿病大鼠血糖血脂炎症的作用与机制[J].中国实验方剂学杂志,2018,24(11):81-86.
[2]CHANG L,Karin M,Mammalian.MAP kinase signalling cascades[J].Nature,2001,410(6824):37-40.
[3]CHEN J Y,WANG C,WANG J,et al.The advanves of MAPK signaling pathwany[J].Chin Med Pharm,2011,1(8):32-34.
[4]George J,Afek A,Abashidze A,et al.Transfer of endothelial progenitor and bone marrow cells influences atherosclerotic plaque size and com-position in apolipoprotein E knockout mice[J].Arterioscler Thromb Vasc Biol,2005,25:2636-2641.
[5]Leor J,Marber M.Endothelial progenitors:a new tower of babel[J].J Am Coll Cardiol,2006,48(8):1588-1590.
[6]Lee H S,Kim S D,Lee W M,et al.A noble function of BAY 11-7082:inhibition of platelet aggregation mediated by an elevated c AMP-induced VASP,and decreased ERK2/JNK1 phosphoryla-tions[J].Eur JPharmacol,2010,627(3):85-91.
[7]陈宁,宋囡,贾连群,等.化瘀祛痰方对AS家兔肝脏线粒体呼吸链酶复合物的影响[J].中国实验方剂学杂志,2018,24(7):171-176.
[8]贾连群,陈文娜,赵秋宇,等.化瘀祛痰方药对动脉粥样硬化模型小鼠肝脏胆固醇代谢相关基因表达调控的影响[J].中华中医药杂志,2014,29(1):214-218.
[9]Nagashima M,Watanabe T,Shiraishi Y,et al.Chronic infusion of salusin-alpha and-beta exerts opposite effects on atherosclerotic lesion development in apolipoprotein E-deficientmice[J].Atherosclerosis,2010,212(1):70-77.
[10]王亚红,秦建国,郭维琴,等.降脂通脉方抗高脂血症及动脉粥样硬化的实验研究[J].中华中医药杂志,2006,21(2):98-100.
[11]朱旑,徐标,高玲,等.糖尿病载脂蛋白E基因缺陷小鼠动脉粥样硬化斑块炎症反应增强[J].中国动脉硬化杂志,2008,16(5):349-352.
[12]Sen U,Moshal K S,Singh M,et al.Homocysteine induced bio-chemical stress predisposes to cytoskeletal remodeling in stretched endothelial cells[J].Mol Cell Bioehem,2007,302(1/2):133-143.
[13]Tyagi N,Moshal K S,Tyagi S C,et al.gamma-aminbuturic acid:a receptor mitigates homocysteine-induced endothelial cell permeabilit[J].Endothelium,2007,14(6):315-323.
[14]Lecleire S,Hassan A,Marrion-Letellier R,et al.Combined glutamine and arginine decrease proinflammatory cyto-kine production by biopsies from Crohn's patients in association with changes in nuclear factor-kappa B and p38 mitogen-acti-vated protein kinase pathways[J].J Nutr,2008,138(12):2481-2486.
[15]刘倩,范颖,李新,等.黄芪葛根配伍调节糖尿病大鼠血糖血脂炎症的作用与机制[J].中国实验方剂学杂志,2018,24(11):81-86.