基于Cav-1/NF-κB通路探讨调脾护心方抗动脉粥样硬化的作用机制
|
摘要
目的:研究调脾护心方对载脂蛋白基因敲除(ApoE-/-)小鼠动脉粥样硬化(AS)的影响,并探讨其作用机制。方法:将40只雄性ApoE-/-小鼠分为空白组、模型组、辛伐他汀组(阳性对照,5 mg/kg)以及调脾护心方低、高剂量组(50、150 mg/kg),每组8只。除空白组小鼠给予普通饲料外,其余各组小鼠均给予高脂饲料以复制AS模型。造模后,各给药组小鼠均灌胃相应药物,空白组和模型组小鼠均灌胃等容生理盐水,每天1次,连续12周。末次给药后,采用分光光度法检测各组小鼠血清总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平,采用硝酸酶还原法检测血清一氧化氮(NO)水平;采用酶联免疫吸附测定法检测血清白细胞介素6(IL-6)、血管细胞黏附分子1(VCAM-1)水平;分离胸主动脉,以苏木精-伊红染色,观察各组小鼠胸主动脉斑块形成情况,并计算校正斑块面积;采用蛋白质印迹法检测各组小鼠胸主动脉组织中核因子κB(NF-κB)p65、小窝蛋白1(Cav-1)、一氧化氮合酶(eNOS)蛋白的表达情况。结果:与空白组比较,模型组小鼠血清TC、TG、LDL-C、IL-6、VCAM-1水平均显著升高,HDL-C、NO水平均显著下降(P<0.01);胸主动脉斑块明显,校正斑块面积显著增大(P<0.01),该组织中NF-κB p65、Cav-1蛋白的相对表达量均显著升高,eNOS蛋白的相对表达量显著下降(P<0.01)。与模型组比较,各给药组小鼠血清TC、TG、LDL-C水平以及辛伐他汀组和调脾护心方高剂量组小鼠血清IL-6、VCAM-1水平均显著下降,各给药组小鼠血清HDL-C、NO水平均显著升高(P<0.05或P<0.01);各给药组小鼠胸主动脉斑块减少,校正斑块面积均显著缩小(P<0.05或P<0.01),该组织中NF-κB p65、Cav-1蛋白的相对表达量均显著下降,eNOS蛋白的相对表达量均显著升高(P<0.05或P<0.01)。结论:调脾护心方可调节血脂水平、降低炎症因子水平、抑制AS斑块的形成,其机制可能与其抑制Cav-1/NF-κB通路有关。
OBJECTIVE:To study the effects of Tiaopi huxin prescription(TPHXP)on the atherosclerosis(AS)of ApoE-/-mice,and to investigate its mechanism. METHODS:Forty male ApoE-/-mice were divided into blank group,model group,simvastatin group(positive control,5 mg/kg)and TPHXP low-dose and high-dose groups(50,150 mg/kg),with 8 mice in each group. Except that blank group was given common diet,other groups were given high-lipid diet to induce AS model. After modeling,administration groups were given relevant medicine intragastrically,and blank group and model group were given constant volume of normal saline intragastrically,once a day,for consecutive 12 weeks. After last medication,the serum levels of TC,TG,LDL-C and HDL-C were determined by spectrophotometry. The serum level of NO was detected by nitrate reduction method. The serum levels of IL-6 and VCAM-1 were determined by ELISA. After separating thoracic aorta,HE staining was used to observe the formation of plaque in the thoracic aorta of mice in each group,and the corrected plaque area was calculated.Western blotting was conducted to determine the expression of NF-κ B p65,Cav-1 and eNOS. RESULTS:Compared with blank group,the serum levels of TC,TG,LDL-C,IL-6 and VCAM-1 were increased significantly in model group,while the levels of HDL-C and NO were decreased significantly(P<0.01). The plaque of thoracic aorta was obvious and the corrected plaque area were increased significantly(P<0.01).The relative expression of NF-κ B p65 and Cav-1 were increased significantly,while the relative expression of eN OS was decreased significantly(P<0.01). Compared with model group,the serum levels of TC,TG and LDL-C in administration groups,the serum levels of IL-6 and VCAM-1 in simvastatin group and TPHXP high-dose group were decreased significantly,while the serum levels of HDL-C and NO were increased significantly in administration groups(P<0.05 or P<0.01). In administration groups,the plaques of thoracic aorta were reduced and the corrected plaque area was decreased significantly(P<0.05 or P<0.01);the relative expression of NF-κB p65 and Cav-1 were decreased significantly,while the relative expression of e NOS was increased significantly(P<0.05 or P<0.01). CONCLUSIONS:TPHXP can regulate the level of blood lipid,decrease the level of inflammatory factors and inhibit the formation of AS plaque,the mechanism of which may be associated with inhibiting Cav-1/NF-κB pathway.
OBJECTIVE:To study the effects of Tiaopi huxin prescription(TPHXP)on the atherosclerosis(AS)of ApoE-/-mice,and to investigate its mechanism. METHODS:Forty male ApoE-/-mice were divided into blank group,model group,simvastatin group(positive control,5 mg/kg)and TPHXP low-dose and high-dose groups(50,150 mg/kg),with 8 mice in each group. Except that blank group was given common diet,other groups were given high-lipid diet to induce AS model. After modeling,administration groups were given relevant medicine intragastrically,and blank group and model group were given constant volume of normal saline intragastrically,once a day,for consecutive 12 weeks. After last medication,the serum levels of TC,TG,LDL-C and HDL-C were determined by spectrophotometry. The serum level of NO was detected by nitrate reduction method. The serum levels of IL-6 and VCAM-1 were determined by ELISA. After separating thoracic aorta,HE staining was used to observe the formation of plaque in the thoracic aorta of mice in each group,and the corrected plaque area was calculated.Western blotting was conducted to determine the expression of NF-κ B p65,Cav-1 and eNOS. RESULTS:Compared with blank group,the serum levels of TC,TG,LDL-C,IL-6 and VCAM-1 were increased significantly in model group,while the levels of HDL-C and NO were decreased significantly(P<0.01). The plaque of thoracic aorta was obvious and the corrected plaque area were increased significantly(P<0.01).The relative expression of NF-κ B p65 and Cav-1 were increased significantly,while the relative expression of eN OS was decreased significantly(P<0.01). Compared with model group,the serum levels of TC,TG and LDL-C in administration groups,the serum levels of IL-6 and VCAM-1 in simvastatin group and TPHXP high-dose group were decreased significantly,while the serum levels of HDL-C and NO were increased significantly in administration groups(P<0.05 or P<0.01). In administration groups,the plaques of thoracic aorta were reduced and the corrected plaque area was decreased significantly(P<0.05 or P<0.01);the relative expression of NF-κB p65 and Cav-1 were decreased significantly,while the relative expression of e NOS was increased significantly(P<0.05 or P<0.01). CONCLUSIONS:TPHXP can regulate the level of blood lipid,decrease the level of inflammatory factors and inhibit the formation of AS plaque,the mechanism of which may be associated with inhibiting Cav-1/NF-κB pathway.
引文
[1] TABAS I,GARCíA-CARDE?A G,OWENS GK. Recent insights into the cellular biology of atherosclerosis[J]. J Cell Biol,2015,209(1):13-22.
[2] PATERAS I,GIAGINIS C,TSIGRIS C,et al. NF-κB signaling at the crossroads of inflammation and atherogenesis:searching for new therapeutic links[J]. Expert Opin Ther Targets,2014,18(9):1089-1101.
[3] ROSS R. Atherosclerosis:an inflammatory disease[J]. N Engl J Med,1999,340(2):115-126.
[4]中国成人血脂异常防治指南修订联合委员会.中国成人血脂异常防治指南:2016年修订版[J].中国循环杂志,2016,31(10):937-953.
[5] HAO CZ,WU F,LU L,et al. Chinese herbal medicine for diabetic peripheral neuropathy:an updated meta-analysis of 10 high-quality randomized controlled studies[J]. PLoS One,2013,8(10):e76113.
[6] DRAB M,VERKADE P,ELGER M,et al. Loss of caveolae,vascular dysfunction,and pulmonary defects in caveolin-1 gene-disrupted mice[J]. Science,2001,293(5539):2449-2452.
[7] BUCCI M,GRATTON JP,RUDIC RD,et al. In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation[J]. Nat Med,2000,6(12):1362-1367.
[8] MINAMINO T,KITAKAZE M,SANADA S,et al. Increased expression of P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation:role of nitric oxide[J]. Circulation,1998,98(17):1721-1727.
[9] PAVLIDES S,GUTIERREZ-PAJARES JL,ITURRIETA J,et al. Endothelial caveolin-1 plays a major role in the development of atherosclerosis[J]. Cell Tissue Res,2014,356(1):147-157.
[10]郑家鼎.从CAV-1探讨护心方对冠心病合并糖代谢异常患者内皮功能研究[D].广州:广州中医药大学,2015.
[11]郭改玲.基于“健脾护心”理论探讨健脾方干预动脉粥样硬化的机制研究[D].沈阳:辽宁中医药大学,2016.
[12]汪志忠,黎红华.动脉粥样硬化与炎性因子[J].中国神经免疫学和神经病学杂志,2011,18(4):296-298.
[13] GARREAN S,GAO XP,BROVKOVYCH V,et al. Caveolin-1 regulates NF-kappa B activation and lung inflammatory response to sepsis induced by lipopolysaccharide[J]. J Immunol,2006,177(7):4853-4860.
[14] FRANK PG,LEE H,PARK DS,et al. Genetic ablation of caveolin-1 confers protection against atherosclerosis[J].Arterioscler Thromb Vasc Biol,2004,24(1):98-105.
[15]中国高血压防治指南修订委员会.中国高血压防治指南2010[J].中华高血压杂志,2011,19(8):701-743.
[16]彭道泉.2016年欧洲心脏病学会/欧洲动脉粥样硬化学会血脂异常管理指南解读[J/D].中国医学前沿杂志:电子版,2017,9(6):8-11.
[17]马悦颖,刘建勋,李澎,等.痰瘀同治方含药血清对ox-LDL损伤的人脐静脉内皮细胞产生NO,caveolin-1和eNOS的影响研究[J].中国中药杂志,2012,37(7):974-978.
[18]钟伟,王永刚,于远望,等.中医防治颈动脉粥样硬化的研究进展[J].中华中医药学刊,2017,35(8):2043-2045.
[19] JIANG W,LI S,MAO W,et al. Effect of Huxin formula on reverse cholesterol transport in ApoE-gene knockout mice[J]. Chin J Integr Med,2012,18(6):451-456.
[2] PATERAS I,GIAGINIS C,TSIGRIS C,et al. NF-κB signaling at the crossroads of inflammation and atherogenesis:searching for new therapeutic links[J]. Expert Opin Ther Targets,2014,18(9):1089-1101.
[3] ROSS R. Atherosclerosis:an inflammatory disease[J]. N Engl J Med,1999,340(2):115-126.
[4]中国成人血脂异常防治指南修订联合委员会.中国成人血脂异常防治指南:2016年修订版[J].中国循环杂志,2016,31(10):937-953.
[5] HAO CZ,WU F,LU L,et al. Chinese herbal medicine for diabetic peripheral neuropathy:an updated meta-analysis of 10 high-quality randomized controlled studies[J]. PLoS One,2013,8(10):e76113.
[6] DRAB M,VERKADE P,ELGER M,et al. Loss of caveolae,vascular dysfunction,and pulmonary defects in caveolin-1 gene-disrupted mice[J]. Science,2001,293(5539):2449-2452.
[7] BUCCI M,GRATTON JP,RUDIC RD,et al. In vivo delivery of the caveolin-1 scaffolding domain inhibits nitric oxide synthesis and reduces inflammation[J]. Nat Med,2000,6(12):1362-1367.
[8] MINAMINO T,KITAKAZE M,SANADA S,et al. Increased expression of P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation:role of nitric oxide[J]. Circulation,1998,98(17):1721-1727.
[9] PAVLIDES S,GUTIERREZ-PAJARES JL,ITURRIETA J,et al. Endothelial caveolin-1 plays a major role in the development of atherosclerosis[J]. Cell Tissue Res,2014,356(1):147-157.
[10]郑家鼎.从CAV-1探讨护心方对冠心病合并糖代谢异常患者内皮功能研究[D].广州:广州中医药大学,2015.
[11]郭改玲.基于“健脾护心”理论探讨健脾方干预动脉粥样硬化的机制研究[D].沈阳:辽宁中医药大学,2016.
[12]汪志忠,黎红华.动脉粥样硬化与炎性因子[J].中国神经免疫学和神经病学杂志,2011,18(4):296-298.
[13] GARREAN S,GAO XP,BROVKOVYCH V,et al. Caveolin-1 regulates NF-kappa B activation and lung inflammatory response to sepsis induced by lipopolysaccharide[J]. J Immunol,2006,177(7):4853-4860.
[14] FRANK PG,LEE H,PARK DS,et al. Genetic ablation of caveolin-1 confers protection against atherosclerosis[J].Arterioscler Thromb Vasc Biol,2004,24(1):98-105.
[15]中国高血压防治指南修订委员会.中国高血压防治指南2010[J].中华高血压杂志,2011,19(8):701-743.
[16]彭道泉.2016年欧洲心脏病学会/欧洲动脉粥样硬化学会血脂异常管理指南解读[J/D].中国医学前沿杂志:电子版,2017,9(6):8-11.
[17]马悦颖,刘建勋,李澎,等.痰瘀同治方含药血清对ox-LDL损伤的人脐静脉内皮细胞产生NO,caveolin-1和eNOS的影响研究[J].中国中药杂志,2012,37(7):974-978.
[18]钟伟,王永刚,于远望,等.中医防治颈动脉粥样硬化的研究进展[J].中华中医药学刊,2017,35(8):2043-2045.
[19] JIANG W,LI S,MAO W,et al. Effect of Huxin formula on reverse cholesterol transport in ApoE-gene knockout mice[J]. Chin J Integr Med,2012,18(6):451-456.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |