补阳还五汤通过调节HUVEC细胞Rho/Rho激酶信号通路作用于动脉粥样硬化的研究
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摘要
目的研究补阳还五汤抗同型半胱氨酸(Hcy)诱导的脐静脉内皮细胞(HUVEC)损伤的作用及其机制。方法采用Hcy造模,将内皮细胞随机分为10组,即空白组(10%空白血清),模型组(Hcy+10%空白血清),补阳还五汤低剂量组(5%含药血清+Hcy)、中剂量组(10%含药血清+Hcy)、高剂量组(20%含药血清+Hcy),10%FBS+Hcy对照组,Rho激酶(ROCK)阻断剂Y27632+Hcy组,靶分子肌球蛋白轻链激酶(MLCK)阻断剂ML-7+Hcy组,丝裂原活化蛋白激酶(MAPK)抑制剂SB203580+Hcy组,细胞外信号调节激酶(ERK)抑制剂PD98059+Hcy组,24 h后,用IP裂解液(含PMSF)裂解细胞。用蛋白质印迹法(Western blot)测定ROCK、MLCK蛋白水平,用RT-PCR技术测定ROCK、MLCK m RNA的表达情况,并用激光共聚焦测定细胞骨架结构蛋白纤维肌动蛋白(F-actin)的变化。结果 Western blot与RT-PCR结果显示:模型组与空白组比较,HUVEC细胞ROCK、MLCK蛋白以及其m RNA水平明显上升,而补阳还五汤含药血清组和抑制剂组对其有明显的下调作用。激光共聚焦检测骨架蛋白F-actin实验中,与空白组比较,模型组细胞应力纤维明显增多;而补阳还五汤各剂量组相对于模型组,应力纤维的形成明显降低。结论补阳还五汤可能通过调节内皮细胞Rho/Rho激酶信号通路,阻止细胞骨架的改变,减轻内皮细胞损伤而发挥其抗动脉粥样硬化的作用。
Objective To explore the effect and mechanism of Buyang Huanwu Tang(BHT) on counteracting human umbilical vein endothelial cells(HUVEC) injury induced by homocysteic acid(Hcy).Methods HUVEC were treated with Hcy for the modeling.HUVEC were randomly divided into 10 groups,namely blank control group(10 % blank serum),model group(Hcy+ 10 % blank serum),FBS group(10 % FBS and Hcy),low-,middle-,and high-dose BHT groups(Hcy+ serum containing BHT at 5 %,10 %,20 % respectively),and inhibitor groups(treated with ROCK inhibitor Y27632,MLCK inhibitor ML-7,MAPK inhibitor SB203580,ERK inhibitor PD98059 respectively together with Hcy).After treatment for 24 hours,the cultured cells were split by IP lysate(including phenylmethyl sulfonylfluoride).Western blot and RT-PCR were used for determining the changes of protein and m RNA expression levels of ROCK and MLCK.Meanwhile,the changes of cytoskeletal structure protein fiber F-actin was determined by laser scanning confocal microscopy(LSCM).Results The results of Western blot and RT-PCR showed that the model group had higher ROCK and MLCK protein and m RNA expression levels than the blank control group.And serum containing BHT and inhibitors could up-regulate the levels of ROCK and MLCK.The results of LSCM examination showed that the amount of stress fiber was increased in the model group as compared with the blank control group,and serum containing BHT had an effect on reducing stress fiber.Conclusion BHT could counteract AS by regulating Rho/Rho-kinase pathway,blocking the changes of the cytoskeletal structure and reducing the endothelial cell injury.
Objective To explore the effect and mechanism of Buyang Huanwu Tang(BHT) on counteracting human umbilical vein endothelial cells(HUVEC) injury induced by homocysteic acid(Hcy).Methods HUVEC were treated with Hcy for the modeling.HUVEC were randomly divided into 10 groups,namely blank control group(10 % blank serum),model group(Hcy+ 10 % blank serum),FBS group(10 % FBS and Hcy),low-,middle-,and high-dose BHT groups(Hcy+ serum containing BHT at 5 %,10 %,20 % respectively),and inhibitor groups(treated with ROCK inhibitor Y27632,MLCK inhibitor ML-7,MAPK inhibitor SB203580,ERK inhibitor PD98059 respectively together with Hcy).After treatment for 24 hours,the cultured cells were split by IP lysate(including phenylmethyl sulfonylfluoride).Western blot and RT-PCR were used for determining the changes of protein and m RNA expression levels of ROCK and MLCK.Meanwhile,the changes of cytoskeletal structure protein fiber F-actin was determined by laser scanning confocal microscopy(LSCM).Results The results of Western blot and RT-PCR showed that the model group had higher ROCK and MLCK protein and m RNA expression levels than the blank control group.And serum containing BHT and inhibitors could up-regulate the levels of ROCK and MLCK.The results of LSCM examination showed that the amount of stress fiber was increased in the model group as compared with the blank control group,and serum containing BHT had an effect on reducing stress fiber.Conclusion BHT could counteract AS by regulating Rho/Rho-kinase pathway,blocking the changes of the cytoskeletal structure and reducing the endothelial cell injury.
引文
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[11]Amano M,Nakayama M,Kaibuchi K.Rho-kinase/ROCK:A key regulator of the cytoskeleton and cell polarity[J].Cytoskeleton,2010,67(9):545-554.
[12]Park JH,Okayama N,Gute D,et al.Hypoxia/alycemia increase sendothelial permeability:Role of s ECsond messengers and cytoskeleton[J].Am J Physiol,1999,277(6pt):C1066-1074.
[13]Erosslinking proteins prot ECst against osmotic stress,ensure cell size,cell shape and motility,and contribute to phagoeytosis and development[J].J Cell Sci,1996,109(11):2679-2691.
[14]Liang JH,Li YN,Qi JS,et al.Peroxynitrite-induced protein nitration is responsible for renal mitochondrial damage in diabetic rat[J].J Endocrinal Invest,2010,33(3):140-146.
[15]Van DOIA,Raterman HG,Nurmohamed MT,et al.Endothelial dysfunction,inflammation,and apoptosis in diabetes mellitus[J].Mediators of Inflammation,2010,2010:792393.
[2]王亚红,郭维琴.动脉粥样硬化中医研究的现状和思路[J].中国医药学报,2002,17(10):624-626.
[3]陈广进,洪永敦,赵静.温阳活血解毒方对Apo E-/-小鼠动脉粥样硬化斑块及斑块内血管新生的影响[J].中药新药与临床药理,2015,26(2):139-144.
[4]刘玉晖,易文凤,游宇.补阳还五汤抗动脉粥样硬化作用与紧密连接蛋白的关系[J].中国实验方剂学杂志,2014,20(24):155-158.
[5]邱顺辉,章常华,高书亮,等.补阳还五汤抗动脉粥样硬化与间隙连接蛋白关系的研究[J].中国实验方剂学杂志,2011,17(18):161-164.
[6]Zhou Q,Liao JK.Rho kinase:an important mediator of atherosclerosis and vascular disease[J].Curr Pharm Des,2009,15(27):3108-3115.
[7]Dong M,Yan BP,Yu CM.Current status of Rho-associated kinases(ROCKs)in coronary atherosclerosis and vasospasm[J].Cardiovasc Hematol Agents Med Chem,2009,7:322-330.
[8]Loirand G,Guérin P,Pacaud P.Rho kinases in cardiovascular physiology and pathophysiology[J].Circ Res,2006,98:322-334.
[9]戴青原,郭涛.氧化应激与糖尿病及动脉粥样硬化研究进展[J].心血管病学进展,2013,34(5):664-668.
[10]Dudek SM,Garcia JG.Cytoskeletal regulation ofpulmonary vascular permeability[J].J Appl Physiol,2001,91:1487-1500.
[11]Amano M,Nakayama M,Kaibuchi K.Rho-kinase/ROCK:A key regulator of the cytoskeleton and cell polarity[J].Cytoskeleton,2010,67(9):545-554.
[12]Park JH,Okayama N,Gute D,et al.Hypoxia/alycemia increase sendothelial permeability:Role of s ECsond messengers and cytoskeleton[J].Am J Physiol,1999,277(6pt):C1066-1074.
[13]Erosslinking proteins prot ECst against osmotic stress,ensure cell size,cell shape and motility,and contribute to phagoeytosis and development[J].J Cell Sci,1996,109(11):2679-2691.
[14]Liang JH,Li YN,Qi JS,et al.Peroxynitrite-induced protein nitration is responsible for renal mitochondrial damage in diabetic rat[J].J Endocrinal Invest,2010,33(3):140-146.
[15]Van DOIA,Raterman HG,Nurmohamed MT,et al.Endothelial dysfunction,inflammation,and apoptosis in diabetes mellitus[J].Mediators of Inflammation,2010,2010:792393.