粉防己碱对先天性膈疝大鼠模型胎仔肺Rho/Rho激酶表达的影响和意义
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摘要
目的探讨中药粉防己碱(TET)产前干预对先天性膈疝(CDH)大鼠模型胎仔肺内RhoA蛋白和Rho激酶表达的影响和意义。方法将孕9.5d的SD雌鼠随机分为对照组、膈疝组和TET组,给予膈疝组和TET组除草醚灌胃建立CDH动物模型。孕16.5d给予TET进行干预。孕21.5d剖宫产取出胎肺,测肺质量/体质量比(Lw/Bw),苏木素-伊红(HE)染色光镜下观察肺发育及肺血管情况,免疫组化和Western blot法检测Rho蛋白A及Rho激酶ROCK1的表达情况。结果膈疝组胎肺明显发育不良,TET组胎肺发育接近对照组。膈疝组肺发育指标Lw/Bw、肺泡面积比(PAA%)及肺血管重构指标管腔面积与血管总面积比值(LA%)明显低于对照组(2.11±0.36 vs.4.24±0.31;33.60±3.12 vs.58.81±2.92;38.58±2.15 vs.61.20±3.23,均P<0.05)。TET干预后Lw/Bw、PAA%、LA%指标较膈疝组明显改善(3.61±0.24 vs.2.11±0.36;42.46±3.68 vs.33.60±3.12;56.07±3.32 vs.38.58±2.15,均P<0.05);膈疝组肺小动脉管壁厚度占血管外径百分比(WT%)和肺小动脉中膜厚度百分比(MT%)均明显高于对照组(26.64±2.41 vs.13.50±1.45;25.98±2.79 vs.16.47±2.07,均P<0.05),TET组WT%、MT%显著低于膈疝组(16.02±2.35 vs.26.64±2.41和17.96±1.95 vs.25.98±2.79,均P<0.05)。免疫组化及Western blot检测提示,RhoA、ROCK1表达由低到高为对照组、TET组、膈疝组(P=0.000)。结论先天性膈疝胎仔肺内存在肺发育不良和肺血管重构,Rho/Rho激酶信号通路参与此过程,产前给予TET可能通过调节Rho/Rho激酶信号通路发挥肺保护作用。
Objective To investigate the effect and significance of the traditional Chinese medicine tetrandrine(TET)prenatal intervention on the expression of RhoA protein and Rho kinase ROCK1 in the fetal lung of congenital diaphragmatic hernia(CDH)rat model.Methods SD female rats with 9.5 dof gestation were randomly divided into the control group,CDH group and TET group.The CDH group and TET intervention group were administered with nitrofen by gavage for establishing CDH model.The TET intervention was given on 16.5 dof gestation.The fetal rat lungs were taken by cesarean section on 21.5 dof gestation and the lung weight/body ratio(Lw/Bw)was measured.The lung development and small pulmonary arterial morphologic changes in HE staining in all groups were observed with microscopy.The protein expression of RhoA and Rho kinase ROCK1 were respectively examined by immunohistochemistry and Western blot.Results In the CDH group,the lungs had obvious maldevelopment and the fetal lung development in the TET group was close to that in the control group.The lung development indicators of Lw/Bw,PAA%,and lung vascular remodeling indicators of lumen area and vascular total area ratio(LA%)in the CDH group were significantly lower than those in the control group(2.11±0.36 vs.4.24±0.31;33.60±3.12 vs.58.81±2.92;38.58±2.15 vs.61.20±3.23,P<0.05),the indicators of Lw/Bw.PAA% and LA% after TET intervention were significantly improved compared with the CDH group(3.61±0.24 vs.2.11±0.36;42.46±3.68 vs.33.60±3.12;56.07±3.32 vs.38.58±2.15,all P <0.05);the ratio of small pulmonary artery wall thickness to vascular external diameter(WT%)and the medium thickness percentage(MT%)in CDH group were significantly higher than those in the control group(26.64±2.41 vs.13.50±1.45 and 25.98±2.79 vs.16.47±2.07,P<0.05),WT%and MT%in the TET group were obviously lower than those in the CDH group(16.02±2.35 vs.26.64±2.41 and 17.96±1.95 vs.25.98±2.79,P<0.05).The immunohistochemistry and Western blot detection indicated that the expressions of RhoA and ROCK1 from low to high were the control group
Objective To investigate the effect and significance of the traditional Chinese medicine tetrandrine(TET)prenatal intervention on the expression of RhoA protein and Rho kinase ROCK1 in the fetal lung of congenital diaphragmatic hernia(CDH)rat model.Methods SD female rats with 9.5 dof gestation were randomly divided into the control group,CDH group and TET group.The CDH group and TET intervention group were administered with nitrofen by gavage for establishing CDH model.The TET intervention was given on 16.5 dof gestation.The fetal rat lungs were taken by cesarean section on 21.5 dof gestation and the lung weight/body ratio(Lw/Bw)was measured.The lung development and small pulmonary arterial morphologic changes in HE staining in all groups were observed with microscopy.The protein expression of RhoA and Rho kinase ROCK1 were respectively examined by immunohistochemistry and Western blot.Results In the CDH group,the lungs had obvious maldevelopment and the fetal lung development in the TET group was close to that in the control group.The lung development indicators of Lw/Bw,PAA%,and lung vascular remodeling indicators of lumen area and vascular total area ratio(LA%)in the CDH group were significantly lower than those in the control group(2.11±0.36 vs.4.24±0.31;33.60±3.12 vs.58.81±2.92;38.58±2.15 vs.61.20±3.23,P<0.05),the indicators of Lw/Bw.PAA% and LA% after TET intervention were significantly improved compared with the CDH group(3.61±0.24 vs.2.11±0.36;42.46±3.68 vs.33.60±3.12;56.07±3.32 vs.38.58±2.15,all P <0.05);the ratio of small pulmonary artery wall thickness to vascular external diameter(WT%)and the medium thickness percentage(MT%)in CDH group were significantly higher than those in the control group(26.64±2.41 vs.13.50±1.45 and 25.98±2.79 vs.16.47±2.07,P<0.05),WT%and MT%in the TET group were obviously lower than those in the CDH group(16.02±2.35 vs.26.64±2.41 and 17.96±1.95 vs.25.98±2.79,P<0.05).The immunohistochemistry and Western blot detection indicated that the expressions of RhoA and ROCK1 from low to high were the control group
引文
[1]BROWNLEE E M,HOWATSON A G,DAVIS C F.The hidden mortality of congenital diaphragmatic hernia:a 20-year review[J].J Pediatr Surg,2009,44(2):317-320.
[2]KEIJZER R,PURI P.Congenital diaphragmatic hernia[J].Semin Pediatr Surg,2010,19(3):180-185.
[3]LIN H,WANG Y,XIONG Z,et al.Effect of antenatal tetrandrine administration on endothelin-1and epidermal growth factor levels in the lungs of rats with experimental diaphragmatic hernia[J].J Pediatr Surg,2007,42(10):1644-1651.
[4]KLUTH D,KANGAH R,REICH P,et al.Nitrofen-induced diaphragmatic hernias in rats:an animal model[J].J Pediatr Surg,1990,25(8):850-854.
[5]BADEJO A M,DHALIWAL J S,CASEY D B,et al.Analysis of pulmonary vasodilator responses to the Rho-kinase inhibitor fasudil in the anesthetized rat[J].Am J Physiol Lung Cell Mol Physiol,2008,295(5):L828-836.
[6]TAKAYASU H,MASUMOTO K,HAGIWARA K,et al.Increased pulmonary RhoA expression in the nitrofen-induced congenital diaphragmatic hernia rat model[J].J Pediatr Surg,2015,50(9):1467-1471.
[7]SOMLYO A P,SOMLYO A V.Ca2+sensitivity of smooth muscle and nonmuscle myosinⅡ:modulated by G proteins,kinases,and myosin phosphatase[J].Physiol Rev,2003,83(4):1325-1358.
[8]FUKUMOTO Y,TAWARA S,SHIMOKAWA H.Recent progress in the treatment of pulmonary arterial hypertension:expectation for rho-kinase inhibitors[J].Tohoku J Exp Med,2007,211(4):309-320.
[9]WATTS KL,COTTRELL E,HOBAN P R,et al.RhoA signaling modulates cyclin D1expression in human lung fibroblasts;implications for idiopathic pulmonary fibrosis[J].Respir Res,2006,7(1):88(1-14).
[10]SUN Y F,WINK M.Tetrandrine and fangchinoline bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells[J].Phytomedicine,2014,21(8/9):1110-1119.
[11]王永刚,刘文英,林涵,等.汉防己甲素对先天性膈疝大鼠模型胎仔肺内表皮生长因子及其受体的影响和意义[J].中国修复重建外科杂志,2006,20(11):1109-1113.
[12]蔡晓辉,王帅,陈保安.汉防己甲素药理作用的研究进展(英文)[J].中国天然药物,2011,9(6):473-480.
[13]RAO M R.Effects of tetrandrine on cardiac and vascular remodeling[J].Acta Pharmacol Sin,2002,23(12):1075-1085.
[2]KEIJZER R,PURI P.Congenital diaphragmatic hernia[J].Semin Pediatr Surg,2010,19(3):180-185.
[3]LIN H,WANG Y,XIONG Z,et al.Effect of antenatal tetrandrine administration on endothelin-1and epidermal growth factor levels in the lungs of rats with experimental diaphragmatic hernia[J].J Pediatr Surg,2007,42(10):1644-1651.
[4]KLUTH D,KANGAH R,REICH P,et al.Nitrofen-induced diaphragmatic hernias in rats:an animal model[J].J Pediatr Surg,1990,25(8):850-854.
[5]BADEJO A M,DHALIWAL J S,CASEY D B,et al.Analysis of pulmonary vasodilator responses to the Rho-kinase inhibitor fasudil in the anesthetized rat[J].Am J Physiol Lung Cell Mol Physiol,2008,295(5):L828-836.
[6]TAKAYASU H,MASUMOTO K,HAGIWARA K,et al.Increased pulmonary RhoA expression in the nitrofen-induced congenital diaphragmatic hernia rat model[J].J Pediatr Surg,2015,50(9):1467-1471.
[7]SOMLYO A P,SOMLYO A V.Ca2+sensitivity of smooth muscle and nonmuscle myosinⅡ:modulated by G proteins,kinases,and myosin phosphatase[J].Physiol Rev,2003,83(4):1325-1358.
[8]FUKUMOTO Y,TAWARA S,SHIMOKAWA H.Recent progress in the treatment of pulmonary arterial hypertension:expectation for rho-kinase inhibitors[J].Tohoku J Exp Med,2007,211(4):309-320.
[9]WATTS KL,COTTRELL E,HOBAN P R,et al.RhoA signaling modulates cyclin D1expression in human lung fibroblasts;implications for idiopathic pulmonary fibrosis[J].Respir Res,2006,7(1):88(1-14).
[10]SUN Y F,WINK M.Tetrandrine and fangchinoline bisbenzylisoquinoline alkaloids from Stephania tetrandra can reverse multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells[J].Phytomedicine,2014,21(8/9):1110-1119.
[11]王永刚,刘文英,林涵,等.汉防己甲素对先天性膈疝大鼠模型胎仔肺内表皮生长因子及其受体的影响和意义[J].中国修复重建外科杂志,2006,20(11):1109-1113.
[12]蔡晓辉,王帅,陈保安.汉防己甲素药理作用的研究进展(英文)[J].中国天然药物,2011,9(6):473-480.
[13]RAO M R.Effects of tetrandrine on cardiac and vascular remodeling[J].Acta Pharmacol Sin,2002,23(12):1075-1085.