补肺益肾方对TGF-β1/BMP-4诱导的肺血管平滑肌细胞TGF-β1/Smad信号通路的影响  

Effect of Bufei Yishen Formula on TGF-β1/Smad Signaling Pathway of Human Pulmonary Arterial Smooth Muscle Cells Induced by TGF-β1 and BMP-4

作　　者：任周新[1,2] 李建生 沈俊岭[3] 梅晓峰 余海滨[3] 冯月[1] REN Zhou-xin;LI Jian-sheng;SHEN Jun-ling;MEI Xiao-feng;YU Hai-bin;FENG Yue(Henan University of Traditional Chinese Medicine （ TCM）, Zhengzhou 450046, China;Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment ＆ Chinese Medicine Development of Henan Province, Zhengzhou 450046, China;First Affiliated Hospital of Henan University of TCM, Zhengzhou 450000, China)

机构地区：[1]河南中医药大学,郑州450046 [2]呼吸疾病诊疗与新药研发河南省协同创新中心,郑州450046 [3]河南中医药大学第一附属医院,郑州450000

出　　处：《中国实验方剂学杂志》2018年第11期126-132,共7页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家中医临床研究基地业务建设第二批科研专项(JDZX2015156)

摘　　要：目的：探讨补肺益肾方对转化生长因子-β1（TGF-β1）/骨形成蛋白-4（BMP-4）诱导的肺血管平滑肌细胞增殖及TGF-β1/Smad信号传导的影响,并探讨其相关的作用机制。方法：应用TGF-β1/BMP-4诱导人肺动脉平滑肌细胞增殖。细胞分为正常组（10%正常大鼠血清）,诱导增殖组（含7.5μg·L^-1TGF-β1与5.0μg·L^-1BMP-4的10%正常大鼠血清）,4%补肺益肾方含药血清（含7.5μg·L^-1TGF-β1与5.0μg·L^-1BMP-4,6%正常大鼠血清及4%补肺益肾大鼠血清）,6%补肺益肾方含药血清（含7.5μg·L-1TGF-β1与5.0μg·L^-1BMP-4,4%正常大鼠血清及6%补肺益肾大鼠血清）及8%补肺益肾方含药血清组（含7.5μg·L-1TGF-β1与5.0μg·L^-1BMP-4,2%正常大鼠血清及8%补肺益肾大鼠血清）。24 h后,显微镜下观察细胞的密度,5-溴脱氧尿嘧啶核苷（5-bromo-2-deoxyuridine,Brd U）法检测细胞增殖,蛋白免疫印迹法（Western blot）检测Smad1,2,3和5以及p-Smad2/3蛋白表达,实时荧光定量聚合酶链式反应法（Real-time PCR）检测纤溶酶原激活物抑制物-1（PAI-1）,结缔组织生长因子（CTGF）,分化抑制因子-1（ID-1）和ID-2 mRNA的表达。结果：TGF-β1/BMP-4作用24 h后,细胞密度增加、细胞增殖率显著增加（P〈0.05）,补肺益肾方抑制TGF-β1/BMP-4诱导的细胞增殖,显示出浓度依赖性,其中8%浓度显著抑制细胞增殖（P〈0.01）。TGF-β1/BMP-4及补肺益肾方均不影响Smad1,2,3和5蛋白的表达;但TGF-β1/BMP-4诱导p-Smad2/3的表达（P〈0.05）,8%补肺益肾方显著抑制TGF-β1/BMP-4诱导的p-Smad2/3表达（P〈0.05）。TGF-β1/Smad2通路的下游效应基因的检测发现,TGF-β1/BMP-4及补肺益肾方均不影响PAI-1,ID-1和ID-2 mRNA的表达;但TGF-β1/BMP-4诱导后,CTGF mRNA表达水平的显著增加（P〈0.01）,8%补肺益肾方抑制TGF-β1/BMP-4诱导的CTGF mRNA的表达（P〈0.05）。结论：补肺益肾方抑制TGF-β1与BMP-4合用诱导的肺动脉平滑肌细胞的增殖,抑制Objective： The purpose of the study was to explore the effect of Bufei Yishen formula(BFYS） on cellular proliferation of pulmonary artery smooth muscle cells induced by transforming growth factor-β1(TGF-β1）/bone morphogenetic protein-4(BMP-4）,furthermore,to explore the effect of BFYS on TGF-β1/Smad signaling pathway in the cells. Method： TGF-β1/BMP-4 was used to induce proliferation of human pulmonary artery smooth muscle cells. The cells were divided normal group(10% normal rat serum）,induced-proliferation group(10% normal rat serum with 7. 5 μg·L^-1 TGF-β1 and 5 μg·L^-1 BMP-4）,4% BFYS serum group(6% normal rat serum with 7. 5 μg·L^-1 TGF-β1 and 5 μg·L^-1 BMP-4 and 4% BFYS rat serum）,6% BFYS serum group(4%normal rat serum with 7. 5 μg·L^-1 TGF-β1 and 5 μg·L^-1 BMP-4 and 6% BFYS rat serum） and 8% BFYS serum group(2% normal rat serum with 7. 5 μg·L^-1 TGF-β1 and 5 μg·L-1 BMP-4 and 8% BFYS rat serum）. After 24 h induction,cell density was observed under a microscope; 5-bromo-2-deoxyuridine(Brd U） was used to detect the cellular proliferation; Western blot was used to detect the Smad 1,2,3,5 and p-Smad2/3 protein expression,and Real-time PCR was used to detect plasminogen activator inhibitor-1(PAI-1）,connective tissue growth factor(CTGF）,dominant negative helix-loop-transcription factors-1(ID-1） and ID-2 mRNA expression. Result： After24 h of TGF-β1/BMP-4 treatment,the cells density was increased under micro-examination with significant increase in cellular proliferation ratio(P〈0. 05）. BFYS inhibited TGF-β1/BMP-4-induced cellular proliferation in a dose dependent manner,with significant decrease in 8% concentration group(P〈0. 01）. Both TGF-β1/BMP-4 and BFYS showed no significant effect on Smad1,2,3 and 5 proteins expression. However,p-Smad2/3 expression was significant increased by TGF-β1/BMP-4 induction(P〈0. 05） and 8% BFYS significantly reduced its expression(P〈0. 05）. For assays of down-scream genes expression of TGF-β1/Smad pathway,TGF-β

关 键 词：转化生长因子-β1(TGF-β1)/骨形成蛋白-4(BMP-4) 补肺益肾方 肺动脉平滑肌细胞 TGF-β1/Smad通路 增殖 

分 类 号：R22[医药卫生—中医基础理论] R24[医药卫生—中医学]