基于TGF-β1/Smad4信号探讨缺氧条件下补阳还五汤促进新生血管成熟的机制

Mechanism of Buyang Huanwutang in Promoting Maturation of Neovascularization Under Hypoxia Condition Based on TGF-β1/Smad4 Signal

目的：研究在缺氧条件下补阳还五汤通过调控转化生长因子-β_1（transforming growth factor-β_1,TGF-β_1）/Smad蛋白4（drosophila mothers against decapentaplegic protein,Smad4）基因信号促进新生血管成熟的分子机制。方法：于体外鼠尾胶基质上种植胸主动脉血管段构建新生血管三维培养模型,随机分为正常组,缺氧模型组,辛伐他汀组,补阳还五汤组。缺氧模型组,常氧条件下培养5 d后缺氧24 h,药物治疗组在缺氧同时分别给予辛伐他汀（10μg·L^-1）和补阳还五汤载药血清（10%）,正常组则常氧条件下培养6 d。利用倒置显微镜观察新生血管生长情况,利用酶联免疫吸附法（enzyme linked immunosorbent assay,ELISA）检测新生血管培养液中TGF-β_1的含量,利用实时荧光定量聚合酶链反应（Real-time PCR）检测新生血管TGF-β_1和Smad4 mRNA的表达,利用蛋白免疫印迹法（Western blot）检测新生血管神经型钙黏蛋白（neural-Cadherin,NCadherin）蛋白表达。结果：各实验组结果显示,培养6 d,倒置显微镜下均可观察到新生血管生成,与正常组比较,模型组中存活的新生血管数量减少（P〈0.05）,给予补阳还五汤干预后存活的新生血管数量增多（P〈0.05）;ELISA结果显示与正常组比较,缺氧模型组TGF-β_1含量明显降低（P〈0.01）,给予补阳还五汤干预后TGF-β_1含量升高（P〈0.05）;Real-time PCR结果显示,与正常组比较,模型组TGF-β_1和Smad4 mRNA表达下调（P〈0.05）,经补阳还五汤治疗后TGF-β_1和Smad4 mRNA表达明显上调（P〈0.05）;Western blot结果显示与正常组比较,模型组N-Cadherin蛋白表达下调（p〈0.05）,补阳还五汤干预后NCadherin蛋白表达上调（P〈0.05）。结论：在缺氧条件下补阳还五汤促进成熟新生血管形成,其机制与激活TGF-β_1/Smad4信号进而启动下游N-Cadherin蛋白合成,形成细胞间紧密连接有关。

Objective： To study the molecular mechanism of Buyang Huanwutang in promoting the maturation of neovascularization by regulating the signal transduction of transforming growth factor-β1（TGF-β1）/drosophila mothers against decapentaplegic protein（Smad4） gene under hypoxia condition. Method： The threedimensional model of vascularization was established by implanting abdominal aorta in Matrigel matrix. There were four groups,namely the normal control group,the hypoxia model group,the Simvastatin treatment group and the Buyang Huanwutang treatment group. After incubation under normoxia condition for 5 days,the model group was given hypoxia intervention for 24 hours,while the treatment groups were respectively given Simvastatin（10 μg·L^-1） and Buyang Huanwutang（10%）,and the control group was incubated under normoxia for six days. The growth of neovascularization was observed under inverted microscope. The content of TGF-β1 in culture medium of neovascularization was detected by enzyme linked immunosorbent assay（ELISA）. The gene expressions of TGF-β1 and Smad4 in neovascularization were detected by Real-time PCR. The protein expression of neural-Cadherin（NCadherin） was detected by Western blot. Result： The results of each experimental group displayed neovascularization under inverted microscope. Compared with the normal group, the number of stable neovascularization was reduced in the model group. After the treatment with Buyang Huanwutang,the number of stable neovascularization was increased,compared with the model group. The results of ELISA showed that the content of TGF-β1 was significantly decreased（P〈0. 01） in the model group,compared with the normal group.After the treatment with Buyang Huanwutang,the content of TGF-β1 was increased（P〈0. 05）,compared with the model group. The results of Real-time PCR indicated that the mRNA expressions of TGF-β1 and Smad4 in the model group were lower（P〈0. 05） than those of the normal group. After the treatment with Buyang Huanwutang,the mRNA expressions of TGF-β1 and Smad4 were higher（P〈0. 05） than those of the model group. The results of Western blot demonstrated that the protein expression of N-Cadherin was lower（P〈0. 05） in the model group than that of the normal group. After the treatment with Buyang Huanwutang,the protein expression of N-Cadherin was higher（P〈0. 05） than that of the model group. Conclusion： Buyang Huanwutang could promote the neovascularization under hypoxia condition. Its mechanism is correlated with the activation of TGF-β1/Smad4 signal,which then starts the synthesis of downstream protein N-Cadherin and forms the close intercellular junction.