补阳还五汤含药血清对人肺动脉内皮细胞间质转化的影响

Effect of Serum Containing Buyang Huanwu Tang on Endothelial-tomesenchymal Transition of Human Pulmonary Artery Endothelial Cells

目的:研究补阳还五汤含药血清对人肺动脉内皮细胞(HPAEC)间质转化(End MT)的调控作用,并从Jagged1/Notch1信号传导进一步分析其作用机制。方法:以53. 36 g·kg-1·d-1剂量灌胃家兔,制备补阳还五汤含药血清,等容积生理盐水灌胃制备空白血清。体外培养HPAEC细胞,转化生长因子-β1(TGF-β1)诱导建立End MT模型,设立空白组(10%空白血清),模型组(10%空白血清+TGF-β1),补阳还五汤含药血清高剂量组(10%含药血清+TGF-β1),补阳还五汤含药血清中剂量组(5%含药血清+5%空白血清+TGF-β1)和补阳还五汤含药血清低剂量组(2. 5%含药血清+7. 5%空白血清+TGF-β1) 5组。采用噻唑蓝(MTT)比色法检测细胞增殖能力,transwell和划痕实验检测细胞迁移能力,免疫荧光观察内皮细胞标志物血小板内皮细胞黏附分子1(CD31),血管内皮钙黏蛋白(VE-cadherin)和间质细胞标志物成纤维细胞特异性蛋白1(FSP1),α-平滑肌肌动蛋白(α-SMA)的表达,蛋白免疫印迹法(Western blot)检测Notch1,Jagged1及CBF1蛋白的表达。结果:与空白组比较,模型组HPAEC细胞增殖能力、迁移能力均显著增强(P <0. 01),CD31和VE-cadherin表达呈下降趋势,FSP1和α-SMA表达呈上升趋势,Notch1,Jagged1和CBF1表达显著上调(P <0. 01)。补阳还五汤含药血清干预后,与模型组比较,细胞增殖能力、迁移能力均明显减弱(P <0. 05,P <0. 01),且CD31和VE-cadherin表达呈上升趋势,FSP1和α-SMA表达呈下降趋势,Notch1,Jagged1和CBF1表达明显降低(P <0. 05,P <0. 01),随着含药血清浓度的增加,作用越明显。结论:补阳还五汤含药血清可部分抑制人肺动脉内皮细胞发生间质转化,这一过程可能与调控Jagged1/Notch1信号有关。

Objective:To investigate the regulatory effect of serum containing Buyang Huanwu Tang on endothelial-to-mesenchymal transition(EndMT)in human pulmonary artery endothelial cells(HPAEC),and make further analysis on its mechanism from the perspective of the signal transduction of Jagged1/Notch1.Method:Rabbit serum containing Buyang Huanwu Tang was prepared by gavage with dosage of 53.36 g·kg-1·d-1,and blank serum was prepared by gavage with same volume of normal saline.The HPAECs cultured in vitro,EndMT model was established by the transforming growth factor-β1(TGF-β1)induced,which were divided into five groups:the control group(10%blank serum),the model group(10%blank serum+TGF-β1),the serum containing high-dose Buyang Huanwu Tang group(10%medicated serum+TGF-β1),the medium-dose group(5%medicated serum+5%blank serum medicated+TGF-β1)and the low-dose group(2.5%medicated serum+7.5%blank serum+TGF-β1).Cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)method.Cell migration was detected by transwell and scratch assay.The endothelial markers platelet endothelial cell adhesion molecule-1(PECAM-1/CD31),vascular endothelial cadherin(VE-cadherin)and the mesenchymal markers fibroblast-specific protein 1(FSP1),α-smooth muscle actin(α-SMA)were observed by immunofluorescence assay.The expression levels of Notch1,Jagged1 and CBF1 were detected by Western blot assay.Result:Compared with the control group,the proliferation and migration abilities of the HPAEC cells in model group were enhanced(P<0.01),and the expressions of CD31 and VE-cadherin were decreased,while the expressions of FSP1 andα-SMA were increased.Further study found that the expressions of Notch1,Jagged1 and CBF1 were up-regulated(P<0.01).After the intervention of the serum containing Buyang Huanwu Tang,compared with model group,the proliferation and migration abilities of cells were decreased(P<0.05,P<0.01),and the expressions of CD31 and VE-cadherin were on the rise,while the expressions of FSP1 andα-SMA were on the decline.The expressions of Notch1,Jagged1 and CBF1 were also significantly lower than those in model group(P<0.05,P<0.01).With the increase of serum concentration,the effect was more obvious.Conclusion:The serum containing Buyang Huanwu Tang can partly inhibit the EndMT in human pulmonary artery endothelial cells,which may be related to the regulation effect of Jagged1/Notch1 signaling.