桔梗总皂苷对小鼠气道平滑肌细胞增殖、迁移及Notch1/ Jagged1通路的影响

Effects of Platycodon grandiflora total saponins on the proliferation, migration and Notch1/Jagged1 pathway of mouse airway smooth muscle cells

关键词:  哮喘  桔梗总皂苷  气道平滑肌细胞  增殖  迁移  Notch1/ Jagged1信号通路

Key words:  Asthma  total saponin of Platycodon grandiflorus  Airway smooth muscle cells  Proliferation  Migration  Notch1/ Jagged1 signaling pathway

摘要:

目的：探讨桔梗总皂苷对小鼠气道平滑肌细胞(mouse airway smooth muscle cells, ASMCs)增殖、迁移及Notch1/ Jagged1信号通路相关蛋白表达水平的影响。方法：分离、培养小鼠原代ASMCs,按照对照组、PDGF组、10 μmol/L桔梗总皂苷组、20 μmol/L桔梗总皂苷组、40 μmol/L桔梗总皂苷组和IL-17+40 μmol/L桔梗总皂苷组随机分组给药；Notch1过表达慢病毒感染ASMCs,按照对照组、PDGF组、PDGF+40 μmol/L桔梗总皂苷组、PDGF+40 μmol/L桔梗总皂苷+pEGF-C1 (Notch1过表达阴性对照质粒)组和PDGF+40 μmol/L桔梗总皂苷+pEGF-C1/Notch1 (Notch1过表达质粒)组分组给药。CCK8、划痕实验分别检测ASMCs的增殖和迁移能力,免疫荧光检测PCAN表达水平,ELISA检测ASMCs上清液中炎症相关因子水平,Western Blot检测ASMCs中Notch1/ Jagged1通路相关蛋白表达水平。结果：不同剂量的桔梗总皂苷均能不同程度地抑制PDGF诱导的细胞增殖、迁移,抑制ASMCs中PCNA荧光表达增强,显著抑制上清液中IL-6、IL-8水平及Notch1、Jagged1、Hes1蛋白表达水平的上调(P<0.05或P<0.01)；与40 μmol/L桔梗总皂苷组比较,IL-17+40 μmol/L桔梗总皂苷组细胞活力、迁移率及上清液中IL-6、IL-8水平均显著升高(P<0.05或P<0.01),PCNA荧光表达强度显著增强(P<0.01),Notch1、Jagged1、Hes1蛋白表达水平显著上调(P<0.05或P<0.01)。与40 μmol/L桔梗总皂苷+pEGF-C1组比较,40 μmol/L桔梗总皂苷+pEGF-C1/Notch1组ASMCs的细胞活力、迁移率及上清液中IL-6、IL-8水平均显著升高(P<0.05或P<0.01),Notch1、Jagged1、Hes1蛋白表达水平显著性上调(P<0.01)。结论：桔梗总皂苷对ASMCs的增殖和迁移有抑制作用,其作用机制可能与下调Notch1/ Jagged1信号通路相关蛋白表达水平有关。

Abstract:

OBJECTIVE To investigate the effects of total saponin of Platycodon grandiflorus on the proliferation and migration of airway smooth muscles (ASMCs) and the expression levels of Notch1/Jagged1 signaling pathway-related proteins in asthmatic mice. METHODS Primary mouse ASMCs were isolated and cultured, and administered in randomized groups according to the control group, PDGF group, 10 μmol/L Platycodon total saponins group, 20 μmol/L Platycodon total saponins group, 40 μmol/L Platycodon total saponins group and IL-17+40 μmol/L Platycodon total saponins group. Notch1 overexpression lentivirus infected ASMCs, according to the control group, PDGF group, PDGF+40 μmol/L Platycodon total saponins group, PDGF+40 μmol/L Platycodon total saponins+pEGF-C1 (Notch1 overexpression negative control plasmid) group and PDGF +40 μmol/L Platycodon total saponins+pEGF-C1/Notch1 (Notch1 overexpression plasmid) group administration. The proliferation and migration ability of ASMCs were detected by CCK8 and scratch assay, the expression level of PCAN was detected by immunofluorescence, the level of inflammation-related factors in ASMCs supernatant was detected by ELISA, and the expression level of Notch1/Jagged1 pathway-related proteins in ASMCs was detected by Western Blot. RESULTS Different doses of Platycodon total saponins can inhibit PDGF-induced cell proliferation and migration to varying degrees, inhibit the enhancement of PCNA fluorescence expression in ASMCs, significantly inhibit the levels of IL-6 and IL-8 in the supernatant and the expressions of Notch1, Jagged1, and Hes1 proteins Level up (P<0.05 or P<0.01). Compared with the 40 μmol/L Platycodon total saponins group, the cell viability, mobility and the levels of IL-6 and IL-8 in the supernatant of the IL-17+40 μmol/L Platycodon total saponins group were significantly increased (P<0.05 or P<0.01), PCNA fluorescence expression intensity was significantly enhanced (P<0.01), Notch1, Jagged1, Hes1 protein expression levels were significantly up-regulated (P<0.05 or P<0.01). Compared with the 40 μmol/L Platycodon total saponins+pEGF-C1 group, the cell viability, migration rate and IL-6 and IL-8 levels in the supernatant of ASMCs in the 40 μmol/L Platycodon total saponins+pEGF-C1/Notch1 group were significantly increased (P<0.05 or P<0.01), and the protein expression levels of Notch1, Jagged1, and Hes1 were significantly increased (P<0.01). CONCLUSION The total saponin of Platycodon grandis inhibited the proliferation and migration of ASMCs, and its mechanism of action may be related to the down-regulation of Notch1/ Jagged1 signaling pathway-related protein expression levels.