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山东大学学报 (医学版) ›› 2021, Vol. 59 ›› Issue (7): 10-18.doi: 10.6040/j.issn.1671-7554.0.2021.0335

• • 上一篇    

骨化三醇对哮喘中TGF-β1所诱导上皮间充质转化的调控作用

张倩,秦明明,何学佳,蔡秋景,张亚民,李庆苏,朱薇薇   

  • 发布日期:2021-07-16
  • 通讯作者: 朱薇薇. E-mail:weiweikeyan@163.com

Effects of calcitriol on EMT induced by TGF-β1 in asthma

ZHANG Qian, QIN Mingming, HE Xuejia, CAI Qiujing, ZHANG Yamin, LI Qingsu, ZHU Weiwei   

  1. Department of Pediatrics, Jinan Central Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250013, Shandong, China
  • Published:2021-07-16

摘要: 目的 探究骨化三醇对TGFβ1所诱导的人支气管上皮细胞(BEAS-2B)上皮-间充质转化(EMT)的影响,为哮喘气道重塑的防治提供理论依据。 方法 筛选TGF-β1作用于BEAS-2B,诱导EMT的最佳时间,将细胞分为空白组与24、48、72 h TGF-β1组;筛选TGF-β1作用于BEAS-2B,诱导EMT的最佳浓度,将细胞分为空白组与0.1、1、10、100 ng/mL TGF-β1组;加入骨化三醇预处理BEAS-2B细胞,将细胞分为空白组、TGF-β1组、骨化三醇组、TGF-β1+骨化三醇组。Western blotting检测各组细胞E-Cadherin、N-Cadherin、p-Akt、p-mTOR的蛋白表达量;Transwell法和划痕试验检测各组细胞的迁移能力。 结果 Western blotting结果显示,空白组与24、48、72 h TGF-β1组细胞E-Cadherin蛋白表达量总体差异均有统计学意义(F=53.245, P<0.001),N-Cadherin蛋白表达量的总体差异有统计学意义(F=54.429, P<0.001);与空白组相比,1、10、100 ng/mL TGF-β1组细胞E-Cadherin蛋白表达量总体差异有统计学意义(F=27.368, P<0.001),N-Cadherin蛋白表达量的总体差异有统计学意义(F=14.272, P<0.001),其中10 ng/mL TGF-β1处理细胞48 h,与空白组比较差异最为显著;TGF-β1可激活PI3K/Akt信号通路相关蛋白的表达,以及诱导BEAS-2B间质标志物的表达,TGF-β1主效应差异有统计学意义(P<0.001);加入骨化三醇预处理,可减弱TGF-β1所激活的PI3K/Akt信号通路相关蛋白的表达,亦可减弱TGF-β1所诱导的BEAS-2B间质标志物的表达,骨化三醇主效应差异有统计学意义(P<0.001),TGF-β1与骨化三醇不存在交互作用(P>0.05);Transwell检测结果和划痕试验结果表明,TGF-β1处理组BEAS-2B细胞迁移能力较空白组增强(P<0.001),与TGF-β1组相比,TGF-β1+骨化三醇组BEAS-2B细胞迁移能力减弱(P<0.001),差异均有统计学意义。 结论 骨化三醇可抑制TGF-β1所诱导的BEAS-2B EMT,从而减轻哮喘气道炎症和气道重塑的发生;这一过程可能与骨化三醇抑制TGF-β1所激活的PI3K/Akt/mTOR信号通路相关蛋白有关。

关键词: 骨化三醇, 转化生长因子-β1, 上皮-间充质转化, PI3K/Akt/mTOR信号通路, 哮喘

Abstract: Objective To investigate the effects of calcitriol on the epithelial-mesenchymal transformation(EMT)of human bronchial epithelial cells(BEAS-2B)induced by transforming growth factor-β1(TGF-β1), and to provide reference for the prevention and treatment of asthma airway remodeling. Methods The optimal time of TGF-β1 acting on BEAS-2B cells to induce EMT was screened, and the cells were divided into blank group, 24 h TGF-β1 group, 48h TGF-β1 group, and 72 h TGF-β1 group. The optimal concentration of TGF-β1 acting on BEAS-2B cells to induce EMT was screened, and the cells were divided into blank group, 0.1 ng/mL TGF-β1 group, 1 ng/mL TGF-β1 group, 10 ng/mL TGF-β1 group, and 100 ng/mL TGF-β1 group. After pretreatment with calcitriol, the cells were divided into blank group, TGF-β1 group, calcitriol group, and TGF-β1+calcitriol group. The expressions of E-Cadherin, N-Cadherin, p-Akt and p-mTOR were detected with Western blotting. The migration ability of cells was detected with Transwell assay and scratch test. Results Western blotting showed statistically significant differences in the expressions of E-Cadherin(F=53.245, P<0.001)and N-Cadherin(F=54.429, P<0.001)in the blank group, 24 h TGF-β1 group, 48 h TGF-β1 group and 72 h TGF-β1 group. The expression of E-Cadherin in the 1ng/mL TGF-β1 group, 10 ng/mL TGF-β1 group and 100 ng/mL TGF-β1 group were significantly different with that in the blank group(F=27.368, P<0.001), and the expression of N-Cadherin was also different(F=14.272, P<0.001), among which the 10ng/ml TGF-β1 group for 48h showed the most significant difference. TGF-β1 induced the expression of PI3K/Akt signaling pathway related proteins and the expressions of interstitial markers in human bronchial epithelial cells, and the effects of TGF-β1 were statistically significant(P<0.001). However, calcitriol attenuated the effects, and the effects of calcitriol were statistically significant(P<0.001). There was no interaction between TGF-β1 and calcitriol(P>0.05). The results of Transwell test and scratch test showed that the migration ability of BEAS-2B cells in TGF-β1 treatment group was increased compared with that in the blank group(P<0.001). Compared with the TGF-β1 group, the TGF-β1+calcitriol group had decreased migration ability(P<0.001). Conclusion Calcitriol can inhibit the EMT bronchial epithelial cells induced by TGF-β1, thereby reducing airway inflammation and airway remodeling in asthma, which may involve calcitriol reducing the TGF-β1-activated proteins of PI3K/Akt/mTOR signaling pathway.

Key words: Calcitriol, Transforming growth factor-β1, Epithelial-mesenchymal transition, PI3K/Akt/mTOR pathway, Asthma

中图分类号: 

  • R725
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