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

doi:10.6040/j.issn.1671-7554.0.2021.0335

摘要/Abstract

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

张倩,秦明明,何学佳,蔡秋景,张亚民,李庆苏,朱薇薇. 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[J]. 山东大学学报 (医学版), 2021, 59(7): 10-18.

ZHANG Qian, QIN Mingming, HE Xuejia, CAI Qiujing, ZHANG Yamin, LI Qingsu, ZHU Weiwei. Effects of calcitriol on EMT induced by TGF-β1 in asthma[J]. Journal of Shandong University (Health Sciences), 2021, 59(7): 10-18.

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