尼洛替尼对二氧化硅刺激HFL-1细胞增殖及胶原合成影响

Effect of nilotinib on silicon dioxide-induced proliferation and collagen synthesis in HFL-1 cells

目的观察尼洛替尼对二氧化硅(SiO2)刺激人胚肺成纤维细胞-1(HFL-1细胞)所致细胞增殖和胶原合成的影响,探讨相关作用机制。方法①以质量浓度为0、5、10、25、50、100 mg/L的SiO2混悬液刺激HFL-1细胞24.0 h,采用免疫印迹法检测转化生长因子-β1(TGF-β1)、C-Abl、血小板衍生生长因子受体(PDGFR)的蛋白表达,筛选后续实验的SiO2剂量。②取HFL-1细胞随机分为6组,对照组不予特殊处理,溶剂对照组细胞予体积分数为0.10%的二甲基亚砜处理;SiO2刺激组细胞予剂量为50 mg/L的SiO2混悬液刺激细胞24.0 h;5、10、15 mmol/L尼洛替尼组细胞先予剂量为50 mg/L的SiO2混悬液刺激细胞24.0 h后,再予相应浓度的尼洛替尼处理24.0 h。采用MTS法检测细胞增殖率,酶联免疫吸附实验检测细胞分泌的TGF-β1水平,免疫印迹法检测细胞内TGF-β1、C-Abl、血小板衍生生长因子(PDGF)、PDGFR和Ⅰ型胶原的蛋白表达水平。结果①后续实验的SiO2剂量设为50 mg/L。②SiO2刺激组和3个尼洛替尼组HFL-1细胞细胞增殖率均高于对照组和溶剂对照组(P<0.05),10、15 mmol/L尼洛替尼组HFL-1细胞细胞增殖率均低于SiO2刺激组(P<0.05)。SiO2刺激组HFL-1细胞TGF-β1分泌水平以及TGF-β1、Ⅰ型胶原、C-Abl、PDGFR、PDGF蛋白相对表达水平均高于对照组和溶剂对照组,15 mmol/L尼洛替尼组HFL-1细胞上述指标均低于SiO2刺激组(P<0.05),5 mmol/L尼洛替尼组HFL-1细胞上述指标与SiO2刺激组比较,差异均无统计学意义(P>0.05);10 mmol/L尼洛替尼组HFL-1细胞仅TGF-β1分泌水平和C-Abl蛋白相对表达水平低于SiO2刺激组(P<0.05)。结论尼洛替尼可抑制SiO2诱导的HFL-1细胞增殖,降低Ⅰ型胶原的表达;该过程可能是通过抑制酪氨酸激酶介导的信号通路实现。

Objective To observe the effects of nilotinib on silicon dioxide(SiO2)-induced cell proliferation and collagen synthesis in human fetal lung fibroblast-1(HFL-1)cells and to explore the related mechanism.Methodsⅰ)HFL-1 cells were induced with different doses of SiO2 suspension(0,5,10,25,50 and 100 mg/L)for 24.0 hours.The expression of transforming growth factor-β1(TGF-β1),C-Abl,and platelet-derived growth factor receptor(PDGFR)was detected by Western blot,and the dose of SiO2 in subsequent experiments was screened.ⅱ)HFL-1 cells were randomly divided into 6 groups:1)the control group:no treatment;2)the solvent control group:cells were treated with 0.10%dimethyl sulfoxide;3)the SiO2 stimulation group:cells were induced with SiO2 suspension at a dose of 50 mg/L for 24.0 hours;4)-6)the nilotinib groups:cells were induced with SiO2 suspension at a dose of 50 mg/L for 24.0 hours and treated with nilotinib at the concentration of 5,10,or 15 mmol/L for 24.0 hours.Cell proliferation was detected by MTS assay.The TGF-β1 protein secreted by cells was measured using enzyme linked immunosorbent assay.The expression of TGF-β1,C-Abl,platelet derived growth factor(PDGF),PDGFR and collagen typeⅠproteins was measured by Western blot.Resultsⅰ)The dose of the SiO2 in the experiments was set to 50 mg/L.ⅱ)The cell proliferation rate of HFL-1 cells in the SiO2 stimulation group and the 3 nilotinib groups was higher than that in control group and solvent control group(P<0.05).The proliferation rates of HFL-1 cells in 10 and 15 mmol/L nilotinib groups were lower than that in SiO2 stimulation group(P<0.05).The level of TGF-β1 and the protein relative expression levels of TGF-β1,collagen typeⅠ,C-Abl,PDGFR and PDGF in HFL-1 cells of SiO2 stimulation group were higher than those in control group and solvent control group(P<0.05).The above indexes of HFL-1 cells in 15 mmol/L nilotinib group were lower than that in SiO2 stimulation group(P<0.05);the above indexes of HFL-1 cells in 5 mmol/L nilotinib group were not significantly different from those in SiO2 stimulation group(P>0.05).The level of TGF-β1 and the relative expression level of C-Abl protein in HFL-1 cells of 10 mmol/L nilotinib group were lower than those in SiO2 stimulation group(P<0.05).Conclusion Nilotinib can inhibit the proliferation of HFL-1 cells and reduce the expression of collagen typeⅠprotein induced by SiO2.This process may be achieved by inhibiting tyrosine kinase-mediated signaling pathway.