1,25-二羟维生素D3对被动致敏人气道平滑肌细胞的抑制作用

目的 检测1,25-二羟维生素D31,25-(OH)2D3]对被动致敏的人气道平滑肌细胞(HASMC)的增殖及其表达基质金属蛋白酶-9(MMP-9)和解整合素-金属蛋白酶33(ADAM33)的影响,探讨其调节支气管哮喘(简称哮喘)患者气道重塑的可能机制.方法用10%哮喘患者血清被动致敏HASMC,以10%非哮喘患者血清为对照.四甲基偶氮唑盐(MTT)法检测不同浓度1,25-(OH)2D3对HASMC细胞增殖活力的变化并确定其有效作用浓度.然后以有效作用浓度的1,25-(OH)2D3预处理HASMC,MTT法测定细胞增殖活力,流式细胞仪测定细胞周期,实时荧光定量PCR及蛋白免疫印迹法分别检测细胞中MMP-9及ADAM33的表达情况.结果 (1)1,25-(OH)2D3在(10-9～10-7)mol/L浓度下能浓度依赖性地抑制被动致敏的HASMC增殖(P<0.05);(2)10-7 mol/L的1,25-(OH)2D3能时间依赖性地抑制被动致敏的HASMC增殖并特异性抑制细胞周期中G1/S的转化;(3)VD组MMP-9及ADAM33蛋白表达较哮喘组分别下降了(63.4±3.6)%和(50.9±2.9)%,但仍显著高于对照组(P<0.01);(4)VD组MMP-9及ADAM33 mRNA表达较哮喘组分别下降了(52.2±2.5)%和(67.8±3.2)%,但仍显著高于对照组(P<0.01).结论 1,25-(OH)2D3能从多个层面抑制被动致敏的HASMC的功能,这可能是其调节哮喘气道重塑的作用机制之一.

Abstract：

Objective To investigate the effects of 1,25-(OH)2D3 on the proliferation of passively sensitized human airway smooth muscle cells(HASMCs) and their expressions of MMP-9 and a disintegrin and metalloprotease 33(ADAM33). Methods HASMCs were passively sensitized with 10% serum from asthmatic patients. MTT colorimetri assay was used to examine the effect of 1,25-(OH)2D3 on cell proliferation at different concentrations(10-10 mol/L, 10-9 mol/L, 10-8 mol/L, 10-7 mol/L).By this way, its optimal inhibitory concentration was determined. And then the effects of 1,25-(OH)2D3 at the optimal concentration on cell proliferation was examined by the same MTT assay and cell cycle analysis by flow cytometry. The expressions of MMP-9 and ADAM33 in HASMCs were studied by real-time quantitative RT-PCR and Western blotting analysis. Results (1)Inhibition of cell proliferation by 1,25-(OH)2D3 was barely detectable at 10-10 mol/L. But with the increasing concentration ranging from 10-9 mol/L to 10-7 mol/L, 1,25-(OH)2D3 markedly inhibited the cell proliferation concentration-dependently and reached the maximum effect at the concentration of 10-7 mol/L.Accordingly,10-7 mol/L was chosen as the optimal concentration of 1,25-(OH)2D3 for the following study. (2)At the concentration of 10-7 mol/L,1,25-(OH)2D3 inhibited the cell proliferation of passively sensitized HASMCs in a time-dependent manner and hampered the G1/S transition. (3)1,25-(OH)2D3 pretreatment attenuated the MMP-9 and ADAM33 protein levels in passively sensitized HASMCs by (63.4±3.6)% and (50.9±2.9)%,respectively (P<0.01). (4)1,25-(OH)2D3 significantly inhibited the MMP-9 and ADAM33 mRNA levels in passively sensitized HASMCs by (52.2±2.5)% and (67.8±3.2) %, respectively (P<0.01). Conclusion 1,25-(OH)2D3 has a direct inhibitory effect on passively sensitized HASMCs in vitro,including the inhibition of cell proliferation and the expressions of MMP-9 and ADAM33,which maybe associated with the beneficial role of 1,25-(OH)2D3 in the prevention and therapy of asthmatic airway remodeling.

Inhibition of 1,25-(OH)2D3 on passively sensitized human airway smooth muscle cells

Objective To investigate the effects of 1,25-(OH)2D3 on the proliferation of passively sensitized human airway smooth muscle cells(HASMCs) and their expressions of MMP-9 and a disintegrin and metalloprotease 33(ADAM33). Methods HASMCs were passively sensitized with 10% serum from asthmatic patients. MTT colorimetri assay was used to examine the effect of 1,25-(OH)2D3 on cell proliferation at different concentrations(10-10 mol/L, 10-9 mol/L, 10-8 mol/L, 10-7 mol/L).By this way, its optimal inhibitory concentration was determined. And then the effects of 1,25-(OH)2D3 at the optimal concentration on cell proliferation was examined by the same MTT assay and cell cycle analysis by flow cytometry. The expressions of MMP-9 and ADAM33 in HASMCs were studied by real-time quantitative RT-PCR and Western blotting analysis. Results (1)Inhibition of cell proliferation by 1,25-(OH)2D3 was barely detectable at 10-10 mol/L. But with the increasing concentration ranging from 10-9 mol/L to 10-7 mol/L, 1,25-(OH)2D3 markedly inhibited the cell proliferation concentration-dependently and reached the maximum effect at the concentration of 10-7 mol/L.Accordingly,10-7 mol/L was chosen as the optimal concentration of 1,25-(OH)2D3 for the following study. (2)At the concentration of 10-7 mol/L,1,25-(OH)2D3 inhibited the cell proliferation of passively sensitized HASMCs in a time-dependent manner and hampered the G1/S transition. (3)1,25-(OH)2D3 pretreatment attenuated the MMP-9 and ADAM33 protein levels in passively sensitized HASMCs by (63.4±3.6)% and (50.9±2.9)%,respectively (P<0.01). (4)1,25-(OH)2D3 significantly inhibited the MMP-9 and ADAM33 mRNA levels in passively sensitized HASMCs by (52.2±2.5)% and (67.8±3.2) %, respectively (P<0.01). Conclusion 1,25-(OH)2D3 has a direct inhibitory effect on passively sensitized HASMCs in vitro,including the inhibition of cell proliferation and the expressions of MMP-9 and ADAM33,which maybe associated with the beneficial role of 1,25-(OH)2D3 in the prevention and therapy of asthmatic airway remodeling.