1,25-二羟维生素D3通过TGF-β1(/Smad2/3)影响的ROS调节气道重塑

Effects of 1,25-dihydroxyvitamin D3 on ROS regulating airwayremodeling through TGF-β1/(Smad2/3)

目的探讨1,25-二羟维生素D3[1,25-(OH)2D3]通过转化生长因子-β1(TGF-β1)/(Smad2/3)对活性氧(ROS)的影响及调节气道重塑的分子机制.方法选取健康雌性Balb/c小鼠24只,随机分为正常组(A组)、哮喘组(B组)、1,25-(OH)2D3+哮喘组(C组).B组和C组于第0、7、14天腹腔注射0.5 mL致敏液(10μg卵清蛋白与2 mg铝制剂),之后用卵清蛋白雾化吸入,于第21~27天每天雾化1次,第28~77天隔天雾化1次制备小鼠支气管哮喘气道重塑模型.C组在每次雾化激发前30 min予以腹腔注射100 ng 1,25-(OH)2D3,实验过程持续至少77 d.收集小鼠肺组织分别用于HE染色、AB-PAS染色、Masson染色分析小鼠气道病理形态改变、气道黏液高分泌及气道重塑,运用计算机图像分析系统评价各组气道重塑情况.用免疫荧光检测气道ROS的表达,Western blot检测TGF-β1、Smad2/3的表达水平.结果B组较A组气道受损明显,可见大量炎性细胞浸润,黏液分泌增加,上皮下胶原沉积明显增多,与B组相比,C组病理形态损伤表现明显缓解,但仍较A组加重;B组较A组ROS,TGF-β1、Smad2/3蛋白的表达水平明显升高.与B组相比,C组ROS,TGF-β1、Smad2/3蛋白的表达降低,但仍较A组升高(P<0.01).结论1,25-(OH)2D3可能通过抑制TGF-β1/(Smad2/3)的表达从而减少ROS水平,达到调节小鼠气道重塑的作用.

Objective To explore the effect of 1,25-dihydroxyvitamin D3[1,25-(OH)2D3]on reactive oxygen species(ROS)through transforming growth factor-β1(TGF-β1)/(Smad2/3)and molecular mechanism of regulating airway remodeling.Methods Twenty-four healthy female Balb/c mice were randomized into control group(group A),asthma group(group B),and asthma treated with 1,25-(OH)2D3 group(group C).The group B and group C were sensitized with ovalbumin via intraperitoneal injection at the day 0,7,and 14,and ovalbumin was also given by aerosol inhalation,once a day from the day 21 to 27,and once every other day from day 28 to 77 to build the airway remodeling model of bronchial asthma of mice.Group C was given 100 ng of 1,25-(OH)2D3 via intraperitoneal injection half an hour before each aerosol challenge,and the experimental process lasted at least 77 days.The lung tissues of mice were collected for HE staining,AB-PAS staining,and Masson staining to analyze the airway pathological changes,high secretion of airway mucus,and airway remodeling status.The computer image analysis system was used to evaluate the airway remodeling.Airway ROS expression was tested by immunofluorescence.The expression levels of TGF-β1 and Smad2/3 were tested with Western blot assay.Results Compared with group A,the airways was significantly damaged in group B with large amount of inflammatory cell infiltration,increased mucus secretion and increased subepithelial collagen deposition.Compared with group B,the pathological and morphological damages were significantly relieved in group C,but still more severe than those of group A.The expression levels of ROS,TGF-β1 and Smad2/3 proteins were significantly higher in group B than those in group A.Compared with group B,the expressions of ROS,TGF-β1 and Smad2/3 proteins were reduced in group C,but still higher than those in group A(P<0.01).Conclusion 1,25-(OH)2D3 may reduce the ROS level by inhibiting the expressions of TGF-β1/(Smad2/3),and thereby regulating the airway remodeling in mice.