The Impact Of1,25（OH）₂D₃on Bleomycin Inducing Pulmonary Fibrosis In Mice

Objective:Cutting the trachea of the mice, and then giving a one-time injectionof bleomycin into mice trachea, to make mouse model of pulmonaryfibrosis, and then pour1,25(OH)2D3into gastric. To observe rat lungpathological changes and measure the amount of lung hydroxyproline,1/3mice in each group will be put tod eath at7th,14thand28thdays aftersurgery, and then study intervention of1,25(OH)2D3on pulmonaryfibrosis. Detect Smad3and Smad7expression of mice lung tissue in eachgroup at different time points, and then investigate1,25(OH)2D3intervention mechanism of pulmonary fibrosis to provide new options forthe treatment of pulmonary fibrosis in clinical drug therapy.Methods:There will be100male SPF C57BL/6mice,10mice will be inmotor only, and divide the remained90randomly into three groups:injecting5mg/kg bleomycin into mice trachea of the treatment group(group A) and the model group (group B); and injecting an equal volumeof saline into the trachea of the control group (group C). Since2daysafter building model, every day,pouring0.5g/kg the castor oil dilution of1,25(OH)2D3into to the stomach of the treatment group.Putting equal volume of castor oil+saline mixture into the stomach of control group.Each group of mice will be executed a third at14th,21thand28thdays.Taking the mice’s the upper lobe and fixing10%neutral formaldehyde,for paraffin section and immunohistochemical staining, and using therest of the lung tissue used liquid nitrogen frozen for determination ofhydroxyproline content. Observing the lung tissue slices under the lightmicroscopy after HE and Masson with double blind principle, and thenaccording to the unified standard to determine the classification andgrading of alveolitis and pulmonary fibrosis. Choosing kit to detecthydroxyproline content, and determining the expression of Smad3,Smad7by immunohistochemical staining method, specific operation shallbe carried out in accordance with the specifications. Control group, modelgroup and treatment group of alveolitis and pulmonary fibrosis in micewill be compared through the rank and inspection comparison；controlgroup, model group and treatment group of hydroxyproline content andSmad3and Smad7expression in mice will be compared by using analysisof variance. All experimental data will be done by SPSS21.0statisticalsoftware, statistical processing, with Significant standardα=0.05.Results:After model establishment, model group mice decrease activity andhave shortness of breath, the treatment group mice also appear afore-mentioned symptoms, but after given1,25(OH)2D3,the symptoms improved when compared with the model group mice, the mice suffermild breathing difficulties and have relatively active. There is mild edemaof lung tissue in treatment group mice at7thdays, edema, congestion andbleeder appear at14thdays,28days lung volume become smaller, whichranges between control group and model group, the quality of a materialbecome harden, scattered nodules can be seen on the surface, but thechanges are less obvious than each the model group at each course. Thealveolitis of treatment group at7thand14thdays, and the pulmonaryfibrosis degree at14thand28thdays are less obvious than the model group(P <0.05); Hydroxyproline levels of treatment group are lower than themodel group’s at14thand28thdays (P <0.01). Smad3expression oftreatment group is lower than those of model group at7th,14thand28thdays (P <0.01), and significantly increases than the control group’s (P <0.01). Smad7expression of treatment group increases more than themodel group’s at14thand28thdays (P <0.05), and is lower than thecontrol group at7th,14thand28thdays (P <0.01).Conclusion:1.After Bleomycin is injected into trachea of mice,it can make thepathological changes of mice lung infammation and fibrosis.2.In secondary interstitial pneumonia induced by bleomycin in mice,the hydroxyproline expression increases, the content of Smad3increases,and levels of Smad7decreases. 3.In secondary interstitial pneumonia induced by bleomycin in mice,1,25(OH)2D3exerts inhibitory effect on pulmonary fibrosis.4.1,25(OH)2D3can effectively restrain high expression of Smad3atthe TGF-beta1/Smad pathway, at the same time promote expressionfactor Smad7which play a negative regulatory role, and suggests that1,25(OH)2D3can play a role of anti-fibrosis and is associated withregulating the Smad protein expression.