The Effects And Mechanisms Of Thalidomide On Bleomycin-induced Pulmonary Fibrosis In Rats

Objective : pumlonary interstitial fibrosis is a chronic progressive disorder characterized by inflammation, fibroblast proliferation and accumulation of extracellular matrix proteins. It can occur as a result of a large number of conditions. Idiopathic pulmonary fibrosis (IPF) is defined as a specific form of chronic fibrotic interstitial pneumonia associated with the histopathological appearance of usual interstitial pneumonia. It is manifested as a restrictive ventilatory defect with impaired gas exchange, culminating in respiratory failure and death. Despite such a poor prognosis, the etiology of IPF remains unknown, and no effective therapeutic strategy has yet been established. Current therapy usually includes corticosteroids with or without a cytotoxic agent. But the effects of the immunosuppressive therapy are limited, and the adverse effects cannot be ignored. Therefore, the establishment of an alternative therapeutic strategy is urgently needed. In vitro studies disclosed that thalidomide (Thal) reduced production of IL-6, TGF-β₁, VEGF, Ang-1, and collagen synthesis from human lung fibroblasts, and both IL-6-dependent proliferation and TGFβ₁-dependent transdifferentiation of the cells, which could be the mechanism underlying the preventive effect of Thal on pulmonary fibrosis.The main purpose of this study is to research the effect of thalidomide on bleomycin-induced pulmonary fibrosis in rat. Explore its possible mechanism by detecting the contents of hypdroxyproline (HYP) in lung tissue, the expression level of TNF-α, TGF-β₁ in lung tissue and blood serum.Method:90 healthy male Sprague-Dawley(SD) rats(provided by Center Experiment Animal of Hebei Medical University) weighting 220±10g were randomly divided into three groups after one week feeding. 1) Control group (group A): 30 rats. They were injected about normal saline (0.1ml/100g) in trachea, and were given intragastric administration with normal saline (1ml/100g) once a time per day after fake establishing model, then were killed randomly on the 7^th day, the 14^th day and the 28^th day (n=10, at each time point). 2) Model group (group B): 30 rats. Pumlonary fibrosis was induced by intratracheal injection of Bleomycin A₅ (5mg/kg). They were given intragastric administration with normal saline (1ml/100g) once a time per day after establishing model, then were killed randomly on the 7^th day (n=9), the 14^th day (n=10) and the 28^th day (n=9). 3) Thalidomide group (group C): 30 rats. They were injected Bleomycin A₅ (5mg/kg) in trachea, and were treated with thalidomide solution (1g of thalidomide dissolved in 100 ml of normal saline) 100mg/kg once a time per day after establishing model, then were killed randomly on the 7^th day, the 14^th day and the 28^th day(n=10, at each time point).The rats were snipped bilateral femoral arteries for collecting the blood until died. The content of TNF-αand TGF-β₁ in blood serum were detected by the method of ELISA(enzyme linked immunosorbent assay). The inferior lobes of right lung were fixed in 4% paraformaldehyde. The levels of TNF-αand TGF-β₁ were measured by immunohistochemical test, and positive area percentage of them were determined by sem quantitative picture analysis. Estimate the degree of alveolitis and pulmonary fibrosis by HE staining, Masson staining and divide by the method of Szapiel into 4 grades.The superior lobes of right lung were made into homogenate to evaluate the concentration of HYP in lung tissue. Statistics work was done with SPSS13.0 statistical software.Results:1 The results of pulmonary pathology: The level of alveolitis of group B was more serious than that in group A on each time point(P<0.01). The level of fibrosis in group B was more serious than that in group A on the 7^th day(P<0.05), the 14^th day and the 28^th day(P<0.01). These indicate that the model of pulmonary fibrosis estabished successfully. The levels of alveolitis and fibrosis in group C were less serious than that in group B. The levels of alveolitis in group C were less serious than that in group B on the 7^th day(P<0.01). The levels of alveolitis and fibrosis in group C were less serious than that in group B on the 14^th day(P<0.01). The levels of fibrosis in group C were less serious than that in group B on the 28^th day(P<0.01).2 The change of the content of HYP in lung tissue: The content of HYP in group B shown an increase tendency accompany with the time and reached a peak on the 28^th day. Compared with group A, there were significant difference on each time point (P<0.01). The values of HYP of the rats in group C were also increased, but took on a low level. The values of HYP in group C were all lower than that in group B on the 7^th day, the 14^th day and the 28^th day(P<0.01).3 The change of the content of TNF-αand TGF-β₁ in lung tissue: The expression of TNF-αand TGF-β₁ in group B was significantly higher than that in group A and group C on the 7^th day, the 14^th day, the 28^th day. There were statistical significance on each time point (P<0.01).4 The change of the content of TNF-αand TGF-β₁ in serum: Except the 28^th day, the expression of TNF-αin group B was significantly higher than that in group A and group C on each time point. There were statistical significance on the 7^th day and the 14^th day (P<0.01). The expression of TGF-β₁ in group B was significantly higher than that in group A and group C on the 7^th day, the 14^th day, the 28^th day. There were statistical significance on each time point (P<0.01).Conclusions:1 Thalidomide can reduce collagen deposition in pulmonary interstitium and possesses some therapeutic effect in bleomycin-induced pulmonary fibrosis models in rat.2 Thalidomide?could inhibit the production of TNF-αand TGF-β₁ ,and then reduced the early Inflammatory reaction , inhibited the late?fibrotic changes. This could be the mechanism underlying the preventive effect and therapeutic effect of thalidomide on pulmonary fibrosis.