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

Objective：Pulmonary fibrosis is a lung disease that is refractory totreatment and carries a high mortality rate. It includes a heterogeneous groupof lung disorders characterized by the progressive and irreversible destructionof lung architecture caused by scar formation that ultimately leads to organmalfunction, disruption of gas exchange, and death from respiratory failure.Pulmonary fibrosis （IPF） represents the commonest and most fatal conditionwith a median survival of3–5years following diagnosis. This reflects the lackof any effective therapy to modify the course of the disease, the pathogenesisof IPF remains unknown, and currently there are no proven effectivetreatments for IPF. Standard antiinflammatory therapies, includingcorticosteroids and cytotoxic agents have shown little efficacy in IPF.Therefore novel therapeutic approaches are urgently required. Thalidomidepossesses anti-inflammatory, immunomodulatory and anti-angiogenicproperties, and has been demonstrated to attenuate bleomycin-induced fibrosisin mice. Its precise mechanism of action remains unclear, although theobserved attenuation in fibrosis following experimental lung injury isaccompanied by a reduction in VEGF expression, suggesting that inhibition ofneovascularization might represent a potential mechanism. IL-6expressionhas also been shown to be reduced in this model suggesting multiple possiblemodes.In this experiment, through detecting the level of TGF-β₁, VEGF in bloodserum and the contents of typeⅠ collagen in lung tissues to observe the effectsof thalidomide and dexamethasone on bleomycin-induced pulmonary fibrosisin rat, and explore the possible mechanism.Method：120healthy Sprague-Dawley（SD） male rats，all provided by Center Experiment Animal of Hebei Medical University，weighting210±10g，after one week feeding,were divided into four groups randomly.1) Controlgroup:30rats（n=10, at each time point）, injected about normal saline（0.1ml/100g） in trachea, and after fake establishing model,the rats were givenintragastric administration with normal saline （1ml/100g） once a time per day,then were killed randomly on the7thday, theβ₁4thday and the28thday.2)Model group:30rats（n=10, at each time point）. Pumlonary fibrosis wasinduced by intratracheal injection of Bleomycin A5（5mg/kg）. Afterestablishing model, the rats were given intragastric administration with normalsaline （1ml/100g） once a time per day, then were killed randomly on the7thday （n=8）, theβ₁4thday （n=10） and the28thday （n=8）.3) Thalidomide group（Thd group）:30rats（n=10, at each time point）. The rats were injectedBleomycin A5（5mg/kg） in trachea, and after establishing model were treatedwith thalidomide solution （1g of thalidomide dissolved inβ₁00ml of normalsaline）β₁00mg/kg once a time per day, then were killed randomly on the7thday, theβ₁4thday and the28thday.3) Dexamethasone group（DEX group）:30rats（n=10, at each time point）. The rats were injected Bleomycin A5（5mg/kg） in trachea, and after establishing model were treated withdexamethasone solution（0.3mg/100g/d）, then were killed randomly on the7thday, theβ₁4th day and the28th day. The rats were snipped bilateral femoralarteries for collecting the blood until died. The levels of transforming growthfactorβ1and Vascular Endothelial Growth Factor in blood serum were detectedby the method of ELISA（enzyme linked immunosorbent assay）. The inferiorlobes of right lung were fixed in4%paraformaldehyde. The expression oftype Ⅰc ollagen were measured by immunohistochemical test,and the averageoptical density of collagen were determined by sem quantitative pictureanalysis. Estimate the degree of alveolitis and pulmonary fibrosis by HEstaining, Masson staining and divide by the method of Szapiel into4grades.Statistics work was done with SPSS13.0statistical software。Results:1The results of pulmonary pathology: Model group: On the same time point, the levels of alveolitis andpulmonary fibrosis was more serious than that in control group（allP<0.01）,This shows that the rat fibrosis model were successfully established.Thalidomide group（Thd group）: The levels of alveolitis and fibrosiswere less serious compared with the model group. And on each time point, thelevels of alveolitis in thalidomide group were less serious（P<0.01）; the levelsof fibrosis in thalidomide group were less serious（P<0.05）.Dexamethasone group（DEX group）: The levels of alveolitis and fibrosiswere less serious than that in model group.On each same time, the levels ofalveolitis in dexamethasone group were less serious than that in model group（P<0.05）.On the7thday the levels of fibrosis were less serious than that inmodel group（P<0.01）, and on theβ₁4thday,the28thday the levels of fibrosiswere also less serious（P<0.05）.While we can not find the significant difference between dexamethasoneand thalidomide group on the same time point （P＞0.05）.2The expression of type Ⅰc ollagen in lung tissue （represented by theaverage optical density）:The expression of type Ⅰc ollagen inmodel group showed an increasetendency accompany with the time and reached a peak on the28thday.Compared with control group, there were significantly differenct on each timepoint （P<0.01）. The expression of typeⅠ c ollagen of the rats inthalidomideand dexamethasone group were also increased, but took on a low level. Theexpression of type Ⅰc ollagen inthalidomide and dexamethasone group wereall lower than that in model group on the7thday, theβ₁4th day and the28thday（P<0.01）. While two groups had no significant difference on the same time（P＞0.05）.3The change of the content of TGF-β₁and vascular endothelial growthfactor in serum:The content of TGF-β₁in model group was significantly higher than thatin control group, thalidomide group and dexamethasone group on the sametime point（P<0.01）. There were no statistical significance on each time point between the content of thalidomide group and dexamethasone group （P＞0.05）.The content of VEGF in model group showed an increase tendencyaccompany with the time and reached a peak on the28th day. On the7th day,theβ₁4th day and the28th day,the content in model group was significantlyhigher than that in control group, thalidomide group and dexamethasone group（P<0.01）. While between the contents of thalidomide group anddexamethasone group, there were no significant difference on the same time（P＞0.05）.Conclusions:1Thalidomide and dexamethasone reduce collagen deposition inpulmonary interstitium and possesses some therapeutic effect inbleomycin-induced pulmonary fibrosis models in rat.2Thalidomide has anti-fibrosis potency similar to dexamethasone inbleomycin-induced pulmonary fibrosis models in rat. Such effect may resultfrom reducing early inflammatory reaction and angiogenesis throughinhibition of cytokines such as TGF-β1and VEGF.