Evaluation Of TNF-α Inhibitor And Gene Expressions Of Cytokines In Pulmonary Fibrosis In Diagnosis And Treatment

PART I Gene expressions of IL-10 and TGF-β₁ in bronchoalveolar lavagefluid and lung tissues in mice with pulmonary fibrosisBackground: The pathogenesis of pulmonary interstitial fibrosis is not fully understood now.A lot of investigations confirmed that the development of pulmonary interstitial fibrosis was related closely to many immune effector cells and the cytokines secreted by them.Especially with the development of cytology and molecular biological techniques ,more and more evidences confirm that a variety of cytokines compose of a multiple network and play an important role in lung injury,inflammatory reaction and subsequent tissue repair,among of which,IL-1,IL-10, TNF-alpha and TGF-beta1 are the cytokines studied deeply, and may play an important role in the stage of pulmonary fibrosis. Objective: To investigate the mRNA expressions of IL-1,TNF-α,IL-10 and TGF-β₁ in bronchoalveolar lavage fluid(BALF) and lung tissues in mice with pulmonary fibrosis in various periods and to study the effects of cytokines on pulmonary fibrosis. Methods: Fifty kunming mice were randomly divided into two groups:experimental group(n=25) and control group(n=25), and poured respectively by bleomycin solutions (5mg/kg) 0.2ml and the same volume of saline. Bronchoalveolar lavage fluid was collected from lungs of the two groups on 3rd,7th,14th,28th and 56th day postinstillation . RT-PCR method was used to analyse the mRNA expressions of IL-1,TNF-α,IL-10 and TGF-β₁ in BALF. Results: The mRNA expressions of IL-1,TNF-α,IL-10 and TGF-β₁ were increased in BALF in experimental group (p<0.01) ,with the maximum of IL-1 and TGF-β₁ on 28th ,IL-10 on 7th and TNF-αon 14th .â‘¡The mRNA expressions of IL-1,TNF-α,IL-10 and TGF-β₁ were increased in lung tissues in experimental group (p<0.01) ,with the maximum of IL-1 on 7th, TNF-αon 14th , IL-1 on 28th and TGF-β₁ on 56th. The expressions of them were keeping in a higher level after that . Conclusion: The periods of mRNA expression of IL-1,TNF-α,IL-10 and TGF-β₁ were different, by which the severity and activities of PF can be judged. They played an important role in the various stages of pulmonary fibrosis. PART II Role and Evaluation of Gene Expresses of Cytokines in BALF in the Patients with Idiopathic Pulmonary FibrosisBackground: Idiopathic pulmonary fibrosis (IPF)is a progressive lung disease characterized by fibroblast proliferation and extracellular matrix remodeling, whose pathogenetic mechanisms are complex and remain to be determined.At present,there haven,t been effective and full-scale methods in evaluating the severity of the disease in clinic. Objective: To investigate whether the mRNA expressions of IL-1,IL-10,TNF-αand TGF-βin bronchoalveolar lavage fluid(BALF) in patients with IPF before and after a month,s therapy with glucocorticosteroid correlate with their clinical symptoms, high-resolution CT (HRCT)and pulmonary function test(PFT). Methods: After diagnosed by the means of HRCT, PFT and transbronchial lung biopsy(TBLB),15 patients with IPF were treated with glucocorticosteroid for a month .Before and after the therapy, the characters of HRCT ,PFT and dyspnea were all scored and integrated and BALF were aquired. RT-PCR method was used to analyse the mRNA expressions of the four cytokines in BALF. Results:â‘ Lung HRCT and clinical symptom integration Before and after the therapy with glucocorticosteroid for a month, there is no significant difference in HRCT integration (p>0.05);However,dyspnea integration manifestly degraded,and there is a significant difference(p<0.01).â‘¡PFT integration Before and after the therapy with glucocorticosteroid for a month,four of PFT indexes (FVC, TLC,FEV1 and Dlco/VA) were lower than those of the control.After therapy,the integration degraded markedly,and there is a significant difference(p<0.05),especially in FVC,FEV1 and Dlco/VA.â‘¢The mRNA expressions in BALF Before and after the therapies with glucocorticosteroid for a month, the mRNA expressions of four cytokines in BALF were higher than those of the control,however, their expressions all degraded after therapy, especially in IL-1 and TNF-α(p<0.01).â‘£The relationship between cytokine and HRCT integration Before therapy with glucocorticosteroid ,there is a positive correlation between HRCT integration and the mRNA expressions of IL-10 and TGF-β, but not of IL-1 and TNF-α. After the therapy, the differences between the four cytokines and HRCT integration are not obsevered.⑤The relationship between cytokine and dyspnea Before and after the therapy with glucocorticosteroid for a month,there is a positive correlation between the mRNA expressions of four cytokines and dyspnea integration(p<0.01).â‘¥The relationship between cytokine and PFT integration Before and after therapy , there is a positive correlation between the mRNA expressions of four cytokines and PFT integration (p<0.05).⑦The relationship between dyspnea and HRCT /PFT integration Before and after therapy, there is a positive correlation between dyspnea and PFT integration(r=0.59, p<0.05),but not between dyspnea and HRCT integration(r=0.27, p>0.05). Conclusion: Dynamic observation of the mRNA expressions of four cytokines in BALF can be helpful to judge the severity of IPF,the sensitivity to corticosteriod therapy and prognosis. PART III Intervention of Etarnecept(TNF-αinhibitor)and Dexamethasone in Pulmonary Fibrosis and Their Constrast StudyBackground:Pulmonary fibrosis is a progressive and usually fatal lung disease, whose pathogenetic mechanisms are complex and remain to be determined. The incidence of the disease increased in the last few years, meanwhile for short of effective therapies,the mortality rate of which also increased .Effective and feasible therapy of pulmonary fibrosis has been an point for sholars home and abroad.,More and more evidences confirm that a variety of cytokines play an important role in the development of the disease, and are related to their overexpression or overbalance in the progress of the disease.TNF-alpha was considered as the initiator of the disorder.Therefore,to inhibit the secretion and bioactivity of the cytokine may be a novel and effective methods in the therapy of the disease. Objective: To investigate the therapeutic effects of etarnecept(TNF-αinhibitor) and dexamethasone in rats with pulmonary fibrosis and to study the effects of cytokines on pulmonary fibrosis. Methods: Ninety-six rats were randomly and equally divided into four groups: control group(N group), model group(C group) , etarnecept group (P group)and dexamethasone group(D group).Except the N group ,the other three groups were instilled with bleomycin solutions and the N group with the same volume of saline. D group , P group , and C group were respectively intraperitoneal injected with dexamethasone(3mg/kg/d), etarnecept (0.4mg/kg, twice a week) and saline 0.5ml/d. Radio-immunity was used to detect the expressions of hyaluronic acid and collagenâ…¢protein in blood serum on 7th ,21th ,28th and 42th .HE and Masson stainings were used to observe the severity of airsacculitis and pulmonary fibrosis in the lung tissue.Results:â‘ The levels of the expression of hyaluronic acid and collagenâ…¢protein were significantly increased in the model group on 7th ,21th ,.28th and 42th and a significant difference was found between the model group and the C group in the four stages(P<0.01); The levels of the expression of hyaluronic acid and collagenâ…¢protein were significantly degraded in D group on 7th ,21th ,.28th and 42th and a significant difference is found between the D group and the model group in the four stages(P<0.05); The levels of the expression of hyaluronic acid and collagenâ…¢protein in the P group were significantly higher than the model group but lower than D group on 7th ,21th ,28th and 42th and a significant difference is respectively found between the P group and the other two groups (the model group,P<0.01and the D group, P<0.01 ).â‘¡In the pathological tissue of the lung ,there is early alveolitis and late fibroblast proliferation and collagen deposition.In contrast to D group and model group , alveolitis is slighter in P group. Conclusion: Etarnecept and dexamethasone were helpful in the therapy of pulmonary fibrosis and Etarnecept outweigned dexamethasone in our experiment..In contrast to dexamethasone, Etarnecept has the advantage of degrading mortality rate ,less side effect and the control of early inflammation.