| Interstitial lung disease is a progressive disorder with an inflammatory cell infiltration, fibroblast proliferation and accumulation of extracellular matrix proteins and collagen for the pathologic characteristics, and finally substitutes normal lung tissue. Recently years, we had carried on the experimental research of preventing and curing of IPF with LTI, and already obtained the initial results. But the mechanism that LTI delays or deteriorates the lung fibrosis is unclear.Chapter 1:Study of the Antagonistic Effects of LTI on Bleomycin-induced Pulmonary FibrosisObjectives On the basis of our research in the past, we utilized an animal model of pulmonary fibrosis through a classic method of intratracheal instillation of bleomycin. The formative procedure of pulmonary fibrosis was interfered through a usage of Leukotriene inhibitor(LTI), Zileuton, so as to investigate the probalble mechanisms of LTI antagonises the bleomycin-induced pulmonary fibrosis in rats.Methods Seventy-two adult male Sprague-Dawley rats were divided randomly into three groups(n=24 in each group):the model group of bleomycin(Group B), the control group(Group C) and the LTI group (Group T). The rats of Group B and Group T were given LTI, Zileuton, at a dose of 100mg/kg/day after a single intratracheal instillation of bleomycin at a dose of 5mg/kg. The rats of Group B were treated with saline instead of Zileuton after the instillation of bleomycin. The rats of Group C were treated with a same volume of saline instead of Zileuton and bleomycin. After the beginning of the experiment,8 rats in each group were sacrificed on the 7th, the 14th and the 28th day respectively. The lungs were harvested for the histopathlogical examination and for the determination of hydroxyproline concentration, and a method of immunohistochemistry was used to detect the expression of a-SMA in the lung tissue.Results (1)The lung coefficients:the lung coefficient of Group B was significantly higher than that of Group C after intratracheal instillation of bleomycin (p<0.01); the lung coefficient of Group T was lower than that of Group B after LTI treatment (p<0.05). (2)The degree Scores of alveolitis:at all time points, the scores of Group B were higher than that of Group C(p<0.01 or p<0.05), the scores of Group T were lower than that of Group B (p<0.05) but higher than that of Group C(p<0.05). (3)The degree Scores of the lung fibrosis (Masson stain):the scores of Group B were higher than that of Group C on the 7th day (p<0.05), the 14th day (p<0.01) and the 28th day (p<0.01); the scores of Group T were lower than that of Group B on the 14th day (p< 0.05) and 28th day (p< 0.01), but were still higher than that of Group C at all the observing time points (p<0.05). (4)The hydroxyproline contents of the lung:on the 14th day and the 28th day, the contents of Group B were significantly higher than that of Group C (p<0.01), the contents of Group T were lower than that of Group B (p< 0.05); on the 28th day, the contents of Group T were significantly lower than that of Group B (p<0.025). (5)The expression degrees of a-SMA in the lung: on the 14th day and the 28th day, the expression degrees of Group B were significantly stronger than that of Group C (p<0.01). The expression degrees of Group T were significantly more weaker than that of Group B on the 14th day(p<0.05) and the 28th day(p<0.01), but the expression degrees of Group T were still significantly stronger than that of Group C (p<0.01)Conclusions 1. Intratracheal instillation of bleomycin can duplicate successfully the animal models of pulmonary fibrosis.2. Leukotrienes participate the formation of prophase alveolitis and anaphase pulmonary fibrosis.3. LTI can decrease the content of hydroxyproline in the lung tissue homogenate, depress the collagen metabolism, alleviate the degrees of prophase alveolitis and anaphase pulmonary fibrosis of the bleomycin-induced pulmonary fibrosis in rats.Chapter 2:Study of the Relationship between LTI and TH1/TH2 Cytokines in Bleomycin-induced Pulmonary FibrosisObjectives (1)To observe the changes of TH1/TH2 cytokines in the formative procedure of bleomycin-induced pulmonary fibrosis in rats. (2)To assess the effect of Leukotriene Inhibitor on IFN-y and IL-4 in the lung tissue. (3)To investigate whether LTI can regulate the unbalance of TH1/TH2 cytokines in bleomycin-induced pulmonary fibrosis.Methods The preparation of a model of bleomycin-induced pulmonary fibrosis in rats is similar to that in part one, the interference of LTI was the same too. The obtained rat right lung tissues of bleomycin-induced pulmonary fibrosis were frozen and stored in the refrigerator. Than, the lung tissue was made to homogenate and an enzyme linked immunosorbent assay was used for the contents of cys-LTs, LTB4, INF-y and IL-4.Results (1)The contents of cys-LTs in the lung tissue:the contents of Group B were significantly higher than that in Group C after intratracheal instillation of bleomycin (p<0.001), but the contents of cys-LTs in the Group T were lower than that in Group B after LTI treatment on the 7th day and the 28th day (p< 0.05), and were significantly lower than that on the 14th day (p<0.025). (2)The contents of LTB4 in the lung tissue:the contents of Group B were obviously higher than that of Group C after intratracheal instillation of bleomycin (p<0.001). Compared with group B, the contents of group T were obviously decreased on the 7th day (p<0.005), the 14th day (p<0.005) and the 28th day (p< 0.025); but compared with the contents of group C, the contents of group T were still obviously higher on the 7th day (p< 0.001), the 14th day (p<0.001) and the 28th day (p<0.05). (3) The contents of INF-?in the lung tissue:the contents of Group B were significantly lower than that of Group C (p<0.001) at all the observing time points. However, the contents of Group T were significantly higher than that of Group B on the 7th day (p< 0.005), the 28th day(p< 0.001), and also were higher than that on the 14th day (p<0.05). (4)The contents of IL-4 in the lung tissue:at all the observing time points, the content of Group B was significantly higher than that in Group C (p< 0.001), the content of Group T was significantly lower than that of Group B (p< 0.001) but was significantly higher than that of Group C (p<0.005) espetially on the 14th day (p<0.005) and the 28th day (p<0.001)Conclusions 1. LTI can decrease the contents of TH1 cytokines, Cys-LTs and LTB4, in the lung tissue of the bleomycin-induced pulmonary fibrosis in rats.2. LTI can increase the contents of TH2 cytokines, INF-?and IL-4, in the lung tissue of the bleomycin-induced pulmonary fibrosis in rats.3. LTI may reduce the degree of the prophase alveolitis of the bleomycin-induced pulmonary fibrosis in rats through modulating the balance of the balance of TH1/TH2 cytokines.Chapter 3:The Influence of LTIon the Expression of Smads, TGF-?Signal Transduction Factors, in Bleomycin-induced Pulmonary FibrosisObjectives To observe the expressive variations of TGF-?1, Smad3, Smad4 and Smad7 through the technique of in situ hybridization (ISH). Meanwhile, observe the influence of LTI on the above signal proteins in the formative procedure of bleomycin-induced pulmonary fibrosis in rats.Methods The preparation of a model of bleomycin-induced pulmonary fibrosis in rats is similar to that in part one, the procedure and method of the interference by LTI are also similar to that in part one.Inferior lobe of left lungs of three group rats were resected. Tissue sections were processed, embedded in paraffin and cut into 5-?m sections. After the tissue sections were fixed, streptomycin avidin -peroxidase (SABC) immunohistochemisty staining was applied to detect the expression of TGF-?1 and ISH using digoxin(DIG)-labelled probes was applied to detect the expression of messenger ribonucleic acid (mRNA) of Smads.Results (1)The expression degrees of TGF-?1 in the lung tissue: at all the time points for observation, the expression degrees of TGF-?1 in Group B and Group T were significantly stronger than that in Group C (p<0.001), the expression degrees on the 7th day were the most strongest. The expression degrees of TGF-?1 in the Group T were decreased than that in Group B (p<0.05). (2) The expression degrees of Smad3 in the lung tissue:Most of Smad3 were expressed in the plasms of macrophagocyte, type?penumonocyte, fibroblasts and the hyperplasia bronchiole epithelium of the lung tissue. There is no change of expressional degrees of smad3 in Group C between the 7th day,14th day and the 28th day. Beginning from the 7th day, the expressional degrees of Smad3 in Group B were increased than that in Group C (p< 0.005) after intratracheal instillation of bleomycin at all three observation time points. It was the most stronger of the expressional degree of Smad3 on the 14th day. Compared with the expressional degree of Smad3 in Group B, the expressional degrees of Smad3 in Group T were decreased (p<0.05) at all the observation time points, but it is still more stronger than that in Group C (p<0.05). (3)The expressional degrees of Smad4 in the lung tissue:Similar with the expressional degrees of Smad3, Most of Smad4 were expressed in the plasma of macrophagocytes, type II penumonocytes, fibroblasts and the hyperplasia bronchiole epithelium cells of the lung tissue. The expressional degree of Smad4 in Group B was significantly increased than that in Group C (p < 0.001) at all three observation time points. It is the most stronger of the expressional degree of Smad3 on the 14th day. Compared with group B, the expressional degree of Smad4 in Group T was decreased (p<0.05) at all three observation time points, but it was still significantly increased than that in Group C (p<0.005).(4)The expressional degrees of Smad7 in the lung tissue:The expressional position of Smad7 was different from that of Smad3 and smad4. Most of Smad7 were expressed in the nuclears of macrophagocytes, type II penumonocytes, fibroblasts and the hyperplasia bronchiole epithelium cells of the lung tissue. Beginning from the 7th day, the expressional degree of Smad7 in Group B was significantly decreased than that in Group C (p<0.001) at all three observation time points. And it was the most lower on the 14th day, but it increased again on the 28th day.The expressional degree of Smad7 in group T was increased than that in the Group B(p<0.05) on the 7th day, the 14th day and the 28th day. However, the expressional degree of Smad7 in the Group T was still lower than that in Group C(p?0.005)at all three observation time points.Conclusions1. The expressional degree of TGF-?1 was increased in the lung tissue of the bleomycin-induced pulmonary fibrosis in rats. LTI may decrease the TGF-?1 expressional lever of the bleomycin-induced pulmonary fibrosis in rats. 2. The expressional degrees of R-Smad (Smad3) and Co-Smad (Smad4) were increased in the plasma of macrophagocytes, type?penumonocytes, fibroblasts and the hyperplasia bronchiole epithelium cells of the lung tissue of the bleomycin-induced pulmonary fibrosis in rats, but the expression of I-Smad (Smad7) was decreased in the neuclear of macrophagocytes, type?penumonocytes, fibroblasts and the hyperplasia bronchiole epithelium cells of the lung tissue. LTI may decrease the expressional degrees of R-Smad (Smad3) and Co-Smad (Smad4) in the plasms of macrophagocytes, type?penumonocytes, fibroblasts and the hyperplasia bronchiole epithelium cells of the lung tissue, and may increase the expressional degree of I-Smad (Smad7) in the neuclear of the above cells. This will delay the formation of the pulmonary fibrosis induced by Bleomycine. |
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