| Objective:The chronic obstructive pulmonary disease combined with pulmonary interstitial fibrosis (PIF-COPD) is an inevitable trend and pathological outcome of the chronic obstructive pulmonary disease,but were different to idiopathic pulmonary fibrosis (IPF).The fibrosis proliferation could be seen in peri-bronchial area,especially in the bronchial under grade VI.The main component of collagen in fibrosis area was identified as typeⅢ.Peri- bronchial fibrosis,interrupt of elastic fibers in bronchial wall caused the bronchial lumen narrowed or collapsed making both airway obstruction and ventilation insufficiency deteriorated.This study aimed to observe the effect of the cigarette and LPS on the chronic obstructive pulmonary disease with pulmonary interstitial fibrosis in rats,and discuss the mechanisms of PIF-COPD,through detecting the level of transforming growth factor-β1 and the tumour necrosis factor-αin the serum and BALF respectively as well as the level of typeⅢcollagen.The correlation between them was analyzed so as to provide the new idea about its prevention,diagnose and the therapy of anti-inflammatory combined with anti-fibrosis of PIF-COPD for clinic.Methods:twrenty-four male Sprague Dawley (SD) rats(provided by Center of Experiment Animal of Hebei Medical University), eight 250-300g, ere randomly divided into three groups:normal control group (group A), 8-day model group (group B),42-day model group (group C).Group B and C were exposed in the box (40cm×50cm×60cm) by five cigarette inhalation respectively one hour each time and two times in one day.In the 1st day and the 15th day,the group B was exposed in tratracheal by LPS (0.2ml/200ug). In the 1st day﹑15th and 28th day,the group C was exposed in tratracheal by LPS.All operations were under the aseptic condition.After anaeathesia with 10% Chloral Hydrate (40mg/Kg) in the abdominal cavity injection,center incisal opening of the neck was made to fully expose the trachea.The rats were punctured from trachea gap of cricoid cartilage,poured slowly LPS,then kept in erect position and revolved gently to distribute physic liquor fully in the lung.Eight rats in each group were sacrificed on 28 and 42 days respectively. The left lung was excised for HE,Mosson Staining and detecting of lung coefficient.The level of typeⅢcollagen was measured by immunohistochemistry,and positive rate of typeⅢcollagendeterminedby sem quantitative picture analysis. Bronchoalveloar lavage fluid was obtained by bronchoalveloar lavage with 0.9% salin in BALF subgroup. BALF was collected and centrifugated (1200rpm, 4℃,10min).The sediment was infused with 0.9% salin to be counted the total leukocytes and PMN numbers.Supernatant fluid was stored in -80℃until processing. The total protein in BALF was determined by using the Comas Blue assay. The pro-inflammatory cytokines TNF-αand TGF-β1 both in serum and BALF.Results:1 In group B ,the total leukocytes and PMN numbers were higher than those of control group (P<0.01).Comparing to control group,the percentage of macrophage decreased in group B and in group C. However,the lymphocyte numbers were higher than control group.2 HE Staining on the lung tissue:The lung of group control rat had no change lesion in every point, but that of group B showed the features of pulmonary emphysema and acute inflammation: the alveolar wall were rupted and there were a few of macrophage and neutrophil in alveolar space ,with fibroblast increasing.The group C, alveolitis relieved, but hemorrhage and edema were still existing, with fibroblast increasing, interval widening obviously, collagen deposited and patches of fibrosis. alveolitis did not relieved obviously. Alveolar structure was destructed with fibroblast aggravated and alveolar space vanished.3 Masson Staining on the lung tissue:There was little collogen which color was blue in the microscope in group A. In group B, there was more collogen than that in group A, which collogen was obviously increased with the time going. In group B and C, this appearance was slightly increaing, and it was less than that in group A.4 typeⅢcollagen in lung tissue percentage:The result of immunohistochemistry suggested that the expression of typeⅢcollagen in lung tissue of group B and C were much higher than group A,and there were significant difference in groups (p<0.01).5 Concentration and percentage of TGF-β1:The concentrations of TGF-β1 in blood serum and in BALF of the model groups on all the time points were all higher than normal group A,but there were no statistic significance (p>0.05). the group C (17.5929) was higher than the group B (16.1554) in blood serum and it (376.929) was higher than the group B(290.946)in BALF too.The tendency of TGF-β1 in blood serum was the same as that of in BALF.6 Content of TNF-αin blood serum and BALF:The concentrations of TNF-αin blood serum of the model B group were higher than group A,there was statistic significance in group B (p<0.05). The concentrations of TNF-αin blood serum of the group C were lower than normal group,there was statistic significance in group C (p<0.05).7 The pulmonary function in different group:In model group B and C,percentage of forced expiratory volume in first 0.3 second to forced vital capacity (FEV0.3/FVC%), peak expiratory flow (PEF),maxium midexpiratory flow (MMF) and dynamic lung compliance (CLdyn) were lower in comparison with control group (P<0.05),While inspiratory resistance (Ri) and expiratory resistance (Re) increased (P<0.05). In model group C, peak expiratory flow (PEF), maxium midexpiratory flow (MMF) and dynamic lung compliance (CLdyn) were lower in comparison with B (P<0.05),While inspiratory resistance (Ri) and expiratory resistance (Re) increased (P<0.05).Conclusions:1 The rats model of the chronic obstructive pulmonary disease combined with pulmonary interstitial fibrosis which were exposed in cigarette and LPS-induced were successful.2 The cigarette and LPS might participate in the formation of the chronic obstructive pulmonary disease with pulmonary interstitial fibrosis through TNF-αand TGF-β1.3 There is the same tendency between the degree of pulmonary and the content of the typeⅢcollagen of the tissue.
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