| Objective:1.To establish the Bleomycin-induced pulmonary fibrosis model though the trachea in rats;2.To observe the effects of CKLF1-C19and hydrocortisone sodium succinate on the Bleomycin-induced pulmonary fibrosis in rats;3.To study the effect of CKLF1-C19and hydrocortisone sodium succinate on the contents of TNF-a and TGF-β1in the rat lungs;4.To study the differences of CKLF1-C19and Hydrocortisone sodium succinate on pulmonary pathology.disparity of the content of TNF-a and TGF-β1in the rat lungs.Method:The sisty healthy male wistar rats were randomly divided into four groups, with15rats in each group, normal group, model group, CKLF1-C19group and hydrocortisone group. Then anesthtizing the rats with abdominal injection of chloral hydrate in a dose of3ml.kg-1, fixing the head and limbs, then intratracheally drpipped with bleomycine to made pulmonary fibrosis model, the method is as follows:open the mouth, pulled out their tongues, while the moment rats breathing, quickly inserted the catheter into the trachea about1-2centimeters with forehead mirror. If can see the liquid surface fluctated with rats breath, the model group, CKLF1-C19group and hydrocortisone group were intratracheally drpipped with0.4%bleomycine0.25ml(5mg.kg-1)one-time, while0.9%saline in the normal group, erected the rats quickly after inject the drug, rotated the rats left and right to made solution well-distributed in the lungs. Treatments were started from the next day, the normal group and the model group were orally given0.9%saline in a dose of2ml, the CKLF1-C19group was orally given CKLF1-C19with the dose of100μg.kg-intraperitoneal hydrocortisone sodium succinate in a dose of25mg.kg-1in the hydrocortisone group.To observe the general condition of the rats (type of figure, mental state, skin, feeding, activity, debris, and so on) and weigh the rats. Five animals were randomly killed by exsanguinating the abdominal aortic in each group at day7,14and28, opening the chest, separating the lungs, observing the lung appearance.and weight the lungs.The left lungs were put into10%neutral formaldehyde solution in a fixed, conwentional paraffin embedding,cut into slices, hematoxylin and eosin-staining, evaluated degree of alveolar inflammation and pulmonary fibrosis, detected the content of TGF-by immunohistochemical method. The right lungs were put into cold normal saline to remove blood, cut into small bricks, then made into10%lung homogenate, lung homogenate was centrifugaled by3000r.min-1and the previous liquid was preserved in-80℃refrigerator, then mensurateg the content of TNF-a by ELISA method.Results:1. General condition of rats:In control group, the action, body figure, skin and feeding of rats were well. In model group, rats were found with different degree of cough and breathing difficulties, poor diet, loss of weight, out of spirit and decrement of activity dull fur without burnish. The rats’of CKLF1-C19group were similar to those in model group at early stage, the situation turned better after being treated after7days, and more better after14days, physical stature, appetite, activity were better than before. There were no obvious difference in rats’of hydrocortisone group and those of CKLF1-C19group.2.General pathological changes of lung:In control group, the rats lung were normal, the lung lobe full, pink, smooth and the flexibility well. The lung volume increased, dull red in colour, a few scattered pointlike bleeding were found in the surface in model group at day7; lung volume decreased than before, poor elastic, in addition to scattered pointlike bleeding, different sizes of gray-white nodules were found at day14; The lung volum were significantly smaller than before, pale, lung hardness were more wore than before, more gray-white nodules were found at day28. The lung lobes partly smooth, dark red, some local different sizes of gray-white nodules, some cattered pointlike bleeding in the edge, some smaller lobectomy, hardness increace could be found in CKLF1-C19group. The findings of lung morphology in hydrocortisone group were similar to those in CKLF1-C19group. 3.Lung coefficient:The lung coefficients of model group, CKLF1-C19group and hydrocortisone group increased more significantly than the control group at day7,14and28(P<0.05); the lung coefficients of CKLF1-C19group and hydrocortisone group decreased more than the model group at day7,14and28(P<0.05), their were no obvious difference in CKLF1-C19group and hydrocortisone group.4. Degree of pulmonary inflammation and fibrosis:There were completely normal parenchymal structures in the control group and no obvious morphological change were found at all observation time point. In model group, the inflammation was very apparent, inflammatory cells were infiltrated priotity with mesangial cells and lymphocytes, fibroblast cells increased, and the alveolar walls were wide at day7; more inflammatory cells were infiltrated, alveolar walls more thick, the alveolar deformated, accompanied by fibroblast cells and smooth muscle cells proliferated, collafen deposited at day14; severe fibrosis manifested by a thickening of alveolar walls, the alveolar space decrease, most of the alveolar disappeared,cell components reduced, the interstitial lung were alternatived by collagen fiber and fibroblast cells, fiber component increased more obviously than before, but the lung inflammatory cells infiltrating reduced at day28. The degree of pulmonary inflammation were decreased significantly in CKLF1-C19group and hydrocortisone group(P<0.05), and there was not obviously difference with the normal group at day28(P>0.05). The pulmonary fibrosis was alleviated compared with those in model group, but more serious than the normal group at day28(P<0.05). there was not obviously difference between the CKLF1-C19group and hydrocortisone group(P>0.05).5. The contents of TNF-a and TGF-β1of the rat lungs:The TNF-a of lung homogenate in the model group, CKLF1-C19group and hydrocortisone group were all higher than that in normal group(P<0.05), the TNF-a in CKLF1-C19group and hydrocortisone group were lower than that in the model group(P<0.05), and the difference of the TNF-a in CKLF1-C19group and hydrocortisone group was not statistically significant. From the day7to28, the TNF-a in CKLF1-C19group and hydrocortisone group were present degressive tendency. The express of TGF-β1protein in control group was weak positived, only in the cytoplasm of a few bronchial mucosal epithelium cells. In model group, the strongest expression of TGF-β1protein was found at day14, and decreased over time trend, mostly located in the cytoplasm of alveolar macrophages, bronchial mucosal epithelium, endothelial cells and fibroblast cells. The TNF-a protein in CKLF1-C19group and hydrocortisone group were lower than that in model group at day14and28(P<0.05), there was no significant statistic difference between the CKLF1-C19group and hydrocortisone group.Conclusions:1. CKLF1-C19peptide and hydrocortisone sodium succinate can partly ameliorate bleomycin-induced pulmonary fibrosis and alveolitis in rats.2. CKLF1-C19peptide and hydrocortisone sodium succinate can partly decrease TNF-a and TGF-β1content of lung, therefore can decrease the lung inflammation and suppress the development of pulmonary fibrosis induced by Bleomycin.3. The difference of the effect of CKLF1-C19peptide and hydrocortisone sodium succinate on ameliorate bleomycin-induced lung inflammation and pulmonary fibrosis was not obvious. |
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