| Objectives:To observe effect of treating rats pulmonary fibrosis induced by BLM on Fei Xian formula, and explore the pathogenesis of pulmonary fibrosis and mechanism of pulmonary fibrosis treated by formula of Fei Xian.Method:According to random number table,80 Wistar male rats, with the weight of 200±20g, were divided into four groups:blank group, model group, hormone group and TCM group, n= 20. Rats were placed in barrier-level animal house to be adaptively fed for 1 week. After tracheal intubation of model group, hormone group, and TCM group, a dosage of lmg/kg of bleomycin was bolus-injected respectively. For the blank group, it was bolus-injected equivalent volume of saline into trachea. Intragastric administration would begin in the second day after modeling. The dose of administration of Fei Xian formula in TCM group was 1.25g/(k·d); the dose of administration of prednisone in hormone group was 3mg/(kg·d); and for model group and blank group, they were given the equivalent volume of 0.5% sodium carboxymethyl cellulose. In the 14th and 28th days, these rats were sacrificed in batches. After anaesthetizing rats with an intraperitoneal injection of 3.5% chloral hydrate 1ml/100g weight, the volume of 2ml of abdominal aorta blood was withdrawn by arterial blood gas needle. It should be sent for inspection within 15 minutes.Open the chest to perfuse the lung tissues. Stop infusing until rat's head, upper limbs, and pulmonary lobes became stiff. Before hematoxylin-eosin (HE) staining and three-color (Masson) staining, a serial preparations should be carried out, like remove of the lungs to neutral formalin for fixation for 24 hours; conventional dehydration, paraffin embedding, and serial tissue sections (thickness of 5μm).HE staining section was divided into non-staining area, dark staining area, and light staining area. The images were pictured by digital microscope of OLYMPUS. Use Image-Pro plus 6.0 to analyze, to measure the area of each part, and choose their average (mm2). Calculate classification of each group's alveolitis and pulmonary fibrosis.Expression of CD34 in vascular endothelial cells of lung tissue sections of rats was detected by immunohistochemistry SABC. The vascular endothelial cells labeled with CD34 antibody were counted the number of microvessel in unit area under microscope, which was called microvessel density (MVD).Expressions of bFGF, VEGF, FLT-1, FLK-1, and ANG-2 were detected by immunohistochemistry SABC. Combined with image analysis, expression of promoting factor of microvascular angiogenesis of each group's rats could be compared.Result:Results of HE staining:There was no obvious pathological change in pulmonary morphology in bland group. In 14d model group, the alveolar septum was obviously widened, also a large number of infiltrated inflammatory cells found in alveolar septum and wall, as well as hyperplasia of fibrous connective tissue, even consolidation. In 14d hormone group, the alveolar septum was widened, and part of alveoli changed into consolidation and large quantities of inflammatory cells infiltrated. Inflammation and fibrosis foci at different degrees could be found in 14d TCM group and also consolidation around the part of small bronchi. Compared with 14d model group, hormone group, and TCM group, inflammatory signs in the same groups of 28d greatly abated. In 28d model group, alveolar septa obviously widened, collagen fibers significantly increased, fibrous tissues distributed in patch, the structure of part alveoli disappeared, shrank, and occluded, and several alveoli expanded in cyst. Compared with 14d model group, infiltration of inflammatory cells in alveolar septum and wall is in greater reduction. Mild fibrosis of alveolar septum was found in 28d hormone group. Fibrosis foci could be seen around the small bronchioles and inflammatory cells appeared in part of alveoli and bronchioles. In 28d TCM group, fibroplasias foci of alveolar septum were less, and the scattered inflammatory cells could be found in alveoli.Results of Masson staining:There was no obvious pathological change in pulmonary morphology in bland group. In 14d model group, alveolar septum widened, and the hyperplasia of blue collagen fibers could be seen around each bronchus and blood vessel. In 14d TCM group, a scanty of fine collagen fibers could be found in alveolar septa and around each bronchus. In 28d model group, a large number of hyperplasia of collagen fibers appeared in alveolar walls and septa, terminal bronchioles, walls of small bronchus, and around respiratory bronchioles. The hyperplasia of collagen fibers is slight in 28d TCM group. In hormone group, the degree of collagen fiber hyperplasia was between model group and TCM group.The result of morphological and quantitative analysis of HE staining:At the fourteenth day, the deep-staining region of hormone group is smaller than that of model group; and there is a significant difference (P<0.05). At the same stage, the deep-staining region of TCM group is much smaller than that of hormone group; and there is a more significant difference (P<0.01). At the fourteenth day, there is no significance to the difference between the light-staining region of hormone group and that of model group (P>0.05). At the same time, the light-staining region of TCM group is much smaller than that of model group; and there is a obvious significant difference (P<0.01). At the 28th day, to the comparison between the deep-staining region of hormone group and that of model group, there is no significance (P>0.05). At this stage, the deep-staining region is much smaller than that of model group, and there is an obvious significant difference (P<0.01). At the 28th day, to the comparison between the light-staining region of hormone group and that of model group, there is no significance (P>0.05). At the same stage, compared with the light-staining region of model group, the region of TCM group is evidently reduced; the difference is obviously significant (P<0.01).MVD result:At the 14th day, MVDs of model group and hormone group are both higher than that of blank group; and there is significance in the differences (P<0.01, P<0.05). The MVDs of model group, hormone group and TCM group at the 28th day are all lower than theirselve at the 14th day; and the differences are all significant (P<0.01, P<0.05, P<0.05). To the comparison between the MVD of hormone group and that of model group, there is no obvious significance in the difference (P>0.05). Compared with the MVD of model group, the MVD of TCM group is reduced at the 14th day, the difference is significant (P<0.05); and it is distinctly reduced at the 28th day, the difference is obviously significant (P<0.01).The results for the expressions of angiogenesis factors:Compared with blank group, the expressions of bFGF, ANG-2 and FLK-1 in model group are all increased obviously; and the differences are all obviously significant (P<0.01). The VEGF expression in model group is increased compared with that in blank group, there is significant meaning in the difference (P<0.05). For FLT-1 expression, that of model group is increased than that of blank group at 14th day, the difference is significant (P<0.05); and distinctly increased at the 28th day, the difference is obviously significant (P<0.01). the expressions of bFGF,ANG-2,FLK-1 in TCM group are all reduced than these expressions in model group, and the differences are all quite significant (P<0.01). The VEGF expression in TCM group is reduced than that in model group, and the differences are all significant (P<0.05). For FLT-1 expression, that of TCM group is increased than that of model group at 14th day, the difference is significant (P<0.05); and distinctly increased at the 28th day, the difference is obviously significant (P<0.01). for the expressions of bFGF,VEGF,FLT-1,FLK-1,ANG-2, there is no significance in the difference between hormone group and model group (P>0.05).The results of qi-blood analysis:Hypoxemia appeared in model group, hormone group and TCM group at both the 14th day and the 28th day. For the PaO2, there is no significance in the difference between the hormone group and model group (P>0.05). However, for this norm, that of TCM is obviously increased than that of model group, and the difference is distinctly significant (P<0.01).Conclusion:1. Successfully modeled the rats with pulmonary fibrosis by injecting bleomycin into trachea in the dose of lmg/kg.2. Bleomycin triggers pulmonary fibrosis of rats; at the early episode, it manifested as pulmonary alveolitis; and pulmonary fibrosis at the end-stage.3. Fei Xian formula can relieve pulmonary alveolitis and pulmonary fibrosis of rats; and it is more effective than prednisone.4. Fei Xian formula can relieve hypoxaemia of rats with pulmonary fibrosis. This may work by improving the function of lung faceing all the vessels.5. The pathological change of microvessel vascularization in the process of pulmonary fibrosis was triggered by bleomycin in rats. The active stage of this change is around the 14th day. Then as pulmonary fibrosis exacerbated, the pathological microvessel vascularization reduced gradually, and the density of microvessel in lung tissue lowered progressively.6. Bleomycin caused the ascending in the expressions of bFGF, VEGF, FLT-1, FLK-1 and ANG-2, which are microvessel angiogenesis-promoting factors in the process of rats pulmonary fibrosis. Then microvessel vascularization was active. Probably, microvessel vascularization is the important point in the process of pulmonary fibriosis. In that case, that restraint the microvessel vascularization may keep down the progress of pulmonary fibrosis radically.7. The mechanism of treating pulmonary fibrosis through Fei Xian fomula may are as follows:restrainting the expressions of microvessel angiogenesis-promoting factors such as bFGF, VEGF, FLT-1, FLK-1 and ANG-2, to keep down microvessel vascularization in the lung tissue, further to relieve the pulmonary fibrosis and slow its progress.8. Fei Xian fomula may work in the way of improving the function of lung facing all the vessels and relieving hypoxaemia in the disease of pulmonary fibrosis to downward the exprssions of microvessel angiogenesis-promoting factors. |
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