Effects Of Zhongyao Recipe On Angiogenesis Of BLM-induced Lung Fibrosis

Idiopathic pulmonary fibrosis (IPF) is one type of idiopathic interstitial pneumonia, whose histopathologic pattern is usual interstitial pneumonia.The disease localizes at the lung, causes diffuse lung changes , harms the lung function and results in dyspnea. Because its accurate pathogenesis is still not clear, people have no good idea about its treatment. Viewing the current therapies, most of all is to improve inflammation, accommodate the immunity. However, there are researches showing in IPF there is not only inflammation and disturbant immunity, but also the aberrant repair after the injury of blood vessel endothelium. The aberrant repair behaves abnormal angiogenesis and remodeling. It is too difficult to obtain ideal effect only depending on the current therapies. Therefore, it becomes very important to explore the more effective methods to treat IPF.Objective: This study aimed to discuss whether applying the Zhongyao recipe which has the ability of protecting blood vessel endothelial cells, can downregulate angiogenic mediators, extenuate the early abnormal angiogenesis and as a result the progress and severity of IPF were decreased.Methods: Fifty-four male Sprague Dawley ( SD ) rats (provided by Center of Experiment Animal of Hebei Medical University),weight 200-250g, were randomly divided into three groups: normal control group(group H), the Bleomycin (BLM) group(group M), and Zhongyao treatment group(group Z). Group H has six rats. By the compartment 3rd, 7th, 14th, 28th day, the other groups were randomly divided into four subgroups, and each subgroup has six SD rats. All operations were under the aseptic condition. After anaesthesia 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 the cricoid cartilage, poured slowly physic liquor into trachea as the dose of 5mg per kilogram weight (normal saline contained BLMA54mg/ml for group M and group Z, the normal saline for group H), then kept in erect position and revolved gently to distribute physic liquor fully in the lung. From the same day ,recipe-drug solution was given to group Z by intragastric administration, once a day .Group H and M were treated with distilled water. At the 3rd, 7th, 14th, 28th day , the rats of group M and Z were killed by blood being drawn from the hearts after being observed the changes of the lung. The blood was immediately anticoagulated and centrifuged, and the plasma was extracted to test the content of ET-1 and vWF. The left lung was made into the common paraffin sections for HE and Masson staining to learn the degree of alveolitis and fibrosis, and for CD31 immunohistochemistry staining to observe angiogesis。The right lung was made into homogenate to evaluate the content of VEGF by ELISA (enzyme linked immunosorbent assay) . The rats of group H were killed at 7 th day, and treated with the same way. Statistics work was done with Spss11.5 statistical software.Results: 1 Content of vWF in plasma (A value was used to reflect indirectedly): there was statistic significance individually between each subgroup and group H (p<0.05).The contents were both higher than that of group H. At the same day, the content of vWF in group Z was lower than that of group M (p<0.05). 2 Content of ET-1 in the plasma (pg/ml): there was statistic significance individually between each subgroup and group H (p<0.05). The contents were both higher than that of group H, and increased continuously with the progress of the disease. At the same time spot, the content in group Z was between that in M and Z. There was no statistic significant differences between Z3 and M3,but according to the average of sample, the content of subgroup Z3(68.498)was lower than that of subgroup M3(69.130). At the other three time spots, the contents in group Z were obviously than that in group M(p<0.05). 3 Content of VEGF in lung homogenate (ng/mgpr): there was statistic significance individually between each subgroup and group H (p<0.05). The contents were both higher than that of group H, and increased continuously with the progress of the disease. The levels were (Z3=11.753;Z7=14.226)( M3=12.982;M7=16.553)in group Z and M. There was no statistic significant differences between the above two pair subgroup(sp>0.05). The contents in subgroup Z14 and Z28 were significantly lower than those in subgroup M14 and M28 (p<0.05). 4 The results of microvascular density: except the 14th day, there was statistic significance individually between group M and group H at the 3rd, 7th and 28th day (p<0.05).Compared with group H, there was statistic significance between the four subgroups of group Z (p<0.05). When it came to the difference of group Z and M, there were no statistic significance at the 3rd and 28th day(p>0.05). But according to the averages of sample, the 3rd abnormal angiogenesis in group Z (9.2717) was less than that in group M (9.3133). At the 28th day, the absence of blood vessles in group Z was better than that in group M ,but worse than that in group H (p<0.05). 5 The results of lung tissue pathology: the degree of alveolitis: compared with group H, the differences were significant in model group and treatment group (p<0.05). Except subgroup Z14 and M14, there were no statistic significance between the other three pair subgroups (p>0.05), but but compared with group M, the degree of aveolitis in group Z was lighter. The degree of fibrosis: Only at the 14th and 28th day, compared with normal control group, there were statistical significance in model group and treatment group(p<0.05). There was statistic significance only between Z28 and M28(p<0.05), while for the other time spots, compared with that in group M, the degree of fibrosis in group Z was little lighter.Conclusion: 1 Bleomycin -induced lung fibrosis in rat is successful. 2 In BLM-induced lung fibrosis, the content of vWF in plasma was increased ,and it showed the vessel endothelium injury existed in the model. The contents of ET-1 in plasma and VEGF in lung homogenate were increased in pulmonary fibrosis. The density of new formed vessles was increased in the early stage ,and gradually decreased with the development of the disease. When fibrosis became very serious, there was no blood vessles almost. 3 Zhong yao recipe could reduce the blood vessel injury, and as a result the contents of ET-1 in plasma and VEGF in lung homogenate were downregulated, and abnormal angiogenesis was decreased. So the degree of fibrosis was consequently reduced.