| ObjectivePulmonary fibrosis(P F) incidence rate showed a rising trend. At present about 16.3/10 million persons and in 3 years the acute worsening incidence was 20.7%. The main pathological feature of PF is the early alveolitis formation and late a lot of fibroblast proliferation, collagen and matrix of accumulation, eventually displacing normal lung tissue structure. The pathogenesis is unclear, the case fatality rate is high, and there is a lack of effective drugs. The early research results showed that the formation of modified Bufei decoction has antioxidant effect of antagonistic fibers. However, the cellular and molecular mechanism of modified Bufei decoction anti pulmonary fibrosis is still unclear. In order to elucidate the modified Bufei decoction on the function of PF and the possible mechanism, the project proposed model by bleomycin induced pulmonary fibrosis in mice, observed pathological changes of the pulmonary fibrosis mouse model through modified Bufei decoction, detected the effect on lung tissue in mice of TNF- alpha, TGF- beta 1 Cat D, LC3 and m TOR expression, studied the effect of modified Bufei decoction on pulmonary fibrosis in mice, investigated its inhibition of pulmonary fibrosis mechanism. Methods&Results1 Different dose of bleomycin induced pulmonary fibrosis in mice model to explore the ideal dose of replication in a mouse model of pulmonary fibrosis.(1) methods: 90 Kunming mice were randomly divided into normal group, sham group, model high(7.5mg/kg), model middle(5mg/kg) and model low(2.5mg/kg) dose group, 18 rats in each group. Model group is injected bleomycin in trachea, sham group is injected same volume of saline. Each animal in 3d, 7d, 14 d was killed, the lung tissue were stained with HE, and observed the pathological changes and mortality of lung tissue.(2) Results:1) Mice with bleomycin induced pulmonary fibrosis model was duplicated successfully.2) Bleomycin injection dose was positively correlated to the degree of pulmonary fibrosis and mortality.3) To establish a mouse model of pulmonary fibrosis: using transtracheal one-time delivery methods, using 5mg/kg replication is dose bleomycin mouse model of pulmonary fibrosis ideal.2 modified Bufei Decoction effection on mice morphological structure by bleomycin induced pulmonary fibrosis, Pharmacodynamics study on anti-fibrosis of mouse pulmonary through modified Bufei decoction(1)Methods: 306 Kunming mice, were randomly divided into sham group, model group, modified Bufei Decoction high, middle, low, and prednisone acetate group and prednisone acetate plus modified Bufei Decoction middle dose group.36 rats in the sham group, 45 rats in rest group. Except the sham group, the other groups were injected with a single intratracheal bleomycin(5mg/kg) to induce pulmonary fibrosis of mice model, the physiological saline sham group single intratracheal injection volume. Administration second days after model, modified Bufei Decoction high, low dose groups were 12g/kg/d, 6g/kg/d, 3g/kg/d dosage, prednisone acetate group according to 0.5mg/kg/d dosage, addition and subtraction supplement dose + acetic acid lung soup in the prednisone group: morning to modified Bufei Decoction by 6g/kg/d dose of prednisone acetate, afternoon according to 0.5mg/kg/d dosage; Model group and sham group were given normal saline. Each animal in the 7th, 14 th, 28 th days of randomly selected 10 rats to be killed, the lung tissues were stained with HE, observed changes in lung tissue pathology, quantitative analysis of images.(2) HE staining showed that the sham group with the alveolar structure complete, clear, alveolar no inflammatory cell infiltration and pulmonary parenchyma lesion; Model group: alveolar septal thickening, alveolar structure disorder, early alveolar cavity for severe inflammatory cell infiltration, advanced fibroblast proliferation, fiber tissues cord like change, the degree of fibrosis progressed with time and further aggravate; As for Modified Bufei Decoction of high, medium, low dose group, prednisone acetate group and modified Bufei Decoction middle dose + prednisone acetate group, alveolar structure is clear, and have local fibrosis in around the bronchi, alveolar sepal thickening, compared to the model group reduced.3 Study on the mechanism of pulmonary fibrosis in mice lung soup addition and subtraction intervention 3 supplement(1) Method:171 Kunming mice were randomly divided into sham group, model group, modified Bufei Decoction group and prednisone group. 36 rats in the sham group, 45 rats in rest group. model group, Bufei Decoction group and prednisone acetate group intratracheal were injected bleomycin to induce pulmonary fibrosis(5mg/kg), sham group was given normal saline in the same condition. Administration medicine after Model in second days, modified Bufei Decoction group according to 12g/kg/d dosage, prednisone acetate group according to 0.5mg/kg/d dosage; Model group and sham operation group were given normal saline. Animals in each group 10 rats were killed with randomly selection at the 7th, 14 th, 28 th days, the lung tissue stained with HE, observed the pathological changes of lung tissue. By immunohistochemistry and Western blot methods, the expression of TGF- beta1 in lung tissues of mice, TNF-alpha, Cat D, LC3 and mTOR, the amount of expression were observed by image analysis software and the average optical density was analyzed.(2)Results:1)The results of immunohistochemistry of TNF- alpha and TGFbeta 1 showed that, the expression was weak in TNF- alpha and TGFbeta 1 in sham group; Gradually enhanced expression in model group, there was significant difference compared with the sham operation group(P<0.01); Modified Bufei Decoction and prednisone group at each time point expression of the same trend with the model group, but significant differences with that of the model group(P<0.01 or P<0.05); To the first 28 d, modified Bufei Decoction group is better than in prednisone group, but no significant difference(P>0.05). Western blot results 14 d mice lung tissue in TGF-beta 1 and TNF-alpha were consistent with the results of immunohistochemistry.2)Cat D immunohistochemical results showed that the sham group is with a small amount of Cat expression of D in bronchial epithelial cells; Bleomycin induced pulmonary fibrosis in mice, Cat D expression was significantly increased, In interstitial lung and bronchial epithelial cells were expressed as time prolonged, and there is a growing trend, Seventh days, 14 days and 28 days compared with the sham operation group had significant difference(P<0.01); Prednisone, modified Bufei Decoction group: Cat D expression in the same trend with the model group, but the expression decreased obviously compared with the model group, compared with model group had significant difference(P<0.01 or P<0.05).3)Based on the analysis of LC3 expression in the lung tissues, we found that LC3 exists in normal lung tissues around the bronchial and vascular, and the protein expression in normal tissues is weak, that autophagy functions within the cell is low; and in lung tissue of pulmonary fibrosis, LC3 was found in the bronchial and vessels except around outside, still visible LC3 is expressed in the cytoplasm, and sham group compared with bleomycin treatment, the expression of LC-3 each time point in mouse lung tissues were significantly increased(P<0.01). Modified Bufei Decoction group compared with the model group, the expression of LC3 decreased obviously, Modified Bufei decoction can reduce excessive expression in lung tissue of pulmonary fibrosis in LC3 mice.4)mTOR immunohistochemical results showed that, the sham group: Protein expression in the bronchi, blood vessels around m TOR, there is little positive expression in cytoplasm. Model group: m TOR expression was significantly increased in the cytoplasm, also has positive expression. Seventh days, 14 days and 28 days compared with the sham operation group, it had significant difference(P<0.01), which revealed that the model group with the degree of pulmonary fibrosis aggravated increased m TOR expression. Modified Bufei Decoction group: the expression trend if the same with the model group, but the expression decreased obviously than those in the model group(P<0.05), suggesting that modified Bufei decoction can increase the activity of mTOR in lung tissue of pulmonary fibrosis mice decreased.Conclusion1 The 5mg/kg bleomycin is ideal replication dose to mouse model of pulmonary fibrosis.2 modified Bufei decoction can improve the mice pulmonary fibrosis infiltration of inflammatory cells, significantly reduce the degree of pulmonary fibrosis, thus form the intervention of pulmonary fibrosis.3 Study on the mechanism of pulmonary fibrosis in mice lung soup addition and subtraction intervention(1)Modified Bufei decoction can inhibit the expression of bleomycin induced pulmonary fibrosis in the lung tissue of mice TNFalpha, TGF- beta 1 protein, the formation of pulmonary fibrosis model in mice significantly reduced the development of alveolitis and pulmonary fibrosis.(2)modified Bufei decoction can relieve bleomycin induced pulmonary fibrosis in mice, the mechanism may be related with the inhibition of excessive expression of Cathepsin D.(3)modified Bufei decoction can reduce excessive expression in lung tissue of pulmonary fibrosis in mice LC3, mTOR, the formation of pulmonary fibrosis. |
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