| Objective The local rennin-angiotensin-aldosterone system (RAAS) has been found to take part in the process of pulmonary fibrosis in some tissue and organs. The experiment is designed to study the effect on pulmonary fibrosis in rats after the blockage of the rennin– angiotensin -aldosterone system at different sites and explore its mechanism;so as to offer some effective theropy method and theory support for the prevention and cure of pulmonary fibrosis.Method Seventy-five SD rats were randomly divided into five groups: normal control group(group N),pulmonary fibrosis model group(group B), captopril group(group C),spironolactone group(S group), losartan group (group L),with 15 rats in each. Group B and every treatment groups received intratracheal instillation of bleomycin (BLM,5mg/kg) to induce pulmonary fibrosis, while group N received normal saline by intratracheal instillation. After the rats wake up, the rats of every groups were perfused into stomach with the same volume fluid (1.5ml) once everyday. Group N and group B were perfused normal saline, group C received captopril 60mg/kg, group L received losartan 10 mg/kg, group S received spironolactone 100mg/kg. In the period of treatments, we observed the general data, increase of weight, death data.Five rats in each group were narcosised on 7, 14, 28 days respectively after intratracheal instillation. Total cell counts and differential analysis of branchoalveolar lavage fluid(BALF) were performed under microscope. After the rats were sacrificed by bleeding, we received lung tissue. Histological changes of the lungs were evaluated by HE stain, Masson′s trichrome stain and hydroxyproline concentration of the lung tissue. The expression of monocyte chemoattractant protein-1 ( MCP-1 ) and tumor necrosis factor-α(TNF-α) was measured by immunhistochemical method with image analyse system. The mRNA expression of MCP-1 was evaluated by RT-PCR quantitative analysis. TUNEL was used to detect the change of cell apoptosis.Results1. General data: Before use the medicine, all the rats of every groups show on significance on general data and weight. In the experiment,for the group of B ,the general data were bad , the increase of weight was slow, three rats had dead during the process. For the group of C,S,L, the general data were better than the group of B significantly, the increase of weight were fast than the group of B, only one rat had dead during the process in each group.2.Analysis in BALF from rats: The total number of cells in the group of B increased,which were significantly higher than that in the group of N(P﹤0.01),there were a degressive trend anon. In the group of C,S,L,the total number of cells were significantly higher than that in the group of N(P﹤0.01),but were significantly lower than that in the group of B(P﹤0.01). The concentrations(%) of macrophages in the group of B descend relatively and were significantly lower than that in the group of N(P﹤0.01)at every time, there were a ascensive trend anon. The concentrations(%)of neutrophils in the group of B were significantly higher than that in the group of N(P﹤0.01)at every time, there were a degressive trend anon. The concentrations(%)of lymphocytes in the group of B were significantly lower than that in the group of N(P﹤0.01)at every time, there were a degressive trend anon. In the group of C,S,L,the concentrations(%) of macrophages were significantly higher than that in the group of N(P﹤0.01)and B(P﹤0.01),and there were a ascensive trend anon. The concentrations(%)of neutrophils were significantly lower than that in the group of B(P﹤0.01),and there were a degressive trend anon, came back the level of the group of N at the 14th,28th day. The concentrations(%)of lymphocytes were significantly lower than that in the group of N(P﹤0.01),but were significantly higher than that in the group of B(P﹤0.01), and there were a degressive trend anon.There were no statistic significant differences in total cell counts and differential analysis in the group of B,C,S,L(P﹥0.05).3. The degree of pulmonary alveolitis and fibrosis :The degree of alveolitis and pulmonary fibrosis of the group of B were in every phase was markedly severe compared with normal contro(lP﹤0.01).The group of C,S,L have the same tendency as the group of B;but the degree of alveolitis and pulmonary fibrosis decreased, compared with the group of B(P﹤0.01).Between C,S,L,there were no significant differences(P>0.05).4.The protein expression of MCP-1: The result of immunohistochemistry suggested that the expression of MCP-1 in lung tissue of the group of B was much higher than that of the group of N at every time(P<0.01). The group of C,S,L have the same tendency as the group of B ,but the MCP-1 concentration were lower than that of the group of B(P<0.01),but there were no significant difference between them(P>0.05).5. The mRNA expression of MCP-1: The result of RT-PCR suggested that the expression of MCP-1mRNA in lung tissue of the group of B was much higher than that of the group of N at every time(P<0.01). The group of C,S,L have the same tendency as the group of B ,but the MCP-1 mRNA were lower than that of the group of B at every time (P<0.01 or<0.05 ),but there were no significant difference between them(P>0.05).6.The protein expression of TNF-α:The result of immunohistochemistry suggested that the expression of TNF-αin lung tissue of the group of B was much higher than that of the group of N at every time(P<0.01). The group of C,S,L have the same tendency as the group of B ,but the TNF-αconcentration were lower than that of the group of B(P<0.01),and there were no significant difference between them(P>0.05).7. Apoptisis index: The apoptosis index of lung cells at every time in the group of B were higher than that in the group of N(P<0.01). The apoptosis index of lung cells in the group of C,S,L were significantly lower than that in the group of B(P<0.01).There were on significant difference between the three treatment groups (P>0.05).8. HYP concentration: HYP concentrations of the group of B were markedly higher then the group of N(P<0.05,P<0.01)and that of B group increased gradually.That of the group of C,S,L took on low level,but increased too,HYP concentration were lower than that of the group of B at every time(P﹤0.01).There were no significant difference in treatment groups(P>0.05).Conclusion1.The rats had received intratracheal instillation with bleomycin (BLM) to induce pulmonary fibrosis models successfully.The results suggested that pulmonary fibrosis of the models were exactly mimicked the clinical procedure of pulmonary fibrosis.2.The local rennin-angiotensin-aldosterone system (RAAS)might take part in the formation of pulmonary fibrosis, the blokage of RAAS can delieve the degree of pulmonary fibrosis.3.The blokage of different sites of RAAS might delieve the degree of pulmonary fibrosis through depressing inflammatory reaction, down-regulate the expression of MCP-1 and TNF-α,delieveing the concentration of HYP and suppressing cell apoptosis.
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