| ObjectivePulmonary fibrosis (PF) is a kind of immune-mediated chronic inflammation diseases, which is characterized by the infiltration of mononuclear macrophage, leucocyte, lymphocyte, proliferation of fibroblast and deposition of fibrosis connective tissue in the alveola interstitial .For the past few years, the incidence and mortality have been elevating in the world. Idiopathic pulmonary fibrosis (IPF) is the prototypical interstitial lung disease. At present, corticosteroid, immunosuppressive agent and anti-fibrosis agent were used for treatment of IPF, but the effects are not satisfying. Therefore, the focus of present study is to explore effective method. This study aimed to observe the effect of hirudo on bleomycin-induced pulmonary interstitial fibrosis in rats, and discuss the potential mechanisms of hirudo curing pulmonary interstitial fibrosis, through detecting the levels of transforming growth factor-β1(TGF-β1) and plasminogen activator inhibitor-1(PAI-1) in lung tissue and blood serum of rats, which to develop the new ideas for clinical study.Methods120 male Sprague Dawley (SD) rats (provided by Center of Experiment Animal of Hebei Medical University) were randomly divided into four groups: normal control group(C group), bleomycin group (B group), hirudo group (H group) and dexamethasone group (D group) . In B,H and D group, PF was induced in SD rats by intratracheal instillation of bleomycin, while C group received was injected NS in trachea. The rats in B and C group received daily NS, in H group hirudo powd(4g/kg) was perfused into stomach and in D group dexamethasone(DEX) 0.5mg/kg was injected in cavum abdominis respectively on the same day. Six rats in each group were sacrificed on 1, 3, 7, 14 and 28 days respectively after intratracheal instillation. The right lung was excised for HE and Messon Staining. The level of TGF-β1 and PAI-1 measured by immunohistochemistry, and positive rate of TGF-β1 and PAI-1 determined by sem quantitative picture analysis. The contents of collagen were detected in inferior lobe of right lung. The levels of TGF-β1 were detected in blood serum and alveolar lavage fluid. Observation the effects of hirudo and analyzed the correlation of TGF-β1 and PAI-1.Results1 HE/Masson Staining on the lung tissueThe lung of normal control group rat had no change lesion in every point, but that of B group showed acute inflammation: there were a few of macrophage and neutrophil infiltration in alveolar space on the 1d; on the 3d macrophage, neutrophil and lymphocyte infiltrating to be increased, with alveolitis making more serious. There were a great deal of poly-cyte and monocyte infiltration in alveolar space and fibroblast begin to proliferation on the 7d, with alveoli normal structure was destructed in severe areas. Alveolitis relieved on the14d, but hemorrhage and edema were still existing, with fibroblast increasing, interval widening obviously, collagen deposited and patches of fibrosis. In 28d- group, alveolitis did not relieved obviously. Alveola structure was destructed with fibroblast aggravated and alveolar space vanished. Alveola interval widening and inflammatory cell infiltrating was lightened obviously in H and D group compared with B group. Pathology of fibrosis was also relieved. While PF was significantly different from normal control just on 14 and 28d. H group has the same tendency as B group; but the degree of alveolitis and pulmonary fibrosis decreased.2 HYP concentrationHYP concentration of B, H and D group were both higher than normal control on 1d-group and 3d-group, but there was no significant difference between the two groups(p>0.05). But that of B group increased gradually from the 7d; that of H group and D group took on low level, but increased too, HYP was lower than that of B group on 7d, 14d and 28d and there was significant difference between them(p < 0.01). HYP concentration of D group was higher than H group on every time, but there was no significant difference between the two groups(p>0.05). 3 PAI-1 in lung tissue percentageThe result of immunohistochemistry suggested that the expression of PAI-1 in lung tissue of B group was higher than that of C group, and the difference at 3~28d were significant (p<0.01). expression of PAI-1 of H group was higher at every point, and there were significant difference at 7~28d (p<0.01). That of D group was higher than that of H group, but there was no significant difference (p>0.05).There was no significant difference on every group at 1d point (p>0.05).4 TGF-β1 concentration in blood serum and bronchoalveolar lavage fluid (BALF)TGF-β1 concentration in blood serum of B group at 3~28d were all higher than normal control group, hirudo group and DEX group(p<0.01). That of B group on 1d was higher than that of the others groups, but there were no significant difference between them. TGF-β1 concentration in blood serum of H group on every time was higher than normal control group(p<0.01); That of D group was a little higher than H group on every time, but there was no significant difference(p>0.05). The change of TGF-β1 in BALF was same as that of in blood serum.5 TGF-β1 in lung tissue percentageThe expression of TGF-β1 of B group was much higher than that of every group, there was significant difference on 3~28d(p<0.01). That of H group was much higher than that of B group at every point, and there was significant difference on 7~28d(p<0.01). The expression of TGF-β1 of H group was higher than that of C group, and there was significant difference on 7~28d(p<0.01). While that of H group was lower than that of D group, but there was no significant difference(p>0.05).6 PAI-1 activity in blood plasmPAI-1 activity of B group, H group and D group has no significant difference compared with C group on 1d-group(p>0.05). That of B group was higher obviously than C group on 3~28d (p<0.01); While PAI-1 activity of H group was lower than that of B group on 3~28d(p<0.01); That of D group was higher obviously than H group on 3~28d (p<0.01).7 The correlation between TGF-β1 concentration in blood serum and PAI-1 in lung tissue percentage was positive correlation (rs=0.936,P<0.01).Conclusions1 Hirudo might be very useful in treating bleomycin-induced pulmonary fibrosis in rats.2 Hirudo can reduce TGF-β1 proteinum expression and degrade PAI-1 activity, this is may be the mechanism of hirudo relieving bleomycin-induced pulmonary fibrosis in rats...
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