Pyrin Recombinant Protein Influnence On Bleomycin Induced Pulmonary Fibrosis In Rats

Objective:To investigate the effect of pyrin recombinant in rodent bleomycin-induced pulmonary fibrosis,and its relation to VEGF/VEGFR2/MMP-9 signaling pathways.Methods:60 SD rats were randomly divided into normal control group(n = 10),bleomycin model group(n = 20),SU5416 positive control group(n = 10),and Pyrin recombinant group(n = 20).Model set up:? normal control group:rats were anesthetized intraperitoneally with 10%hydrated trichloroacetaldehyde(3.5mL/kg),then fixed on an operating plate with supine position.The neck median fissure was cut and exposed to the trachea,and the saline was injected into the trachea(0.2 mL/kg).? bleomycin model group:after anesthetization,a dose of bleomycin(5mg/kg dissolved in 0.2 ml saline)was injected into the trachea.?Pyrin recombinant protein group:after anesthetization,a dose of bleomycin(5mg/kg body dissolved in saline 0.2 ml)was injected into the trachea.Two days after the injection,Pyrin recombinant protein(100 ?g/kg per day dissolved in 0.2 ml saline)was injected into trachea at a fixed time every morning for 14/28.day(depending on the subgroup),until the day before sacrifice.? SU5416 positive control group:after anesthetization,bleomycin(5mg/kg in 0.2ml saline)was injected one-time in the trachea,with intraperitoneal injection of SU5416(50mg/kg in 0.2 mL saline).Biopsies were taken at 14 and 28 days in all groups.The degree of alveolitis and pulmonary fibrosis were observed by HE staining and MASSON staining.The expression of VEGF,VEGFR-2,MMP-9 and mRNA were examined by immunohistochemistry and RT-PCR respectively.Result:The histopathological changes in lung:no alveolitis and pulmonary fibrosis were observed in the normal control group.The alveolar structure was clear and complete.In all other groups,macrophage infiltration was prominent among the alveolar and pulmonary interstitial with various inflammatory cells(i.e.eosinophils,neutrophils,lymphocytes and macrophages).Several fibrous lesions were also identified with collagen fiber deposition around the alveoli and trachea.The degree of alveolitis was peaked at day 14 and reduced at day 28,with on-going deposition of extracellular matrix(ECM).HE staining showed that in the bleomycin model group the alveolitis was most severe always,while the normal control group had no alveolitis(P<0.05).In the Pyrin recombinant protein group,the alveolitis was significantly increased at day 14 and day 28,compared with the normal control group,but not as severe as the bleomycin model group(P<0.05).There was no significant difference in alveolitis between day 14 and day 28.Masson staining showed that the bleomycin model group had the most severe fibrosis always,while the normal control group had no obvious pulmonary fibrosis(P<0.05).The Pyrin recombinant protein group has lower pulmonary fibrosis than the bleomycin model group(P<0.05)at day 14 and day 28.There was no significant difference in pulmonary fibrosis between day 14 and day 28(P>0.05),though the degree of fibrosis looked increasing.Immuno-histological Staining(VEGF,VEGFR2,MMP-9):At day 14,VEGF,VEGFR2 and MMP-9 in the lung tissue of the normal control group were expressed in a small amount,but prominent in the bleomycin model group(P<0.05).SU5416 positive control group and Pyrin recombinant protein group had significantly higher expression than normal control group(P<0.05),but lower than the bleomycin model group(P<0.05).At day 28,SU5416 positive group and Pyrin recombinant protein group still had significantly higher expression than normal(P<0.05)and lower than the bleomycin model group(P<0.05).mRNA expression(VEGF,VEGFR2,MMP-9):In the bleomycin model group,SU5416 positive control group and Pyrin recombinant protein group,VEGF,VEGFR2,MMP-9 were significantly higher than those in the normal control group P<0.05).The expression at day 28 were slightly higher than day 14 with no statistical significance(P>0.05).The expression of these mRNAs in normal control group were relatively stable.In Pyrin recombinant protein group,the expression was significantly lower than the bleomycin model group on day 14 and day 28(P<0.05).Conclusion:1.Pyrin recombinant protein may inhibit neovascularization in pulmonary fibrosis by down-regulating VEGF/VEGFR2/MMP-9 signaling pathway.2.Pyrin recombinant protein may inhibit the ECM deposition by down-regulating VEGF/VEGFR2/MMP-9 signaling pathway.3.Pyrin recombinant protein may have an anti-pulmonary fibrosis effect by down-regulate the expression of VEGF/VEGFR2/MMP-9 signaling pathway.