Role Of Osteopontin In Rats With The Pulmonary Fibrosis After Acute Lung Injury Induced By Lipopolysaccharide

Objective:To investigate the expression and function of osteopontin (OPN) in lung tissue establishing the pulmonary fibrosis after acute lung injury induced model of rats induced by lipopolysaccharide (LPS) injected through Intraperitoneal.To investigate the expression of hydroxyproline, TGF-21, I and type III collagen mRNA levels in lung tissue.To explore the status and role of OPN in the pulmonary fibrosis in rats after acute lung injury induced by lipopolysaccharide.Methods:①approaches in establishing in the pulmonary fibrosis caused by acute lung injury after lipopolysaccharide in rats model in rats:90 male Wistar rats of SPF(Specific pathogen Free) class were randomly divided into 3 groups; (1) control group,n=30; (2)ALI group, n=30;(3) intervention group, n=30. Group ALI:LPS (5mg.kg-1.d-1,1mg LPS was dissolved in 1ml per sterile saline)was injected though intraperitoneal to rats at 0 day,1 day,2 day, normal saline (0.5ml) were injected through intraperitoneal 5 minutes after LPS injection. Intervention groups:LPS (5mg.kg-1.d-1, lmg LPS was dissolved in lml per sterile saline)was injected though intraperitoneal to rats at 0 day,1 day,2 day, and OPN antibody (Anti-OPN-Ab) (0.5ml, titer 1:32) were injected through intraperitoneal 5 minutes after LPS injection.; Control group:normal saline instead of LPS and Anti-OPN-Ab; Each group's 5 rats were randomly sacrificed at the 4th,8th and 24th hour and ay 7th,14th,28 th day respectively. At the same time keeping the specimenes to measure.②Alveolitis and fobrosis were observed by HE and Masson staining and scored.③Hydroxyproline in the tissue of lung were assessed.the specific operation was strictly according to kit instructions.④TGF-β1 in lung tissue was assessed by immunohistochemistry.⑤Real-time quantitative reverse transcription polymerase chain reaction(FQ RT-P CR) was performed to detect collagenⅠ,ⅢmRNA expression in lung tissue.⑥Expression of OPN in lung tissue bomogenatein was analysised by western blotting,and the results were expressed by OPN/β-actin.Result:①Lobular structure and alveolar structure were normal in NS group;alveolitis septal edema,minflammation cell infiltration and fibrosis were not found. At 8th hour a great deal of inflammation cells infiltration,alveolar wall thickening and blue collagen fibers into the alveolar were observed in ALI group; at 7th day Normal alveolar structural damaged,alveolar closured,fuse, fibrosis formated by blue collagen fibers and inflammatory cells in ALI group;at 14th day the alveolar was still heavier,but inflammation cells were significantly reduced,the number of fibroblasts and matrix in alveolar septum were obviously increased; At 28th day the inflammation relieved,pulmonary fibrosis increased,parts of alveolar space was disappeared and formed sereve fibrosis. Compared with ALI group the pathology of intervention group was significantly reduced.②the expression of hydroxyproline content, TGF-β1,ⅠandⅢcollagen mRNA showed a progressive increase in lung tissue of ALI group and intervention groups compared with the NS group,the most obvious expression was at 28th day (P<0.05),and there was a significant difference compared with the NS group(P<0.05),but the expression of intervention groups significantly decreased compared with the ALI group (P<0.05).③The expression of OPN in rat lung tissue:Western bolt results showed:A small amount expression of OPN in NS group, whereas the expression of OPN was rised with time increased in ALI group and Intervention group, the most obvious expression of 28 days;further analysis suggested by the software, at the same time points showed no significant difference in OPN between ALI group and the intervention group (P>0.05),(See Figure 35-40, Table 7).Conclusion:①The pulmonary fibrosis after ALI model of rats can be successfully established by Hpopolysaccharide injected through Intraperitoneal.. The various changes of lung injury and pulmonary fibrosis were remarkable and typical at the time of 8th hour and 28 th day.②The early stage of ALI, the expression of OPN, hydroxyproline, TGF-21,ⅠandⅢcollagen mRNA were increased, pulmonary fibrosis stage increased more significantly.③OPN can promote expression of TGF-β1, hydroxyproline,ⅠandⅢcollagen mRNA in ALI, and promote the formation of pulmonary fibrosis after ALI.