The Effect Of IGFBPrP1 On Lung Tissues In SD Rats And Beagles

Backgroud:Insulin-like growth factor binding protein-related protein 1(IGFBPr P1)also known as insulin-like growth factor binding protein 7(IGFBP7),is a secreted glycoprotein which expressed in a wide variety of human tissues and organs.IGFBPr P1 has been implicated in cellular processes including cell differentiation,cell adhesion,stimulation of prostaglandin synthesis,angiogenesis,cell growth and survival,senescence and apoptosis even cancerization.Liu et al discovered that IGFBPr P1 was a novel molecule of hepatic fibrogenesis,it stimulated transforming growth factor beta 1(TGFβ1)production in fibrotic livers,which may be the main pathway of hepatic fibrosis induced by IGFBPr P1,and TGFβl also can stimulate IGFBPr P1 secretion.TGFβ1 belongs to the transforming growth factors family.It serves to regulate cell differentiation,cell adhesion,immunomodulation,trauma-repair,inflammation and the formation and degradation of ECM.TGFβ1 has been recognized as the strongest meditor in the pathogenesis of fibrosis and related to lung and liver fibrosis closely,so we speculate that IGFBPr P1 may have effects on lung tissues.Similar reports were rare at present,therefore,we investigate the effects of IGFBPr P1 on lung tissues through establishing the model by injecting Ad-IGFBPr P1 to SD rats’ caudal vein and subcutaneous injection of TAA to beagles.Objective:To investigate the effect and mechanism of IGFBPr P1 on lung tissues in SD rats and beagles.Methods:1.60 healthy male SD rats,five to six weeks old,weighting 120~150g,were randomly divided into group A: normal group(n=10,the same doses of saline was injected into caudal vein once),group B: Ad-EGFP group(n=25,Ad-EGFP of 4×109pfu was injected into caudal vein once)and group C: Ad-IGFBPr P1 group(n=25,Ad-IGFBPr P1 of 4×109pfu was injected into caudal vein once).Lung tissues were obtained for analysis at week 1,2,4,6 and 9.2.Eighteen healthy male beagles,eight-month-old,weighing 9~11 kg,were randomly assigned to three groups of 6 each.Beagles in group D(nomal group)had a free access to food and water for 12 weeks.Beagles in group E(TAA group)were given subcutaneous injection of TAA(12 mg/kg,twice per week)lasting for 12 weeks.Beagles in group F(TAA+IGFBPr P1-Ab group)were given subcutaneous injection of TAA(12mg/kg,twice per week)for 12 weeks,and IGFBPr P1-Ab(5 μg/kg,once per week)were injected into portal vein from week 5 at the same time.Lung tissues of all beagels were obtained for analysis at week 12.The methods of group C and E can cause hepatic fibrosis to SD rats and beagles successfully.HE staining and Sirius Red staining were observed to investigate the pathomorphological changes and deposition of collagen fibers in lung of SD rats and beagles.The expressions and distribution of IGFBPr P1、TGFβ1 、 fibronectin(FN)and interleukin-1β(IL-1β)of lung tissues in SD rats and IGFBPr P1 of lung tissues in beagles were detected by immunohistochemical staining(IHC staining).Results:1.The results of SD rats1.1 The HE staining showed that there were intact structure with normal alveolar wall and alveolar cavity of the lung tissues of rats in the normal group and Ad-EGFP group.And rats infected 1 week after the injection of Ad-IGFBPr P1 appeared a few neutrophilic granulocytes and moacrophages in part of alveolar space.With the increasing time,there were desquamation of bronchial ciliated columnar epithelial cells and infiltration of a lot of inflammatory cells in the alveolar septum and peribronchial.Rats infected 4 weeks after injection of Ad-IGFBPr P1 showed that there existed infiltration of a lot of inflammatory cells in the alveolar septum,peribronchial and alveolar septa,mainly lymphocytes and macrophages.Meanwhile,alveolar wall and alveolar septa were gradually thickening.Rats infected 6 weeks after injection of Ad-IGFBPr P1 showed that inflammatory cells decreased gradually and alveolar wall and alveolar septa thickened visibly.Rats infected 9 weeks after injection of Ad-IGFBPr P1 showed the increase of collagen fibers in lung,even part of the alveolar structural damaged and alveolar fused.1.2 Sirius Red staining showed that collagen fibers in lung of rats in Ad-IGFBPr P1 group increased comparing with normal group and Ad-EGFP group,which were gradually increased with the increasing time of injection of Ad-IGFBPr P1.The area percentage of collagen fibers increased significantly in Ad-IGFBPr P1 group of 9 weeks compared with normal group,Ad-EGFP group and Ad-IGFBPr P1 group of 1,2,4and 6 weeks(P<0.01).1.3 The results of IHC staining in lung tissues of SD rats.(1)In normal group and Ad-EGFP group,the expression of IGFBPrP1 could be observed rarely in the bronchial ciliated columnar epithelial cells cytoplasm,which was light yellow.In Ad-IGFBPr P1 group,IGFBPr P1 expression presented deeper and larger brown particles scattered in the bronchial ciliated columnar epithelial cells cytoplasm and macrophages cytoplasm and the expression of IGFBPr P1 peaked(3.55 ± 0.18)at 2 weeks after injection and IGFBPr P1 level later gradually downregulated.The protein level of IGFBPr P1 increased significantly in Ad-IGFBPr P1 group of 2,4 and 6 weeks compared with normal group,Ad-EGFP group and Ad-IGFBPr P1 group of 1 and 9 weeks(P<0.01).(2)In normal group and Ad-EGFP group,there were rare expression of IL-1β in the cytoplasm of macrophages,and the expression of IL-1β in Ad-IGFBPr P1 group increased significantly than that in normal and Ad-EGFP group,which was tan in colour.The level of protein IL-1β increased from 1 week of Ad-IGFBPr P1 group,which peaked(1.94±0.03)at 4 weeks after the infection and then reduced with the time extended.The protein level of IL-1β increased significantly in Ad-IGFBPr P1 group of 2,4 and 6 weeks compared with normal group,Ad-EGFP group and Ad-IGFBPr P1 group of 1 and 9 weeks(P<0.01).(3)TGFβ1 expressed in the same location with IGFBPr P1,and the expression of TGFβ1increased with the increase of the degree of the lesion and peaked at 9 weeks(2.38±0.01).The expression of TGFβ1 increased in Ad-IGFBPr P1 group of 1,2,4,6 and 9 weeks compared with normal and Ad-EGFP group(P<0.01).(4)The positive expression of FN located in the cytoplasm of bronchial ciliated columnar epithelial cells.The positive rate of FN in Ad-IGFBPr P1 group was higher than that in Ad-EGFP and normal group.The protein level of FN increased with the increase of TGFβ1,and peaked(1.23±0.05)at 9 weeks of Ad-IGFBPr P1 group.The level of protein FN increased significantly in Ad-IGFBPr P1 group of 9 weeks compared with normal group,Ad-EGFP group and Ad-IGFBPr P1 group of 1,2,4and 6 weeks(P<0.01).2.The results of Beagles2.1 The HE straining showed that intact structure with normal alveolar wall and alveolar cavity of the lung tissues was observed in normal group and pathomorphological changes and deposition of collagen fibers were observed in TAA group with infiltration of a lot of inflammatory cells and thicker alveolar wall and alveolar septa.Comparing with TAA group,the degree of pathomorphological changes and the content of collagen fiber decreased in TAA+IGFBPr P1-Ab group.2.2 The Sirius Red straining showed that collagen fibers in lung of beagles in TAA group increased comparing with normal group,and that in TAA+IGFBPr P1-Ab group was less than TAA group and more than normal group(P<0.01).2.3 The IHC staining showed that the expression of IGFBPrP1 protein in TAA group(1.39±0.20)and TAA+IGFBPr P1-Ab group(1.05±0.16)increased comparing with that in normal group(0.50±0.14),and that in TAA+IGFBPr P1-Ab group was less than TAA group(P<0.01).Conclusions:1.IGFBPr P1 contributes to the inflammatory injury and fibrosis of the lung tissues in SD rats,and it can also play a role in the pathogenesis of inflammatory injury and fibrosis of the lung tissues in beagles induced by TAA;2.IGFBPr P1 may be an non-tissue-specific pathogenic factor.