The Effect Of The Insulin-like Growth Factor System In CLD And Its Mechanism

IntroductionChronic lung disease(CLD) is presently defined as the need for oxygen therapy either at 28 days of age or 36 weeks postmenstrual age. Because of the more advanced and intensive respiratory support provided for compromised children,the supplement of alveolar surfactant and additionally the overall improved survival of preterm babies, the incidence of CLD has increased continuously in the past decade.CLD has been the most difficult problem in neonatal intensive care unit and the hottest topic in international neonatology, so it is very important to study the etiology and pathology of CLD and investigate the useful treatment of CLD. Animal experiment and clinical studies indicate that CLD is associated with cytokine,oxygen free radicals,inflammation and the imbalance of extracellular matrix synthesis and degradation. The expressions and effects of peptide growth factors in CLD are unknown. Earlier studies has demonstrated that some growth factors are involved in CLD, such as PDGF-AA,PDGF-BB,KGF,CTGF,VEGF,IGFs. Several lines of evidence indicate that the insulin-like growth factor system(IGFs) is an important regulator of growth,mitogenesis and differentiation and plays a role in lung development and lung injury. At present, studies about the expression and effect of the insulin-like growth factor system are mainly based on adult animals and adult fibrotic lung diseases, the expression and effect of the insulin-like growth factor system in CLD remains unknown. In our experiment, we establish CLD model by way of neonatal rats persistent inhalation high concentration oxygen. The purpose of our experiment is to investigate the expression and effect of the insulin-like growth factor system and to open out the mechanism of the insulin-like growth factor system in CLD and the intracellular signal transduction pathway of IGF-I using fetal rat lung fibroblast in vitro. Our results give a new club to illustrate the pathogenesis of CLD and the theory basis for clinical supplement of rhIGF-I.Material and methods1,Animal modelsFull-term neonatal Wistar rats were randomly exposed to hyperoxia(hyperoxia group) and room air(room air group) within 12 hours after birth. Hyperoxia group animals were exposed to 80%～90% oxygen in a transparent Plexiglas chamber in which oxygen concentration was monitored three times daily, the humidity maintained at 60%～70%, the temperature maintained at 25℃～26℃, the CO₂ level maintained at 0.5%. Pups were fed by lactating fosters. To avoid oxygen toxicity of the foster mothers, foster mothers were rotated daily between hyperoxia group and room air group. Room air controls were raised in the same room as hyperoxia-exposed animals and maintained under normal vivarium conditions. There was no statistically significant difference in body weight between the animals enrolled in the different groups.2,Lung tissue preparationPups(n=8) were anesthesized with an intraperitoneal injection of 10% Chloral Hydrate(0.3ml/100g bodyweight) at different time points(1,3,7,10,14 and 21 days). The thoracic cavity was opened and the right lungs were removed, snap-frozen in liquid nitrogen and stored at-80℃until use for real-time RT-PCR. For histology and immunohistochemical studies, the left lungs were fixed in 4% paraformaldehyde for 24 h at 4℃, and embedded in paraffin after dehydration in a graded alcohol series and xylene.3,Lung morphology examination and radical alveolar counts(RAC)Paraffin sections(5μm) from the left lung were cut mounted on slides and stained with hematoxylin and eosin. According to the Jakkukla's method, RAC(×100) were measured in lung sections chosen at random from eight animals in each group. Sections were viewed under bright field optics and were counted five times every section.4,Quantification of secondary crest formationFive-micrometer sections were cut from approximate center of the paraffin block, mounted on slides, and stained with Gomori's stain for elastic fibers. Five nonoverlapping microscopic fields were chosen on each slide and photographed. According to the Kathleen's method, secondary crests(×200) were identified by their characteristic structure: small ridges that contain elastic fibers and extend out from both sides of primary alveolar septa. These ridges subsequently subdivide the lung saccules into alveoli. Crests were differentiated from alveolar septa by using the definition of alveolus as a polygonal structure whose depth exceeds its diameter.5,Immunohistochemical analysis(SABC)Immunohistochemical analysis for IGF-I,IGF-IR,IGFBP-5 and collagen Iwere performed using rabbit anti-rat IGF-I antibodies,rabbit anti-rat IGF-IR antibodies,rabbit anti-rat IGFBP-5 antibodies,rabbit anti-rat collagenâ… antibodies(boster, wuhan, China). Rabbit anti-rat IGF-I antibodies,rabbit anti-rat IGF-IR antibodies,rabbit anti-rat IGFBP-5 antibodies and rabbit anti-rat collagenâ… antibodies were used at 1: 75 dilution,1: 100 dilution,1: 150 dilution and 1: 100 dilution respectly. Results: brown granules in the cytoplasm were considered as positive result. Sections were viewed using Metamorph/Dplo/Bx41 Image Processing System(×400). Eight sections were chosen from each time point randomly and five images were chosen from each section. Staining intensity was analyzed using Meta Morph software and was recorded as integrated optical density(IOD).6,In situ hybridizationFor the expression of tropoelastin mRNA(boster, wuhan, China)at alveolar wall, tissue sections 5μm thick were cut from paraffin-embedded specimens. Results: brown granules in the cytoplasm were considered as positive result. Sections were viewed using Metamorph/Dplo/Bx41 Image Processing System(magnification=×400).Eight sections were chosen from each time point randomly and five images were chosen from each section. Staining intensity was analyzed using Meta Morph software and was recorded as integrated optical density(IOD).7,Lung IGF-I IGF-IR,IGFBP-5 and collagenâ… mRNA expression and fetal rat lung fibroblast ERK1,ERK2,AKT,collagenâ… and tropoelastin mRNA expression(RT-PCR)Total RNA was isolated from lung tissue homogenates and cell homogenates and first strand cDNA synthesis was performed according to TRizol and M-MLV Reverse Transcriptase methods. IGF-I,IGF-IR,IGFBP-5,collagen I,ERK1,ERK2,AKT,tropoelastin andβ-actin Primers were designed with the Primer 5.0 software package according to the National Center for Biotechnology Information(NCBI) medline gene sequence. Data were analyzed with the G: BOX Detection System and quantified using the comparative threshold cycle method withβ-actin as a housekeeping gene reference. We studied at least 8 animals per experimental group.8,Cell culture of fetal lung fibroblastPrimary cultures of fetal rat lung fibroblasts were prepared using differential adherence as described previously with some modification. Rat pups were delivered aseptically from 19-d gestation timed pregnant Wistar rats. The lungs were removed, minced, and incubated with trypsin(0.25%) and trypsin activity was stopped by adding H-DMEM with penicillin(100U/ml), streptomycin(100U/ml), and 10% fetal calf serum. The adherent cells are predominantly fibroblasts and are termed primary fibroblast cultures in this study. This method yields primary cultures of fibroblasts having 85%～90% purity as judged by morphology and vimentin immunostaining. Cells were grown in 5% CO₂/95% air at 37℃. Cells from passage 3 were used for these studies and were grown to 80% confluence for each experiment. Cell culture experiments were repeated at least six times. Fetal rat lung fibroblasts were washed three times in serum-free DMEM and then cultured in serum-free medium for 24h before experiments to decrease the effect of binding proteins present in the serum. Cells were treated with rhIGF-I(0-500 ng/ml, peprotech, USA) for 24h and rhIGF-I(200ng/ml)for 30min, 1h, 2h, 6h, 12h, 24h, 48h, 72h. In experiments in which fetal rat lung fibroblasts were pretreated with the MAPK pathway inhibitor and the PI3-K pathway inhibitor, cells were incubated in serum-free medium for 24h, then treated with PD98059(1mg, sigma, USA) or LY294002(1mg, sigma, USA) with or without rhIGF-I(100ng/ml) for 24h. At the end of each experiment, the medium was removed, and cells were washed twice with cold PBS. Cells were then scraped and processed for analysis of mRNA.9,Statistical analysisValues are expressed as mean±SEM. Differences between groups were analyzed with the an paired Student's t-test using SPSS 11.5,correlation analysis using pearson correlation. Statistical differences between groups within an experiment were determined by ANOVA and Dunnett's test for multiple comparisons to a control group. P-values<0.05 were considered statistically significant.Results1. The expression of IGF-I in CLD is dynamic, the expression of IGF-I protein is consistent with that of IGF-I mRNA, the expression of IGF-I decreases from 3 days to 10 days after hyperoxia exposure and then increases at 14 days and 21 days, IGF-I immunoreactivity is localized primarily to alveolar and interstitial macrophages, myofibroblasts, interstitial mesenchymal cells around airway and alveoli, and alveolar epithelial cells.2. The expression of IGF-I R in CLD is dynamic, the expression of IGF-I R protein is consistent with that of IGF-I R mRNA, the expression of IGF-I R decreases from 3 days to 10 days after hyperoxia exposure and then increases at 14 days and 21 days, IGF-I R immunoreactivity is localized primarily to alveolar epithelial cells, myofibroblasts, perivascular interstitial mesenchymal cells, interstitial mesenchymal cells around airway and alveoli, vessel endothelial cells, and perivascular and peribronchial smooth muscle cells. 3. The expression of IGFBP-5 in CLD is dynamic, the expression of IGFBP-5 protein is consistent with that of IGFBP-5 mRNA, the expression of IGFBP-5 increases from 3 days to 10 days after hyperoxia exposure and then decreases at 14 days and 21 days, IGFBP-5 immunoreactivity is localized primarily to bronchial and alveolar epithelial cells, myofibroblasts, perivascular interstitial mesenchymal cells and interstitial mesenchymal cells around alveolar septa. The expression of IGFBP-5 mRNA correlates negatively with IGF-I mRNA.4. The expression of collagenâ… and tropoelastin in CLD is dynamic, the expression of collagenâ… and tropoelastin decreases from 3 days to 10 days after hyperoxia exposure and then increases at 14 days and 21 days.5. The expression of IGF-I mRNA correlates positively with tropoelastin mRNA. The expression of IGF-I and IGF-I mRNA correlates positively with collagenâ… and collagenâ… mRNA.6. RhIGF-I(10～200ng/ml) increases collagenâ… mRNA abundance in fetal rat lung fibroblast in a dose-dependent manner.7. RhIGF-I(200ng/ml for 30min, 1h, 2h, 6h, 12h, 24h) increases collagenâ… mRNA abundance in fetal rat lung fibroblast in a time-dependent manner.8. RhIGF-I(10～500ng/ml) increases tropoelastin mRNA abundance in fetal rat lung fibroblast in a dose-dependent manner.9. RhIGF-I(200ng/ml for 30min, 1h, 2h, 6h, 12h, 24h, 48h, 72h) increases tropoelastin mRNA abundance in fetal rat lung fibroblast in a time-dependent manner.10. LY294002 can block the rhIGF-I-dependent increase in collagenâ… mRNA abundance partly, but PD98059 has no effect.11. LY294002 and PD98059 can block the rhIGF-I-dependent increase in tropoelastin mRNA abundance.Conclusion1. IGF-I and IGF-I R act as the important accelerant of alveolar development in CLD, IGFBP-5 is the inhibitory factor to the activity of IGF-I 2. IGF-I is one of the most important growth factors involved in the development of CLD, its mechanism is to influence the synthesis of elastin and collagenâ… .3. RhIGF-I increases collagenâ… mRNA and tropoelastin mRNA abundance in fetal rat lung fibroblast by way of MAPK-MEK-ERK2 pathway or PI3-K-AKT pathway.4. RhIGF-I increases collagenâ… mRNA abundance in fetal rat lung fibroblast by way of PI3-K-AKT pathway.5. RhIGF-I increases tropoelastin mRNA abundance in fetal rat lung fibroblast by way of MAPK-MEK-ERK2 pathway and PI3-K-AKT pathway.