The Role Of Epithelial Mesenchymal Transition In Hyperoxia-induced Lung Injury And Interventional Effect Of IGF-1

Background:Oxygen therapy is essential to neonatal intensive care and an important measure, but the oxygen belongs to gas drugs, also have adverse reactions. With the development of the neonatal rescue technology and lung surface active substance, the incidence of bronchial pulmonary dysplasia (BPD) increased year by year. Inhaled high concentration oxygen for a long time can cause pulmonary inflammatory change and pulmonary fibrosis and induced bronchopulmonary dysplasia(BPD), Which seriously affected the neonatal survival rate and survival quality[1].The mechanism of BPD was known clearly. BPD was caused by breathing machine capacity, oxygen toxicity. BPD Can cause abnormal lung tissue repairation [2]. BPD prevention become a research hotspot, There has no drugs for the prevention and treatment of bronchial pulmonary dysplasia in the clinical curative.Lung development need many signal molecules of the epithelial cells and mesenchymal cells secreted[3]. Lung development process included five stages [4], including (1) Embryonic period;(2) Adenoid period; (3) Catheter period; (4) Vesicle period;(5)Alveolar period. A number of studies have shown that lung development and lung damage repairation process need growth factors.High oxygen Inhaled can cause the changes of structure and function in the lung. The changes in the main structure cell can lead to abnormal pulmonary fibrosis in the lung tissue. The high oxygen may lead to abnormal lung development. Proliferation and repairation of alveolar epithelial cells were inhibited in pulmonary fibrosis. A number of fibroblasts cells hyperplasiaed and aggregated. ECM deposited. II alveolar epithelial cells (AECII) involved in the process of growth, development, damage and repair in the lung. Alveolar type II epithelial cells (AEC II) was considered to repair injury and conversed into alveolar type I epithelial cells (AEC I).Alveolar epithelial cells (AECs) has a great phenotypic plasticity. The function has been gradually recognized [5]. High concentration of oxygen inhalated can cause acute or chronic lung injury. Lung injury included alveolar structure damage and increased permeability and fibrosis lesions. AECII is the key to repair lung injury cells. Under the factors, AEC II conversed into AECI differentiation was restrained. The alveolar type II epithelial cells proliferated and released a lot of cytokines, inflammatory factors. Epithelial mesenchymal transition happened and induced the formation of pulmonary fibrosis [6]. Epithelial mesenchymal transition was the progress of epithelial markers missed, mesenchymal markers appeared[7]. Fibroblasts cells may be an important source of pulmonary fibrosis through EMT pathway [8].IGF-1 can inhibit apoptosis and have other biological activities. Its function is through the IGF-1 receptor (IGF-1R) mediated. IGF-1 combinated with IGF-1R by the regulation of IGFBPs [9].The mechanism of IGF 1 can promote cell DNA synthesis and metabolism, prompting cells into G1 phase. Veness-Meehan [10] found that the lungs of IGF 1 increased and followed the high oxygen exposure time.IGF-1 involved in the progress of cell growth and lung injury structure reconstruction.We researched results show that the endoplasmic reticulum stress (ERS) not only can induce cell apoptosis, also affect the change in the phenotype of cells. IGF-1 can effectively restrain high oxygen induced with the newborn rat lung inflammation and lung epithelial cell apoptosis. Endoplasmic reticulum stress can activate the P13K/Akt, Akt can lead to GSK-3 β. GSK-3β involved in lung epithelial-interstitial transformation (EMT).First part, we use the newborn rats to make the model of high oxygen lung injury. Through the intervention of IGF 1, we observe pathology changes and the expression of inflammatory factor in the lung tissue the lung injury. We explore the mechanisms IGF-1 which inhibits the lung cell apoptosis and lung injury induced by high oxygen. Second part, we explore the regulation function of epithelial mesenchymal transformation mechanism in the progress of high oxygen lung injury occurred. The results of the study can provide the prevention and treatment to high oxygen lung injury and experimental basis for the clinical application of IGF-1.Part oneEffects of IGF-1 to inflammatory cytokines in lung tissue of newborn rat with hyperoxia-induced lung injuryObjective:To investigate effects of IGF-1 to inflammatory cytokines in lung tissue of newborn rat with hyperoxia-induced lung injury.Methods:Newborn Wistar rats were randomly divided into air control group, hyperoxia model group and hyperoxia model+IGF-1 group. According to the dosing (NS) time every group was divided into four groups 1 day,3 days,7 days and 14 days. Rats were killed by hypreoxia expoxure on 1 day,3 days,7 days and 14 days. Measure lung tissues wet and dry ratio; take the lung tissues of injury with HE staining, observe lung cell apoptosis with nuclear staining combined by TUNEL; expression levels of TNF-α、 IL-1β、IL-6 in the lung tissue were detected by ELISA.Results:1.The lung wet/dry (W/D) proportion in hyperoxia group on the 3rd,7th and 14th days after expoxure to hypreoxia was significantly increased with the control group. The lung wet/dry (W/D) proportion in the hyperoxia +IGF-1 group was less than hyperoxia group on the 3rd,7th and 14th days.2. HE staining results:In the air control group rats, Lung tissue structure was normal and observed with no pulmonary interstitial edema and less inflammatory cells infiltration under light microscopy. High oxygen+NS group rats were exposed to high oxygen 3 days, we observed that the small blood vessels in the lungs expanded, bronchi wall mild edema, alveolar epithelium swelling, inflammation cells exudated with mainly neutrophils and lymphocytes. After 7days, alveolar decreased significantly, alveolar wall thickness, inflammatory cells infiltration increased obviously. After 14 days, the above change was more obvious, and pulmonary fibrosis was appeared. High oxygen+IGF-1 group was exposure to high oxygen for 3 days,7 days,14 days, witch the pathological changes were significantly reduced than high oxygen+NS group on the same day.3. Measured pulmonary apoptosis with TUNEL staining:A small amount of apoptosis positive cells in the lung tissue were found in the air control group; High oxygen +NS group rats were exposed by high concentration oxygen for 1 day,3 days,7 days and 14 days, apoptosis positive cells were observed in the lung tissue of and apoptosis index were higher than in normal control group(P< 0.05). Apoptosis index in the high oxygen+ IGF 1 group was significantly decreased than in the high oxygen+NS group with the same day (P<0.05)4. Expression levels of TNF-α, IL-1β,IL-6 by ELASA method:High oxygen +NS group of rats were exposed with high oxygen for 1 day,3 days,7 days and 14 days, expression levels of TNF -α, IL-1β,IL-6 in the lung tissue were significantly higher than in air control group(P< 0.05). With high oxygen exposure time prolonged, the expression levels of TNF-α, IL-1β,IL-6 gradually increased. The expression level reached its peak on the 7 days. The expression levels of TNF-α, IL-1β,IL-6 gradually reduced to the 14 days. High oxygen+IGF-1 group rats were exposed to high oxygen for 3 days,7 days,14 days. The expression levels of TNF-α, IL-1β, IL-6 in high oxygen+ IGF-1 group were obviously decreased with the high oxygen+NS group and were significantly higher than in air control group(P< 0.05).Conclusion:1. High concentrations of oxygen can induce acute lung injury and pathological changes in the lung tissue of newborn rats. With high oxygen exposure time extended, the pathological changes showed more obviously.2. IGF-1 can reduce pulmonary edema, inhibit inflammatory cytokines and prevent apoptosis in newborn rats with the high concentrations of oxygen exposure.3. IGF-1 has a protective effect of high oxygen lung injury in newborn rats.Part twoThe role of epithelial mesenchymal transition in hyperoxia-induced lung injury and interventional effect of IGF-1 in newborn ratObjective:To investigate the role of epithelial mesenchymal transition in hyperoxia-induced lung injury and interventional effect of IGF-1 in newborn ratMethods:Newborn Wistar rate were randomly divided into air control group, hyperoxia model group and hyperoxia model+IGF-1 group. According to the dosing (NS) time every group was divided into four groups Iday,3 days,7 days and 14 days. Rats of the model group were prepared to make hyperoxia-induced lung injury model. Rats were killed on 1day,3 days,7 days and 14 days. After modeling, lung tissues wet and dry ratio were measured; taken the lung tissues of injury with HE staining, observed lung fibrosis by Masson staining; expression levels of Slug, Snaill, GSK3β,p-GSK3β, Akt, p-Akt, a-SMA in the lung tissue were detected by Western blotting method.Results:1. The lung wet/dry (W/D) proportion in hyperoxia group on the 3rd,7th and 14th days after expoxure to hypreoxia was significantly increased with the control group. The lung wet/dry (W/D) proportion in the hyperoxia+IGF-1 group was less than hyperoxia group on the 3 days,7 days and 14 days..2. HE staining results:In the air control group rats, Lung tissue structure was normal and observed with no pulmonary interstitial edema and less inflammatory cells infiltration under light microscopy. High oxygen+NS group rats were exposed to high oxygen 3 days, we observed that the small blood vessels in the lungs expanded, bronchi wall mild edema, alveolar epithelium swelling, inflammation cells exudated with mainly neutrophils and lymphocytes. After 7days, alveolar decreased significantly, alveolar wall thickness, inflammatory cells infiltration increased obviously. After 14 days, the above change was more obvious, and pulmonary fibrosis was appeared. High oxygen+IGF-1 group was exposure to high oxygen for 3 days,7 days,14 days, witch the pathological changes were significantly reduced than high oxygen+NS group on the same day.3. Measured lung fibrosis by Masson staining:In air control group rats on the 1 day, 3 days,7 days and 14 days, normal alveolar structure, uniform size, few of collagen deposition were observed. In the high oxygen+NS group rats with high oxygen for 1day alveolar structure was normal. For 3 days, wide alveolar septa, alveolar structure parts, seepage, bronchi and blood vessels can have a small amount of collagen fibers deposition. After 7 days and 14 days, alveolar structure damaged significantly, bronchial wall thickened, collagen fibers deposition increased, fibrosis appeared. High oxygen+IGF-1 group rats were exposed to high oxygen for 7 days,14days. The lung pathology changes of alveolar structure damaged, bronchial wall thickened, collagen fibers deposition, fibrosis appeared redused with high oxygen model group in the same day.4. Expression levels of Slug, Snaill, GSK3β, p-GSK3β,Akt, p-Akt, α-SMA by Western blotting method:The expression levels of Slug, Snaill, GSK3β,p-GSK3β, Akt, p-Akt,αa-SMA in the normal control group rats were less. High oxygen+ NS group rats exposed to high oxygen for 3 days,7 days, The expression levels of Slug, Snaill, GSK3β, p-GSK3β, Akt, p-Akt, a-SMA increased more than th air control group (p< 0.05). When high oxygen was exposed to 14 days, the expression levels of Slug, Snaill, GSK3β, p-GSK3β, Akt, p-Akt, α-SMA Slug in the lung tissue increased more obviously (p< 0.05); High oxygen+IGF-1 group rats were exposed to high oxygen for 1 day,3 days, the expression levels of Slug, Snaill, GSK3β, p-GSK3β, Akt, p-Akt, α-SMA were no significant difference with the high oxygen+NS group(P> 0.05). When high oxygen+ IGF-1 group rats were exposed to high oxygen for 7 days,14 days, the expression levels of Slug, Snaill, GSK3β, p-GSK3β, Akt, p-Akt, α-SMA significantly decreased with the high oxygen+NS group on the same day(P< 0.05).Conclusion:1. High concentration of oxygen can induce pulmonary edema, inflammation. With the high oxygen exposure time extended, the pathological changes showed more obviously.2. High oxygen exposure can cause fibrosis changes in the lung tissue of newborn rats. When the high oxygen exposure time prolonged, fibrosis degree became more obviously.3. High oxygen exposure can change the epithelial cells phenotypic in the newborn rats, promote EMT. The expression levels of Slug, Snaill, GSK-3β,p-GSK -3β,Akt, p-Akt, α-SMA protein in the lung tissue increased gradually.4. IGF-1 can reduce pulmonary edema with high oxygen exposure caused and inhibited the expression levels of Slug, Snaill, GSK-3β, p-GSK -3β,Akt, p-Akt, a-SMA protein. IGF-1 has a protective effect of hyperoxia-induced lung injury.