The Study On Dynamic Changes Of AQP,SP And Occludin Expression In Pulmonary Alveolar Epithelia And Their Effects On Chronic Lung Disease Induced By Hyperoxia In Newborn Rats

IntroductionClinical investigations showed that hyperoxia - induced lung injury had become one of the main complications of intensive newborn care, which could even develop to the severe sequela -chronic lung disease(CLD).The causes and mechanisms of CLD are not completely clear now and the common opinion is that pulmonary immaturity, oxygen toxicity, baro/volutrauma as a consequence of mechanical ventilation and infections are important factors.Normal pulmonary anatomic structure and physiological mechanisms help to maintain constant water content in pulmonary mesenchyma and keep an ideal moisture in the alveoli, which are essential for the fulfillment of the lung functions. If under some conditions the amount of extravascular fluid in the lung tissues increases so much even leading to exudation into alveoli, disturbance of physiological function, i. e. pulmonary edema, will occur.Carter found that in rats hyperoxia exposure for 60h could damage pulmonary vascular endothelium and alveolar epithelium and lead to pulmonary edema. A study of Matthay et al in 2002 showed that the changes of the barrier function of alveolar epithelium after acute lung injury were closely related to the occurrence and development of pulmonary edema. Under the function of pathological factors, alveolar epithelium was damaged, intercellular connection was broken, epithelial permeability increased and fluid exuded into alveolar cavity;and at the same time fluid transportation mechanism in alveolar epithelium was disturbed, fluid clearance in alveoli was failed and then alveolar edema was resul-ted. In Matthay's opinion, the barrier function of alveolar epithelium plays a critical role in the pathogenesis of acute lung injury. The damage extent of alveolar barrier determines the severity of acute lung injury and the restoration extent of alveolar barrier confirms the prognosis of the patient. The alveolar epithelial barrier consists of type I and type II alveolar epithelial cells ( AEC I and AEC II) and intercellular connections.Alveolar epithelial cells II(AEC II) are the main stem cells in the embryonic lungs and their proliferation and differentiation to AEC I leading to the reepithelization and restoration of air/blood barrier is an important repairing way for lung injury. Surfactant protein ( SPs) , produced mainly by AEC II, have many biological effects, such as reducing pulmonary surface tension, promoting fluid absorption, prevention of alveolar collapse and edema, maintaining the normal size of alveoli and the dilation condition of alveoli at the end of exhalation. The expression and signal regulation mechanism of SPs in hyperoxia - induced lung injury were rarely reported.Aquaporin ( AQP) are a newly founded group of membrane transportation proteins related to water permeability, whose discovery revealed the regulation of water transmembrane transportation. A study showed that the alveolar water permeability decreased 10 times in AQP, and AQP5knockout rats;Towre found that in adenovirus infected mice AQP, and AQP5 expression reduced not only in infected lung tissues but also in the whole lung. This study first demonstrated that AQP, and AQP5 were related to lung infection and edema by influencing fluid transportation;In acute lung injury models induced by endotoxin both AQP, and AQP5 expression decreased when lung edema occurred;An animal study found hyperoxia exposure (FiO20.9) for 7 days could reduce AQP expression and further reduction occurred at the 14th day. It is still unclear whether reduced AQP, and AQP5 expression occurred at the beginning period of hyperoxia exposure leading to abnormal transmembrane transportation and accumulation of water in lung tissues. And the signal regulation mechanism of AQP expression was also unknown.Platelet - derived growth factor ( PDGF) can promote cellular mitosis and has chemotactic function. PDGF - A and PDGF - B are two types of polypeptide ?growth factors and play their biological effects through platelet derived growth factor receptor ( PDGFR). In the development process of lung tissues PDGFR are divided into two types, aand (3, and play their biological roles by transmitting signals into cellular nucleoli after combination with their ligands. Some studies found that PDGFR - aand pare closely related to lung fibrosis at the middle and late period of hyperoxia - induced CLD. But the effects of PDGFR at the early period of pulmonary alveolar edema are rarely reported.As previous studies showed, hyperoxia — induced changes of alveolar epithelial barrier were related with the occurrence and development of lung edema. Hyperoxia could damage alveolar epithelia, break intercellular connection, increase epithelial permeability and result in fluid leakage into alveolar cavities. Although the critical role of alveolar epithelial barrier in the pathogenesis of a-cute lung injury has been certificated, the role of the alveolar epithelial dense connection including its molecular structure and regulation mechanism has not been demonstrated.In this study, we produced hyperoxia - induced CLD models in newborn rats and dynamically observed the morphology changing rules of alveolar epithelia at the early stage of hyperoxia - induced lung injury by immunohistochemistry and molecular biology methods. We also observed the dynamic changes of alveolar epithelial barrier. The dynamic changing rules of AQP and its possible downstream signal pathway were studied at both protein and mRNA levels. And preliminary research about pulmonary edema mechanism at the early stage of hyperoxia - induced lung injury has been done in order to provide experimental basis to accomplish the pathogenesis and preventive and therapeutic methods of CLD in neonates.Materials and MethodsI. Animal modelWithin 12 hours of birth, newborn rats were randomly divided into 4 groups with different FiO2: experimental group 1 ( FiO20. 8) , experimental group 2 ( FiO20. 6 ) , experimental group 3 ( FiO20. 4 ) and control group ( FiO20. 21).Rats of control group were remained in room air. Hyperoxia exposure were done in Plexiglas chamber into which oxygen was continuously delivered to achieve a constant level of 80% , 60% and 40% oxygen monitored daily with an oxygen monitor(OM -25ME, USA). Oxygen and room air were filtered through Sodal-ime to keep CO2 levels below 0.5%. Temperature and humidity were maintained at 25 -26T! and 60% -70% respectively. Chamber was opened for 0.5h daily to switch nursing mother and change water, add food, clean dirty cages and record survival and body weight daily. Nursing mothers were rotated between oxygen exposed and room air litters every 24h to avoid oxygen toxicity in the mothers and to eliminate maternal effects between groups.II. Tissue preparation8 rats of each group were killed on day 1, 3, 5, 7 and 14. Lung tissues were collected and stored respectively according to the following methods;fixed in 4% formaldehydum polymerisatum for immunohistochemistry study;fixed in 2. 5% glutaral for ultrastructure examination;put in the Rnase -free Eppendorf tubes and stored at - 80T! freezer for reverse transcription polymerize chain reaction ( RT - PCR) exam.III. Experiment methods1. Microscope exam: (l)lung histological study(2)width of interalveolar septa (Universal Imaging Porporation, USA ) (H)radical alveolar counts ( RAC ).2. Electronic microscopy: the observation of the ultrastructures of AEC II and alveolar barrier function through lanthanum nitrate tracing technology.3. Measurement of lung wet/dry weight ratio ( W/D ) , protein content in BALF by ultraviolet/visible spectrophotometer and alveolar epithelial permeability (AEP) with Evans blue.4. Immunohistochemistry: signal transducers and activators of transduction 3 ( STAT3 ) , extracellular signal - regulated kinane ( ERK^ ) , aquaporinl (AQPj), aquaporin5 ( AQP5), Platelet - derived growth factor A ( PDGFA) , Platelet - derived growth factor B (PDGFB) , platelet derived growth factor receptor a(PDGFRa) and platelet derived growth factor receptor p(PDGFRp).5. Reverse transcription polymerase chain reaction (RT -PCR);the detection at mRNA level of surfacant proteins (SPs) , AQPj ,AQP5 and occludin.6. Western Blotting: the detection at the protein level of AQPj and AQP5.IV. Statistical analysisSPSS 11.5 was used to perform statistical analysis, with all data expressed as ( x ± s ). Statistically significant differences in the mean values among multiple groups were analyzed using ONE WAY ANOVA analysis and correlation between two variables was analyzed with Spearman analysis.ResultsI. General status of the newborn rats after hyperoxia exposureThe hyperoxia rats were didnt present dyspnea after one day of oxygen exposure, but began to present fatigue, dyspepsia and slight dyspnea after being detached from oxygen supplementation from the 3th,5th or 7th day. On the 14' day the situation got worse, such as body weight loss, severe dyspnea with cyanosis and shaking head after detachment from oxygen for 5 — lOmin and even death.II. The lung morphology of the newborn rats after hyperoxia exposure1. Pathological findings: In the control group, the alveolar structure was irregular, the terminal air space size was small and the alveolar septum was thick on day 1 -3 of the experiment, but on day 5-14 the alveolar structure became more regular, the size of alveolus was equal and the alveolar septum was thinner. For oxygen - exposed groups inflammation cells infiltration and protein and RBC exudation could be seen in alveolar sac on day 3-5 and the situation became worse on day 7 - 14, where enlarged alveolar sac, irregular alveolar structure , increased interstitial cells quantity, thicker interstitium and alveolus fusion even could be seen. On the whole, the severity of pathological changes was parallel to the concentration of oxygen and the duration of exposure.2. The dynamic changes of alveolar septum and RAC: No significant differences about the alveolar septum and RAC could be seen between experimental groups and control group on dayl -3. But from day5 the alveolar septum became markedly thicker and RAC decreased continuously compared with those in control group.3. Ultrastructure changes of AECII: There were normal microvilli (Mv) , mitochondria (Mi) and lamellar bodies(LB) in the AECII of control group. Experimental groups showed fewer Mv, swelled Mi, decreased electronic density and emptied LB on day7 -14.lanthanum nitrate tracing technology: In control group there was no lanthanum nitrate granules in the basal membrane of AECII. In the experimental groups, widened and swelled alveolar septa could be seen and there were some lanthanum nitrate granules in the dense connection, AECII and alveolar septa.III. The dynamic study of AEP and SPs transcription in hyperoxia induced CLD1. The dynamic changes of lung wet/dry weight ratioCompared with control hgroup, lung W/D ratios in the experimental groups increased with significant differences on day5 - 14 ( p < 0. 05 ). On the whole W/D ratios increased with the oxygen concentration elevated.2. The dynamic changes of protein content in BALFThe protein contents of BALF in the experimental groups were significantly higher than that in control group and increased as the oxygen concentration elevated and exposure time prolonged.3. The dynamic changes of AEPAEP in the experimental groups were much higher than that in control group with the significant difference on day3 - 7 and increased as the oxygen concentration elevated.4. The dynamic changes of SPs mRNA expressionIn general, there was no significant difference of SPs mRNA expression between experimental groups and control group on dayl - 3, but significantly higher expression of SPs (SPA, SPB, SPC) mRNA could be seen during day5 -14.5. The dynamic changes of STAT3 protein expressionIn the lung tissues STAT3 protein mainly expressed in alveolar epithelial cells, bronchial epithelial cells, pulmonary vascular endothelial cells and inflammatory cells and sited mainly in the cytoplasm with some expression in the nucleus. Brown granules indicate positive expression. From day 3 the expression of STAT3 in the experimental groups began to increase compared with that incontrol group and the difference was much more marked on day5 and day7 ( p < 0.05).6. The relationship between SPs mRNA and STAT3 protein expression The expression of SPB mRNA in the experimental group was positively related to the expression of STAT3 protein, respectively(r =0.892, P <0.001).IV. The dynamic changes of AQP, and AQP5expression in alveolar epithelial cells of hyperoxia induced CLD1. AQPj and AQP5 protein expressionImmunohistochemistry results showed that AQPj protein was expressed in AECII and capillary endothelial cells and AQP5 protein was expressed in AEC -I with brown staining in the cytoplasm and cellular membrane.Western blotting got the similar results as those in immunohistochemistry. Both AQPjand AQP5were positively expressed in the normal lung tissues and remained at a relatively high level. But their expression in the experimental groups began to decrease after oxygen exposure and the decreasing extent was positively parallel to the oxygen concentration and exposure duration.2. AQPj and AQP5 mRNA expressionOn the whole, AQPj and AQP5 mRNA expression in the experimental groups began to decrease after oxygen exposure and the decreasing extent was positively parallel to the oxygen concentration and exposure duration.3. The expression of ERK1/2The expression of ERK1/2in the lung tissues began to increase after oxygen exposure, especially on day 3-14 for experimental group 1 and on day5 -14 for experimental group 2 and 3.4. The relationship between ERK1/2 expression and AQPj and AQP5 mRNA expressionThe expression of ERK1/2was positively related to the expression of AQPj mRNA (r= 0.627,p<0.001).V. The dynamic changes of PDGF and occludin expression in the lung tissues of hyperoxia induced CLD1. Oxygen exposure could increase the expression of PDGF - B and PDGFR - pin the lung tissues especially on day 7 and 14. But the difference of PDGF - A and PDGFR - aexpression between different groupswas not significant.2. In experimental groupl, the expression of occludin mRNA began to decrease on day 3 and kept in this lower stage during oxygen exposure( p < 0.05 ). In experimental group 2 and 3, the similar tendency could be seen.3. The expression of occludin mRNA was positively related to that of PDGF -B (r= -0.796,p<0.001).Conclusions1. Hyperoxia induced CLD model in newborn rats could mimic the occurrence, development and pathology of CLD in premature infants.2. Increased AEP could be seen at the early stage of hyperoxia induced lung injury;the lung injury became worse with higher oxygen concentration and longer exposure duration.3. At the early stage of hyperoxia induced lung injury JAKs - STAT3 pathway could be activated to play a protective role and the signal for the synthesis and secretion of SPB might be transmitted through STAT3 pathway.4. The dynamic changes of AQPj and AQP5 expression at both protein and mRNA levels showed that in hyperoxia induced lung injury AQPj and AQP5 expression decreased and closely correlated to the severity of pulmonary edema, indicating that AQPs might take part in the formation and absorption of pulmonary edema at the early stage of hyperoxia induced lung injury.5. The signals activating the synthesis and secretion of AQP in hyperoxia induced lung injury might be transmitted by MAPK pathway.6. The increment of PDGF - B expression in the lung tissues of newborn rats after oxygen exposure could be one of the mechanisms to influence the synthesis and distribution of occludin and result in alveolar edema.