| IntroductionNeonatal chronic lung disease (CLD) is one of the most frequent and serious clinical problems associated with premature birth in neonatal intensive care units (NICU). Although the pathogenesis of CLD is unclear, it probably involves responses to damage inflicted on the immature lung by oxygen therapy, mechanical ventilation and airway inflammatory responses, and hyperoxia is thought to be the most important reason and high-risk factor.In the normal lung, the alveolar epithelium comprises two principal cell types. It has been shown that the changes of AEC II , including morphotype, number and functions, are present in animals with hyperoxia-induced lung injury. At present, studies about mechanisms of impaired development of alveolar are focused on: 1) whether apoptosis of alveolar epithelial cells is involved in the formation of CLD. It has been demonstrated that hyperoxia can induce significant apoptosis of AEC II in vitro; And the longer hyperoxia exposed, the more apoptotic signals appeared in the term-neonatal mice. 2) Influence of hyperoxia on proliferation of alveolar epithelial cells. Proliferation of AEC II was inhibited by hyperoxia in most vitro experiments, while the contradictory results were found in premature baboon with hyperoxia and mechanical ventilation-induced CLD. These researches had obtained some preliminary knowledge about mechanism of CLD. But there are so many problems needed resolved. Such as whether apoptosis has relation with pathological changes in CLD? Which ways are apoptotic signals transduced? And proliferation of AEC II is inhibited or induced by hyperoxia? And so on.KGF (keratinocyte growth factor) is a member of fibroblast growth factor (FGF) family. KGF has been reported to regulate several functions of alveolar epithelium, including proliferation of alveolar epithelial cells, synthesis of surfactant proteins (SPs) , and wound closure in airway epithelium. KGF was also shown in adult rodents to attenuate oxygen-induced lung injury. Accepted mechanisms involved in protection of KGF are mainly described as following: First, KGF may exert its protective effect by stimulating proliferation of AECII, which could maintain or restore an intact epithelial surface. Second, KGF may decrease lung injury in part by augmenting expression of surfactant proteins. Third, KGF may limit hyperoxia-induced increases in lung epithelial cell permeability. Finally, KGF may also reduce hyperoxia-induced DNA damage and ap-optosis.Based on premature rats with hyperoxia induced CLD, investigated the morphologic changes, apoptesis and expression of its main signaling factors, and proliferation and differentiation of alveolar epithelial cells (proliferation marked with alkaline phosphatase and differentiation marked with aquaporin-5) , with morphologic, immunohistochemistric and molecule biologic technologies.The aims of this study were at mechanisms of impaired alveolar epithelium growth and protective effects of KGF on premature rats with hyperoxia-induced CLD. We try to provide more information of CLD and KGF.Material and Methods1. Animal modelPremature rat pups were delivered by cesarean section at gestational age 2Id. All surviving premature pups ( average survival rate was 58% ) were pooled together and randomly distributed to the group of surrogate mother rats. Four experimental groups were set up: controlled and model groups which received daily injection of physiologic saline, and KGF and treated groups which received 1.0 mg/kg/day rhKGF for the first 3 days of exposure followed by daily doses of 0.5 mg/kg until the end of the experiment. All injection were subcutaneous in the upper back area at injection volume of 5jxl/g body weight. Themodel and treated groups were exposed to hyperoxia ( ^95% 02) in 1. 75m3 clear Plexiglas exposure chambers and the two air groups were maintained in chambers open to room air. The 02 concentration was monitored several times a day, as were C02 ( < 0. 5% }, temperature (22 ~ 25^ ) and humidity (50 ~ 70% ). Hyperoxia exposures were continuous at 2. OL/min, except for a daily 45 min internal required for weighing and injecting the pups, switching the mother rats from 02 to air litters and vice versa.2. Sample collection and treatmentPups were killed by an intraperitoneal inject of pentobarbital sodium and exsanguinated by aortic transaction on different times points. The thorax was opened and lung tissue was collected and stored respectively according to the following methods; some samples was fixed in 4% formaldehyde polymerisatum and some in 2. 5% glutaral and residual sample in the Eppendorf tubes was stored at - 80*C (Rnase-free) freezer. i3. Experimental methods and Analysis marker1) Light microscopy (OLYMPUS BX50): pathology of lung tissue; radical alveolar counts (RAC); ratio of alveolar and septa area (A/S).2) Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-DIG nick end labeling (TUNEL) : the observation of apoptotic cells.3) Electronic microscopy (JEM1200Ex): the observation of ultra structuresof AEC1I.4) Immunohistochemistry: expression of pCaspase3, phosphatidylinositoB-kinase (PI3K) , and extracellular signal-regulated kinase (ERK1/2).5) Reverse transcription polymerase chain reaction (RT-PCR, PE9600): expression levels of surfactant proteins (SP-A, SP-B, SP-C, SP-D), Bax, Bcl-2 and Fas mRNA.6) Real-time PCR (ROCHE light Cycling): expression of alkaline phos-phatase (ALP) and aquaporin-5 (AQP5) mRNA.4. Statistical analysisAll data were expressed as the x ± 8, ANOVA, t-tset and Spearman analysis were carried out using SPSS11.5 statistical software.Result1. Morphologic changes in premature rats with hyperoxia-induced CLD Pathological morphology: Inflammation cells infiltration were seen in alveolar space and interstitial area on day 3, alveolar number and septum were decreased, interstitial cells and alveolar internal surface area were increased after day 7 and more pronounced until day 21.2. Mechanisms of impaired lung development in premature rats with hyperoxia-induced CLD1) The changes of the number of apoptotic cells: the number of apoptotic cells in model group increased significantly on day 7, with peak on day 21.2) Ultrastructure changes of AEC II : Model group showed: swelled Mi on day 1, emptied LB on day 3, increased matachromatic granules of nuclei on day * 7, disappeared organelle, dissolved nuclei on day 21.3 ) Signaling pathways of AEC II :(DpCaspase3 was enhanced on 7d(7d p = 0.004,14d%21d p <0.001) in model group, and kept on rising till 21d(Oneway ANOVA,F = 138.92, p < 0.001). Changes of pCaspase3 correlated positively with the number of apoptotic cells (r = 0.542, p < 0.01). ?Bax mRNA of model group was higher than controlled ones (p < 0.05) since 3d (3d p = 0.02; 7d p = 0. 001,14%21d p < 0. 001), while Bcl-2 was lower (p < 0.05) on 7,14 and 21d(7d p =0.047,14N21d p <0.001); (|)There was no difference of Fas mRNA between these two groups (p > 0.05).4) Changes of proliferation and differentiation in premature rats with hyperoxia-induced CLD:In model group, expressions of ALP mRNA were lower on Id and 3d (Id p <0.001, 3d p = 0. 041) , and higher on 14 and 21d( 14d p = 0.019,21d p <0.001);expression of AQP5 mRNA kept on reducing from 7days to 21 days and in positive correlating with RAC (r = 0.511, p = 0.01).3. Protective effects of KGF on premature rats with hyperoxia-induced CLD The changes of histology in treated group were lightened with lowered A/Sand increased RAC.4. Protective mechanisms of KGF on premature rats with hyperoxia-inducedCLD1) Effects of KGF on AEC E apoptosis in premature rats with hyperoxia-in-duced CLD(pCompared with model group, the number of apoptotic cells was lower on 7d, 14d, and 21d in treated group(7dp=0.005,14dN21dp<0.001).(D There was no difference for Bax mRNA between treated and model groups(p >0.05) ; and Bcl-2 mRNA was increased on 3d and kept on rising to 21d(3dp=0.014,7~21dp<0.001) in treated group.(^Expression of PI3K was higher in treated group at all time points than in model group( Id p <0.05,3 ~21d p <0.01).2) Effect of KGF on proliferation and differentiation in premature rats with hyperoxia-induced CLD(Din treated group,levels of ALP mRNA were higher significantly, but changes of AQP5 mRNA were delayed and didnf t reached the levels of air-controlled.?Expression of ERK (1/2) was higher in treated group at all time points than in model group.3) Compared with model group, levels of SPs mRNA (p < 0.05) were increased at different extents in treated group.Conclusion1. Inhibition of alveolar development is the main pathological changes of premature rats with hyperoxia-induced CLD.2. Excessive apoptosis, inhibition of AEC II proliferation, and disorder of differentiation from AEC E to AEC I may be the main causes which lead to the formation of abnormal alveolar epithelial growth.3. Apoptosis of alveolar epithelial cells is involved in the formation of CLD, and its main signaling pathway may be initiated by imbalance of Bax/Bcl-2, and activation of caspase3 may be the key role of AEC E apoptosis.4. There is an inhibition of proliferation in earlier phrase and stagnatation of differentiation during the whole process in AEC E of premature rats with hyper-... |
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