Related Study On Effect Of Calcitonin Gene-related Peptide In Hyperoxia Induced Lung Injury And Sonic Hedgehog Pathway

BackgroundContinuous oxygen therapy with higher concentration can induceacute lung injury (ALI) or bronchopulmonary dysplasia (BPD), whichseriously threaten to the health of children. There are not effectiveprevention and cure methods to ALI and BPD at home and abroad. Butstudies suggest that the etiologies are closely associated with thematuration and injury/repair of the lungs. The diffuse and non-specificinflammations in lungs can lead to remodeling of airway and fibrosis ofalveolar, which are the most important risk factor and the final pathologycharacteristics in ALI and BPD. The satisfactory repair of lung injury is therestoration of both the structure and function by the nature of the cellsinstead of the proliferation of fibroblasts. Alveolar epithelial cells typeⅡare stem cells and can differentiate into alveolar epithelial cells typeⅠbutlack of proliferation ability. Calcitonin gene-related peptide (CGRP) is aneuropeptide with general regulating function. Shh signaling pathway is closely related to proliferation and differentiation in cells. Shh signalingpathway probably is regulated by the neuropeptide and affect theproliferation and reconstruction of AECⅡ in vitro and in vivo, which maybecome a new entry point of the basic theoretical innovation and clinicalpractice in hyperoxic induced lung injury.Objective1Set up the method of isolation, purification and primary cell cultureof alveolar epithelial cells typeⅡ (AECⅡ) from preterm rats for the effectstudy of CGRP on hyperoxia induced AECⅡ injury and its signal pathway.2Explore the influence of95％oxygen and the protective effect ofcalcitonin gene-related peptide(CGRP)on alveolar epithelial cellstypeⅡ(AECⅡ).3Investigate the effect of Calcitonin Gene-Related Peptide(CGRP) onprimary alveolar epithelial cells typeⅡ (AECⅡ) and the expressions ofSonic hedgehog (Shh) signaling pathway components,Smo and Gli1, uponthe exposure of hyperoxia.4Study the protection of Calcitonin gene-related peptide (CGRP)treatment on hyperoxia-induced ALI symptoms in neonatal rats.5Investigate the expressions of Smo and Gli1signaling molecules inlungs of newborn rats exposed to prolonged hyperoxia and explore the roleof Sonic hedgehog (Shh) signaling pathway in hyperoxia-induced lung injury.Methods1Isolated AECⅡ from the Sprague-Dawley (SD) fetal lungs of SPFrats with19days gestational age. The pregnant rats were anesthetized bychloral hydrate. Use the way of uterine incision delivery to pick out thefetal rats. The fetal lungs were isolated from the fetal rats. Digested theLung tissues by trypsin and collagenase-1, then the AECⅡ were purifiedwith the methods of different centrifugal force and repeated attachment.The dulbecco's modified eagle's medium (DMEM/F12) complemented with10%fetal calf serum were used to cultured the AECⅡ. Trypan blueexclusion was used to assessed AECⅡ viability. The cells purity wasevaluated by modified papanicolaou staining. AECⅡ were also identifiedby transmission electron microscopy. And the cellular growth status andmorphologic changes of AECⅡ were observed with inverted phasecontrast microscope.2AECⅡ were isolated and purified from premature rats and exposedto air (21%oxygen), air+CGRP, air+CGRP+CGRP8-37, hyperoxia(95%oxygen), hyperoxia+CGRP and hyperoxia+CGRP+CGRP8-37for24hrespectively. Morphologic change of AECⅡ was observed under theinverted phase contrast microscope. The apoptosis ratio of AECⅡ,production of intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase(SOD) in AECⅡ weredetected by flow cytometry or spectrophotography. The mRNA expressionsof surfactant protein C (SPC) in AECⅡ were also detected by real-timequantitative polymerase chain reaction (RT-qPCR).3AECⅡ were isolated and purified from premature rats and exposedto air (21%oxygen), hyperoxia (95%oxygen), hyperoxia+CGRP andhyperoxia+CGRP+CGRP8-37for24h respectively. The mRNAexpressions of Smo and Gli11in AECⅡ were detected by real-timequantitative polymerase chain reaction (RT-qPCR), and the proteinsexpression of Smo and Gli11were also detected by western blot.4Newborn Sprague-Dawley rats were randomly divided into threeexperimental treatment groups: normoxia, hyperoxia and hyperoxia withCGRP. Hyperoxia groups were subjected to conditions of oxygen levels inexcess of95%and treated with alternating daily doses of either normalsaline or CGRP for14days. Subsequently, rat lung pathology wasexamined using hematoxylin and eosin (HE) staining under lightmicroscopy, with further studies revealing the presence of bothmalondialdehyde (MDA) and superoxide dismutase (SOD) in homogenizedlung tissues. Furthermore, tumor necrosis factor-α (TNF-α), interleukin-6(IL-6) and endogenous αCGRP mRNA were quantitated by RT-qPCR, andthe protein expression of transforming growth factor-β1(TGF-β1) wasanalyzed by western blot analysis. 5Neonatal rat pups were placed in chambers containing room air oroxygen about95%for14days after birth. The rats were sacrificed at3,7or14days and their lungs removed and subjected to hematoxylin and eosin(HE) staining. Smo and Gli11were observed by immunohistochemistry,and western blotting was utilized to assess the dynamic expressions of Smoand Gli1protein.Results1One of pregnant rat lungs can obtain the number of AECⅡ about(8.1±1.1)×106the survival rate is about (95.5±1.6)％.the purity is about(95.6±2.1)％. The microvillus on the cell membrane and the lamellarbodies in cytoplasm were observed under electron microscope. AECⅡattached the bottom after cultured12~18h. The exponential growth phaseof the AECⅡ was24h~48h after culture. After72h, AECⅡ becameattached cells decreased and died.2Under the95%oxygen condition, the apoptosis ratio of AECⅡ, theproduction of ROS and MDA in AECⅡ were significantly increased, butthe levels of SOD, SPC mRNA in AECⅡ were decreased comparison withair group(P＜0.05). After the intervention of CGRP, the apoptosis ratio,production of ROS and MDA in AECⅡ were down regulated, however, theSOD was up regulated, and the SPC mRNA in AECⅡ were also increasedobviously(P＜0.05).CGRP8-37could block the biological effects CGRP. 3Under the95%hyperoxia condition, the mRNA and proteinsexpression of Smo and Gli1in AECⅡ were decreased comparison with airgroup(P＜0.05). After the intervention of CGRP, the mRNAs and proteinsof Smo and Gli11in AECⅡ were increased obviously(P＜0.05).CGRP8-37could down regulate these mRNAs and proteins by blockthe biological effects of CGRP.4Hyperoxia could induce server histomorphological changes inneonatal lung tissue after exposed to high concentration oxygen. And theMDA was unregulated, the SOD was down regulated, the mRNAexpressions of TNF-α andIL-6were also increased. CGRP treatmentsignificantly alleviates ALI symptoms, preventing both reductions inneonatal body weight and histomorphological changes to neonatal lungtissue. CGRP treatment for exposure to hyperoxic conditions resulted in theinhibition of MDA activation, increased SOD activation, decreased mRNAexpression of TNF-α, and reduced expression of TGF-β1. The endogenousmRNA expressions of αCGRP were no difference in each group.5Significant histo-morphologic changes were showed in thehyperoxia-exposed lungs at3,7and14days of age by HE staining. Smoand Gli1proteins were observed in bronchial epithelial cells, alveolarepithelial cells, and vascular endothelium cells, and partly showed on thefibrotic tissue in lung interstitium. The results of immunohistochemistryand western blot showed that the expression of Smo was significantly higher in the hyperoxia-exposed lungs at7and14days,and Gli1wassignificantly elevated at14days. But low Smo and Gli1were detected innormal air group.Conclusion1It has been known that the methods of different centrifugal force andrepeated attachment are the best ways to culture the primary AECⅡ. Itcould get the best cells number, purity and vitality of AECⅡ. These cellscould be used for the experiments in vitro about AECⅡ. The purity andvitality of AECⅡ perhaps could be further elevated by improve the keysteps in this methods.2The hyperoxia of95%oxygen suppressed the growth of AECⅡ, andresults in dysfunction of the cells. CGRP could partly protect AECⅡ fromoxidative stress injury and promote the cells proliferation.3The situation of Shh signal pathway maybe inhibited by hyperoxiain AECⅡ. CGRP could partly protect AECⅡ from oxidative stress injuryand promote the cells proliferation, which might be associated with theactivation of Smo and Gli1in Shh signal pathway.4Hyperoxia-induced ALI is likely associated with both the oxidativestress and the inflammatory response induced by high oxygen conditions.CGRP treatment may play a protective role in the prevention and treatmentof hyperoxia-induced ALI in neonatal rats by inhibiting these processes. 5Exposure of neonatal rat to prolonged hyperoxia results in acutelung injury and arrested lung development. Smo and Gli1are up-regulatedat time points preceding the lung injury, and Shh signal pathway seems tobe involved in the pathogenesis of hyperoxia-induced lung injury andbronchopulmonary dysplasia.