Relationship Of HoxA5 And HoxB5 Genes And Alveolar Epithelial Cell Dysreepithlialization In CLD Induced By Hyperoxia In Neonate Rats

IntroductionChronic lung disease (CLD) of prematurity is the most frequent and severe complication that is caused by inhaling concentration oxygen, mechanical ventilation therapy or lung infection. Bronchopulmonary dysplasia (BPD) is one of chronic lung diseases first described in 1967 as a complication of therapy for premature infants with hyaline membrane disease. At the same time treatment with high concentration of oxygen was thought to be a major contributor to its development. Since administration of supplemental oxygen as one of supportive therapies elevates oxygen concentrations, that may cause pulmonary oxygen toxicity, and even develop to CLD. Improvements in respiratory care and management had allowed infants of more than 30 wk of gestation to survive their respiratory distress syndrome, even before the widespread useof exogenous surfactant therapy. This had led to an increased survival of very immature infants (BW<1000g), who were at most risk of developing bronchopulmonary dysplasia.(reaching 30%-40% abroad, upgrading tendency domestic). In total about 10%-15% CLD infants died with respiratory failure due to lung functional disturbance, because of deficiency of effect monitoring and treating means. Consequently the survival have to rely on oxygen or mechanical ventilation for a long term.Pulmonary fibrosis has happened followed with alveolar epithelial cell (AEC) damage which is the main pathology characteristic. Formerly, lots of studies tried to illuminate the mechanism of the alveolar epithelial cell impairment (necrosis or apoptosis) and the pathological process of fibrous tissue substituting AEC. In order to prevent CLD many experiments have been done to relieve the degree of AEC injury and the amount of collagen deposition. Unfortunately there has no effective measures.So far the development of the research about CLD treatment is still on the slow pace. It is just the inreversible pulmonary fibrosis after the normal alveolar epithelial cell defeated in remodeling that impacts the infants'respiratory function. Why the damaged AEC couldn't be repaired but substituted by fibrous tissue? There is no answer for it. So we assume that studying the reparation of AEC and the mechanism of its regulation might be the break to resolve the problem about the normal alveolar epithelial cell defeated in remodeling.The alveolar epithelial cells consist of typeⅠ(AEC-Ⅰ) and typeⅡ(AEC-Ⅱ)cells. It has been confirmed that typeⅠalveolar(epithelial)cells (AEC-Ⅰ) are the noble cells which can't be regenerated, proliferated and differentiated. Meanwhile typeⅡalveolar (epithelial) cells (AEC-Ⅱ) are the main stem cells in the lung. If AEC-Ⅱare cultured in vitro they will lose their specific marker SPC while expressing aquaporin5 (AQP5) which is the specific marker of AEC-Ⅰ. After 3H-TDR tagged animal endosomatic AEC-Ⅱfor 2 days, the labelled AEC-I would be found near the AEC-Ⅱ. It can be seen that if the alveolar epithelial cells are damaged it would depend on AEC-Ⅱto proliferate and differentiate to accomplish the epithelial remodeling as AEC-I hasn't the ability of regeneration and reparation.For the past few years the function of homeobox genes (Hox genes) in embryonic development and differentiation has been one of the hot topics all around the world because of Hox genes'conservatism in evolutionary process. The study about hox genes in normal lung development and its functionary in pneumocyte differentiation is in early stage. Different Hox genes are expressed in different regions during embryonic lung development which are important to lung cell lines differentiation and maturation. For example, there is an intimate correlation between hoxa5 and hoxb5 and branching morphogenesis, and so is hoxb7, hoxb8 and hoxb5 and lung bud development. Several reports have described that the normal expression of hoxb5 is concerned with congenital pulmonary sequestration and the excessive expression of hoxb5 is concerned with primary pulmonary hypertension and emphysema. The mechanism of the regulation of hox genes expression in the post-lung has not been clear. Hox genes are also expressed in the neonatal lung,. The expression of hoxa5 is the most intensive followed by hoxb5. The two genes are mainly located in alveolar epithelial cells, and AEC-Ⅰis the ultimate differentiated cell.So we assume that the decrease of expression of hoxa5 and hoxb5 might be the cause of dysdifferentiation of AEC-Ⅱto AEC-Ⅰin the hyperoxia induced CLD.We have a hypothesis:hoxa5 and hoxb5 may be two of the chief genes that control AEC-Ⅱdifferentiated to AEC-Ⅰ.The abnormal expression of them leads to failure of the injured AEC-Ⅰreparation, and results in the ultimate pathology of restrained reepithlialization at last and fibrous tissue substituting the epithelium. If the hypothesis was correct we could illuminate the time-space model of hoxa5 and hoxb5 genes'expression in the normal and CLD rats'lungs but also we could find out that hox genes might play a role in the regulation of the AEC-Ⅱdifferentiation. Thus it will provide experimental and theoretical basis to accomplish the pathogenesis and preventive and therapeutic methods of CLD in premature.Materials and MethodsⅠAnimal modelThe present study was performed in accordance with the guidelines provided by the Experimental Animal Laboratory of Shengjing Hospital China Medical University. Within 24 hours after birth, pups were randomly redistributed to the newly delivered mothers. Term neonatal Wistar rats were divided randomly into two groups.A gruop is an experimental group,which was exposed to oxygen. B group is AIR group that served as controls and were kept in air and not subject to hyperoxia. The pups of hyperoxia exposure groups were contained in glass chambers in which the oxygen was infused continuously to achieve 85% oxygen concentration which was monitored with an oxygen monitor(OM-25ME, USA) twice daily. CO2 was removed by soda lime absorption to keep CO2 levels below 0.5%(Dapex Gas Monitor, USA). Temperature and Humidity was maintained at 20℃-25℃and 50%-70%respectively. We opened the chambers for 0.5h to exchange nursing mothers between air and O2-exposed groups every 24 hours to prevent maternal O2 toxicity and eliminate maternal effects between groups. Meanwhile change water, add food, clean dirty cages and record body weight. All animals were raised in the same room and all other conditions were the same.ⅡTissue preparationOn postnatal 1d,3d 7d,14d,21d. Each group of hyperoxia as well as air rats were anesthetized by intraperitoneal injection of 5% chloral hydrate (6ml/kg), and put to death. The thorax was opened and the lungs were resected. The right lung tissues were put in the Rnase-free Ependorf tubes, and immediately frozen in liquid N2 for mRNA, Enzyme linked immunoadsorbent assay(ELISA), and so on. The left lung tissues were fixed in 4%formaldehydum polymerisatum which contained 0.1% DEPC(0.1MPBS, pH7.0-7.6), then were dehydrated, embedded in wax within 24 hours.ⅢExperiment methods1.The appearances and weight were monitored daily in each experiment groups.2.The changes of the lung pathology(1)For lung pathology assessment, lung sections from the left lobes were stained with HE (hematoxylin and eosin).(2)Ratio of alveolar and septa area(A/S).(3)Radical alveolar counts(RAC).(4)Electronic microscopy:the observation of the ultrastructures of AECII.3.Using Immonohistochemistry and Reverse transcription polymerase chain reaction(RT-PCR) to detect protein and mRNA of AQP5 and SPC expression in the lung.4.The measurement of protein and gene level of HoxA5 and HoxB5 dynamic expression in the lung tissue(1)Immonohistochemistry:the detection of intensity and position of the protein expression in lung tissue about HoxA5 and HoxB5(2)RT-PCR:the detection at mRNA level of HoxA5 and HoxB5 in the lung tissue.(3)Hybridization in situ:the detection of intensity and position of the mRNA expression in lung tissue about HoxA5 and HoxB5(4)Western-blot:the detiction at the protein level of HoxA5 and HoxB5 in the lung tissue of neonates rats.IV Statistical analysisSPSS versionl3.0 was used to perform statistical analysis with all data expressed by (mean±SD). Statistically significant differences in the mean values among multiple groups were analyzed using One Way ANOVA analysis.The mean value of two groups were analyzed with Independent-Samples T Test. Correlation between two variables was analyzed with Spearman analysis. Statistically significant differences set P<0.05.ResultsⅠGeneral status of rats in experimental groups and control groupsThe hyperoxia rats were didn't present dyspnea after one day of oxygen exposure. Tachypnea was observed on the 3rd day after they were detached from oxygen supplementation. After the 7th day of oxygen exposure they began to appear fatigue, pale, and to present tachypnea and cyanosis for different degrees. After 14 days some even had to rely on high oxygen and become worse with the time gone. The control groups didn't have the appearances above.At the beginning there was no difference in average birth weight between the two groups. From 7 days of oxygen exposure, weights of the experimental groups showed a significant decrease compared with control groups at the sams time period(P<0.05).ⅡComparision of lung pathomorphism of the two neonatal rats' groups1. The pathological changes of the lung tissue:In the control groups,on the 1st day of the experiment, it was observed that the alveolar structure was irregular, terminal air space size was rather small, and the alveolar septum was thick. On the 3rd day, the alveolar structure of each group was more regular, the size of alveolus was equal, and alveolar septum was thinner, but in the experimental group, there were a few inflammatory cells exuded out, and blooding interstitial cells increased. On the 7th day the alveolar size was equal in room air rats, while the terminal air space size of the oxygen-exposed rats became large, and there was inflammatory response and more interstitial cells. On 14th day and 21st day, air groups continued to develop alveolization, the alveolar became more regular, the size of alveolus was equal and the alveolar septum was thinner; but in O2 groups, the terminal air space size grew significantly large, secondary septum decreased, the quantity of alveolar reduced with alveolus fusion, interstitial cells increased, and alveolar septa was thicker. On the whole the severity of pathological changes was parallel to the duration of exposure.2. The dynamic changes of ratio of alveolar and septa area(A/S) and Radical alveolar counts(RAC):No significant differences about A/S and RAC could be seen between experimental groups and control groups on day 1-3(p>0.05). But there was a significant difference between the experimental groups and the control groups on the 7th and 14th day(p<0.01). On the 21st day, A/S increased to the highest level and RAC decreased to the lowest level.3. Ultrastructure changes of AECⅡ:There were large normal microvilli (Mv) on the surface of AECⅡ, the structure of the cell was in integrity, the nucleus was ellipse, nuclear membrane was complete, and chromoplasm was distributed evenly; There were mitochondrias(Mi) and large number lamellar bodies(LB) in the intracytoplasm. After exposure hyperoxia for one day, some mitochondrias were swelling. LB began to be emptied on the 3rd day; and on the 7th day the number of Mv was decreased,the main chromoplasm was heterochromatin, Mi swelled and LB was vacuolization, but the nucleus was complete. On the 14th day large number of microvilli outpocketed from the membrane and several of them ablated. Mitochondrial cristae broke and some of them dissolved. On the 21st day there was karyopyknosis and cell nucleus dissolved,and d Mi, LB and Mv disappeared.Ⅲ.The dynamic changes of SPC and AQP5 expression in the lung tissue of hyperoxia induced CLD1. The dynamic changes of SPC and AQP5 protein expression in the neonate rat lung after being exposed in hyperoxiaImmuonhistochemical studies shows SPC is an identification marker of AECⅡ, which is expressed in kytoplasm of the typeⅡalveolar epithelial cells in the lung. Compared with the control group, SPC expression in the experimental group decreased significantly on the 3rd day (P<0.05) while it increased obviously on the 14th and 21st day (P<0.05),but there was no difference on the 1st and 7th day. AQP5 is an identification marker of AECI, which is expressed in kytoplasm and cell membrane of the typeⅠalveolar epithelial cells in the lung. On the 1st and 3rd day, there was no difference of its expression, but on the 7th,14th and 21st day its expression in the experimental group decreased significantly (P<0.05)2. The dynamic changes of SPC and AQP5 mRNA expression in the neonate rat lung after being exposed in hyperoxiaThe expression tendency of SPC mRNA was not in a straight line.Compared with the control group, SPC mRNA expression in the experimental group decreased significantly on the 3rd day (P<0.05),while it increased obviously on the 7th,14th and 21st day (P<0.05) and its relative amount is more than that in the control group, but there was no difference on the 1st day. AQP5 mRNA expression in the experimental group decreased gradually, and its change slowed down on the 14th and 21st day. The level of AQP5mRNA in the control group increased gradually and its tendency was gentle. There was significant difference between the two groups except the 1st day.IV Empirical study of the dynamic changes of HoxA5 and HoxB5 genes expression in the lung tissue of hyperoxia induced CLD1. The dynamic changes of HoxA5 and HoxB5 protein expression in the neonate rat lung after being exposed in hyperoxiaImmuonhistochemical results showed that HoxA5 protein was mainly expressed in alveolar epithelial cells, mesenchymal cells and vascular endothelial cells, and small amounts of them expressed tunica mucosa bronchiorum cells in the lung. With the alveolization development, the tendency of HoxA5 protein expression was more obvious in alveolar epithelial cells. In the control group, the level of HoxA5 protein expression was high and there was no significant difference from the 1st to 3rd day(p> 0.05), and its expression was steady and kept on a high level after 7 days. Compared with the control group, HoxA5 protein expression in the experimental group began to decrease on the 7th day and got to the lowest level on the 14th day, and it remained on a low level on the 21st day (P<0.05),while there was no significant difference between the 1st and 3rd day(p>0.05). HoxB5 protein was mainly expressed in alveolar epithelial cells, tunica mucosa bronchiorum cells and vascular endothelial cells, and small amounts of them expressed in mesenchymal cells of the neonate rat lungs. With the alveolization development, more HoxB5 protein was obviously expressed in the alveolar septum and cristae. In the control group, the level of HoxB5 protein expression was high and there was no significant difference between the 1st and 3rd day, it reached the peak on the 7th day, and its expression was steady and kept on a high level on the 14th day and 21st day. Compared with the control group, there was no significant difference of HoxB5 protein expression between the 1st and 3rd day(p>0.05). HoxB5 protein expression decreased gradually on the 7th day, and the difference of HoxB5 protein expression was significant between the two groups on the 7th,14th and 21st day (P<0.05)Western blotting got the similar results as those in imunohistochemistry. In the control group, the level of HoxA5 protein expression was high and there was no significant difference between the 1st and 3rd day(p>0.05), HoxA5 protein expression increased on the 7th,14th and 21st day. In the experimental group, the level of HoxA5 protein expression was high on the 1st and 3rd day, HoxA5 protein expression decreased and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05). In the control group, the level of HoxB5 protein expression was high on the 1st and 3rd day, HoxB5 protein expression increased on the 7th,14th and 21st day. In the experimental group, the level of HoxB5 protein expression was high on the 1st and 3rd day, HoxB5 protein expression decreased gradually and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05), and the protein expression of HoxB5 was almost negative on the 21st day.2 The dynamic changes of HoxA5 and HoxB5 mRNA expression in the neonate rat lung after being exposed in hyperoxiaRT-PCR results showed:In the control group, the level of HoxA5 mRNA expression was high on the 1st and 3 rd day, and HoxA5 mRNA expression increased on the 7th,14th and 21st day. In the experimental group, the level of HoxA5 mRNA expression was high on the 1st and 3rd day, HoxA5 mRNA expression decreased gradually and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05) in the neonate rat lungs. There was no significant difference of HoxB5 mRNA expression which expressed at a high level between the experimental group and the control group on the 1st and 3rd day (P>0.05). And there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05). In the experimental group, HoxB5 mRNA expression decreased gradually and there was significant difference between the two groups on the 7th,14th and 21st day (P<0.05)Hybridization in situ results showed that HoxA5 mRNA was expressed mainly in alveolar epithelial cells, mesenchymal cells and vascular endothelial cells. Small amounts of them expressed tunica mucosa bronchiorum cells in the lung which expressed both in karyoplast and kytoplasm. HoxA5 mRNA was mainly located in alveolar septum cells and alveolar epithelial cells. With the alveolization development, its expression was more obvious in alveolar epithelial cells. In the control group, the level of HoxA5 mRNA was high and there was no significant difference between the 1st and 3rd day(p>0.05). Its expression was steady and kept on a high level after 7 days. Compared with the control group, HoxA5 mRNA expression in the experimental group began to decrease on the 7th day, it remained at a low level on the 21st day(P<0.05) while there was no significant difference between the 1st and 3rd day(p>0.05). HoxB5 mRNA was mainly expressed in alveolar epithelial cells, tunica mucosa bronchiorum cells and vascular endothelial cells, and small amounts of them expressed in mesenchymal cells of the neonate rats lung. With the alveolization development, HoxB5 mRNA was expressed more and more obviously in the alveolar septum, junction and cristae. In the control group, the level of HoxB5 mRNA expression was high and there was no significant difference between the 1st and 3rd day (P>0.05), it reached the peak on the 7th day, and its expression was steady and kept on a high level on the 14th day and 21st day. Compared with the control group, there was no significant difference of HoxB5 mRNA expression between the two groups on the 1st day (p> 0.05). HoxB5 mRNA expression began to decreas on the 3rd day, the difference of HoxB5 mRNA expression was significant between the two groups on the 7th,14th and 21st day (P<0.05)Concludsion1.Pathological findings charactered by the arrested alveolar development and deposited fibres in newborn rats after prolonged hyperoxia-exposed are consistent with those of CLD of prematurity in human beings.2. SPC is expressed mainly in the typeⅡalveolar epithelial cells in the lung. AQP5 is expressed in the typeⅠalveolar epithelial cells in the lung. AQP5 is also expressed in the tunica mucosa bronchiorum and small vessels submucosa.3. The number of AEC-Ⅱdecreased in early stage of CLD, then it would keep in a higher level,while the number of AEC-Ⅰdecreased gradually which was the changes of alveolar epithelial cells in the impaired lungs induced by hyperoxia.4. AEC-Ⅱis the stem cells of AEC-Ⅰ, and so we suppose that there would be dysdifferentiation of AEC-Ⅱto AEC-Ⅰin the hyperoxia induced CLD. 5. HoxA5 and HoxB5 genes are expressed in the normal neonate rats. In the period of alveolization, the tendency of the two genes expressed amount is upgraded. Both of the two genes are expressed in alveolar epithelial cells especially in the alveolar septum junction and cristae.6.The expression of HoxA5 and HoxB5 genes is abnormal in the impaired lungs induced by hyperoxia. With the extended exposure time, the amount of their expression deceased gradually.7. At the early stage of hyperoxia induced lung injury alveolar epithelium is damaged and alveoli of lung reparation is blocked. HoxA5 and HoxB5 genes might regulate AEC-Ⅱdifferentiated to AEC-Ⅰ.