The Signal Transduction Mechanism For Neuropeptide Substance P Regulating Repairing Of Alveolar Epithelial Type Ⅱ Cells From Preterm Rats After Hyperoxia-induced Lung Injury

Hypoxemia is a common clinical feature of critical neonates and severe hypoxemia can threat their lives. Oxygen therapy is an important method to improve hypoxia condition of neonate. In neonate especially preterm infant, pulmonary developmental immaturity leads to pulmonary interstitial and al-veolus differential insufficency and pulmonary elastic fibrous and connective tissular dysplasia. Long-time exposure to hyperxia will result in alveolar epithelial cells damage, death, acute lung injury (ALI) and even to bron-chopulmonary dysplasion (BPD) caused by respiratory function failure. In the past few years, the incidence of BPD raised yearly in the world. Usually, pulmonary maldevelopment and hypofunction are accompanying with the survivors. Up to now, there is no definited effective prevention and cure approach globally. However, vast of clinical data indicate hypoxemia and prolonged oxygen therapy are high risk factors for BPD. Furthermore, the etiopathogenisis of BPD is closely related to hyperxia-induced lung injury. Currently, the domestic and overseas studies focus the following aspects: Ⅰ.To attempt to retrieve impaired AECⅡfunction by pulmonary surfat-cant-associated protein A (SP-A). However, the biologic extract of SP-A is a difficult task and the long-term effectiveness is not satisfied.Ⅱ. Some scholars tried to differentiate epithelium stem cell into AECⅡand transplant the AECⅡinto patients with lung injury. Unfortunately, some key tech-niques such as cell differentiation, culture and growth regulation in vivo could not be solved successfully.Ⅲ. To utilize growth factor medicines produced by genetic engineering technology. Although epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) were successfully used in body surface repairing and reconstruction of patients suffered from wound and burn, these growth factors have not been applied in the active repairing and reconstruction during lung injury.Ⅳ. Transforming growth factorβ(TGF-β) and interferonγ(INF-γ) plays a positive and negative role in abnormal transconformation of myofibroblast, respectively. However, the rivalry mechanism of the both factors is still unknown.Ⅴ. Although the role of extracellular matrix (ECM) accumulation in the development of pulmonary fibrosis was confirmed, there is still no effective method to prevent ECM accumulation.Ⅵ. For the a series of un-certain pathophysiological change in AECⅡrepairing and reconstruction after lung injury, many therapy methods such as changing mechanical ven-tilation pattern, hormone, surface active substance replacement therapy and immunotherapy achieved little. We reviewed the past studies and found the studies mainly focused on how to block the damage of harmful factors to AECⅡ. However, the studies on how to protect and promote AECⅡre-pairing and reconstruction are very few. Recent study indicated that survival and apoptosis change of alveolar epithelial typeⅡcells (AECⅡ) might involve in the development and turnover of hyperxia-induced lung injury, which affected the repairing after lung injury. Pulmonary epithelial cells injury may be alleviated if the change of AECⅡapoptosis is intervented at early stage, which can reverse lung injury and block subsequent pulmonary interstitial hyperplasia and pulmonary fibrosis. Now, active repairing theory on AECⅡis a hot spot. Looking for new regulatory factors for AECⅡac-tive repairing is becoming an new point for preventing hyperxia-induced lung injury. Recently, the role of sensor neuropeptides (Ne) transmitters that are secreted by pulmonary neuroendocrine cell (PNECs) has been focused. Ne is a cellular regulatory factor or extracellular messenger molecular. It is one of the signal substances recognized by nerve, endocrine and immunity systems jointly. Meanwhile, Nerv e-Hu mor-Im munity network regulates the process of wound repairing. SP was first found to be a neuropeptide in tachykinin family, and it distributes widely in airway endothelial cell layer, pulmonary vessel, trachea, bronchus smooth muscle and around gland and bronchus ganglion. Recent studies suggest SP plays a key role in prolifera-tion, migration and differentiation of impaired cells. Meanwhile, SP has significant proliferation and differentiation effect on keratinocyte, smooth muscle cell, osteoblast and endothelium. SP expresses abundant in respiratory or-gans. However, the repairing effect of SP on hypoxia-induced lung injury is still unknown.Apoptosis is a cellular active death manner under gene regulation, which frequently involves some signal pathways. If SP is an new regulatory factor for AECⅡ, then what is the regulatory mechanism? Mitogen acti-vated protein kinases (MAPKs) pathway including Extracellular sig-nal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38 kinase is a junction and common access of information transfer pathway such as cell proliferation and differentiation. However, whether SP can promote AECⅡrepairing after hypoxia-induced lung injury and the regulatory mechanism is related to MAPK pathway or not is still not understood.In this context, as an important sense neuropeptides transmitter, SP may alleviate hypoxia-induced lung injury via MAPK pathway. It is a feasible approach to prevent and cure hypoxia-induced acute lung injury(ALI) and BPD. In present study, primary cultured premature rats AECⅡwas selected for the following study. First, we established cellular oxidative damage patternl in vitro, and then observed the effect of SP on proliferation and survival of AECⅡat different time. Finally, the related MAPKs regulatory mechanism was investigated. PartⅠ. Isolation, purification, culture and identifica-tion of primary AECⅡof preterm ratsBackgroundOxygen therapy is a commonly and effective measure for neonate acute respiratory failure(ARF). However, long-time hypoxia exposure leads to oxidative stress- induced lung injury. Hypoxia (>95% oxygen) exposure is a main pathogenic factor that causes oxidative stress-induced lung injury. Hypoxia-induced lung injury is a complicated pathophysiological event involving disseminated alveolitis, repairing and reconstruction after lung injury. Fibroblast not normal alveolar epithelium replaces the impaired ep-ithelium in the process of lung repairing, which results in the disorder of endogenous alveolar epithelial restoration after injury.Alveolar epithelial repairing dependents on the proliferation and differentiation of alveolar ep-ithelial stem cell- AECⅡ. AECⅡcan cover the surface of alveolus and completes the repairing. AECⅡis comparatively smaller and cuboidal. The amount is about 60% of total alveolar epithelial cells. It can transform into alveolar type I epithelial cells (AECI) by proliferation, spreading and mi-gration, which restores normal morphology and function of alveolar epithe-lium. Lung tissue is mainly consist of AECⅡand lung fibroblast (LF) in the late stage of fetal lung development. Therefore, isolation, purification and primary culture of AECⅡand LF in fetal lung is crucial to study pulmonary disease of preterm infant in vitro. Nevertheless, it is difficult to isolate and purify primary AECⅡfrom preterm rats. Meanwhile, AECⅡcan not be passage cultured and the culture condition is comparatively more strict. In present study, we managed to obtain enough and pure AECⅡfor next needs by isolation, purification, culture of primary AECⅡof preterm rats. ObjectiveTo establish the isolation, purification, culture methods for primary AECⅡof preterm rats and provide enough and pure AECⅡto investigate related survival, apoptosis and signal transduction mechanism under hy-poxia exposure.MethodsThe adult specific-pathogen-free (SPF) Sprague-Dawley( SD ) rats used in this study were obtained from the Experimental Animal Center of Third Affiliated Hospital of Third Military Medical University (Chongqing, China). Rats were raised in a cage with an appropriate proportion (1:1) between female and male. Gravidity was confirmed if vaginal plug was seen at the second morning. Pick out the premature rats from the pregnant rats at 19 day and isolated AECⅡquickly. Briefly, the pregnant rats were anesthe-tized by chloral hydrate at 19 day. Uterine-incision delivery was used to pick out the infant rats and isolated infant lung. Non-lung tissues such as trachea and bronchus were cut into a 1mm3-size. The cells were digested by 0.25% trypsogen for 25-30 min.Complete medium was added to suspend the di- gestion. Then, the cell suspension was filtered by a 100 screen openings-grit. Centrifuged at 800 rpm for 10 min and discarded the supernatant. 0.1% collagenase I was added to digest the precipitation for 45 min. Centrifuged at 800 rpm for another 10 min and removed the supernatant. The precipitation was resuspended in dulbecco's modified eagle's medium(DMEM/F12) complemented with 10% fetal calf serum, 100 U/ml penicillin and 100μg/ml streptomycin. The cells were inoculated in a plastic flask. The adherent cells were LF, and non-adherent ones were AECⅡ. Removed AECⅡand centrifuged at 800 rpm for 10 min. Then inoculated in an new plastic flask. The ma-nipulation was repeated three times to purify AECⅡ. Finally, the non-adherent cells were inoculated in a 6-well plate (1.5×106 /ml). Cultured for 12 h and discarded the non-adherent cells. Inoculated AECⅡin another 6-well plate or 96 well plate. Replaced fresh culture medium and cultured for another 12 h for use. Cells viability was assessed by trypan blue exclusion. Modified papanicolaou staining was employed to evaluate cells purity. The cells were identified under transmitting electron microscopy (TEM). Meanwhile, we observed cellular character and morphologic change at 12, 24 and 48 h after the inoculation.ResultsI. (36±5)×106 AECⅡcould be obtained from every 3-5 preterm rats (19 d).II. Trypan blue exclusion assay showed the cells viability was more than 90%.III. Cells purity was confirmed to be more than 90% by modified papani-colaou staining.IV. Typical structure of AECⅡ, lamellar body, was observed under TEM.V. AECⅡgrew well at 12-24 h. More particles appeared in cytoplasm. Cells began to elongate at 48 h along with decreased particles and emergence of vacuolus.ConclusionIn present study, we observed primary AECⅡfrom preterm rats(19d) was in a fusion status approximately at 12 h. The cellular proliferation and metabolism was enhanced at 24-48 h after the primary culture, and the growth status is best. Therefore, productive, pure and active primary AECⅡcould be obtained for study in vitro in this period.PartⅡ. Effect of hypoxia exposure and intervention of SP on typeⅡalveolar epithelial cells of preterm ratsBackgroundAs an adjunctive therapy, oxygen therapy is employed to enhance saturation of blood oxygen and improve tissular hypoxia condition. Me- chanical ventilation of high concentration oxygen(>95% oxygen, hyperxia) is a common therapy for the treatment of respiratory failure(RF) especially acute respiratory distress syndrome(ARDS). However, prolonged exposure to hyperxia will result in pulmonary oxidative stress-injury. It will cause impaired pulmonary development and injury of preterm infants, which lead to BPD in preterm infants. Therefore, how to promote repairing after hy-perxia-induced lung injury and prevent and cure BPD is a conspicuous issue in clinic. Compared with AECⅡ, AECⅠis more susceptible to suffer from hyperxia-induced injury. For AECⅡis an important cell in lung tissue, it can transform into AECⅠand repair the impaired alveolus when alveolus are damaged. At this moment, proliferation and transformation of AECⅡis needed to repair the impaired alveolus structure. Recently, studies indicated that hyperxia is responsible for repatternling and abnormal growth in key stage of pulmonary development. Among it, hyperxia-induced apoptosis is a primary factor. However, the role of apoptosis in hyperxia-induced lung injury is still not known. Meanwhile, majority of the studies indicated hy-perxia-induced apoptosis apoptosis was positively related to lung injury degrees. In addition, study on the relationship between apoptosis and pul-monary fibrosis indicated that apoptosis of alveolus and bronchiole epithe-lium may be responsible for pulmonary fibrosis. In view of this, we specu-late that BPD after hyperxia-induced lung injury can be prevented and cured if some measures are adopted to suppress the apoptosis of AECⅡ. Studies in the past mostly focused on to how to block the damage of harmful factors to AECⅡ. They included the effect of immunogenic factors such as inflammatory cells and cytokines on wound repairing. However, few studies are pay attention to on how to protect and promote repairing and reconstruction of AECⅡ, which ignores the regulation of Nerve- Hu-mour-Inmmunity network to the process of repairing. Therefore, searching new regulatory factors for AECⅡactive repairing is becoming an new point for preventing hyperxia-induced lung injury. Ne is a cellular regulatory factor or extracellular messenger molecular. It is one of the signal substances recognized by nerve, endocrine and immunity systems jointly. Meanwhile, Nerve-Humor-Immunity network regulates the process of wound repairing. Recently, study suggested that sensory neuropeptide SP plays an important role in proliferation, migration and differentiation of impaired cells. In the study on skin wound healing, SP was not only confirmed to initiate nerve source inflammatory reaction in the early stage of healing but closely related to proliferation, regeneration and scarring of impaired cells. SP in airway is mainly from the erminatio of sensory nerve c- fiber. It distributes widely in airway endothelial cell layer, pulmonary vessel, trachea, bronchus smooth muscle and around gland and bronchus ganglion. Neuroendocrine cell, smooth muscle cell, eosinophile granulocyte, lymphocyte and alveolar macrophage in airway all can synthesize and secrete SP. Special receptor for SP distributes in smooth muscle, submucosal gland and blood vessel endo- thelium. Some inflammatory cells also can express SP receptor. SP is rich in respiratory organs. However, the role of SP in lung injury is seldom reported in domestic and oversea study. In present study, we investigated the effect of hyperxia exposure and SP intervention on proliferation and survival of AECⅡat different time. Meanwhile, we observed dynamic change of SP when exposed to hyperxia at different time.ObjectiveⅠ. To establish oxidative damage patternl for primary AECⅡof preterm rats in serum-free medium by hyperxia exposure.Ⅱ. To observe effect of hyperxia exposure and SP intervention on mor-phology, survival and apoptosis of AECⅡat different time and inves-tigate time-effect relationships between hyperxia exposure, SP inter-vention and AECⅡinjury degrees.Ⅲ. To establish animal patternl for hyperxi- induced lung injury and assay SP.MethodsI. Isolated and purified AECⅡfrom preterm specific-pathogen free(SPF) Spraque-Dawley(SD) rats. Trypan blue exclusion assay and modified papanicolaou staining was used to identify the cells viability and purity, respectively. The AECⅡwere seperated into the following groups: air group (21% oxygen), hyperxia group (95% oxygen), SP + air group and SP + hyperxia group. For SP groups, 1×10-6 mol/L SP was added before the exposure to air and hyperxia. Then, the cells were exposed to air and hyperxia for 12, 24 and 48 h, respectively. The morphologic changes of AECⅡwere observed under TEM. Respectively, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay and flow cytometry(FCM) was used to determine the proliferation and apoptosis rate to confirm the time-effect relationship between cell damage and hyperxia exposure time and the effect of SP intervention on the injury of AECⅡII. Uterine-incision delivery was used to pick out the preterm rats from the pregnant rats at 21 day (22 d for full term ) and isolated AECⅡquickly. The isolated AECⅡwere randomly divided into air group(21% oxygen) and hyperxia group(95% oxygen). The cells were exposed to air and hyperxia for 3, 7 and 14 d, respectively. Then, SP in lung tissue was assayed at different time, respectively.ResultsI. Compared with air exposure group, slightly widened intercellular space and reflect augmented vacuolus appeared at 12 h after hyperxia expo-sure. Widened intercellular space, smaller cells, increased intracellular vacuolus, part shrinked cell nucleus and decreased intracellular parti-cles-lamellar bodies presented at 24 h after hyperxia exposure. At 48 h, plenty of rounding and shrinked cells, non-adherent cells, floating cells and debris in the supernate could be seen. Typical apoptotic cell (smaller volume, shrinked cytoplasm, invaginated membrane and ka-ryopyknosis) and apoptotic body surrounding were observed under TEM. With the prolong of hyperxia exposure time, the cellular mor-phologic damage was aggravate compared with air group. Interestingly, the organs damage was alleviated by the intervention of SP at the same hyperxia exposure time compared with hyperxia group.II. MTT assay indicated that the proliferation activity of AECⅡwas sig-nificantly decreased at 12, 24 and 48 h after hyperxia exposure com-pared with air group. The survival of AECⅡdecreased remarkably(P < 0.05). The survival rate was decreased gradually along with the expo-sure time. There were significant differences among groups(P < 0.05). However, the proliferation activity of AECⅡin SP+ hyperxia group was lower than that in air group, whereas the activity was obviously enhanced compared with simple hyperxia group.III. FCM results suggested that the apoptosis of AECⅡin hyperxia group increased gradually along with the prolong of hyperxia exposure time compared with air group(P < 0.05). Although apoptosis rate in SP group was higher than that in air group, it was obviously lower than that in hyperxia group.IV. The SP product in lung tissue was significantly decreased compared with air group at 3, 7 and 14 d after hyperxia exposure, respectively(P < 0.01). Furthermore, SP level decreased remarkably along with hyperxia exposure time. There was a statistical difference between SP+hyperxia group and simple hyperxia group(P < 0.01).ConclusionI. Hyperxia induced the damage of AECⅡin a time-dependent manner.II. It was confirmed that hyperxia induced the apoptosis of AECⅡby FCM and TEM.III. SP intervention could decrease hyperxia-induced AECⅡapoptosis and enhance the cells viability, suggesting SP could attenuate the oxidative stress on AECⅡand might have a protective effect on AECⅡunder oxidative stress.IV. Hyperxia could result in dynamic change of SP product, suggesting hyperxia exposure led to SP loss at the end of c-fiber of airway sensory nerve and regional decreased SP might be associated with the injury degrees of AECⅡ.PartⅢ. The signal mechanism for hyperxia exposure and SP intervention regulating the repair-ing of AECⅡfrom preterm ratsBackgroundAfter long-time hyperxia uptake, non-specific changes including in-creased alveolar capillary permeability, increased alveolus effusion, in-flammatory damage, fibrin deposition and decreased pulmonary surfactant activity will lead to oxidative stress-induced lung injury. Meanwhile, BPD is a common complication after hyperxia therapy in preterm infant.We have confirmed that hyperxia induced the injury of AECⅡin a time-dependent manner in PartⅡ. Hyperxia exposure could induce the apoptosis of AECⅡ. SP intervention could decrease the cells apoptosis rate and increase survival rate after hyperxia exposure, which might have a protective effect on AECⅡunder oxidative stress. Apoptosis is a cellular active death pattern involving some signal pathways frequently. SP is an new regulatory factor for AECⅡ, and then what is related molecular me-chanism? MAPK pathway plays a key role in mediating hyperxia-induced lung injury. There are 3 members in MAPKs including ERKs, JNKs and p38 kinase. MAPK is an important channel by which different extracellular signals transduct from cellular surface into nucleus. It can mediate cell apoptosis induced by the activation under stimulation. MAPK is a junction and common access of information transfer pathway such as cell prolifera-tion and differentiation.Studies have confirmed that hyperxia exposure and H2O2 stress could trigger persistent activation of activator protein-1 (AP-1), MAPK family members p38 and JNK, which mediated hyperxia-induced cell swelling death and apoptosis. Moreover, cell survival rate could be significantly im-proved after the adding of specific blocker. Some study suggested that tretinoin could attenuate hyperxia-induced lung injury in preterm rats via regulating MAPK pathway. MAPK pathway was involved in signal transduction of AECⅡapoptosis under hyperxia-induced oxidative stress, duction of AECⅡapoptosis under hyperxia-induced oxidative stress, and it promoted the apoptosis of AECⅡ. Whether SP can affect the survival and apoptosis of AECⅡvia MAPK pathway is still unknown. In this part, we further to investigate the effect of hyperxia and SP intervention on the damage of primary cultured AECⅡ.In view of this, we expected to confirm MAPK pathway participation in lung injury and related regulatory mechanism.ObjectiveI. To establish oxidative damage patternl for primary AECⅡof preterm rats in serum-free medium by hyperxia exposure.II. To observe effect of hyperxia exposure and SP intervention on mor-phology, survival and apoptosis of AECⅡat different time and inves-tigate time-effect relationships between hyperxia exposure, SP inter-vention and AECⅡinjury degrees.III. To understand the activation of MAPK in AECⅡapoptosis under hy-perxia-induced oxidative stress and SP intervention, and investigate the effect of MAPKs on AECⅡapoptosis and the regulation of SP inter-vention to MAPKs pathway.MethodsIsolated and purified AECⅡfrom preterm specific-pathogen free(SPF) Spraque-Dawley(SD) rats. Trypan blue exclusion assay and modified pa-panicolaou staining was used to identify the cells viability and purity, respec- tively. The AECⅡwere divided into the following groups: air group (21% oxygen), hyperxia group (95% oxygen), SP + air group and SP + hyperxia group. For SP groups, 1×10-6 mol/L SP was added before the exposure to air and hyperxia. Then, the cells were exposed to air and hyperxia for 12, 24 and 48 h, respectively. Western blot was used to assay the expressions of total phosphorylated ERKs, JNKs and p38 at different exposure time.ResultsI. The phosphorylated ERK(p-ERK) was induced and expressed rapidly after hyperxia exposure. Respectively, the level of p-ERK in hyperxia group was significantly higher than that in air group at 12, 24 and 48 h after hyperxia exposure. The p-ERK protein was expressed best at 48h. The expression in SP+hyperxia group was more than that in hyperxia group at 12, 24 and 48 h, respectively.II. The phosphorylated p38 (p- p38) was induced and expressed rapidly af-ter hyperxia exposure. Respectively, the level of p- p38 in hyperxia group was significantly higher than that in air group at 12, 24 and 48 h after hyperxia exposure. The p- p38 protein expressed best at 48 h. The expression in SP+hyperxia group was less than that in hyperxia group at 12, 24 and 48 h, respectively.III. The phosphorylated JNK (p- JNK) was induced and expressed rapidly after hyperxia exposure. Respectively, the level of p- JNK in hyperxia group was significantly higher than that in air group at 12, 24 and 48 h after hyperxia exposure. The p- JNK protein expressed best at 48 h. The expression in SP+hyperxia group was less than that in hyperxia group at 12, 24 and 48 h, respectively.ConclusionIn present study, we found hyperxia exposure could induce expressions of MAPKs in AECⅡrapidly. The expressions levels reached peaks at 48 h after hyperxia exposure.Respectively, the cells apoptosis rate and survival rate in SP+hyperxia group was obviously lower and higher than that in hy-perxia group at the same time, suggesting MAPKs signals mediated the apoptosis of AECⅡ. The intervention of SP had a potential protective effect on AECⅡunder oxidative stress via activating extracellular signals and suppressing the activation of JNK and p38.