The Signal Pathway And Role Of Sildenafil In Meconium-Induced Acute Lung Injury In Neonatal Rats

Background and objectivesMeconium aspiration syndrome (MAS) is a serious respiratory disorder in term and post-term neonates. Its incidence is 1.2-2.0% in liveborn infants, while its mortality reaches 3-12%. Meconium obstructs peripheral airways and induces lung injury characterized by increasing of pro-inflammatory cytokines, reactive oxygen species and apoptosis. All these aforementioned substances subsequently participated in pulmonary surfactant impairment, parenchymal disruption, edema and alveolar hemorrhage.MAS is a complex progress interacted by many factors. Inflammatory factors, oxidative stress injury, apoptosis and so on, involve in the occurrence and development of ALI. The extracellular signal is transmitted to the nucleus, through the signal pathway which regulates the expression of genes and proteins of many cytokines. We hope to find new intervention drugs through the study of signal pathway of ALI.People used to choose adult rats, adult guinea pigs, newborn rabbits and newborn pigs to build the MAS model. There are three problems:first, the adult animal organs have been mature, can not fully reflect the pathophysiological process of neonatal; secondly, tracheotomy may be fatal to animals; third, animals need ventilator after tracheal incision. Tracheotomy and ventilator may cause stress lung injury. The process is complex and the success rate is low. We established the MAS model with 14-21 days old neonatal rats by orotracheal intubation and tracheotomy, compared the succeeding rate, lung wet/dry weight ratio and pathological score. At the same time, We tested different concentrations of meconium to find the appropriate concentration to establish MAS model in neonatal rats.Tumor necrosis factor-a (TNF-a) has an important role in the development of MAS. In the pathological process of MAS, TNF-a is one of the earliest and most important cytokines. In addition to its own strong inflammatory effect, it also has a function of stimulate the release of multiple inflammatory factors. The stimulation of meconium can activate a series of cytokines including TNF-a, cause oxidative stress injuries and promote a variety of cell apoptosis in lung through the signal pathway.Mitogen activated protein kinase (MAPKs) is a serine/threonine protein kinase regulates the activity of transcription factor and the expression of genes related to cytokines and inflammatory mediators by phosphorylate corresponding cellular protein. MAPKs, the central of the multiple signaling pathways, receive the signal conversed and transferred by membrane receptors and bring it into the cell nucleus, and play key role in many signal pathways about cell proliferation. The MAPKs pathway mainly includes P38 pathway, JNK pathway, ERK1/2 pathway and ERK5 pathway. In mammals, ERK1/2 is widely present in various tissues, and is involved in the regulation of cell proliferation and differentiation. Various growth factor receptors are required to complete the signal transduction process by ERK1/2 activation. The JNK family is a key molecule in the signal transduction induced by various stress molecules, which is involved in the cellular stress response. P38 mediates inflammation, apoptosis and so on. Therefore, MAPKs signal pathway play an important role in ALI induced by MAS.Phosphatidylinositol-3-kinase (PI3K) is an important part of intracellular signal pathway. It is involved in the regulation of cell functions such as proliferation, differentiation, apoptosis, and glucose transport and so on. Serine/threonine protein kinase (Akt), also known as Protein Kinase B (PKB), is the main downstream effectors of PI3K. Akt can activate protein translation, enhance cell growth through phosphorylating downstream target proteins. It has anti-inflammatory, anti-apoptotic and cell protective effect.Sildenafil is a selective phosphodiesterase type 5 (Phosphodiesterase-5, PDE5) inhibitor. It can inhibit cGMP (Cyclic guanosine, monophosphate, cGMP) hydrolysis, increase the concentration of cGMP. So it can selectively reduce pulmonary artery pressure. According to MAS guidelines, Sildenafil has been used to treat secondary pulmonary hypertension. Recent study found that Sildenafil also can improve oxidative stress and inhibit apoptosis. There are many studies of Sildenafil on acute lung injury, acute respiratory distress syndrome, lung ischemia reperfusion injury and so on. There is no relevant research about the effect of Sildenafil on ALI caused by MAS.We established an ALI model induced by meconium in neonatal rats and studied the pathological damage, inflammatory factors, oxidative stress, and apoptosis. We compared the effect of sildenafil with dexamethasone. Furthermore, we studied the role of MAPKs (p38, ERK1/2, JNK) and PI3k/Akt signal pathway on ALI and try to clear the regulation effect of Sildenafil on signal pathway.There are three parts:Part 1 Establish animal model of meconium-induced acute lung injury in neonatal rat by orotracheal intubationPart 2 Sildenafil ameliorated meconium-induced acute lung injury in a neonatal rat modelPart 3 The signal pathway and role of sildenafil in meconium-induced acute lung injury in neonatal ratsMethodsPart148 healthy newborn male Wistar rats,14-21 days old, were divided randomly into three groups:18 rats (group A) were tracheal intubated after tracheostomy and given ventilation; 24 rats (group B) were orotracheal intubated.,6 rats (group C) was control group. Group B was divided into 4 groups including saline control group (group BN), low concentration group (group BL), middle concentration group (group BM), high concentration group (group BH). There are 6 rats in each group. Rats of group A, B had endotracheal intubation after intraperitoneal anesthesia. Group A were treated by traditional tracheotomy, while Group B were given orotracheal intubation. Meconium (2ml/kg) were given after intubation. The concentration of meconium: 40mg/ml (group A); 30mg/ml (group BL); 60mg/ml (group BM); 90mg/ml (group BH). The rats of group BN received saline (2ml/kg).6 hours later, all rats were sacrificed. Lung pathology examined by light microscopy and lung wet/dry ratio (W/D) was evaluated.Part 224 neonatal Wistar rats were randomly divided into group M (meconium), group S (sildenafil), group D (dexamethasone) and group C (control). Acute lung injury was induced by endotracheal instillation of human meconium (60 mg/ml,2 ml/kg). After meconium instillation, rats in group S and D received sildenafil (25 mg/kg, ig.) and dexamethasone (1 mg/kg, ig.), respectively; while rats in group C and M both received saline (2 ml/kg, ig.). Rats were sacrificed at six hours for histopathological, biochemical, immunohistochemical and TUNEL analyses of lung damage indicators. The activity of MPO, SOD, content of MDA、NO、TNF-α, cAMP and cGMP were detected by ELISA method. Bcl-2 and Bax protein expression were detected by immunohistochemistry and Western blot, while the genes of TNF-a, Bcl-2 and Bax with qRT-PCR.Part 348 neonatal rats, were randomly divided into 8 groups:C (saline), M (meconium), S(Sildenafil), SB (P38 inhibitor), PD (ERK inhibitor), SP (JNK inhibitor), W (PI3K inhibitor), and D (DMSO). Each group was given intraperitoneal injection (5ml/kg). the rats of C, M and S group were given PBS, and the others were given corresponding inhibitors diluted by PBS. Group SB:SB203580 (lmg/kg); Group PD: Group PD95809 (10mg/kg); Group SP:SP600125 (6mg/kg); Group W:Wortmannin (1mg/kg); Group D:DMSO (50mg/kg). All the rats had endotracheal injection (2ml/kg), group C:saline; other groups:meconium (60mg/ml). Then group S received sildenafil (25 mg/kg, ig.) while other groups both received saline (2 ml/kg, ig.). In order to prevent the influence of oxygen, all the rats inspirated air during the experiment. Rats were sacrificed six hours later for histopathological, ELISA, immunohistochemical analyses to detect the content of TNF-a and the xpression of p-P38, p-JNK, p-ERK1/2 and p-Akt protein.Statistics analysisStatistics analysis was performed using SPSS statistical software (SPSS Science, Chicago, USA). Data were expressed as mean ± standard deviation (SD). All parameters were evaluated by one-way analysis of variance (ANOVA) test, counting material by Chi-square test. A P value<0.05 was considered statistically significant.ResultsPart11 Success rate3 rats (16.67%) in group A survived to 6 hours after tracheal intubation.22 (91.67%) rats in group B survived.2 rats of group BH died within 6 hours after extubation. The difference of success rate between group A and group B was statistically significant (X=24.01, P<0.01).2 W/D of lung tissureThe difference of lung tissure W/D between 6 groups was statistically significant (F=24.48, P<0.01). Lung tissure W/D in the group C and group BN were lowest, then from low to high:group BL, group BM, group A and group BH (P<0.01). The difference between group A and group BH was no significant difference (P>0.05).3 Pathological examinationThe difference between 6 groups was statistically significant (F=51.31, P<0.01). The lung histopathological score of the group BH and group A were the highest, and there was no significant difference between group BH and group A(P>0.05). Followed by the group BM and group BL, then group BN and C. There was no significant difference between the group C and group BN(P>0.05).Part 21 Pathological damageLung injury score significantly increased in the M group compared with the C group (P<0.05). However, these scores statistically decreased after Sildenafil and dexamethasone treatment (both, P<0.01). There was no significant difference between the S and D groups.2 Inflammatory cytokine levels in lung tissues, oxidative and nitrative injuriesMPO activity increased to a great extent in the M group, compared with the C group. Lungs from rats treated with Sildenafil exhibited markedly decreased MPO activity compared with the M group. Sildenafil could also significantly downregulate the mRNA and protein expression of TNF-a in a similar way. MDA levels significantly increased in the M group compared with the C group. However, reduced MDA activities were reversed after administration of Sildenafil and dexamethasone. There were no significant differences were observed between the S and D groups. Lung tissue SOD activity was found to be significantly decreased in rats subjected to meconium. The M group exhibited an elevated NO concentration compared with the C group. Elevated NO levels were significantly lower in the S and D groups, compared with the M group.3 ApoptosisMeconium resulted in the increased expression of pro-apoptotic Bax (protein and mRNA) and decreased level of anti-apoptotic Bcl-2 (protein and mRNA). However, elevated Bax protein and mRNA expression was significantly inhibited following Sildenafil and dexamethasone treatment (both P<0.05, compared with the M group). In TUNEL assay, the M group revealed the greatest apoptotic index among all groups, while the S and D groups exhibited a significant decrease in apoptotic index, compared with the M group.4 Effect of Sildenafil on cAMP and cGMPBoth Sildenafil and dexamethasone increased cAMP and cGMP levels in lung tissues, compared with the M and C groups. Noticeably, Sildenafil triggered cGMP to the greatest extent, while dexamethasone induced cAMP the most.Part 31 Pathological damage The difference between eight groups was statistically significant (F=28.23, P<0.01). Lung injury score significantly increased in the SB、SP and S group compared with the C group. These scores statistically lower than the M, D, W and PD groups. The histopathological score of the C group was the lowest. There was no significant difference between the group M, D, W and PD (P>0.05).2 Serum TNF-a concentration detectionThe serum TNF-a concentration of the group W and PD were the highest, followed by the group M and D, then the group S、PB and SP. The TNF-a concentration of the group C was the lowest. The difference between eight groups was statistically significant (F=31.87, P<0.01).3 Expression of p-P38, p-JNK, p-ERK1/2 and p-Akt protein in lung tissue3.1 p-P38 proteinAfter the treatment of meconium, the positive cells remarkably increased in the group M and group D. However, the expression of p-P38 protein was significantly inhibited by Sildenafil and SB203580 (both P<0.01, F=48.28, compared with the M group).3.2 p-ERKl/2 proteinMeconium resulted in the increased expression of p-ERK protein. But the activities were further increased after administration of Sildenafil (compared with the M group, P<0.05). After the treatment of PD98509, the protein quantity decreased. The difference between the five groups were significant (F=124.28, P<0.01).3.3 p-JNK proteinAfter the treatment of meconium, the positive cells remarkably increased in the group M and group D, but decreased after the treatment of SP600125 and Sildenafil. Protein quantity of p-JNK in the SP and S group was decreased compared with the group M and D. There were significant difference between the five groups (F= 119.96, P<0.01).3.4 p-Akt proteinImmunohistochemical detection showed that the expression of p-Akt protein increased after the treatment of meconium. In the group S, more positive cells can be found. The protein quantity of p-Akt decreased in the group W after the treatment of wortmannin, while increased after exposure to Sildenafil (F=57.26, p<0.01).Conclusions1 Traditional tracheotomy intubation may cause fatal harm stress and lung injury to neonatal rats so the success rate is low. The experiment result may be affected. We establish MAS model in neonatal rats by orotracheal intubation. The success rate wad higher than the control group.60mg/ml meconium solution can lead to extensive and typical pathological changes.2 Inflammatory cytokines, oxidative stress and apoptosis were involved in the pathogenesis of acute lung injury in neonatal rats induced by meconium aspiration. Sildenafil can inhibit the expression of inflammatory factors, reduce oxidative stress injury; inhibit apoptosis, thereby reducing the pathological damage of ALI caused by MAS. Sildenafil may play a biological effect through increasing the content of cGMP and cAMP.3 MAPKs (P38, ERK1/2, JNK) and PI3K/Akt signaling pathways play important roles in the pathological process of ALI induced by MAS. P38 MAPK and JNK MAPK may increase the expression of inflammatory cytokines such as TNF-a to improve the damage. ERK MAPK and PI3K/Akt can reduce the expression of TNF-a and thus reduce the pathological damage. Sildenafil can affect the signal pathway by inhibitting the expression of p-P38 and p-JNK protein in lung tissue and increasing the expression of p-ERK and p-Akt protein, and have protective effect on ALI induced by MAS.Innovation and significance1. This study establish neonatal rat model of MAS through direct orotracheal intubation. The operation is simple and the success rate is high.2. We explored that Sildenafil have protective effect in ALI through reducing the expression of inflammatory cytokines and oxidative stress, regulating apoptosis and other aspects. This study supply theoretical basis for the clinical application of Sildenafil.3. We defined that the MAPKs (P38、JNK、ERK1/2) and PI3K/Akt signal pathways were involved in the ALI induced by MAS. Sildenafil may reduce the pathological damage of ALI through effecting the MAPKs and PI3K/Akt signaling pathway.