The Study On The Role Of Methylation Of P16 Gene Promotor On Lung Fibrosis In Premature Rat With Hyperoxia-induced CLD

Chronic lung disease(CLD) is the most common complication in premature infant after long-term exposure to high-concentration oxygen inhalation and mechanical ventilation therapy and/or infection.Though the mechanisms of it have not been known yet,the pathological termination of alveolar epithelium injure in early stage and irreversible pulmonary interstitial fibrosis in advanced stage has been generally acknowledged.In recent years,with obvious increasing of the survival of very low birth weight infants,the incidence of CLD is also increasing year by year.At present, CLD has been the most important disease threatening health and life of premature infant.Although its pathogenic mechanisms are not fully understood,higher levels of long-term supplemental oxygen is thought to be the most important reason and highrisk factor resulting in CLD so far.Topics on the mechanisms of lung fibrosis in premature infant with CLD induced by hyperoxia become the most challenging in domestic and international study.The main pathological changes of CLD in premature infant are proliferation of lung fibroblast(LF),deposition of extracellular matrix(ECM) and fibrosis of lung tissue.Nowadays accepted pathogenic mechanisms of CLD are mainly described as following:1.Because of the deficient antioxidant system of premature baby(for example deficient superoxide dismutase) oxygen free radicals are overproduced after inspiring higher concentration of supplemental oxygen and lead to pulmonary lesions of oxidative stress.2.The functionary consequence of cytokines:more and more evidence indicates that the development of CLD is closely correlated with coordination of multiple cytokines.Among of them cytokines involved in promoting inflammation and fibrosis include interleukin-1(IL-1),IL-6,IL-8,tumor necrosis factor-α(TNF-α) and so on.These substances cause the process of inflammatory reaction,recruit and stimulate inflammatory cells.In addition,cytokines such as IL-4,IL-10,TGF-β1, VEGF and so on play roles in anti-inflammation,promoting fibrosis and regulating neovascularization.3.Dynamic imbalance of ECM also participates in the development of CLD.Matrix metalloproteinase(MMPs) is a family of proteolytic enzyme,which mainly degradates ECM and plays an important role in lung development process. Collagenase MMP-1 and MMP-8 degradate I type collogen which is the major structural protein of ECM.MMP-2 and MMP-9 degradateⅣtype collogen which is the essential component of basal membrane of lung.Tissue inhibitor of metalloproteinase(TIMPs) regulates the activity of MMPs.If the levels of MMPs decreases or the levels of TIMPs mentioned above increases will both make ECM deposited and fibrosis developed.P16 gene is also called multiple tumor supresser gene,which directly affects cell cycle and suppresses cellular proliferation.,found as an important anti-oncogene in the most early stage and thought to be an important regulating factor of cell cycle.In recent years,it has become the hot study in the field of life science.During tumorous research,it has been found that abnormal methylation of p16 gene promoter has an obvious inhibitory effect on its gene expression,which may control gene expression at transcriptional level,and accordingly cause silence of corresponding gene expressed. The results of considerable information of research indicate that the inactivation of p16 caused by methylation of p16 gene promoter is closely correlated with many kinds of malignant tumors,intestinal metaplasia,psoriasis,postburn cicatricial formation and senescence and so on.Then,during the development of CLD in premature infant,it has not been yet studied and elucidated that whether lung fibrosis induced by abnormal proliferation of lung interstitial fibroblast has a similar controlling process of gene.So our study is based on research in the past,the model of CLD induced by hyperoxia in premature rat and lung interstitial fibroblast thought to be objects,we investigate the mechanism of action of p16 gene in lung fibrosis from tissue,cellular and molecular level,explore pathophysiological mechannism of lung fibrosis in premature infant with CLD from a new point of view and try to redesign and find the effective way of preventing and curing CLD.Materials and Methods1.Animal modelPremature pups were delivered by cesarean section at 21 days of gestational age and pool together in 2-3 litters and then randomly redistributed to surrogate mother rats. In the same experiment,the pups of experimental group were placed in 90%oxygen, and pups of control group remained in room air(21%oxygen).Nursing mothers were rotated between oxygen-exposed and room air litters every 24 hrs to avoid oxygen toxicity and to eliminate maternal effects between groups.Oxygen exposures were done in 3-ft~3 plexiglas chamber into which oxygen was continuously delivered at 2.0L/min.Continuous flow achieved a constant level of 90%oxygen and the carbon dioxide concentration was less than 0.5%.Temperature and relative humidity were maintained at 22℃～27℃and 50%～70%respectively.For a daily 30min period,the chamber was opened to allow provision of fresh food and water,weighing of the rat litter,and exchange the surogate mothers between the two groups.2.Sample collection and treatmentRats of the two groups on postnatal 3d,7d,14d and 21d were anesthetized by an intraperitoneal injection of 5%chloral hydrate(6ml/kg) and put to death.The thorax was opened and lung tissue was resected.The left lung tissue was fixed in 4% formaldehydum polymerisatum,then was dehydrated,embedded in wax within 24 hrs in order to prepare 5μm tissue section.The right lung tissue was put in the Rnase- free Ependorf tubes and stored at -80℃freezer for detecting of mRNA,methylation and so on.3.Isolation,primary culture and identification of LFs from premature rat lungsWistar premature rats were randomly selected to be anesthetized by an intraperitoneal injection of 5%chloral hydrate(6ml/kg) and killed on postnatal 3d,7d, 14d and 21d.Lungs were immediately resected under sterile condition.The lung tissue was digested by trypsin.LFs were isolated and purified through centrifugate and recurrent adherence,then cultured.The nature of the cultures was identified by vimentin staining.LFs were passaged according to the ratio of 1 to 2 or 3.The third passage of LFs was experimented.4.Experiment methods and Analysis markers(1) The changes of lung pathology were compared by lung sections from the left lobes stained with HE.At the same time,the degrees of lung fibrosis were assessed.(2) The levels of typeⅠcollagen in lung tissue were detected by Enzyme linked immunoadsorbent(ELISA).(3) The expressions of p16 protein in lung tissue were observed and detected by immunohistochemistry and Western-blot.(4) The methylation of p16 gene promoter in lung tissue was detected by MSP and semi-nested PCR.(5) The methylation of p16 gene promoter of LFs was detected by semi-nested PCR.(6) The levels of p16 mRNA in lung tissue and LFs were detected by RT-PCR.(7) The protein expressions of p16 and proliferating cell nuclear antigen(PCNA) in lung tissue and LFs were detected by immunohistochemistry. (8) The vigor of LFs proliferation was assessed by methyl thiazolyl tetrazolium (MTT).5.Statistical analysisSPSS version was used to perform statistical analysis.Numeration data was analyzed by chi-square test,all numeration data expressed as means±SD was analyzed by t test.Spearman and Logistic regression were performed to analyze correlation. Statistically significant differences were set by P less than 0.05.Results1.The pathological changes of lung tissue(1) The changes of morphology in lungsThe terminal air space size was irregular and small and the alveolar septum was relatively thick in control groups on 3 day after the experiment,but in experimental groups there was small blood vessel dilitated and congested,blooding,interstitial cell increased.On 7 day the appearance of lung alveoli became regular and their sizes were well-distributed in control groups,while in experimental groups Intra-alveolar hemorrhage,edema,inflammatory cell infiltration and increased interstitial cells were observed.At the same time,the terminal air space was dilatated.On 14day and 21day alveolar structure in control group continued to be alveolization in progress,however, in experimental groups the alveolar septum was thicker with alveoli fusion and disorganization,the terminal air space was dilatated obviously and the quantity of alveoli decreased.(2) Score of lung fibrosisThere was no significant difference between the two groups on 3day and 7day(P＞0.05).But on 14day and 21day the score in experimental groups was significantly more than that of in control groups(P＜0.01). 2.The levels of typeⅠcollagen in lung tissueThere was no significant difference between the two groups on 3day and 7day after the experiment(P＞0.05),While on 14day and 21day the levels of type I collagen in experimental groups were significantly more than that of in control groups(P＜0.05 onl4day,P＜0.01 on 21day).3.The dynamic changes of p16 protein in lung tissueImmunohistochemical study shows that p16 protein was mainly expressed in lung interstitial fibroblasts,then bronchial epithelial cells,alveolar epithelial cells,vascular smooth muscle cells,vascular endothelial cells and alveolar macrophages were also observed in the two groups.However,There was no significant difference between the two groups on 3day but p16 expression in experimental groups was significantly less than that of in control groups on 7day,14day and 21day(P＜0.05 in immunohistochemical study;P＜0.01 on 14day and 21day in Western-blot detection ). The analysis tendency of p16 protein was coincident between immunohistochemical study and Western-blot.4.Methylation of p16 gene promotor in lung tissueMethylation of p16 gene promotor in lung tissue was detected by two methods of semi-nested PCR and MSP.The two results tendency was coincident.The occurrence of methylation increased in experimental groups with the time of oxygen exposure,but there was no methylation in control groups.The rate of hypermethylation detected by semi-nested PCR was more than that of detected by MSP but there was no statistical significance(P＞0.05).5.Methylation of p16 gene promotor in lung fibroblastThere was no methylation in control groups at different time point.In experimental groups the rate of methylation increased with the time of oxygen exposure,there was statistical significance compared with that in control groups (P＜0.01). 6.The levels of p16 mRNA in lung tissue and LFsThe levels of p16 mRNA in lung tissue and LFs in experimental groups were significantly downregulated on 7day,14day and 21day(P＜0.05 on 7day,P＜0.01 on 14day and 21day in lung tissue;P＜0.05 on 7day,P＜0.01 on 14day,P＜0.05 on 21day in LFs)but there was no statistical significance on 3day(P＞0.05).The expression of p16 mRNA in LFs was correlated with methylation(t =3.45,P＜0.01,OR-0.846).The expression of p16 mRNA and protein was significantly downregulated in sample with methylation(P＜0.01).7.The expression of p16 and PCNA in LFs(1) The expression of p16 in control groups compared with that in experimental groups there was no statistical significance on 3day(P＞0.05).However,the expression of p16 in experimental groups was significantly downregulated on 7day,14day and 21day compared with that in control groups(P＜0.05 on 7day,P＜0.01on 14day,P＜0.05 on 21day).(2) The expression of PCNA in LFs was significantly higher on 7day,14day and 21day in experimental groups compared with that in control groups(P＜0.05 on 7day, P＜0.01 on 14day and 21day).But there was no significant difference in the expression of PCNA between the two groups on 3day(P＞0.05).The levels of p16 mRNA was significantly negative correlation with the expression of PCNA on 14day and 21day(r=-0.886,P＜0.05;r=-0.928,P＜0.01).8.The vigor of LFs proliferationThe levels of OD in LFs was significantly higher on 7day,14day and 21day in experimental groups compared with that in control groups(P＜0.05).But there was no significant difference in the level of OD between the two groups on 3day(P＞0.05). Conclusion1.The p16 mRNA and protein expression in lung tissue was downregulated in premature rat exposed to oxygen.Its downregulation was closely correlated with the development of lung fibrosis.2.Hyperoxia may induce abnormal methylation of p16 gene promoter in lung tissue of premature rat,the rate of p16 gene methylation gradually increased with the time of oxygen exposure.3.Hypermethylation of p16 gene induced by hyperoxia may be one of the mechanisms of p16 mRNA and protein downregulaed.4.Abnormal methylation of p16 gene promoter induced by hyperoxia resulted in downregulation of p16 gene,which made cell cycle regulation of LFs disordered and in the end proliferation of LFs may be one of the mechanisms of lung fibrosis in premature rat with CLD.