A Study On The Roles Of ERK1/2 Protein Signaling In Proliferation,Transformation And Migration Of Lung Fibroblasts In Neonatal Rats With BPD

Objective:Although the survival rate of premature infants,especially very-low-birth-weight?VLBW?infants,has improved significantly alongside the widespread development of the mechanical ventilation,the improving parenteral nutrition therapy and the widespread use of pulmonary surfactants,the consequent bronchopulmonary dysplasia?BPD?in premature infants due to long-term depending on the oxygen has become the major disease threatening the health and life of premature infants,which affects nearly 10,000 neonates each year in the United States.There is still no effective preventive and therapeutic approaches against BPD due to its elusive pathogenesis,leading to mortality rates of 10-15%among infants within one year after birth due to respiratory failure as a result of severe pulmonary fibrosis?PF?.Besides,there is a need for long-term reliance upon oxygen or even mechanical ventilation due to pulmonary dysfunction among infants who survived.The pathological outcome of advanced pulmonary interstitial fibrosis is indubitable,despite the pathogenesis of BPD not being fully understood at present.Pulmonary interstitial fibrosis is mainly characterized by the over-proliferation of lung fibrolasts?LF?that accumulates in the pulmonary interstitium or replaces the normal respiratory epithelium.The proliferation and replacement of fibrous tissue following tissue injuries are important pathophysiological processes of tissue repairs.Therefore,the timely and modest regulation of LF proliferation without affecting tissue repair and inducing fibrosis due to hyperplasia has become the key to preventing fibrosis.This issue has to be addressed by targeting the regulatory mechanism of LF proliferation.Mitogen-activated protein kinases?MAPK?are serine/threonine protein kinases present in most cells and serve as an important signaling system that induces cellular responses in eukaryotic cells by transducing extracellular signals into cells.The mammalian MAPK family comprises 11 members that can currently be further divided into 6 classes,i.e.,extracellular signal-regulated protein kinase?ERK?1/2,ERK3/4,ERK5,ERK7/8,c-Jun amino-terminal kinase?JNK?1/2/3 and p38s??/?/??ERK6?/??.ERKs are the earliest known and most characteristic MAPK family members for proliferation,transformation and differentiation of cells.The sustained ERK activation promotes the transition of cells from G₁ phase to S phase and is closely related to tumorigenesis.Therefore,we speculate that there might be causal relationships between“hyperoxia,activation of ERK1/2 protein signaling pathway,abnormal increase in LF proliferation,as well as BPD and PF”.On the basis of our previous studies,the primary culture of LF isolated from neonatal rats with hyperoxia-induced BPD was experimented in this study to observe the effects of hyperoxia on the proliferation of LF and the biosynthesis of type I collagen,to investigate the dynamic changes in ERK1/2 signaling pathway during the onset and development of BPD and PF via various methods,as well as to study from multiple perspectives the in vitro regulation of proliferation,transformation and migration of LF by ERK1/2 activators and inhibitors.This study aimed to provide new clues for clarifying the mechanism of BPD and PF,as well as to lay theoretical and experimental bases for further exploration of new effective approaches for an effective prevention of BPD among preterm infants.Methods:1.Construction of animal models and cultivation of LFThe newborn Wistar rats were randomly divided into hyperoxia or control air groups and selected from each group on Day 3,7 and 14 after the experiment.Their left lung tissues were subjected to morphological observation and immunohistochemical?IHC?staining,while the right lung tissues were subjected to Western blot and PCR assays.LFs were isolated and purified on Day 3,7 and 14 too.And LF from just newborn rats were added with drugs and divided into four groups additionally:Control group:Blank control;PD98059 group:Added with the ERK inhibitor,i.e.,PD98059 at10 nmol/mlL;EGF group:Added with the ERK activator,i.e.,epidermal growth factor?EGF?at 20 ng/mL;EGF+PD98059 group:Added with PD98059 and EGF at one-hour interval.Those cells were harvested after 24 hours.2.Experimental Methods and Test Indicators?1?Study on the Effects of Hyperoxia on the Proliferation of LF and the biosynthesis of Type I Collagen?1?Flow cytometry:To determine the cell cycle phases of LF in each group.?2?IHC staining:To determine the expression level of proliferating cell nuclear antigen?PCNA?.?3?ELISA:To determine the type I collagen protein level in the supernatant of LF culture in each group.?4?Real-time PCR:To determine the mRNA expression level of type I collagen in LF.?2?Dynamic Changes in ERK1/2 Expression in Lung Tissues and Fibroblasts From Each Group?1?IHC staining:To determine the protein expression level of p-ERK1/2 in lung tissues and fibroblasts from each group.?2?Western blot assay:To determine the protein expression level of ERK1/2 and p-ERK1/2 in lung tissues and fibroblasts from each group.?3?Real-time PCR:To determine the mRNA expression level of ERK1 and ERK2in lung tissues and fibroblasts from each group.?3?In Vitro Study on the Effects of ERK Activator and Inhibitor on LF?1?IHC staining,Western blot and Real-time PCR assays were employed to determine the protein and mRNA expression levels of ERK1/2 in LF from each group,respectively.?2?Cell Counting Kit-8?CCK-8?assay:To determine the proliferation of LF in each group.?3?Flow cytometry:To determine the cell cycle phases of LF from each group.?4?IHC staining:To determine the expression level of PCNA in LF from each group.?5?IHC staining,Western blot and Real-time PCR assays were employed to determine the expression of?-smooth muscle actin??-SMA?in LF from each group,so as to reflect the transformation status of cells.?6?Transwell migration assay:To determine the migration ability of cells from each group.3.Statistical AnalysesThe data were analyzed using SPSS 13.0 statistical software and expressed as Mean±SD.The pairwise comparison of quantitative data was carried out using t-test,while the multiple-group comparison was carried out using one-way analysis of variance?ANOVA?.The significance level used was 0.05.Results:1.Study on the LF Proliferation and the Biosynthesis of Type I Collagen in Each Group?1?Changes in cell cycle phases:The flow cytometry analysis showed that the7-day hyperoxia group has a lower proportion of cells in the early phase of DNA replication?G₀/G₁ phase?and a higher proportion of cells in the DNA replication phase?S phase?than its corresponding air group?p<0.05?.This trend became even more pronounced on Day 14 of hyperoxia exposure?p<0.01?.?2?Expression of PCNA:There was no significant difference in the cell proliferation rate between the 3-day hyperoxia group and its corresponding air group.The 7-day hyperoxia group has a relatively higher cell proliferation rate than its corresponding air group?p<0.01?,while the 14-day hyperoxia group has a significantly higher cell proliferation rate than its corresponding air group?p<0.01?.There was no significant difference in the cell proliferation rate between air groups.?3?The protein level of type I collagen in cell culture supernatant:The protein level of type I collagen on Day 7 of hyperoxia exposure began to increase compared to the control group?p<0.05?,and this trend became even more pronounced on Day 14 of hyperoxia exposure?p<0.01?.?4?The mRNA expression of type I collagen in LF from each group:The 3-day,7-day and 14-day hyperoxia groups have significantly higher mRNA expression levels of type I collagen than their corresponding control groups with statistically significant difference?p<0.01?.2.Dynamic Changes in ERK1/2 Expression in Lung Tissues and Fibroblasts From Each Group?1?Determination of p-ERK1/2 Protein Expression via IHC Assay:There was no significant difference in the tissue and cellular expression p-ERK1/2protein between the 3-day hyperoxia group and its corresponding air group?p>0.05?.However,the p-ERK1/2 protein expression level began to increase and increased significantly on Day 7?p<0.01?and Day 14?p<0.01?of hyperoxia exposure compared to their corresponding air groups.?2?Determination of ERK1/2 and p-ERK1/2 Protein Expression via Western blot Assay:There was no significant difference in the tissue and cellular expression of p-ERK1/2 protein between the 3-day hyperoxia group and its corresponding air group?p>0.05?,but the p-ERK1/2 protein expression began to increase and increased significantly on Day 7?p<0.01?and Day 14?p<0.01?of hyperoxia exposure compared to their corresponding air groups,respectively.Besides,there was no significant difference between groups in the ERK1/2 protein expression level?p>0.05?.?3?Determination of ERK1/2 mRNA Expression via Real-time PCR Assay:There was no significant difference between groups in ERK1 and ERK2 mRNA expression levels?p>0.05?.3.In Vitro Study on the Effects of ERK Activator and Inhibitor on LF?1?Changes in the ERK1/2 Expression Level:?1?IHC assay:The cellular p-ERK1/2 expression level increased significantly 24hours after being added with EGF?20 ng/mL??p<0.01?,and the p-ERK1/2 expression level could be reduced via the addition of PD98059?10 nmol/mL??p<0.01?.?2?Western blot assay:The cellular p-ERK1/2 expression level increased significantly 24 hours after being added with EGF?20 ng/mL??p<0.05?,and the p-ERK1/2 expression level could be reduced via the addition of PD98059?10 nmol/mL??p<0.01?.There was no significant difference between groups in the ERK1/2 protein expression level?p>0.05?.?3?Determination of ERK1/2 mRNA expression via real-time PCR assay:There was no significant difference between groups in the mRNA expression levels of ERK1and ERK2?p>0.05?.?2?Effects of ERK Activator and Inhibitor on LF Proliferation:?1?Determination of Changes in Cell Proliferation via CCK-8 Colorimetric Assay:The results of CCK-8 assay showed that PD98059 group and EGF+PD98059group have significantly higher cell proliferation rates than control group after 6 hours and 12 hours following the addition of PD98059?p<0.05?.On the other hand,the cell proliferation rate of EGF group began to increase 24 hours after the addition of EGF?p<0.05?,during which the inhibitory effect achieved in PD98059 group peaked?p<0.01?,while EGF+PD98059 group also displayed inhibitory activities against cell proliferation?p<0.01?.?2?Flow Cytometric Determination of Cell Cycle Phases:The results showed that the EGF-treated LF displayed a significant increase in proliferation,whereby the proportion of G₀/G₁ phase cells decreased significantly?p<0.01?,whereas the proportion of S phase cells increased significantly?p<0.01?.However,the addition of PD98059?10 nmol/mL?could inhibit the cell proliferation?p<0.01?and arrest cells in the G₀/G₁ phase,thereby antagonizing the proliferation-promoting effect of EGF?p<0.01?.?3?Expression of PCNA:It can be seen that the expression of PCNA in LF increased significantly following the EGF treatment?p<0.01?,while the addition of PD98059 could significantly inhibit the expression of PCNA in normal LF?p<0.01?.?3?Effects of ERK Activator and Inhibitor on LF Differentiation:?1?Determination of?-SMA Protein Expression via IHC Assay:The cellular?-SMA protein expression level increased significantly 24 hours after the addition of EGF?20 ng/mL??p<0.01?,and the?-SMA expression level could be reduced via the addition of PD98059?10 nmol/mL??p<0.01?.Hence,PD98059 could significantly inhibit the effect of EGF?p<0.01?.?2?Determination of?-SMA Protein Expression via Western blot assay Assay:The cellular?-SMA protein expression level increased in EGF group compared with the control group?p<0.01?.and the?-SMA expression level could be reduced in EGF+PD98059 group compared with EGF group?p<0.01?.Hence,PD98059 could significantly inhibit the effect of EGF?p<0.01?.?3?Determination of?-SMA mRNA Expression via Real-time PCR Assay:The cellular?-SMA mRNA expression level increased significantly 24 hours after the addition of EGF?20 ng/mL??p<0.01?,and the?-SMA mRNA expression level could be reduced via the addition of PD98059?10 nmol/mL??p<0.01?.Hence,PD98059 could significantly inhibit the effect of EGF?p<0.01?.?4?Effects of ERK Activator and Inhibitor on LF Migration:The transwell migration assay showed that the migration ability of cells was significantly enhanced 12 hours after the addition of EGF?p<0.01?,and PD98059?10nmol/mL?could significantly inhibit the migration ability of cells.Therefore,PD98059could significantly inhibit the effect of EGF?p<0.01?.Conclusions:1.Hyperoxia could induce cells to enter the S phase and significantly accelerate cell proliferation.2.Hyperoxia may lead to BPD in rats with PF by promoting the formation of LF and increasing the secretion of type I collagen.3.Hyperoxia could induce the phosphorylated activation of ERK1/2 protein in LF of neonatal rats,but there were no significant changes in the gene expression levels.The activated p-ERK1/2 may be the key to the over-proliferation of LF.4.The in vitro study on the effects of ERK activator and inhibitor on LF has confirmed ERK1/2 as important signaling molecules in regulating the proliferation,transformation and migration of LF.