| BackgroundsBronchopulmonary dysplasia (BPD) is a chronic lung disease, with a high morbidity and mortality in the preterm infants, especially in VLBWI (very low birth weight infant, VLBWI) and ELBWI. (extremely low birth weight infant,ELBWI). With the development of perinatal medicine, the survival rate of premature increases, it is the cause of prolonged hospitalization with serious social and economic consequences. Therefore, to investigate how to prevent BPD and reduce the incidence is the important topic.Although the pathogenic and genetic basis of BPD remains incompletely understood, many studies suggest that the inflammation-mediated damage and the oxidative stress play an important role in the development of BPD. A lot of cytokines, such as tumor necrosis factor-alpha(TNF-a), interleukin-8(IL-8), interleukin-1β(IL-1β), interleukin-6(IL-6), intercellular adhesion molecule 1 (ICAM-1) and nuclear factor-κB(NF-κB) were proposed to involve in lung inflammation, closely related to the pathogenesis of BPD.BPD is a multifactorial disease, its etiology and pathogenesis is very complex,involving many aspects of hyperoxia, mechanical ventilation, prenatal infection and postpartum inflammation, cause there hasn’t been an effective way for the preventionand treatment. Currently recognized as, oxidative stress and inflammatory reaction play an important role in the pathogenesis of BPD. Oxidative stress response is regulated by the imbalance between ROS generation and antioxidant enzyme degradation of ROS. Under physiological circumstances, there is a delicate balance between the human body ROS generation and antioxidant defense system. The balance will be broken when the over expression or inadequate clearance of ROS was out of the metabolic control of cells, resulting the oxidative stress injury.It is well known that apoptosis is critical for normal lung development and function(39,40) and plays a key role in remodeling of lung tissue by clearing excess epithelial and mesenchymal cells after injury(41,42). Abnormal apoptotic activity may contribute to the pathophysiology of many lung diseases, including BPD. Kazzaz et al reported that apoptosis has been clearly induced in the lungs of mice hyperoxia exposure to hyperoxia(43). Normally, the lung alveolar epithelium forms a tight barrier to restrict the movement of proteins and liquid from the interstitium into the alveolar spaces. If the alveolar epithelium is damaged, both the permeability of endothelial cells and epithelial cells would change, and it could lead to major alveolar flooding with high-molecular-weight proteins, with prolonged changes in gas exchange.Pre-B-cell colony-enhancing factor (PBEF), was first identified by Samal in 1994 as a putative cytokine, which was involved in the maturation of B-cell precursors in the presence of IL-7,also named NAMPT. PBEF is synthesized and secreted by many kinds of cells, such as activated lymphocytes, epithelial cells, neutrophils. Recently, it has been demonstrated that PBEF exerted proinflammatory activities by upregulating the production of the proinflammatory cytokines IL-1β, TNF-a, IL-6 in human monocytes. Ye and colleagues reported the first findings of PBEF expressed in lung tissues and overexpressed in acute lung injury(ALI) Furthermore, PBEF was considered to be associated with the regulation of inflammation and endothelial barrier dysfunction in ALI. Su identified that PBEF stimulates ROS generation in endothelial cells, and antioxidants blocked PBEF-induced NF-κB activation and cell adhesion molecules(CAM) expression. PBEF expression is up-regulated and provide an important function during the development of a variety of inflammation-related diseases including acute lung injury, sepsis, rheumatoid arthritis, atherosclerosis. Based on these results, we speculate that PBEF might be a potential modulator in the pathogenesis of BPD.The EA.hy926 cell line is a permanent human endothelial cell line, which was derived from the fusion of human umbilical vein endothelial cell (HUVEC) line and A549 cell line. The EA.hy926 cell line expresses highly differentiated functions that are characteristic of lung adenocarcinoma cells and vascular endothelium. Xu and colleagues used these alveolar endothelial cells to construct the alveoli in vitro model. EA.hy926 cells have been widely applied in the study of inflammation, oxidative stress and protein expression.In the current study, we aimed to explore the role of PBEF in BPD and its mechanism of action by establishing an in vitro cell model of BPD. PBEF siRNA was used to knockdown PBEF in EA.hy926 cell to observe the regulation of PBEF in inflammation and apoptosis caused by hyperoxia.Objectives1. To establish the BPD cell model in vitro.2. To explore the role of pre-B cell colony-enhancing factor (PBEF) in thehyperoxia model of EA.hy926 cellsMethods1.EA.hy926 cell cultures were divided into four groups:the air group as the blank control, the hyperoxia group, the hyperoxia plus PBEF siRNA group and the hyperoxia plus scramble siRNA group. The air group were cultured for 24,48 and 72 h in a 5% CO2,95% air,37℃ incubator. The hyperoxia group were cultured for 24, 48 and 72 h in the 37℃ triple gas mixture. The hyperoxia plus PBEF siRNA group were transfected by PBEF siRNA, and cultured for 24,48 and 72 h in the 37℃ triple gas mixture. The hyperoxia plus scramble siRNA group were transfected by scramble siRNA, and cultured for 24,48 and 72 h in the 37℃ triple gas mixture.2.The cell viability and the reactive oxygen species (ROS) were measured by the assay kit.3.The protein and mRNA expression levels of PBEF, interleukin(IL)-8 and tumor necrosis factor-alpha(TNF-a) were also detected by corresponding methods.Results1. CCK8 assay revealed that hyperoxia treatment could significantly reduce the cell viability by 16% after 24 h,17% after 48 h, and 21% after 72 h compared with the air group, while the cell viability of hyperoxia plus PBEF siRNA group was significantly higher than that of the hyperoxia group (P<0.05)2. The ROS levels of the hyperoxia group and and the hyperoxia plus PBEF siRNA group were significantly higher than that of the air group at 24,48 h and 72 h (P<0.01), while the ROS level of the hyperoxia plus PBEF siRNA group was significantly lower than that of the hyperoxia group (P<0.01).The ROS levels of the hyperoxia group at 48,72 h were significantly higher compared with the ROS level at 24 h (P<0.01).3. Real-time PCR analysis showed that the PBEF,IL-8,TNF-a mRNA expression levels of the hyperoxia group were significantly higher than the air group at each time point(P<0.01), while the mRNA expression level was significantly reduced in the hyperoxia plus PBEF siRNA group compared with the hyperoxia group at each time point(P<0.01). In the hyperoxia plus PBEF siRNA group, it can be shown that PBEF siRNA knocked down PBEF mRNA expression in EA.hy926 cell(Fig3.more than 65% decrease,**P<0.01). scramble siRNA alone had no effect on the PBEF expression. The TNF-a and PBEF gene expression levels of the hyperoxia group and the hyperoxia plus PBEF siRNA group at 48,72 h were significantly higher than the gene expression levels at 24 h(P<0.01). The PBEF gene expression levels of the hyperoxia group and the hyperoxia plus PBEF siRNA group at 72 h were significantly higher than the gene expression levels at 24,48 h(P<0.01). No significantly differences were seen between the hyperoxia group and the hyperoxia plus scramble siRNA group at any time point.4. The results from western blot analysis revealed that the protein expression of PBEF, IL-8, TNF-a in the hyperoxia group was increased compared with the air group(P<0.01), while the protein expression of PBEF,IL-8,TNF-a in the hyperoxia plus PBEF siRNA group was significantly lower than that of the hyperoxia group(P<0.01). In the hyperoxia plus PBEF siRNA group, it can be shown that PBEF siRNA knocked down PBEF protein expression in EA.hy926 cell(Fig4.more than 65% decrease,**P<0.01). Scramble siRNA alone had no effect on the PBEF expression. The TNF-a and PBEF protein expression levels of the hyperoxia group and the hyperoxia plus PBEF siRNA group at 48 h,72 h were significantly higher than the protein expression levels at 24 h (P<0.01). No significantly differences were seen between the hyperoxia group and the hyperoxia plus scrabmle siRNA group at any time point.Conclusions1. This study demonstrate that hyperoxia significantly induced PBEF, IL-8 and TNF-a expression at both the mRNA and protein levels in EA.hy926 cells. Inflammation and oxidative stress are the hall-marks of the pathogenesis of BPD2. The knockdown of PBEF expression by PBEF siRNA significantly blunted TNF-a and IL-8 mRNA and protein production and attenuated hyperoxia induced ROS level in EA.hy926 cells. PBEF may play a key role as a pro-inflammatory cytokine in the dysregulation of alveolar epithelial cell barriers in the development of BPD. These results lend further support to the potential of PBEF to serve as a diagnostic and therapeutic target in future studies of BPD. |
