| BackgroundsBronchopulmonary dysplasia(BPD)is a chronic lung disease that seriously threatens the health of premature infants.It is very common in premature infants,and the incidence rate increases with the decrease of gestational age.It usually accompanied by serious complications,which greatly affects the survival and quality of life of children.The occurrence and development of BPD are affected by various factors,including hyperoxia,mechanical ventilation,infection and so on.Up to now,there is no effective treatment.The prevention and treatment of BPD has always been an important content of research.At present,it is recognized that the most important factor is the oxidative stress and inflammatory reaction caused by long-term mechanical ventilation and high concentration oxygen therapy.Oxidative stress is essentially the destruction of the balance of oxidation-antioxidation system.Both mechanical ventilation and hyperoxia exposure can induce a large amount of reactive oxygen species(ROS)in lung tissue.At the same time,premature infants’antioxidant mechanism is not mature.So that a large amount of ROS accumulate in the body.ROS is a highly active oxidant,which can cause damage to DNA,protein and lipid structure.Afterwards it affects gene expression,protein function and normal metabolic activity of cell tissue,and then causes damage to its structure and function.In addition,oxidative stress can also activate a variety of pro-inflammatory factors and inflammatory mediators,which induce inflammatory cascade reactions,lead to cell damage and apoptosis,and ultimately lead to the occurrence of BPD.Caffeine is currently widely used in the prevention and treatment of apnea in premature infants.It has been found that it can protect the lung from injury and effectively reduce the incidence of BPD through various way.However,its mechanism of action is not clear,and we needs further study to prove whether it plays an antioxidant and anti-inflammatory role.A variety of signal pathways and cytokines also play an important role in the occurrence and development of BPD.In recent years,a vascular factorepidermal growth factor-like domain 7(EGFL7),which is specifically secreted by vascular endothelial cells and can specifically act on vascular endothelium,has been found.EGFL7 can not only regulate the formation of blood vessels,but also reduce the expression of some inflammatory factor,like TNF-α、ICAM-1 and so on,and then reduce the inflammatory reaction.Notch pathway is an evolutionarily highly conserved signal pathway widely existing in various species,which can regulate the process of proliferation,differentiation and apoptosis by mediating and regulating the interaction between cells.The research found that Notch signal pathway can negatively regulate the proliferation of endothelial cells and the formation of blood vessels,participate in the regulation of the development and maturation of bronchi and alveoli,and also play an important role in oxidative stress.In addition,EGFL7 can also block the transmission of Notch signal pathway and inhibit the expression of its downstream genes.Whether EGFL7 can regulate the expression of Notch signaling pathway and participate in the occurrence and development of BPD,whether it can participate in the process of oxidative stress and inflammatory damage,and whether caffeine regulate this process need further research.Objectives1.To explore the antioxidant and anti-inflammatory effects of caffeine on EA.hy926 cells damaged by oxidative stress,and to provide theoretical basis for the use of caffeine in the prevention and treatment of BPD.2.To explore whether the role of caffeine involves EGFL7 and Notch signaling pathway,and to find the target of caffeine action at the molecular level,so as to provide a new therapeutic direction for the prevention and treatment of BPD in clinical practice.Methods1.The oxidative stress injury model was established by hydrogen peroxide stimulation.2.Experimental grouping The experiment was divided into four groups:control group,H2O2 group,H2O2+caffeine(2.5μM)Group,H2O2+caffeine(5μM)Group.Firstly,the cells of each group that have changed the serum-free DMEM medium were put into the incubator for 24 hours.Then add H2O2(400μM)to the groups except the control one for 1 hour incubating in the incubator.After that add 2.5 μM concentration of caffeine to H2O2+caffeine(2.5μM)Group,and add 5μM concentration of caffeine to H2O2+caffeine(5 μM)Group.Finally all the groups were placed in the incubator for 24 hours.3.The cell survival rate and the levels of SOD,CAT,GSH,IL-6,TNF-α in each experimental groups were measured with the detection kit.4.The protein expression levels of EGFL7,Notch 1 and Notch2 in each experimental group were determined by Western blotting.5.SPSS 26.0 software was used for analysis.The data is expressed by mean± standard deviation(X±S),and the significance of difference is tested by one-way ANOVA.P<0.05 indicates that the difference is statistically significant.Results1.Hydrogen peroxide decreased the survival rate of EA.hy926 cells in a concentration-dependent manner(P<0.01)and the semi-inhibitory concentration(IC50)is 400 μM.There was no significant cytotoxicity in the range of 1-5μM of caffeine concentrating to the cells(P>0.05),but it could improve the survival rate of cells injured by oxidative stress(P<0.01).2.Hydrogen peroxide significantly decreased the levels of SOD,CAT and GSH in cells(P<0.05),while caffeine significantly increased the levels of SOD,CAT and GSH in cells damaged by oxidative stress(P<0.05).3.Hydrogen peroxide significantly increased the levels of IL-6 and TNF-α in cells(P<0.01),while caffeine significantly decreased the levels of IL-6 and TNF-α in cells damaged by oxidative stress(P<0.01).4.Hydrogen peroxide significantly down-regulated the expression of EGFL7 protein in EA.hy926 cells(P<0.05)and up-regulated the expression of Notch1 and Notch2 protein(P<0.05).While caffeine significantly up-regulated the expression of EGFL7 protein(P<0.01)and down-regulated the expression of Notchl and Notch2 protein(P<0.01).Conclusions1.Caffeine can increase the level of cell antioxidant enzymes to achieve antioxidant effect,reduce pro-inflammatory factors to reduce inflammatory reaction,and protect cells from oxidative stress and inflammatory damage.2.Caffeine may prevent and treat BPD by regulating the expression of EGFL7 and Notch signal pathway related receptors. |
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