Related Study On The Effect Of Calcitoniongene-relatedpeptide In Type Ⅱ Alveolar Epithelial Cell Of Premature Rats Exposed To Hyperoxia And Notch Signaling Pathway

Background Bronchopulmonary dysplasia(BPD)is a common chronic pulmonary disease in surviving premature.In recent years,with the development of perinatology,the survival rate of premature infants increased significantly,as a result,BPD incidence is on the rise.Oxygen therapy is a common clinical treatment,however,inhalation of highly concentrated oxygen for a long time can lead to hyperoxic lung injury(HLI).HLI aggravates the occurrence and development of BPD.The pathological changes of BPD include abnormal alveolarization and immature lung tissue abnormalities,which are closely related to pulmonary cell proliferation.Type II alveolar epithelial cell(ACE II),as the alveolar epithelial stem cell,can be differentiated into TypeⅠ alveolar epithelial cell(ACEⅠ),of which the proliferation and differentiation can not only repair the normal structure of alveoli but also the function.Calcitonin gene-related peptide(CGRP)is a neuropeptide with a wide regulation performance that plays an important role in the proliferation of various types of epithelial and endothelial cells.Notch signaling has been verified that it plays a broad and critical regulatory role in the differentiation,proliferation,apoptosis and a range of other physiological and pathological processes in the vast majority of cells.Existing studies have shown that CGRP has a protective effect on AECII subjected to hyperoxia injury.Therefore,discussion on the effect of CGRP on Notch signaling pathway of AECII subjected to hyperoxia injury and the role of Notch signaling pathway in CGRP could provide new ideas for basic research and clinical prevention and treatment to HLI and BPD.Objective 1 Toinvestigate the oxidative stress and apoptosis of AECⅡ in premature rats exposed in 95% volume fraction of oxygen as well as its impact on Notch signaling pathway.2 Whether Notch signaling pathway involved in the protective effect of CGRP on hyperoxia injured AECⅡwas discussed by using Notch pathway blockerandsilencing the Notch1 gene with si RNA interference.It may provide new ideas for basic research and clinical prevention and treatment to HLI and BPD.Methods 1 Fetuses were taken out from pregnancy SPF-level Sprague-Dawley on the 19 th day by cesarean section,and the lungs were removed,cut and slaked,in which AECⅡ was purified by the methods of differential centrifugation and differential attachment to perform the primary culture of AECⅡ.The activity of AECⅡ was detected by automatic cell analyzer;AECⅡ was detected by immunohistochemistry and transmission electron microscopy;the growth of AECⅡ was observed by inverted phase contrast microscope.The AECII hyperoxia model of premature rats was established by the airtight instrumentwhich was ventilated 95%oxygendeveloped by our laboratory.2 In the relative studies of Notch pathway blockade,the primary AECⅡ of premature rats were isolated and purified,and then randomly divided into 6 groups: Group A: normoxia group: cells were placed in a airtight instrument with oxygen volume fraction of 21%,then cultured in CO2 Cell incubator.Group B: hyperoxia group: cells were placed into a airtight instrumentwhich was ventilated 95%oxygen,then cultured in CO2 Cell incubator.Group C: hyperoxia + CGRP group: CGRP was added into the cell culture medium in advance and then hyperoxia treatment was performed.Group D: hyperoxia + CGRP + CGRP8-37 group: CGRP and CGRP inhibitor CGRP8-37 were added in advance,and the others steps were as same as those of hyperoxia group.Group E: hyperoxia + DAPT group: Notch pathway blocker DAPT was added in advance,and the others steps were as same as those of hyperoxia group.Group F: hyperoxia + DAPT + CGRP group: DAPT and CGRP were added in advance,and the others steps were as same as those of hyperoxia group.The morphologic changes of ACEⅡ were observed after culture for 24h;by using flow cytometry,ROS,apoptosis rate of cells were measured;the activity of malondialdehyde(MDA)and superoxide dismutase(SOD)were detected;the expression level of receptor Notch1 m RNA of Notch pathway was tested by using Q-PCR;the level of HERP protein,Notch downstream target gene,was detected by using Western blot method.3 Three Notch1 interference sequences and primers were composed in advance,in which the sequence with the most powerful interfering effect was chosen to transfect AECⅡ with plasmid as the carrier to establish the si RNA interference model of AECⅡNotch1 gene in premature rats.4 In relative studies of si RNA interference,the primary AECⅡ was isolated and purified,and then randomly divided into five groups:(1)hyperoxia group;the cells were cultured in 95%O2 environment;(2)blank plasmid transfection Group: blank plasmid was transfected and then cultured in hyperoxic environment;(3)si RNA interference group: cultured in hyperoxic environment after si RNA interference;(4)blank plasmid + CGRP group: blank plasmid was transfected and then cultured in culture medium by adding CGRP in hyperoxic environment;(5)si RNA interference + CGRP group: cultured in culture medium by adding CGRP in hyperoxic environment after si RNA interference;The apoptotic rate,oxidative index,Notch1 m RNA expression and HERP protein were measured after 24 hours of culture.Result 1.AECII primarily cultured by the method of our laboratory has a better production,AECII,under the light microscope,shows a polygonal island-like distribution,of which cells are closely connected and the nucleus is obvious,there are a large number of granular substances in the cytoplasm;the results detected by automatic cell analyzer shows that the total cell number isolated from each rat was(8.0±1.5)×106,in which the l survival rate is about(98±1.5)%.AECII accounted for(97±2.0)% of total cell number,shows the purity and activity of AECⅡ qualified.Microvilli can be seen on the cell membrane and a large number of osmiophilic lamellar bodies can be seen in the cells under transmission electron microscopy.Immunohistochemistry showed that the AECII cytoplasm was brown.2.In comparison with normoxia group,as for AECⅡ exposed in hyperoxic environment for 24 h,the apoptosis rate increased significantly,ROS and MDA rose obviously and SOD activity decreased significantly(P<0.05),and at the same time,the expression of the receptor Notch1 m RNA of AECⅡ Notch pathway and the protein expression of target gene HERP decreased significantly(P<0.05).3.In comparison with hyperoxia group,CGPR interference could raise the expression of Notch1 m RNA and HERP(P<0.05),and at the same time,could reduce the apoptosis rate of AECⅡ,lower the ROS and MDA as well as increase the SOD activity(P<0.05).CGRP8-37 could interdict the aforesaid biological functions of CGRP.4.The expression of Notch1 m RNA and the protein expression of were further decreased after the interference by using the Notch pathway blocker DAPT,and the apoptosis rate was further increased,the ROS and MDA was further risen and SOD activity was further decreased in AECⅡ exposed in hyperoxic environment for 24h(P<0.05);while aforesaid changes could be relived after the performance of CGRP interference(P<0.05),and the expression of Notch1 m RNA,the receptor of Notch pathway,and target gene HERP were increased at the same time(P <0.05).5.There was no statistical difference in apoptosis rate,ROS,SOD and MDA between the hyperoxia group and the blank plasmid tranfection group(P>0.05),compared with the blank plasmid transfection group,the expression of Notch1 m RNA and the protein expression of HERP were significantly decreased in AECⅡ subjected to hyperoxic injury in the si RNA interference group(P <0.05),oxidative damage was aggravated and apoptosis rate of cells increased;The expression of Notch1 m RNA and the protein expression of HERP in blank plasmid transfection + CGRP group and si RNA interference + CGRP group were higher than those in groups without CGRP(P <0.05),and the apoptotic rate was lower.Conclusion 1.AECⅡ differentiated and purified by using differential centrifugation and differential adherence methods has large quantity,high purification and excellent activity,which is appropriate for cellular experiments.2.Hyperoxia(95% volume fraction)could lead to oxidative damage of AECⅡ,promote apoptosis of AECⅡ,decrease the expression of Notch1 m RNA and target gene HERP;blocking Notch pathway can further decrease the expression of Notch1 m RNA and HERP,and aggravate the oxidative damage,which showed that Notch participated in the biological effect caused by hyperoxia on AECII.3.CGRP could reduce the apoptosis of AECII subjected to hypoxia injury and reduce its oxidative damage,so as to protect the hyperoxia-induced injured lungs.At the same time,CGRP could promote the expression of Notch1 m RNA and target gene HERP,CGRP8-37 could interdict the aforesaid biological functions of CGRP and down-regulatethe Notch1 m RNA and HERP levels.while blocking Notch pathway can reduce the rotective effect of CGRP.which manifested that promoting the expression of Notch signaling pathway may be one of the mechanisms of CGRP to protect AECII subjected to hypoxia injury.4.we established the model of AECII Notch1 si RNA interference in premature rats by lipofection method,which effectively down-regulated the expression of Notch1 and target gene HERP,and Notch1 gene silencing can increase the apoptosis rate and oxidative stress of hypoxia injured AECII.Addition of CGRP can partly up-regulatethe Notch1 m RNA and HERP levels andreduce the hypoxia injury of AECII.It was further verified that the Notch signaling pathway may be involved in the protective effect of CGRP on AEC damage induced by hyperoxia.