| Objective 1.To explore the effect of CGRP on AECⅡ apoptosis in mice exposed to hyperoxia.2.To explore whether the inhibitory effect of CGRP on AECⅡ injury and apoptosis under hyperoxia exposure involves the AMPK / p53 / mTOR response axis.3.To explore the role of CGRP in inhibiting AECⅡ damage and apoptosis during the process of cell autophagy.Methods Mouse lung type II epithelium(cell line MLE-12)was used as the experimental research object.MLE-12 cells were cultured in DMEM / F12 medium containing 5% FBS.When the cells reached 80% fusion,the ratio was 1:3 The subcultured MLE-12 cells will be expanded.Randomly divided into three parts according to three parts: the first part(normal oxygen group,hyperoxic group,normal oxygen CGRP intervention group,hyperoxic CGRP intervention group,hyperoxic CGRP treatment antagonist group),the second part(hyperoxic +CGRP intervention Control group,hyperoxia + CGRP + P53 overexpression group,hyperoxia + CGRP + P53 interference group),the third part(hyperoxia +CGRP intervention control group,hyperoxia + CGRP + 3-MA intervention group,hyperoxia + CGRP + Rapamycin intervention group)After 24 hours in vitro culture,the growth morphology of MLE-12 cells was observed under an inverted phase contrast microscope.Flow cytometry was used to detect the apoptosis rate of MLE-12 cells in each group.Western blot was used to detect apoptosis-related proteins(Caspase-3,Bcl-2),Pathway-related proteins(AMPK,P53,MTOR),autophagy protein(Becline-1)expression levels.Results 1.Compared with the normoxic group,the MDA and apoptosis rates of AECⅡMLE-12 cells in the hyperoxic group increased significantly(P <0.05),and the survival rate decreased;compared with the hyperoxic group,the MDA and apoptosis rates in the hyperoxic CGRP intervention group were significantly reduced(P <0.05),the survival rate increased;CGRP antagonist(CGRP8-37)can block the biological effect of CGRP.2.Compared with the hyperoxic CGRP intervention group,the expression levels of pathway-related proteins P53,AMPK and anti-apoptotic protein Bcl-2 in the hyperoxic + CGRP + P53 interference group were significantly reduced,and pathway-related proteins MTOR protein and apoptosis protein Caspase The expression level of 3 was significantly increased(P <0.05),and the apoptosis rate increased;compared with the hyperoxic CGRP intervention group,the hyperoxia+ CGRP + P53 overexpression group,pathway-related proteins P53,AMPK,MTOR and apoptosis-related proteins The expression levels of Caspase-3 andBcl-2 did not increase or decrease(p> 0.05),and the apoptosis rate did not change significantly.3.Compared with the hyperoxia CGRP intervention group,the hyperoxia + CGRP+ 3-MA intervention group,the expression levels of apoptosis protein Caspase-3and autophagy protein Becline-1 increased significantly,and the expression level of anti-apoptotic protein Bcl-2 Significantly decreased(P <0.05),cell apoptosis rate increased,survival rate decreased;compared with hyperoxic CGRP intervention group,hyperoxia + CGRP + rapamycin intervention group,apoptosis-related proteins Caspase-3,Bcl-2 There was no significant change in the expression level of the autophagy protein Becline-1(p> 0.05),and there was no significant increase or decrease in cell apoptosis rate and survival rate.Conclusions 1.Hyperoxic injury causes apoptosis of MLE-12 cells,CGRP reagent can effectively protect the apoptosis effect of hyperoxia on MLE-12 cells.2.The AMPK / p53 / mTOR response axis was inhibited after interfering with the expression of P53 gene,and CGRP failed to inhibit the apoptosis of MLE-12 cells by hyperoxia.3.Calcitonin gene-related peptide mediates AMPK / p53 / mTOR response axis to activate cell autophagy to reduce AECⅡ damage and apoptosis. |
