| Objective To observe the pathological changes of Notch signaling pathway in the repair of lung injury after hyperoxia exposure and intervention with calcitonin gene-related peptide at different time,and to explore the protective effect of calcitonin gene-related peptide on hyperoxia lung injury and its mechanism through the changes of Notchl,heRP,Hes protein content and gene expression level of some members of Notch signaling pathway.Methods The neonatal premature rats were exposed to 95% oxygen to induce hyperoxia-induced lung injury.They were divided into six groups: air group,hyperoxia group,hyperoxia + CGRP group,hyperoxia + MWl67 group,hyperoxia + CGRP + CGRP 8-37 group,hyperoxia + CGRP + MWl67 group.The pathological changes of lung tissue were observed by HE staining and light microscopy on the 3rd,7th and 14 th day,and the pathological scores of lung injury were obtained.The distribution and expression of Notch1 in lung tissue were detected by immunohistochemistry.At the same time,the content of Notch1,heRP and Hes protein and the expression of Notch1 in lung tissue were detected by Western blot and Q-PCR.Results 1.Pathological changes of lung tissues in each group:(1)In air control group,the lung tissues were more regular and the cells arranged neatly on the 3rd,7th and 14 th day.(2)In the hyperoxia group,a few inflammatory cells were observed on the 3rd day,alveolar septum was broken obviously on the 7th day,interstitial edema and alveolar fusion were observed,trachea and blood vessels were destroyed extensively on the 14 th day,and the wall of the tube was thickened.(3)Hyperoxia + MWl67 group: Alveolar septal rupture and alveolar fusion were observed in lung tissue on the 3rd day after hyperoxia exposure,and inflammatory cell infiltration,a large number of vacuoles and interstitial edema were observed on the 7th day.On the 14 th day,extensive destruction of capillaries and marked thickening of tracheal wall were observed.(4)Hyperoxia + CGRP group: trachea and blood vessel were normal on the 3rd day after hyperoxia exposure;alveolar septal rupture,alveolar fusion and inflammatory cell infiltration were observed on the 7th day;fibrosis and thickening of trachea and vascular wall were observed on the 14 th day.(5)Hyperoxia + CGRP + CGRP 8-37 group: on the 3rd day of hyperoxia exposure,a small amount of alveolar septum rupture,alveolar fusion and inflammatory cell infiltration were observed;on the 7th day,a large number of vacuoles,alveolar interstitial edema and inflammatory exudation were observed;on the 14 th day,tracheal and vascular destruction and wall thickening were observed.(6)Hyperoxia + CGRP + MWl67 group: On the 3rd day after hyperoxia exposure,a little inflammatory exudation was observed in lung tissue;on the 7th day,alveolar septal rupture,alveolar fusion,interstitial edema;on the 14 th day,trachea and blood vessels were destroyed and the wall of the tube was thickened.2.The pathological scores of lung injury in each group:(1)The pathological scores of lung injury in preterm rats exposed to hyperoxia for 3,7 and 14 days were higher than those in the air group at the same time point(P < 0.05),and the difference was significant.Compared with the hyperoxia group,the pathological score was the lowest on the 3rd day and the highest on the 14 th day,suggesting that the pathological score was positively correlated with the time of hyperoxia exposure.(2)The pathological scores of lung injury in hyperoxia + MWl67 group on the 3rd,7th and 14 th day were higher than those in hyperoxia group at the same time point(P < 0.05).(3)The pathological scores of lung injury in the hyperoxia + CGRP group were lower than those in the hyperoxia group at the same time point(P < 0.05)on the 3rd,7th and 14 th day,and the difference was significant.(4)The pathological scores of lung injury in hyperoxia+CGRP+CGRP 8-37 group on the 3rd,7th and 14 th day were higher than those in hyperoxia+CGRP group at the same time point(P<0.05).(5)The pathological scores of lung injury in hyperoxia+ CGRP+MWl67 group on the 3rd,7th and 14 th day were higher than those in hyperoxia+CGRP group at the same time point(P<0.05).(6)The pathological scores of lung injury in hyperoxia + CGRP + MWl67 group on the 3rd,7th and 14 th day were lower than those in hyperoxia group at the same time point(P < 0.05).3.The distribution and expression of Notch1 were detected by immunohistochemistry.Notch1 was expressed in the lower airway,alveolar epithelial cells and vascular endothelial cells,mainly in the airway epithelial cells.The expression of Notch1 positive cells in hyperoxia group decreased on the 3rd,7th and 14 th day compared with that in air group at the same time point.The Notch1 positive cells in the hyperoxia + MW 167 group decreased on the 3rd,7th and 14 th day compared with the hyperoxia group at the same time point.The Notch1 positive cells in the hyperoxia + CGRP group increased at the 3rd,7th and 14 th day compared with the hyperoxia group at the same time point.Notch1 positive cells in the hyperoxia + CGRP + CGRP 8-37 group decreased on the 3rd,7th and 14 th day compared with those in the hyperoxia + CGRP group at the same time point.Notch1 positive cells in the hyperoxia + CGRP + MW 167 group decreased on the 3rd,7th and 14 th day compared with those in the hyperoxia + CGRP group at the same time point.4.Western blot and Q-PCR were used to detect the changes of Notch1,heRP and Hes protein content and expression level in lung tissues of each group:(1)The expression of Notchl,heRP and Hes in hyperoxia group was significantly lower than that in air group at the same time point on the 3rd,7th and 14 th day(P < 0.05).(2)The expression of Notchl,heRP and Hes in hyperoxia + MWl67 group decreased further on the 3rd,7th and 14 th day than that in hyperoxia group at the same time point(P < 0.05).(3)The expression of Notch1,heRP and Hes in the hyperoxia + CGRP group increased significantly on the 3rd,7th and 14 th day compared with the hyperoxia group at the same time point(P < 0.05).(4)The expression of Notch1,heRP and Hes in hyperoxia + CGRP + CGRP 8-37 group decreased significantly on the 3rd,7th and 14 th day compared with that in hyperoxia + CGRP group at the same time point(P < 0.05).(5)The expression of Notch1,Hes and heRP in hyperoxia + CGRP + MW 167 group decreased significantly on the 3rd,7th and 14 th day compared with that in hyperoxia + CGRP group at the same time point(P < 0.05).The expression of Notch1,heRP and Hes in hyperoxia + CGRP + MW 167 group had no significant difference on the 3rd,7th and 14 th day compared with that in hyperoxia group at the same time point(P > 0.05).Conclusion 1.After hyperoxia exposure,the expression of Notch1,heRP and Hes was down-regulated,and pathological changes were observed in lung tissues,suggesting that Notch signaling pathway was involved in the occurrence and development of BPD.2.CGRP can significantly reduce the degree of lung pathological damage in premature rats after hyperoxia exposure,suggesting that CGRP may be a protective factor for hyperoxia lung injury.3.CGRP may play a protective role in hyperoxia-induced lung injury by promoting Notch signal expression.Moreover,Notch signaling pathway may only be one of the mechanisms by which CGRP exerts its effects on hyperoxia-induced lung injury.. |
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