Effect And Mechanism Of Hypoxia On Cleft Palate Through HIF-1α/BNIP3

Research background and purpose:Non-syndromic cleft lip with/without cleft palate(NSCL/P)is a common congenital developmental defect,and its etiology can be divided into genetic factors and environmental factors.Environmental factors include maternal smoking during pregnancy,chronic diseases and living in the plateau areas and so on.One of the common effects is that they lead to fetal hypoxia.HIF-1α(hypoxia inducible factor-1α)is a nuclear transcription factor that mediates the adaptive response to hypoxia when stimulated by hypoxia.Abnormal expression of HIF-1α during pregnancy may lead to congenital malformations,but its role in NSCL/P remains unclear.Bcl-2 19-kDa interacting protein 3(BNIP3)is an important downstream gene of HIF-1α and has been found to promote autophagy in recent years.Autophagy is an evolutionarily conserved mechanism related to the degradation and recovery in eukaryotes,which can maintain the homeostasis of the intracellular environment and is crucial for the proliferation and survival of cells.Abnormal autophagy in the embryonic stage may affect the fusion of the palate embryo and lead to cleft palate.The structure and development process of zebrafish are largely conserved in human,making it possible to be used as an animal model for craniofacial development research.Its tolerance to hypoxia gives it a unique advantage in hypoxia research.In this experiment,the zebrafish was used as the animal model.The hypoxic cleft palate model was constructed to observe the effects of different hypoxic environments on the phenotype of cleft palate,detect the expression of HIF-1α/BNIP3,and preliminarily explore the changes of subsequent autophagy.Experimental methods:(1)Study on tolerance of zebrafish embryos to hypoxic environment:A zebrafish hypoxia model was constructed and its livability,hatchability,embryo heart rate and deformity rate were detected.(2)Effect of hypoxic environment on maxillofacial deformity of zebrafish:Alcian blue staining and autofluorescence of TG(osx:nlsGFP)transgenic zebrafish strains were used to measure the length of hyoid,mandible and ethmoid palate of zebrafish embryos,and acridine orange staining was used to detect the apoptotic cells in maxillofacial region.(3)Regulation mechanism of HIF-1α/BNIP3 signaling pathway on abnormal development of zebrafish maxillofacial under hypoxic environment:RT-qPCR was used to detect the changes of hif-1α mRNA and bnip3 mRNA expression in different hypoxia durations.Immunohistochemical fluorescence staining was used to detect the expression and location of Hif-1α and Bnip3 proteins.RT-qPCR was used to detect the expression of autophagy-related genes beclin-1 and lc3Ⅱ.(4)Analysis of Hif-1α binding sites and functions throughout the zebrafish genome:The raw data of ChIP-seq sequencing of zebrafish with high Hif-1α expression and wild-type zebrafish were downloaded from the database for GO and KEGG analysis.Find molecules related to the development of the palate.Experimental results:(1)In the hypoxia environment,the livability and hatchability of zebrafish embryos are decreased,and the heart rate and deformity rate are increased.(2)The hyoid,mandible and ethmoid palate of zebrafish in the maxillofacial region were shortened and developed abnormally under hypoxic environment.(3)After 4 h of hypoxia stimulation,hif-1α mRNA expression was increased,then decreased.Compared to the normal oxygen group,the hif-1α mRNA expression shows a small difference at 48h of hypoxia.Immunohistochemical staining showed fluorescence of Hif-1α protein near the ethmoid palate.After 4 h of hypoxia,the expression of bnip3 mRNA was increased rapidly.Until 48 h of hypoxia,the expression of bnip3 mRNA in the hypoxia group was slightly lower than that in the normoxia group.The fluorescence at the margin of bone were visible in the immunohistochemistry experiment,and the expression of Bnip3 protein at the margin of bone was increased.After 4 h of hypoxia stimulation,the expression of beclin-1 mRNA and lc3Ⅱ mRNA were increased.(4)GO enrichment analysis found that Hif-1α played an important role in embryo development,and KEGG analysis found that Hif-1α was enriched in autophagy.Searching of 194 genes involved in embryo development showed that there were significant peaks at sox9,bmp4,mdm2,and there were enhancers or promoters near them.Conclusion:(1)Zebrafish embryos could tolerate hypoxia and could be used as animal models for hypoxia research.(2)Hypoxic environment can cause abnormal development of zebrafish maxillofacial skeletons.(3)Under hypoxic environment,HIF-1α/BNIP3 pathway is involved in maxillofacial skeletal dysplasia by inducing autophagy.(4)Hif-1α participates in the process of embryo development,and is enriched in the autophagy pathway.sox9,bmp4 and mdm2 may be related to cleft palate caused by hypoxia.