| Goldfish belongs to the carp family,Carassius auratus.As a variety ofCrucian carp,goldfish is deeply loved by the public and has a good market prospect for its various shapes and colors.Lamellae of goldfish and other fishes have plasticity in different dissolved oxygen environments.Under the condition of normoxia,there are embedded cells between lamellaes,and the surface area of gill is small;When the amount of dissolved oxygen in water decreased,the interlamellar cell mass(ILCM)decreased or disappeared,the epithelial cells of gill filaments became thinner,the base of gill lamellae all appeared,and the surface area increased about 7.5 times,so as to improve the absorption ability of gill to dissolved oxygen in water.This process is called gill remodeling,which is thought to be related to cell apoptosis and cell abscission,but its mechanism needs further study.Hypoxia inducible factors(HIFs)are key transcription factors that mediate the response of cells to hypoxia.They regulate the gene expression of cells to adapt to hypoxia conditions by reducing oxygen consumption and increasing oxygen supply.It is speculated that HIF-1 may play an important role in the process of gill remodeling.Adhesion is an E-cadherin mediated intercellular adhesion structure.It not only connects adjacent cells in turn,maintains the normal morphology and polarity of epithelial cells,but also participates in intercellular and intracellular signal transduction.The expression of E-cadherin is regulated by HIF-1 and its target genes twist and snail in mammal cells.In order to explore the role of HIF-1/E-cadherin in the process of gill remodeling in goldfish,this experiment mainly uses fluorescence quantitative PCR,Western blot,immunofluorescence and enzyme activity assay to carry out research,the contents are as follows:1.Analysis of the expression of E-cadherin induced by hypoxia30 healthy goldfish of similar size purchased from local market were divided into 6 groups,which were divided into normal oxygen group(6-8 ppm),normal oxygen+HIF-1α stabilizer dmog group(40 mg/m L),low oxygen group(1.8 ppm),low oxygen+proteasome inhibitor bortezomib group(1 mg/m L),low oxygen+HIF-1 α antisense nucleic acid group(33 mg/m L),and placed in 15℃ water for 8 hours after drug injection.After anesthesia with MS222,the whole gill tissue was dissected and the remaining tissue was stored in liquid nitrogen.After 4% PFA fixation,the gill tissue was dehydrated,dried and coated.It was observed by electron microscope that ILCM cells in gill tissue of goldfish decreased or disappeared after hypoxia treatment for 8 hours or 12 hours.The treatment of HIF-1 α stabilizer dmog could promote ILCM decrease in gill tissue of goldfishin normoxia,while the treatment of bortezomib and HIF-1α antisense nucleic acid could inhibit hypoxia induced-shrink of ILCM,indicating that hypoxia could affect the process of gill remodeling of goldfish through HIF-1.Immunofluorescence,histochemistry and Western blot analysis showed that the E-cad protein levels in gill tissues of goldfish increased with treated with dmog or hypoxia treatment,but there was no significant difference between HIF-1 α antisense nucleic acid group,bortezomib group and hypoxia group.The 2000 bp promoter region of E-cadherin gene was cloned and bioinformatics analysis of the promoter transcription site was carried out.It was found that there may be transcription binding sites such as twist,GCM-1 and hoxb9 in the promoter region sequence of E-cad in goldfish.Meanwhile,hypoxia treatment can improve the expression of snail and E-cad,but has no effect on twist.These results indicate that hypoxia and HIF-1 can promote the expression of E-cad in gill tissue of goldfish,which is different with the widely reported EMT process of cancer cells.2.The effect of E-cad on the apoptosis in gill of goldfishApoptosis is mainly divided into mitochondrial pathway and death receptor pathway.In the mitochondrial pathway,the expression level of Bim and caspase-3 related to apoptosis in goldfish gill is significantly higher than that in other tissues,indicating that goldfish Gill has a high level of mitochondrial apoptosis signal.Among the three subtypes of BIM protein(BimEL,Bim L and Bim S),bortezomib treatment can effectively increase the expression of BimEL and inhibit the hypoxia-induced increase of BimS protein level.Meanwhile,both bortezomib treatment and HIF-1α antisense nucleic acid treatment reduced caspase-3 level in gill tissue of goldfish.In death receptor apoptosis pathway of mammalian cell,E-cad/β-Catenin can combine with death receptor DR4/DR5 to promote cell apoptosis.The expression level of caspase-8 in gill tissue of goldfish was significantly increased by hypoxia,while the expression level of death receptor ligand trail had no significant difference between different treatment groups.We speculated that the expression level of E-cadherin(not ligand trail)affect caspase-8 activity.Bortezomib treatment reduced caspase-8 activity,β-catenin expression and cyclin D-1 mRNA level of β-Catenin target gene in gill tissue of goldfish,indicating that the increase of E-cadherin expression in gill tissue induced by hypoxia may promote the apoptosis pathway of death receptor,and bortezomib can block this process by inhibiting β-Catenin level.In conclusion,hypoxia can induce the expression of E-cad in gill tissue of goldfish through HIF-1,and activate the apoptosis pathway of death receptor in ILCM cells.Bortezomib can inhibit the apoptosis mitochondrial pathway through BIM,and block the apoptosis death receptor pathway through β-Catenin,so as to regulate the process of gill morphological remodeling. |
