The Study Of PI3K-nos2b Involved In Zebrafish Embryo Vascular Development Regulated By Simulated Microgravity

With the rapid development of the world's aerospace projects, it is longer and longer that human beings stay in outer space. In recent years, studies have shown that such environmental changes can cause local abnormal angiogenesis and cardiovascular system reconstruction. Astronauts were under microgravity environment for a long time, ultimately leading to a variety of cardiovascular diseases. Microgravity can regulate cell morphological structure and function and affect the cardiovascular system development. Over the years, more and more researchers begin to focus on the effects of microgravity on cardiovascular system development and have reported some signal regulation pathways successively. However, what key molecular signals and pathway were changed to affect cardiovascular development under microgravity environment and related mechanobiology mechanism has not been fully elucidated in any study till now.The present study used the MG-IIA type ground microgravity effect simulation system manufactured by the Chinese academy of sciences institute of mechanics. Transgenic zebrafish embryos(Flk1: GFP) with green fluorescent markers of vascular endothelial cells were used as the research model of response to the ground simulated microgravity.Then simulated microgravity(simulated microgravity, SM) treatment through the rotation of zebrafish embryos. Observe the effects of simulated microgravity on zebrafish vascular development in vivo real-timely and its molecular mechanism carries on the preliminary exploration.The following are the main research contents and results of the present paper:(1)The influence of simulated microgravity on zebrafish embryonic vascular development.The embryos that were post-fertilization to 24 hours and 36 hours, and treated by SM from 12 hpf to 24 hpf and 36 hpf were collected. The control group was cultured under the condition of 1g normally and SM group was cultured in MG-IIA type bioreactor.The SM group, control group's conditions of the zebrafish embryos phenotypic changes and the vascular development by microscope was observed The experimental results show that the SM has no effect on the survival rate of zebrafish, but reduces the heart rate significantly. And treated to 36 hpf in microgravity significantly influenced local abnormal vascular development of zebrafish embryos, promoted the development of caudal vein plexus, angiogenesis and reconstruction. The vascular network complexity was improved. This indicates that SM has an effect on development of zebrafish blood vessels.(2) nos2 b participates in simulated microgravity on vascular development of zebrafish embryos.At first, the NO content in the total proteins of zebrafish embryos was measured in SM treatment of 36 hpf.The results show that the NO content in the total proteins of zebrafish embryos in SM treatment was significantly increased relative to the control group. It can be preliminarily ascertained that NO signal was involved in microgravity on vascular development of zebrafish embryos. Then inject nos2 b MO and inject nos2 b MO with microgravity respectively and observed vascular development in 36 hpf. Experimental results shown that when nos2 b gene was knocked down, the abnormalities by SM could be rescue, indicating that nos2 b was involved in SM regulated zebrafish early vascular development process.(3)PI3K-nos2 b signaling pathways was involved in the simulated microgravity on vascular development of zebrafish embryos.1)PI3K was involved in zebrafish early vascular development. In the experiment, we used the specific PI3 K inhibitors LY294002 to treat zebrafish embryos to 36 hpf. Results show that the development of treated zebrafish embryos with inhibitor was significantly suppressed compared with the control group, internode vascular was disordered, tail vein plexus had burst.2)PI3K was involved in SM on zebrafish vascular development. The experiment is divided into three groups: control group, SM treated to 36 hpf, SM treated to 36 hpf with the specific PI3 K inhibitors LY294002. From the results of the phenotype, qPCR and whole embryo in situ hybridization, it showed that zebrafish embryonic vascular development abnormally treated with SM could be partly rescued by PI3 K inhibitor. This demonstrates that the PI3 K really was involved in SM on zebrafish vascular development process.3)PI3K-nos2 b signaling pathways was involved in zebrafish vascular development by SM. Respectively inject nos2 b mRNA and inject nos2 b mRNA with the specific PI3 K inhibitors LY294002.From the results of the phenotype, q PCR and whole embryo in situ hybridization, it showed that vascular development treated with LY294002 can be inhibited and nos2 b mRNA could be partly rescue this phenomenon.In conclusion, this study shows that SM can make zebrafish vascular reconstruct and enhance angiogenesis. On the other hand, PI3K-nos2 b signaling pathway was also involved in SM on zebrafish vascular development. This study not only preliminary illustrates the molecular mechanism of vascular development regulated by simulated microgravity, but also provide a new therapeutic targets for cardiovascular disease caused by microgravity.