Study On Embryonic Developmental Toxicity Of Zebrafish Induced By Ionizing Radiation And Radiation Protective Effect Of Phenformin

With the wide application of nuclear science and technology in the fields of medicine and health,industry and agriculture,life science and so on,radiation not only brings convenience to human beings,but also increases the risk of exposure to radiation.Ionizing radiation can cause radiation damage to the body,especially to the cardio-cerebrovascular system.Therefore,deepening the understanding of the mechanism of body damage caused by radiation is more helpful to find reasonable protective measures and provide theoretical basis for safe and effective radiation protective agents.In this paper,zebrafish,a new model organism,was used as the research object to explore the effects of different ionizing radiation on embryonic development.at the same time,the molecular mechanism of radiation injury was discussed,and phenformin was used to intervene.The protective effect and possible mechanism of zebrafish embryonic neurodevelopmental toxicity induced by radiation were evaluated.The response effects of zebrafish embryos to different doses of X-ray irradiation were different.Low-dose X-ray irradiation had little effect on the development of zebrafish embryos,but high-dose(more than 4 Gy)radiation significantly increased the mortality and deformity rate of zebrafish embryos,and decreased the ability of autonomous movement and heart rate,which had a significant effect on the development of zebrafish embryos.Irradiation caused a significant increase in ROS content and apoptosis in embryonic cells,and the number of apoptosis increased with the increase of radiation dose.Subsequently,we used RT-qPCR to detect the expression of important genes(bax,bcl-2,caspase-9,caspase-3,p53,puma,Apaf-1,and mdm2)in apoptosis-related signaling pathway,and preliminarily discussed the possible pathway of apoptosis induced by radiation.The results showed that high dose X-ray irradiation significantly increased the expression of pro-apoptotic gene bax,caspase-9,caspase-3,p53 and puma,while decreased the expression of anti-apoptotic gene bcl-2,mdm2.Different radiation types can induce significant differences in gene expression patterns.Next,we used the same 2 Gy physical dose irradiation to explore the molecular mechanism of carbon ion and proton induced developmental toxicity.In addition,we focused on Wnt signal pathway closely related to development and intervened with HLY78,an agonist of Wnt signal pathway,to evaluate the possible role of Wnt signal pathway in radiation-induced developmental toxicity.The results showed that the Wnt signal was significantly down-regulated after carbon ion radiation,and the Wnt signal reactivated by HLY78 could alleviate the radiation-induced developmental toxicity.However,Wnt signal was not significantly down-regulated after proton radiation,while HLY78 had little effect on proton radiation-induced developmental toxicity.These results suggest that HLY78 has a significant protective effect on the developmental toxicity of zebrafish embryos induced by carbon ion radiation,but has no significant effect on the developmental toxicity induced by proton radiation.Therefore,Wnt signal may be the initial molecular pathway involved in carbon ion-induced developmental toxicity rather than proton-induced developmental toxicity.This experimental study evaluated the protective efficacy of the anti-hyperglycemic/anti-neoplastic agent phenformin against radiation-induced developmental toxicity in zebrafish embryos.Zebrafish embryos pretreated with 25 ?M phenformin 1h before x-ray irradiation were compared to irradiation-only embryos for mortality,hatching rate,morphology,spontaneous movement,heart beat,larval swimming,activities of the antioxidant enzymes superoxide dismutase(SOD)and catalase(CAT),malondialdehyde content(MDA,a byproduct of membrane lipid oxidation),and acetylcholinesterase(AChE)activity.In addition,expression levels of multiple genes related to neural development and apoptosis(sod2,bdnf,ache,p53,bax,and bcl-2)were compared by RT-PCR and associated protein expression levels by western blotting.Pretreatment with phenformin increased hatching rate,spontaneous movement,heart beat,and larval motor activity,decreased mortality and malformation rate,increased SOD,CAT,and AChE activities,and reduced MDA compared to irradiation-only embryos.The mRNA expression levels of anti-apoptotic sod2,bdnf,ache,and bcl-2 were enhanced while mRNA expression of p53 and pro-apoptotic bax were reduced in the phenformin pretreatment group.Further,p53,Bax,and ?-H2AX(a biomarker of DNA damage)were downregulated while Bcl-2 and BDNF were upregulated by phenformin pretreatment.Taken together,this study supports the protective efficacy of phenformin against radiation toxicity in zebrafish embryos by suppressing oxidative stress and ensuing apoptosis.