Melatonin Ameliorates Developmental Abnormalities And Circadian Rhythm Disturbances In Zebrafish Induced By Low Dose γ Radiation

Objective:As one of the four environmental pollution sources,ionizing irradiation has been paid more and more attention to its effect on biological clock.This study evaluated the protective effects of melatonin on irradiation-induced morphogenesis abnormalities and biorhythm disturbances in zebrafish embryos,and explored the mechanism.From the perspective of biological rhythm,it provides scientific basis for irradiation control.Methods:The wild type zebrafish of AB line were selected as animal model,and the zebrafish were treated with low dose ionizing irradiation of 0.10Gy with bioirradiator at 2.0 hours after the fertilized eggs developed,and1×10^-5mol/L melatonin was added immediately after the irradiation.The biological effects of zebrafish in different groups were observed at different levels.1.Morphological and developmental observation:the gross morphological and developmental observation of the juvenile fish was made under the microscope,and the body length,eye area,spine width and caudal fin length of the zebrafish were measured when the juvenile fish developed to 3 dpf.2.Behavioral monitoring:Zebrafish behavior recorder was used to observe the swimming behavior of juvenile fish when they grew to 6 dpf,and analyze the cumulative swimming distance,swimming time and average speed of 50-hour swimming behavior.Meanwhile,the peak value,time phase and period of swimming behavior are calculated.3.Dopamine detection:the fish dopamine（DA）elisa kit was used to detect the melatonin expression level of zebrafish when they developed to 6 dpf.4.Melatonin detection:fish melatonin（MLT）elisa kit was used to detect the melatonin expression level of zebrafish when they developed to6 dpf.5.Detection of apoptosis level:Acridone orange staining was used to analyze the apoptosis level of zebrafish after development to 3 dpf.6.m RNA expression level detection:RT-qPCR was used to detect the m RNA expression levels of key genes,motion-related genes,melatonin-relatedgenes,apoptosis-relatedgenesand antioxidation-related genes in zebrafish embryos.7.CRY1 protein expression detection:Western blot was used to detect CRY1 protein expression in zebrafish embryos.Results:1.The body length,eye area,spinal width and caudal fin length of zebrafish were analyzed with Image J software.The results showed that the body length,eye area,spinal width and caudal Fin Length of zebrafish in irradiation group were increased compared with those in control group（P<0.05）,and those in irradiation+melatonin group were decreased compared with those in irradiation group（P<0.05）.2.When the young zebrafish developed to 3 dpf,the zebrafish were dyed with acridine orange,and it was found that the fluorescence of zebrafish after low dose ionizing irradiation was brighter and more,and the fluorescence intensity was stronger.Fluorescence decreased and fluorescence intensity weakened after melatonin treatment.3.The results of zebrafish swimming behavior showed that compared with the control group,the cumulative swimming distance,the cumulative swimming time and the average swimming speed of zebrafish in the irradiation group increased（P<0.05）,and the circadian rhythm time-phase Advance,peak value increases and the cycle shortens.However,in the irradiation+melatonin group,these indexes were all decreased（P<0.05）.4.The levels of melatonin and dopamine in zebrafish 6 dpf were detected by enzyme-linked immunosorbent assay（Elisa）.Compared with the control group,the expression of MLT was decreased and the expression of DA was increased in the irradiation group（P<0.05）.MLT expression increased and DA expression decreased in irradiated zebrafish treated with melatonin（P<0.05）.5.RT-qPCR results showed that:（1）the expression levels of bmal1,clock1a,per1a and cry3a were up-regulated in zebrafish exposed to irradiation（P<0.05）,while there was no significant change in the expression levels of cry2 and perb（P>0.05）.Compared with the irradiated group,there were down-regulated expression changes of these genes in zebrafish in the irradiation+melatonin group（P<0.05）.（2）exercise-related genes:compared with the irradiation group,the expression levels of th,mao and dbh in zebrafish in the irradiation group were up-regulated（P<0.05）,and those in the radiation plus melatonin group were down-regulated（P<0.05）.（3）melatonin-related genes:compared with the control group,the melatonin synthesis-related genes aanat1,Tph1a and Asmt2 were up-regulated in both mtnr1ab and mtnr1c expression levels in irradiated zebrafish（P<0.05）,however,the expression level of irradiation+melatonin was significantly lower than that of irradiation-only zebrafish（P<0.05）.（4）apoptotic/anti-apoptotic genes:compared with the control group,the expression levels of Bax,caspase-3,p53,mdm2 and Bcl-2 were increased in the radiation group（P<0.05）,and decreased in the irradiation+melatonin group（P<0.05）.（5）oxidative/antioxidant genes:compared with the control group,the m RNA transcription levels of Gpx1,CAT,and sod2 were up-regulated in the irradiation group（P<0.05）,and down-regulated in the irradiation+melatonin group（P<0.05）.6.The expression of CRY1 protein in zebrafish was detected by Western blot after 6-DPF was randomly collected.The results showed that the expression of CRY1 protein in zebrafish larvae was up-regulated after 0.10 Gyγ-irradiation（P<0.05）.After Melatonin treatment,the expression of CRY1 protein was down-regulated（P<0.05）.Conclusions:1.Low dose ionizing irradiation of 0.10 Gy can promote the growth and development of zebrafish and its swimming behavior,and appropriate exogenous melatonin can effectively reduce the excitatory effect of low dose ionizing irradiation.2.γ-irradiation at 0.10 Gy dose decreased the expression of endogenous melatonin in zebrafish,but the expression of melatonin synthesis rate-limiting enzyme gene and melatonin receptor gene increased,which may be due to the compensatory mechanism.3.Exogenous melatonin can effectively improve the anti-apoptosis and anti-oxidation ability of zebrafish induced by low dose ionizing radiation.4.The circadian rhythm disorder of zebrafish swimming behavior was caused by 0.10 Gy low dose ionizing irradiation and exogenous melatonin can effectively regulate circadian rhythm disorders.