Role Of Autophagy Regulated By PIK3C3/Vps34 Gene In Neurodevelopmental Toxicity Of Zebrafish Larvae Induced By AlNPs

Objective:To elucidate the role of autophagy in the neurodevelopmental toxicity of zebrafish larvae induced by alumina nanoparticles(AlNPs),and to explore the regulatory role of PIK3C3/Vps34 in the neurodevelopmental toxicity induced by AlNPs.Methods:In this study,we used zebrafish embryo-larvae stage as a model.Six hours post fertilization(6hpf)embryos of the same batch were randomly divided into control group and control group,3MA group,AlNPs group(100 mg/L)and AlNPs+3MA group(3MA concentration of 2.5 mmol/L),each group selected 30 zebrafish embryos and added 0.025% DMSO.After 144 hpf of exposure,the morphological changes of embryos and larvae and the general toxicity of larvae were observed,including hatching rate,mortality rate and deformity rate,motor behavioral indicators(motor ability in dark,thigmotaxis,and photophobic response),oxidative stress indicators(fluorescence intensity of reactive oxygen species,activities of superoxide dismutase and lactate dehydrogenase),and Beclin1,LC3? and Vps34 genes expression changes.In addition,in order to elucidate the physiological function of PIK3C3/Vps34 and determine its regulatory role in autophagy,we used antisense morpholino oligonucleotide(MO)to block the expression of PIK3C3/Vps34 gene in zebrafish embryos.The embryos fertilized with 6hpf were randomly divided into control group,negative control group,Vps34 gene knockdown group(Vps34 MO concentration was 1 mmol/L),AlNPs group,AlNPs+Vps34 gene knockdown group to observe the effects of Vps34 gene on neural behavior,oxidative damage and autophagy related genes of zebrafish larvae induced by AlNPs.Moreover,Nissl staining was used to detect the changes in the number of nerve cells in the brain of each group.Results:(1)Morphological changes of zebrafish embryos and larvae: the embryos in AlNPs group developed slowly at 12 hpf and 24 hpf,showing no obvious somite formation and tail extension,however,the development of embryos in AlNPs group improved at24 hpf after adding 3MA,which showed obvious tail extension,and no edema or pyknosis.There was no significant difference in mortality,hatching rate and deformity rate among the groups.(2)Movement behavior of zebrafish juveniles: compared with control group,3MA group and AlNPs+3MA group,the average speed,moving distance and tactile degree of AlNPs group were decreased(P<0.05).In the panic escape reflex experiment under strong light stimulation,the speed of each group was decreased in the light period,while the speed of AlNPs group decreased more quickly under light(P<0.05).(3)Detection of oxidative stress level of zebrafish larvae: SOD activity in AlNPs group was lower than that in control group,3MA group and AlNPs+3MA group(P<0.05),while ROS fluorescence intensity and LDH activity in AlNPs group were higher than those in control group,3MA group and AlNPs+3MA group(P<0.05).(4)Detection of autophagy related genes in zebrafish larvae: the gene expression of Beclin1,LC3? and Vps34 increased in AlNPs group(P<0.05),but decreased after adding 3MA(P<0.05)(5)After microinjection of zebrafish embryos,the movement behavior of larvae were detected: the average speed,moving distance,and tactile degree of larvae in AlNPs group were lower than those in control group,negative control group,and AlNPs+Vps34 group(P<0.05);Compared with the control group and negative control group,the average speed and moving distance of Vps34 group were decreased(P<0.05),while tactile degree were increased(P<0.05)compared with AlNPs group;Compared with the control group and negative control group,the average speed and moving distance of AlNPs+Vps34 group were decreased(P<0.05),but the tactile degree had no significant difference(P>0.05).(6)The oxidative stress level of zebrafish larvae after microinjection of embryos:compared with control group,negative control group and AlNPs+Vps34 group,SOD activity in AlNPs group and Vps34 group decreased(P<0.05),while LDH activity increased(P<0.05);compared with Vps34 group,SOD activity in AlNPs group decreased(P<0.05),while LDH activity increased(P<0.05).(7)After microinjection of zebrafish embryos,the number of nerve cells in the brain of larvae were detected: compared with the control group,negative control group and AlNPs+Vps34 group,the number of nerve cells in AlNPs group and Vps34 group decreased(P<0.05);compared with Vps34 group,the number of nerve cells in AlNPs group decreased(P<0.05).(8)Detection of autophagy-related genes in zebrafish embryos after microinjection:compared with the control group and negative control group,the expression of Vps34 gene in the microinjection group was down regulated(P<0.05);The expression of ULK1,Beclin1,LC3? and PARK2 in the AlNPs group were higher than that of control group,negative control group,and Vps34 group(P<0.05),however,when Vps34 gene was knocked down,the expression of these genes in AlNPs group were decreased(P<0.05);The levels of BCL2L1 and VDAC1 genes in AlNPs group were lower than those in control group,negative control group,Vps34 group,and AlNPs+Vps34 group(P<0.05),while the expression of these genes in AlNPs+Vps34group were higher than that in control group and negative control group(P<0.05).In addition,compared with control group and negative control group,the expression of Beclin1,LC3? and PARK2 genes in Vps34 group was decreased,ULK1,BCL2L1,and VDAC1 were up-regulated(P<0.05).Conclusion:(1)AlNPs induced excessive autophagy caused early neurodevelopmental toxicity in larval zebrafish,while 3MA can reduce oxidative damage caused by AlNPs and down-regulate the expression of autophagy-related genes,and significantly improve the early neurodevelopmental toxicity caused by AlNPs exposure.(2)PIK3C3/Vps34 gene plays an important role in regulating autophagy.(3)PIK3C3/Vps34 plays a key regulatory role in AlNPs induced excessive autophagy in zebrafish larvae,which is essential for normal neural function.