Apoptotic Mechanisms Of Propofol-Induced Developmental Toxicity In Zebrafish Embryos

Objective:General anesthetics during early pregnancy can cause neurological damage and long-term behavioral/cognitive impairment.However,the adverse influence on embryo development induced by propofol is unclear.We used embryonic zebrafish to explore the effects of propofol on embryonic and larval growth and development,and the related apoptotic mechanism.Methods:20 healthy adult zebrafish(10 males and 10 females)aged 3-4 months were provided by the National Zebrafish Resource Center.The fish were paired and fertilized eggs were collected.At 6 hours post-fertilization(6 hpf),normal developing embryos were selected and randomly divided into a control group and different concentrations of propofol groups,with 50 embryos in each group.The control group embryos were placed in the culture dish containing E3 medium and incubated at 28.5℃ in a biochemical incubator.The propofol group embryos were placed in a culture dish containing E3 medium,with dimethyl sulfoxide(DMSO)added to a final concentration of 0.014%,and propofol solution added to achieve a final concentration of 1,2,3,4,or 5 μg/mL.The embryos were then incubated at 28.5℃ in a biochemical incubator,and at 48 hpf the medium was replaced with propofol-free E3 medium.At 24 hpf,spontaneous tail movements of zebrafish were observed under a stereomicroscope,and at 48 hpf,their response to stimulation was observed.The survival rates of zebrafish in each group were recorded from 24 to 96 hpf,and the most suitable concentration of propofol was determined.The body length,malformation and injury rates,and heart rate of zebrafish in the control and optimal propofol concentration groups from 24 to 96 hpf were observed.The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)was used to detect cell apoptosis in zebrafish in both groups.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect mRNA expression of 18 apoptosis-related genes(casp3a,casp3b,casp6,casp7,casp8,casp9,tp53,baxa,baxb,bida,bcl2,bcl2l1,mcl-1a,mcl-1b,fadd,pmaip1,bbc3,and cflara)in zebrafish,with actbl and ef1 as reference genes.Whole-mount in situ hybridization(WISH)was used to detect the spatial expression of casp3a,casp3b,casp9,and baxb in 48 hpf zebrafish larvae.Results:The survival rate of embryos in the control group and the 1 μg/mL and 2μg/mL propofol groups did not change significantly.However,a significant dose-dependent decrease in survival rate was observed from 24 hpf in the 3 μg/mL,4 μg/mL,and 5 μg/mL propofol groups(P<0.05).The spontaneous movement of embryos in the propofol groups was completely inhibited during this stage,while the control group embryos exhibited spontaneous movement 2-5 times per minute at 24 hpf.Embryos in the 2 μg/mL propofol group hatched significantly later at 72 hpf(P<0.05),and from 24 to 96 hpf,the heart rate of zebrafish in the 2 μg/mL propofol group was significantly lower than that in the control group,with higher rates of deformities and damage and delayed body length development(P<0.05).The main deformities and tissue damage observed in the zebrafish in the 2μg/mL propofol group were poor development of the tail fin,superficial pigmentation,edema,bleeding,and spinal deformities.The number of apoptotic cells in 12 hpf,48 hpf,and 72 hpf embryos treated with propofol increased significantly,with upregulated mRNA expression levels of casp3a,casp3b,casp9,and baxb genes related to the intrinsic apoptotic pathway mainly in the head and tail regions of zebrafish.qRT-PCR revealed that 48 hpf was the most significant stage affected by zebrafish apoptotic genes,and WISH also detected a significant increase in the expression of four apoptotic genes in the head and tail regions of 48 hpf embryos in the 2 μg/mL propofol group.Conclusions:Our study found that zebrafish embryos exposed to propofol exhibited significant growth retardation and organ damage,which may be related to abnormal expression of casp3a,casp3b,casp9,and baxb genes in the propofol-induced intrinsic apoptosis pathway.However,the effects and mechanisms of anesthetics on development require further investigation.This zebrafish model enables us to comprehensively and meticulously explore the effects of propofol-induced developmental toxicity on cell apoptosis and further develop potential protective strategies against general anesthesia toxicity.The study of the mechanism of propofol-induced zebrafish cell apoptosis during development is expected to provide valuable evidence for exploring the effects of anesthetics on children during non-obstetric and neonatal surgery,and improve the clinical safety of anesthesia-related procedures.