| Objective:To study the developmental toxicity and cardiotoxicity of propofol exposure to zebrafish juvenilesMethods:In this study,zebrafish embryos were exposed to different concentrations(0.05,0.1,0.5,1,5,10,and 20 μg/ml)of propofol,and the hatching rate(48,72 and 96hpfl)was measured at each time point.96hpf),mortality(24,48,72,96 and 120 hpf),deformity rate(24,48,72,96 and 120 hpf),heart rate(24,48,72 and 96hpf),etc.,paraffin sections to observe different concentrations propofol exposure group,the incidence of pericardial edema in zebrafish juveniles at different time points(72,96 and 120 hpf).Samples were collected at 120 hpf,and detected by in situ hybridization experiment and quantitative reverse transcription polymerase chain reaction(qRT-PCR)to evaluate the effect of propofol on the expression of genes related to heart development and function in juvenile zebrafish.Results:Exposure to propofol reduced the survival rate and embryonic hatching rate of zebrafish embryo.In the 0.5 μg/ml treatment group,the mortality rate increased significantly from 96 hpf(p<0.01),while in the 1,5,10,and 20μg/ml treatment groups,the mortality rate increased significantly from 24 hpf;The mortality of the 1 μg/ml and 5μg/ml treatment groups increased with development time;in the 10μg/ml and 20 μg/ml treatment groups,almost all embryos died at 24 hpf.In addition,in the 1 μg/ml and 5 μg/ml propofol treatment groups,the rate of embryo deformity increased in a concentration-dependent manner,and the hatching rate decreased in a concentration-dependent manner.Observe the different types of malformations after propofol administration.The proportion of pericardial cysts increases with the increase of concentration and the prolonged exposure time,and the heart rate decreases with the increase of propofol concentration.The qRT-PCR method was used to detect the effect of propofol on the expression of genes related to heart development and function Compared with the control group,the propofol group functional genes atp2b3b,atp2b2,kcnj8 were up-regulated,and atp2a1l and nppa were down-regulated,which were related to heart development.Among the expressed genes,compared with the control group,vmhc,cmlc,myl4 and myh6 in the propofol group were all up-regulated to varying degrees,and the myl2b gene was down-regulated.Conclusion:The results of the study indicate that propofol exposure will reduce the survival rate and embryonic hatching rate of zebrafish,and produce significant toxicity to the development and heart of zebrafish.Propofol exposure can change the expression of genes related to heart development and function.Objective:To explore the signaling pathways of propofol-induced heart development defects in zebrafish embryosMethods:In this study,transgenic zebrafish Tg(cmlc:EGFP)and wild AB zebrafish expressing enhanced green fluorescent protein(EGFP)in the heart were selected.Male zebrafish and female zebrafish were paired,8 hours after fertilization,Transgenic zebrafish Tg(cmlc:EGFP)and wild AB zebrafish were treated with 0.5,1,and 5 mg/L propofol and DMSO was used respectively,paraffin tissue sections and HE staining to observethe heart morphology and heart structure changes in each group,and the transcriptome sequenced Methods To analyze the changes of zebrafish heart transcriptome after exposure to 5mg/L propofol,and use qRT-PCR to verify changes in cardiac development-related and Wnt and Notch signaling pathway-related gene expression.Results:Propofol exposure can cause obvious cardiac development abnormalities in the embryonic heart of Tg(cmlc:EGFP)transgenic zebrafish,including prolonged pericardium,increased pericardial cysts,and significant enlargement of the atrioventricular space.The zebrafish embryos in the propofol treatment group had reduced atrial and ventricular overlap capacity and reduced cyclization,especially in the high-concentration treatment group(5μg/ml).The results of paraffin section and HE staining showed that as the concentration of propofol exposure increased,the myocardial wall became thicker,the atrium and ventricular cavity decreased,and the heart structure was damaged.These damages included morphological abnormalities and structural abnormalities;and they were obvious dose dependent.Compared with the control group,treatment of zebrafish embryos with different concentrations of propofol(0.5μg/mL,1μg/ml and,5μg/ml)can lead to obvious cardiac developmental abnormalities,with a higher incidence of pericardial cysts.Zebrafish heart transcriptome sequencing results after exposure to 5μg/ml propofol showed that propofol exposure affected many signal pathways related to cardiac development,among which Notch and Wnt signal pathways were significantly enriched as key signal pathways for cardiac development;Combining qRT-PCR to detect the key molecules of Notch and Wnt signaling channels and transcriptome sequencing results suggest that compared with the control group,the expression of myl2b,atp2a1l,nppa,notch1a and wnt2 in the experimental group was significantly reduced(p<0.01),and the expression of cacalda and wnt6a Significantly increased(p<0.01).Conclusion:Propofol exposure may cause damage to zebrafish heart development by interfering with Notch and Wnt signaling pathways.Objective:To investigate the effect of propofol on heart rate by affecting zebrafish sv2a expression and GABAergic neuron activityMethod:Treat zebrafish embryos with 1 μg/ml propofol 8 hours after fertilization,treat zebrafish with GABA receptor inhibitor(primdone)and vesicle secretion inhibitor(levetiracetam)at 48hpf,and observe Heart rate changes at72,96,and 120hpf.QRT-PCR and ELISA were used to detect whether propofol exposure interfered with the expression of genes related to GABA metabolism.Use crispant technology to knock out the zebrafish sv2a gene(sv2a KO),measure the heart rate changes of zebrafish juveniles,use GABA receptor inhibitor(primdone)and vesicle secretion inhibitor(levetiracetam)to sv2a KO mutant Perform treatment to verify whether propofol inhibits zebrafish heart rate through sv2a and GABA.Conclusion:The Zebrafish embryos exposured to 1μg/ml propofol were treated with GABAreceptor inhibitor(Prindone)and vesicle secretion inhibitor(Levetiracetam)at 48hpf.The results showed that at 96 and 120hpf,Both Prindone and levetiracetam can significantly improve the decrease in heart rate caused by propofol exposure(p<0.01),indicating that the secretion of GABA rate-limiting enzymes and vesicles are involved in the process of propofol exposure to reduce the heart rate of zebrafish embryos.Zebrafish embryos were treated with propofol(1 μg/ml),and samples were collected at 96hpf.The results showed that the expression of the rate-limiting enzymes gad1a and gad2 synthesized by GABA increased significantly(p<0.001),and the expression of gadlb did not change significantly;The expression of contact vesicle protein sv2a increased(p<0.01);GABA receptors did not change significantly,and the GABA concentration increased after propofol treatment(p<0.01).At 48,72 and 96hpf,the heart rate of the sv2aKO mutant was significantly higher than that of the wild type.Knockout of sv2a can reduce the GABA level(p<0.01);Inject the sv2aKO mutant mRNA into zebrafish single-cell fertilized eggs,compared with the treatment group exposed to propofol,the heart rate can be significantly increased(p<0.01).Conclusion:Propofol reduces heart rate of zebrafish by enhancing sv2A gene expression and promoting GABA release... |
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