| Objective:To explore the roles and possible mechanisms of nr1d1 in the autism-like behavior of zebrafish larvae induced by BPA exposure.Methods:1.The effect of BPA exposure on the expression and rhythm of nr1d1.Zebrafish embryos were placed under different treatment conditions and cultured to 72 hpf,and continued to be cultured in the systematic water to 96 hpf;after 96 hpf,samples from each treatment group were collected at CT 4,CT 10,CT 16,and CT 22 respectively(after 72 hpf,keep avoiding Light),using quantitative PCR(q-PCR)to detect the transcription level and rhythmic oscillation of the zebrafish clock gene nr1d1.2.Zebrafish larvae autism-like behavior experimentPut zebrafish embryos under different treatment conditions,culture them to 72 hpf,and culture them in the systematic water to 13 dpf to conduct social behavior and tactile behavior experiments respectively.After 10 minutes of adaptation,changes in the autismlike behavior of zebrafish larvae in different treatment groups were evaluated under continuous 1h light conditions.3.Detection of autism-related genes in zebrafish larvaeZebrafish embryos were placed under different treatment conditions and cultured to 72 hpf,and continued to be cultured in the systematic water to 96 hpf;after 96 hpf,samples from each treatment group were collected at CT 4,CT 10,CT 16,and CT 22 respectively(after 72 hpf,keep avoiding Light),and q-PCR was used to detect zebrafish autism-related genes(α-nrxn2a,β-nrxn2a)transcription and rhythmic oscillation.4.Detection of indicators related to redox balanceZebrafish embryos were placed under different treatment conditions and cultured to 72 hpf,and continued to be cultured in the systematic water to 96 hpf;after 96 hpf,samples from each treatment group were collected at CT 4,CT 10,CT 16,and CT 22 respectively(after 72 hpf,keep avoiding Light),q-PCR was used to detect the transcription and rhythm of the key genes(nrf2,gpx,gst,sod1,sod2)in the antioxidant system;the enzyme-linked immunosorbent assay(ELISA)was used to detect the antioxidative activity of different treatment groups in zebrafish.Oxidation-related enzymes(GPx,GST,SOD)content and rhythmic oscillation.5.Detection of inflammatory indicators in zebrafishZebrafish embryos were placed under different treatment conditions and cultured to 72 hpf,and continued to be cultured in the systematic water to 96 hpf;after 96 hpf,samples from each treatment group were collected at CT 4,CT 10,CT 16,and CT 22 respectively(after 72 hpf,keep avoiding Light).The transcription and rhythm of interleukin 1β(il-1β)were detected by q-PCR;the recruitment and migration of neutrophils were observed by Tg(mpo:EGFP)transgenic zebrafish.6.Detection of zebrafish astrocyte activationThe Transgenic zebrafish Tg(gfap:EGFP)embryos were placed under different treatment conditions and cultured to 72 hpf,and continued to be cultured in the systematic water to 96 hpf;after 96 hpf,the effects of different treatment groups on the activation of zebrafish astrocytes were observed at CT 4,CT 10,CT 16,and CT 22(after 72 hpf,keep avoiding Light),and the fluorescence intensity was semi-quantified using Image J software.7.E/I balance related index detectionThe Transgenic zebrafish Tg(dlx5a/dlx6a:EGFP)embryos were placed under different treatment conditions and cultured to 72 hpf,and continued to be cultured in the systematic water to 96 hpf;after 96 hpf,the effects of different treatment groups on the activation of GABA neurons were detected at CT 10 and CT 22(after 72 hpf,keep avoiding Light),and the fluorescence intensity was semi-quantified using Image J software.Zebrafish embryos were placed under different treatment conditions and cultured to 72 hpf,and continued to be cultured in the systematic water to 96 hpf;after 96 hpf,samples from each treatment group were collected at CT 4,CT 10,CT 16,and CT 22 respectively(after 72 hpf,keep avoiding Light),the transcription and rhythmic oscillation of key genes(gad1b,glsb,gls2a,gls2b)in the process of glutamate and GABA synthesis were detected by q-PCR,and the contents of glutamate and GABA were detected in CT 10 and CT 22.Results:1.Changes in the circadian rhythm gene nr1d1 in zebrafish:It was found that BP A can disrupt the rhythm of nrldl,and the transcription decreased significantly.The nr1d1 agonist GSK4112 can restore its rhythm and transcription.2.In the autism-like behavior of zebrafish,zebrafish larvae in the BPA group developed autism-like behavior,manifested as social deficits and reduced responsiveness to tactile stimuli,and this effect was particularly evident at night.The nr1d1 agonist GSK4112 can alleviate the autism-like behavior induced by BPA.3.Changes in autism-related genes in zebrafish:It was found that BPA could disrupt the rhythm of the autism-related genes α-nrxn2a and β-nrxn2a in zebrafish larvae and significantly increase the transcription.The changes in the transcription of autismrelated genes and the disruption of rhythm returned to normal levels.4.Redox balance and rhythm changes in zebrafish:It was found that BPA can lead to a significant increase in ROS levels and a change in rhythm,and the transcription of nrf2,gpx,gst,sod1,and sod2 in the antioxidant system were significantly reduced and the rhythm was disturbed.Through ELISA detection,it was found that the rhythms of antioxidant-related enzymes GPx,GST and SOD were disrupted and their contents decreased significantly.Nr1d1 agonist GSK4112 can restore the redox imbalance and rhythm disorder caused by BPA.5.Changes in inflammatory response in zebrafish:By using Tg(mpo:EGFP)transgenic zebrafish,it was found that BPA can lead to increased recruitment and migration of neutrophils and disordered rhythm,and using q-PCR,it was found that the rhythm of il-1β transcription was disrupted and the amount of transcription was significantly increased.The nr1d1 agonist GSK4112 can restore the inflammatory response and rhythm disruption induced by BPA.6.Zebrafish astrocyte activation:BPA exposure significantly enhanced the fluorescence intensity of transgenic zebrafish Tg(gfap:EGFP),which proved that astrocyte activation was significant and neuroinflammation occurred.The nr1d1 agonist GSK4112 can restore BPA-induced activation of astrocytes and disruption of their rhythms.7.Changes in the E/I balance of zebrafish:BPA exposure disrupted the release of glutamate and GABA,the activation of GABA neurons,and the transcription of related genes,resulting in an imbalance of E/I in zebrafish.The nr1d1 agonist GSK4112 restores BPA-induced E/I imbalance.Conclusion:This experiment found that exposure to BPA at the embryonic stage of zebrafish can lead to autism-like behavior in larvae.BPA interferes with the transcription level and rhythmic oscillations of the clock gene nr1d1,on the one hand,the redox rhythm is unbalanced,the inflammatory factor il-1β is released in large quantities,and astrocytes are activated,thereby inducing the inflammatory response in the zebrafish brain;on the other hand,it leads to the imbalance of excitation-inhibition in the nervous system.By disrupting the rhythmic oscillations of these biological processes,it ultimately leads to autism-like behavior in zebrafish larvae. |
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