Molecular Mechanism And Significance Of Sonic Hedgehog Signaling Pathway In Retinal Dysplasia Caused By PCB Exposure

In recent years,with the increasing of environmental pollution,the occurrence of children's eye diseased has gradually increased,which caught more and more attention.Polychlorinated biphenyls?polychlorinated biphenyls,PCBs?are one of the most common environmental pollutants,with high toxicity,semi-volatility and bioaccumulation characteristics.It has been reported that PCBshave a serious influence on the development of children's eyes,especially retinal development.Children retinal development is closely related to Shh?sonic hedgehog?signaling pathway.It can affect the differentiation of retinal progenitor cells,arrange the photoreceptor cells and detemine retinal development layer structure.In this study,we started with the Shh signaling pathway which was closely related to children's retinal development to explore the mechanism of the abnormal retinal development caused by PCBs exposure.Therefore,the retinal developmentmodel animal — — zebrafishwas selectedfirstly.zebrafish visual behavior?optomotor response?and Shh signaling pathway related gene expressions after PCBs exposure were then studied.Finally,the RGC-5 cell line were used to verify the role of Shhsignal pathway in the abnormal retinal development caused by PCBs exposure.Part?: Exposure to PCB₁₂₅₄ caused abnormal OMR in the zebrafish larvaeObjective: To explore the zebrafish larvae optomotor response?OMR?characteristics and the optimal experimental conditions,in order to build an experimental platform of zebrafish larvae OMR and study the OMR of zebrafish larvae after exposed to PCB₁₂₅₄ for 7 days after fertilization?dpf?.Methods: A zebrafish larvae OMR device was established to form continuous visual stimuli produced by black and white bars moving flatly at a constant speed.The behavioral characteristics of zebrafish larvae were accordingly observed.Furthermore,the optimal experimental conditions were confirmed by watching the larval behaviors under different spatial frequency or moving speed.Then,zebrafish were exposed to PCB₁₂₅₄ at concentrations of 0.125,0.25,0.5 and 1 mg/L.A blank group and 0.01% methanol group were set up.After exposed for 7 days,the visual behavior of zebrafish was assessed using optomotor response?OMR?tests.Results: When the grating was moving,the zebrafish larvae performed four behavior responses: positive swimming,opposite swimming,immobility,and hesitant swimming.Only a positive swimming indicated a sensitive response.When the grating speed was set to 25.0 cm/s,the proportion of positively swimming fish increased initially and then decreased as grating spatial frequency was progressively increased to 0.040,0.050,0.100,0.200,and 0.800 LP/mm,respectively;and there was a peak value?73.0%?at 0.200 LP/mm.When the spatial frequency was 0.200 LP/mm,the proportion of positively swimming fish increased initially and then decreased as the speed was progressively increased to 12.5/25.0/50.0/75.0 cm/s,and a peak value?72.5%?was observed at 25.0 cm/s.At this situation,the OMR sensitive ratio decreased significantly in 0.5 mg/L and 1 mg/L group?P< 0.05?.Conclusion: Zebrafish larvae had their unique OMR behavior features.The optimal grating conditions for zebrafish larvae OMR test are spatial frequency of 0.200 LP/mm and speed of 25.0 cm/s.Besides,exposed to 0.5 mg/L and 1 mg/L concentrations of PCB₁₂₅₄ can cause abnormal OMR indicating that it is necessary to strengthen the monitoring and control of adverse influences of environmental toxin on the children eyes development.Part ?:Effects of PCB₁₂₅₄ exposure on the expression of Shh signaling pathway related gene expressionsObjective: To study the changes of Shh sigling pathway related gene expressions after PCB₁₂₅₄ exposure in zebrafish.Methods: Zebrafish embryos were separately exposed to 1.0,0.5,0.25,0.125mg/L PCB₁₂₅₄,0.01% methanol?as a control for the PCBs cosolvent?and blank control.Embryos were assessed at 4 dpf and 7cdpf for changes of the Shh signaling pathway related gene expressions by RT-PCR.The expressions of Shha and ptch1 protein were detected by immunofluorescence and Western blotting.Results: RT-PCR showed that: 1)4 dpf,the expression of Shha m RNA declined obviously in the PCB₁₂₅₄-treated larvae at concentrations of 0.5 mg/L and 1 mg/L compared with their controls?p<0.05?,ptch2 m RNA expresions increased obviously in these larvae compared with their controls?p<0.05?.2)7dpf,the expression of Shha,Shhband gli1 m RNA declined obviously in the 0.5 mg/L and 1 mg/L PCB₁₂₅₄-treated larvae compared with their controls?p<0.05?.The expression ofgli2 m RNA declined obviously in the PCB₁₂₅₄-treated larvae at concentrations of1 mg/L compared with their controls(p<0.05,while ptch2 m RNA expresions increased obviously in the 0.5 mg/L and 1 mg/L PCB₁₂₅₄-treated larvae compared with their controls?p<0.05?.Immunofluorescence showed that Shha and ptch2 protein mainly expressed in photoreceptor cells.1)4 dpf,the expression of Shha protein decrease slight with the concentrations of PCB₁₂₅₄ increasing,while the expression of ptch2 increase slight accordingly.2)7dpf,the expression of Shha protein decreased slight at 0.125 mg/L and 0.25 mg/L PCB₁₂₅₄ exposure and obviously at concentrations of 0.5-1.0 mg/L. However,ptch2 protein expression increased in these groups.Conclusion:Exposure to PCB₁₂₅₄ inhibited the Shh signaling pathway,suggesting that it may be the possible mechanism of PCBsexposure on retinal damage.Part III: Effect of PCB₁₂₅₄ exposure on the Proliferation,Differentiation,Apotosis and Shh Signaling Pathway of RGC-5 cellsObjective: To explore the effect of PCB₁₂₅₄ exposure on the proliferation,differentiation and apotosis and Shh signaling pathway of RGC-5cells in vitro.Methods:The RGC-5 cells were exposed to different concentrations of PCB₁₂₅₄,respectively.A cell viability CCK-8 assay was used to assess the proliferation of cells.The distributions of cell cycle were analysed by flow cytometry.The cells apotosis were measured by caspase-3 activity.We performed RT-PCR to evaluate the relative expression of Shh signaling pathway related gene.Shhsignaling pathway-related proteins were detected by Western blot and immunofluorescence.Results: 1.Compared with controls,PCBs exposure inhibited the proliferation and cell cycles in S-phase.The apototsis of RGC-5 cell after PCBs exposure was significantly increased.2.RT-PCR: 24 h,the expression of Shha,gli1,gli2 m RNA declined obviously in the PCB₁₂₅₄-treated larvae at concentrations of 0.125mg/L,0.25 mg/L,0.5 mg/L and 1 mg/L compared with their controls?p<0.05?,ptch2 m RNA expresions increased obviously in 1mg/L treated larvae compared with their controls?p<0.05?.48 h,the expression of Shham RNA declined obviously in the 0.25mg/L,0.5 mg/L and 1 mg/L PCB-treated larvae compared with their controls?p<0.05?.The expression of gli1,gli2 m RNA declined obviously in the PCB₁₂₅₄-treated larvae at concentrations of 0.125mg/L,0.25mg/L,0.5mg/L and 1 mg/L compared with their controls?p<0.05?,while ptch2 m RNA expresions increased obviously in the 1 mg/L PCB₁₂₅₄-treated larvae compared with their controls?p<0.05?.3.Immunofluorescence and western-blot showed that the expression of Shha protein decreased obviously at concentrations of 0.25mg/L,0.5mg/L and 1mg/L compared with their controls.However,ptch2 protein expression increased in 1 mg/L PCB₁₂₅₄-treated larvae.The expression of gli1,gli2 protein decrease obviouslyin all PCB₁₂₅₄-treated larvaeConclusion: PCB₁₂₅₄ exposure might damage the development of retina by Shh signaling pathway,which provided evidence to prevent the eye diseases caused by environmental pollutants.