| Paralytic shellfish toxins(PSTs)are neurotoxins that exist in various seafood shellfish tissues.Saxitoxin(STX)is one of the most toxic poisons in PSTs.Excessive consumption of STX will cause toxic hazards of nerve paralysis,with high mortality and no specific antidote for it.The acute toxic mechanism of STX has been relatively clear,it mainly inhibits nerve conduction by affecting Na+ channels.However,its chronic and subchronic toxicity is still not clear.In the early stage,combined with the dietary consumption survey of Shenzhen population,the research group conducted a dietary exposure risk assessment of PSTs for Shenzhen residents,and found that Shenzhen residents have the risk of acute poisoning and may be exposed to low-dose PSTs for a long time.To provide clues for the mechanism of chronic neurotoxicity of STX,Quantitative proteomics technology was used to screen the differential protein expression profile of hippocampus of mice was exposed by subchronic low-dose STX,and further to explore the role of related signal pathways at the cellular level.This study was conducted in regard to the following three parts.1.Establishment and evaluation of an animal model of cognitive impairment in C57BL/6NJ mice induced by chronic and low-dose exposure to STXMethods:Six-month old C57BL/6NJ mice were randomly divided into four groups,each consisting of 16 mice.The group of STX alone was treated with 1.5μg/kg of STX through drinking water for periods of 3 and 6 months,with no chemical in the drinking water provided to the corresponding control groups.After the termination of chemical exposure,the learning,memory and cognitive functions of mice were evaluated by the tests of electric jump platform,new object recognition and Morris water maze test.The pathological changes in the brain tissue from some mice were evaluated following HE staining,and the expression of phosphorylated tau protein in hippocampus was determined at different exposure durations.Results:The short-term and long-term memory and cognitive function in the mice subjected to subchronic and chronic STX expousre were decreased.Pathological examination showed that STX caused disordered arrangement and irregular shape of pyramidal cells in hippocampal CA1 subregion,where the number of neurons was decreased.The expression of phosphotylated tau protein in hippocampal tissue in each STX group was higher than that in its control group,with more intensive changes in 6-month STX than in 3-month STX group(P<0.05).Conclusion:The animal model of cognitive impairment in C57BL/6NJ mice induced by STX at a low-dose under chronic and subchronic was successfully established.2.Proteomics of hippocampus in mice with the cognitive impairment following low-dose and subchronic exposure to STXMethods:Proteins were extracted from the hippocampus of mice exposed to STX for 3 months,then the differentially expressed proteins were revealed by quantitative proteomics,and further verified by Western blot assay.Results:A total of 5225 proteins were identified in the two groups,of which 2437 proteins were intersected between 6 exposure/control pairs.The differentially expressed proteins were screened according to sufficient volume of changes(ratio<0.83 or>1.2).The results showed that 58 proteins were down-regulated and 29 proteins were up-regulated.After KEGG analysis and Western blot verification,we found that Arsa was down-regulated and smpd3 was up-regulated,which appeared to be involved in sphingolipid metabolism pathway;while Ppp1c and YAP1 proteins in Hippo signaling pathway were up-regulated,with the changes in 6-month STX group being more intensive than in 3-month STX group.Conclusion:Sphingolipids metabolic pathway and Hippo signaling pathway might play a role in subchronic and chronic low-dose STX exposure-induced toxicity.3.Exploration of the mechanism for the involvement of Hippo signaling pathway in the biologic effects of in mouse neuroblastoma(N2A)cells induced by STXMethods:N2a cells were treated with STX at concentrations of 5,50,and 500 nM for 24 h.The levels of protein expression of Ppp1c,YAP1,phosphorylated tau,Aβ 1-40 and Aβ1-42 were determined by Western blot or ELISA,the rate of apoptosis was analyzed by flow cytometry.The localization of YAP1 visualzed by immunofluorescence,which showed that YAP1 was primarily expressed in the nucleus and cytoplasm,thus the protein level of p-YAP1-S127 was further explored by Western blot assay,to understand whether the Hippo signaling pathway was activated.Results:The levels of proteins such as Ppp1c,YAP1,phosphorylated tau in N2A cells were elevated by STX treatment,and along with the increase in STX concentration,the rate of apoptosis increased gradually.Meanwhile,following STX treatment,the protein expression of YAP1 and p-YAP1-S127 increased with YAP1 localized in the cytoplasm.Conclusion:STX may induce the damage of neuron-originated cells by activating Hippo signaling pathway. |
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