| Objectives:Neurological diseases such as post-traumatic stress syndrome,depression and Alzheimer’s disease are all related to varying degrees of amnesia,which bring heavy burden to patients,families and society.In this study,C.elegans was used as the model organism to construct a model of C.elegans forgetting pathogen bacteria.Forgotten by proteomics analysis process involved in signaling pathways,specifically Arp2/3 complex in pathogenic forgotten regulation and analysis in the process of forgetting Arp2/3 complex gene expression changes and the changes of cytoskeleton,reveals c.elegans forgotten mechanism of pathogenic bacteria,to forget the drug development and application of related diseases provide clues.Methods:C.elegans was used as the model organism to establish a model of pathogen forgetting.The proteomic changes of C.elegans on pathogenic bacteria PA14 were detected by Label-free quantitative proteomics.Through GO and KEGG enrichment analysis,the regulatory pathway of the Arp3/3 complex was selected as the main screening target.Using C.elegans pathogen amnesia model to study the mechanism of the ARP2/3 complex:RNAi,qPCR,microinjection,calcium ion fluorescence and other techniques were used to clarify the expression changes of candidate genes before and after the removal of pathogenic signals,clarify the changes of calcium flow in C.elegans pathogenic forgetting neurons,clarify the location of Arp2/3 complex in C.elegans tissues,and observe the dynamic changes before and after the removal of pathogenic signals.To explore the molecular mechanism of C.elegans cytoskeleton changes regulating pathogen amnesia behavior.The data were sorted out by Microsoft Excel,processed by ImageJ,mapped by Graphad Prism 8.4.0,and analyzed by SPSS 21.0.P<0.05,the difference was statistically significant.Results:According to the forgetting model analysis,the forgetting time of the pathogen of C.elegans was 2 hours.The quantitative determination of proteome before and after forgetting,KEGG and GO analysis showed that the differently expressed proteins were enriched in the following processes:Arp2/3 signal pathway,heme a biosynthesis,acid complex assembly,mitotic chromosome condensation and so on.When PA14 infected C.elegans for 4 hours and pathogen forgetting for 2 hours,2.38%and 5.83%of the differential proteins were subcellular located in the cytoskeleton,respectively.After interfering with the expression of Arp2/3 complex genes arx-1,arx-2,arx-3,arx-5and arx-7,the time of pathogen forgetting was shortened,and the mRNA levels of Arxmurl,arx-2,arx-3,arx-5 and arx-7 proteins were significantly decreased after 1 hour of removal of pathogenic bacteria.Through the localization of Arp2/3 complex by arx-2::mcherry in C.elegans,the expression of arx-2::mcherry complex was observed in head neurons,pharynx,abdominal nerve cord and dermis,and obvious microfilament reticulation was observed.During amnesia,the fluorescence of arx-2::mcherry in head neurons decreased.Calcium ions were labeled with green fluorescence to detect the changes of calcium flow fluorescence intensity of C.elegans before and after the removal of pathogen PA14.The fluorescence signal decreased during the removal of PA 14 after the removal of pathogen PA 14,When M9 was removed,it was found that there was no significant difference in fluorescence signal during the removal of M9.Conclusions:C.elegans has pathogen amnesia behavior and the forgetting time is 2h.The Arp2bin3 complex participates in the process of pathogen amnesia.The expression of calcium ion in nematode decreased after removal of pathogen PA14.During the process of pathogen amnesia,the expression of differential protein subcellular cytoskeleton increased,the expression of Arp2/3 complex genes arx-1,arx-2,arx-3,arx-5 and arx-7 was down-regulated,and the down-regulation of Arp2/3 complex gene expression inhibited cytoskeleton nucleation,resulting in pathogen amnesia behavior. |
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