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Objective:To explore the key pathways and genes involved in microglia inflammation through transcriptome sequencing and bioinformatics analysis.Methods:BV2 cells were stimulated by lipopolysaccharide to establish microglia inflammation model. The levels of IL-6 and TNF-α were detected by ELISA and RT-qPCR. The established microglia inflammation model was sequenced by transcriptome sequencing, and the differentially expressed genes were screened by bioinformatics method. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes were performed. The protein-protein interaction network of differentially expressed genes was constructed by using string database, and the protein-protein interaction network was visualized by using Cytoscape software. The protein interaction network module was extracted by using MCODE app. The hub gene was extracted by using cytohubba app and was verified through RT-qPCR. We conducted enrichment analysis of hub genes, predicted their targeted miRNAs and interacting drugs.Results:The microglia inflammation model was successfully established and verified by ELISA and RT-qPCR. We screened 434 differentially expressed genes by bioinformatics analysis of transcriptome sequencing results. GO analysis showed that these differentially expressed genes were mainly concentrated in cellular response to cytokine stimulus, inflammatory response, regulation of response to external stimulation. KEGG analysis showed that these differentially expressed genes were mainly concentrated in Chemokine signaling pathway, TNF signaling pathway, IL-17 signaling pathway. We constructed the protein interaction network of these differentially expressed genes, and carried out module analysis and extraction of hub genes. Most of hub genes are located in module 1, and the seed gene of module 1 is S1pr1. Hub genes include S1pr1, Cxcr4, Cx3cl1, Cx3cr1, Cxcl10, Cxcl2, Ccl4, Ccl5, Ccl9, Fpr1. RT-qPCR results showed that compared with the culture medium group, the mRNA expressions of S1pr1, Cxcr4, Cx3cl1 and Cx3cr1 were down-regulated, and the mRNA expressions of Cxcl10, Cxcl2, Ccl4, Ccl5, Ccl9 and Fpr1 were up-regulated in the LPS group. The enrichment analysis of hub genes mainly focused on chemokine-mediated signaling pathway, Class A/1 (Rhodopsin-like receptors), cell chemotaxis and so on. Drugs and miRNAs that may interact with hub genes were predicted. Conclusion:Through transcriptome sequencing and bioinformatics analysis of microglia inflammation model, differentially expressed genes were screened, hub genes and seed genes were extracted, which will help us further understand the molecular mechanism of microglia inflammation and provide potential targets for the treatment of related diseases.
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Objective To discover novel conopeptides which are the antagonists of neuronal nicotinic acetylcholine receptors (nAChRs) in order to contribute to the development of novel analgesic drugs and neuropharmacological probes.Methods Based on the conserved untranslated region and intron of A-superfamily conotoxins,a novel α-conotoxin Lt1.1 was cloned from Conus litteratus.The peptide-resin was synthesized using the solid-phased method and was cleaved.The resulting linear peptide was oxidized by air to give the product containing disulfide bridges.The folding product was finally purified by HPLC.The disulfide bond connectivity was determined using the two-step oxidative folding methods.The cRNA of rat nAChRs was expressed on the membrane of Xenopus oocyte.Membrane currents were recorded using the two electrode voltage-clamp technique.Results A novel α-conotoxin designated as Lt1.1(GCCSHPACNVNNPDIC-NH2) was cloned and its disulfide connectivity was C1-C3,C2-C4.Lt1.1 selectively inhibited the α3β2 and α3β4 nAChRs with an IC50 of 166.76 and 190.00 nmol/L,respectively.Conclusion Lt1.1 is a novel 4/7 α-conotoxin that selectively targets α3β2 and α3β4 nAChRs.
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Objective To clone a new conotoxin Bt14.10 from Conus betulinus derived from the South China Sea, synthesize the peptide , and to determine linkage of its disulfide bridges .Methods The genomic DNA was extracted from C.betulinus venom duct while the Bt14.10 sequence was cloned using primers designed based on the untranslated region and intron.The peptide was then synthesized using solid-phase method and folded into the target product whose disulfide bridge connection was further determined by two-step oxidative folding .Results A novel conotoxin designated as Bt 14.10 (CAHSVPGMHPCKCNNTC-NH2) was obtained,the disulfide connectivity of which was C1-C3,C2-C4.Conclusion Bt14.10 is a new A-superfamily conotoxin and has a distinct loop spacing pattern between cysteines in A-superfamily conotoxins.