| Conopeptides are a class of neurotoxin peptides which are secreted by Cone snail for prey.According to the highly conserved signal peptide sequences,conopeptides can be divided into A-,B1-,B2-,B3-,C-,D-,E-,F-,G-,H-,I1-,I2-,I3-,J-,K-,L-,M-,N-,01-,02-,03-,P-,S-,T-,V-and Y-superfamilies,etc.Conopeptides also can be further divided into ????????????????? and conantokins-subfamily,etc,according to the conservative cysteine frames and pharmacological targets.Conopeptides have the advantages of unique structure,high diversity,high activity and high selectivity,can specifically bind to various ion channels,receptors and their subtypes.Thus,they can be used for neural pharmacological molecular probe,diagnostic reagents or drugs.Eb1.6 belongs to the 4/7 subfamily of alpha-conoeptides and consists of 16 amino acids and two disulfide bond(the disulfide bond is "1-3,2-4").Our previous results showed that Eb1.6 exhibited a potent analgesic activity for neuropathic pain.In this study,we will detect the molecules involved with pain by the technology of realtime PCR and western blot.After the administration of doses of Eb1.6(i.m.)in PNL model rat,the expression of genes and proteins,such as C-fos,ATF3,GABA,PICK 1,AMPA,PKC-y,GAP-43,NGF-p,TRPV1,TPH1 mRNA and BDNF,NGF-?,GABA,AMPA,C-fos,CAMKII-?,CAMKII-p and CAMK-IV were determined.Far western and magnetic bead adsorption technology were used to discover the target molecules.The distribution of Eb1.6 in mice brain and rat sciatic nerve was also detected by Fluorescent labeled-Eb1.6.Con-T[M8Q]is a variant of Con-T,which belongs to the conantokins family targeting specific subtypes of N-Methyl-D-aspartate(NMDA)receptors.Our previous experiments showed that Con-T[M8Q]exhibited the potent inhibitory activity to the morphine dependent mice.The genes or proteins involved in NMDAR pathway,such as pNR2B,NR2B,pERK,C-fos,CAMKII-?,CAMKII-?,CAMK-IV in morphine dependent were determined primarily,but data were not complete.Thus,the above molecules were determined again in this assay.In addition,other molecules related with morphine dependence,such as the mRNA of the nNOS,PKC-?,AMPA,NR2B,and the protein of the nNOS,PKC-? were also investigated.These study will provide the basis for discovering the targets of Ebl.6 and Con-T[M8Q],reaction mechanism and related application.The results are as follows:(1)Compared with the saline group,the different doses of Eb1.6 significantly elevated the BDNF,NGF-? gene expression,but the same effects were observed for the expression of NMDA gene at the high dose of Eb1.6(1.25mg/kg)or for ATF3,GAP-43,PICK1 gene at high(1.25mg/kg)and mediate dose(250ug/kg),respectively;Eb1.6 significantly decreased the expression of the gene and protein of AMPA and GABA as well as the expression of CaMKII-? protein;no significant effect on the gene of C-fos and PKC y a as well as the protein of C-fos,CAMKII-a,CAMK-IV were observed.(2)The primary results of Far Western experiment showed that there were one or more molecules might bind to Eb1.6 in rat hippocampal tissue.(3)The distribution of fluorescent-labeled Ebl.6 showed that some molecules binding to Ebl.6 might exist in mice hippocampus and rat sciatic nerve.(4)After the injection of different doses of Con-T[M8Q]in morphine dependent mice,the expression of protein and gene of CAMKII-a,CAMKII-?,CAMKIV,NR2B in hippocampal tissue decreased with a dose-dependent mode compared with Ifenprodil.The gene expression of nNOS and PKC-? as well as the protein expression of C-fos,pERK also decreased,but no significant effect was observed in the AMPA gene expression. |
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