Study Of The Hsp70-binding Proteins In Hippocampus Of Rats Induced By Sub-chronically Benzo[a]pyrene

Objective:The domestic and international research shown that Benzo[a]pyrene(B[a]P) have neurotoxicity. Exposure to B[a]P can lead to the human and animal learning and memory damage. As a molecular chaperone, Heat shock protein70(Hsp70) were possibly involved in neurotoxicity mechanism of B[a]P. However, The exact mechanisms are not clear. This study detect and analysis the Hsp70-interacting proteins of hippocampus in rats, and to explore the neurotoxicity mechanism of benzo[a]pyrene.Methods:According to the results of behavioral tests, healthy adult male SD rats, adult were randomly divided into4groups:untreated group, blank control group, olive control group, and B[a]P group, which were administered intra-peritoncally for90days. The Morris water maze test was perfomed to test the learning and memory abilities of rat. The expression level of Hsp70was detected by Western-blot. Later, immunoprecipitation and Thermo LTQ Orbitrap Velos MS were applied to detect the Hsp70-interacting proteins. Confocal microscopy analisis was further performed to characterize the interaction between Hsp70and the Hsp70-interacting proteins.Results:The Morris water maze test showed, the escape latencies of navigation in B[a]P group was significantly higher than blank control group and olive control group(P<0.05). And the frequency of crossing platform and the swimming time of platform quadrant and the swimming time percentage of platform quadrant in B[a]P group were significantly lower than blank control group and olive control group (P<0.05).The results of Westem-blot showed that the expression levels of Hsp70in B[a]P group was significantly lower than blank control group and olive control group (P<0.05).The results of immunoprecipitation and Thermo LTQ Orbitrap Velos MS showed that respectively410,482and609proteins were combined with Hsp70in untreated group, control group and B[a]P group. These proteins were categorized into11groups based on the function, including skelemin, chaperonin, signal transduction, DNA damage, repair-related protein, metabolism, apoptosis, oxidative stress, neoplasms, neural development, neurotransmitter generation and transfer, unknown function protein.333proteins were combined with Hsp70in untreated group, control group and B[a]P group, including CaMK Ⅱα subunit, PKC a type, PKC β type, PKA a subunit, PKA β subunit, MAPK1, MAPK3, glutamate receptor2precursor, calmodulin,14-3-3protein, glutathione S-transferase P, ubiquitin carboxyl-terminal hydrolase isozyme L1, neuromodulin, synaptotagmin-1, synaptotagmin-2, synaptophysin, cytochrome c oxidase subunit Ⅱ, neural cell adhesion molecule1, and so on. Compared with the untreated group,118new proteins were combined with Hsp70in control group after learning and memory training, including neurogranin, catenin beta, calcineurin, glycogen synthase kinase-3beta, cytochrome c, and so on. However,46proteins had no more combine with Hsp70, including syntaxin-7, voltage-dependent anion-selective channel protein2, and so on. Compared with control group,198new proteins were combined with Hsp70in B[a]P group, including F-actin-capping protein subunit beta, metallothionein-3, Complexin-1, Complexin-2, and so on. However,94proteins had no more combine with Hsp70, including glutamate receptor1precursor, voltage-dependent calcium channel gamma-8subunit, dihydropyrimidinase-related protein4, neurogranin, glutamate decarboxylase, and so on.The confocal microscopy had observed coexpression phenomenon of Hsp70and PKC, PKA, NMDAR1, NMDAR2A, NMDAR2B.Conclusion:1、 Sub-chronic exposure to benzo[a]pyrene could damage learning and memory and inhibit the expression of Hsp70in rat hippocampus.2、 Hsp70coulde be involved in signal pathway, cell apoptosis, oxidative stress, neural development, neurotransmitter generation and transport through regulate the Hsp70-bingding proteins in rat hippocampus. Hsp70coulde be involved in hippocampal LTP through bind with CaMK Ⅱ, PKC, PKA, MAPK1, MAPK3, glutamate receptor2precursor, calmodulin, neuromodulin, synaptotagmin-1, synaptotagmin-2, synaptophysin, and so on. Sub-chronic exposure to B[a]P inhibiting the expression of Hsp70in rat hippocampus may effect the normal physiological process and the process of learning and memory. 3、 Neurogranin may related to the formation of spatial learning and memory after training. Hsp70binding with Neurogranin may involved in its generation, transport, fold and play function.4、 Sub-chronic exposure to B[a]P effecting the kind of the Hsp70-binding proteins may related to the neurotoxicity mechanism of B[a]P, especially.....and so on. Meanwhile, B[a]P inducing new Hsp70-binding proteins coulde be involved in the mechanism of oxidative stress and damage repair and so on.