具有脑保护作用的化合物W026B的蛋白质组学研究和靶点发现（英文）
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摘要
W026B是一种对小鼠脑缺血再灌注(I-R)损伤具有保护作用的新化合物,但其具体的作用机制尚不明确。本研究应用蛋白质组学观察了W026B在I-R损伤脑组织中对蛋白表达的影响,并试图揭示其潜在的靶点。通过蛋白质组学共检测到4852种蛋白,在脑I-R模型组和W026B治疗组中发现了42种发生显著变化的蛋白,这些蛋白大多与免疫炎症、代谢、神经保护以及细胞增殖和细胞结构有关。经Westernblotting验证,在五种候选蛋白中有三种蛋白表现出与蛋白质组学检测结果一致的改变。我们选择RGS17作为进一步研究的对象,利用siRNARGS17敲低RGS17蛋白后加重了脑损伤,并抵消了W026B的保护作用。W026B可与RGS17结合(KD:6.04×10~(–6)mol/L)。RGS17的下调加重了I型代谢型谷氨酸受体激动剂(I型m GluRs)诱导的Neuro-2a细胞损伤,并抵消了W026B的保护作用。综上所述,W026B通过影响多种蛋白保护大脑免受I-R损伤,RGS17可能是W026B的靶点之一,也是潜在的脑I-R损伤治疗靶点。受W026B影响的RGS17调节的G蛋白上游受体可能是I型mGluRs。本研究为W026B的进一步研发和潜在的临床应用提供了有益的证据。
W026B is a new compound that has a protective effect on cerebral ischemia reperfusion(I-R) injury in mice, while its specific mechanism is still unknown. In this study, proteomics was used to observe the effect of W026B on protein expression in brain I-R tissue, and to reveal its potential target. A total of 42 significantly altered proteins were identified in both brain I-R model and W026B treatment from 4852 proteins detected by proteomics, and most of these proteins were related to immunity and inflammation, metabolism, neuroprotection as well as cell proliferation and cell structure. Western blotting analysis showed that three out of five selected proteins showed consistent alteration with the proteomics. Regulator of G protein signaling 17(RGS17) was selected for further study, and its knockdown by siRNA RGS17 aggravated brain injury and abolished the protective effect of W026B. W026B could bind with RGS17(K_D: 6.04×10~(–6) mol/L). The knockdown of RGS17 aggravated Neuro-2 a cell damage induced by group I metabotropic glutamate receptors(mGluRs) agonist, and abolished the protective effect of W026B. In conclusion, W026B protected brain against I-R injury by affecting diverse proteins. RGS17 might be one of its targets and a potential therapeutic target of brain I-R injury. The upstream receptor of G protein, which was regulated by RGS17 and affected by W026B, might be group I m GluRs. This study provided useful evidence for the further R&D and the potential clinical application of W026B.
W026B is a new compound that has a protective effect on cerebral ischemia reperfusion(I-R) injury in mice, while its specific mechanism is still unknown. In this study, proteomics was used to observe the effect of W026B on protein expression in brain I-R tissue, and to reveal its potential target. A total of 42 significantly altered proteins were identified in both brain I-R model and W026B treatment from 4852 proteins detected by proteomics, and most of these proteins were related to immunity and inflammation, metabolism, neuroprotection as well as cell proliferation and cell structure. Western blotting analysis showed that three out of five selected proteins showed consistent alteration with the proteomics. Regulator of G protein signaling 17(RGS17) was selected for further study, and its knockdown by siRNA RGS17 aggravated brain injury and abolished the protective effect of W026B. W026B could bind with RGS17(K_D: 6.04×10~(–6) mol/L). The knockdown of RGS17 aggravated Neuro-2 a cell damage induced by group I metabotropic glutamate receptors(mGluRs) agonist, and abolished the protective effect of W026B. In conclusion, W026B protected brain against I-R injury by affecting diverse proteins. RGS17 might be one of its targets and a potential therapeutic target of brain I-R injury. The upstream receptor of G protein, which was regulated by RGS17 and affected by W026B, might be group I m GluRs. This study provided useful evidence for the further R&D and the potential clinical application of W026B.
引文
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[11]Zhang,Z.J.;Zhang,G.X.;Sun,Y.W.;Szeto,S.S.W.;Law,H.C.H.;Quan,Q.;Li,G.H.;Yu,P.;Sho,E.;Siu,M.K.W.;Lee,S.M.Y.;Chu,I.K.;Wang,Y.Q.Tetramethylpyrazine nitrone,a multifunctional neuroprotective agent for ischemic stroke therapy.Sci.Rep.2016,6,37148.
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[13]Shah,F.A.;Park,D.J.;Koh,P.O.Identification of proteins differentially expressed by quercetin treatment in a middle cerebral artery occlusion model:A proteomics approach.Neurochem.Res.2018,43,1608-1623.
[14]Shui,M.Y.;Liu,X.Y.;Zhu,Y.J.;Wang,Y.Y.Exogenous hydrogen sulfide attenuates cerebral ischemia-reperfusion injury by inhibiting autophagy in mice.Can.J.Physiol.Pharmacol.2016,94,1187-1192.
[15]Zhang,G.C.;Chen,L.;Yang,L.L.;Hua,X.D.;Zhou,B.Q.;Miao,Z.G.;Li,J.Z.;Hu,H.;Namaka,M.;Kong,J.M.;Xu,X.S.Combined use of spatial restraint stress and middle cerebral artery occlusion is a novel model of post-stroke depression in mice.Sci.Rep.2015,5,16751.
[16]Wi?niewski,J.R.;Zougman,A.;Nagaraj,N.;Mann,M.Universal sample preparation method for proteome analysis.Nat.Methods 2009,6,359-362.
[17]Hu,Z.Y.;Bian,X.L.;Liu,X.Y.;Zhu,Y.J.;Zhang,X.Y.;Chen,S.Z.;Wang,K.W.;Wang,Y.Y.Honokiol protects brain against ischemia-reperfusion injury in rats through disrupting PSD95-nNOS interaction.Brain Res.2013,1491,204-212.
[18]Zhang,H.P.;Wang,F.;Kranzler,H.R.;Anton,R.F.;Gelernter,J.Variation in regulator of G-protein signaling17 gene(RGS17)is associated with multiple substance dependence diagnoses.Behav.Brain Funct.2012,8,23.
[19]Soundararajan,M.;Willard,F.S.;Kimple,A.J.;Turnbull,A.P.;Ball,L.J.;Schoch,G.A.;Gileadi,C.;Fedorov,O.Y.;Dowler,E.F.;Higman,V.A.;Hutsell,S.Q.;Sundstr?m,M.;Doyle,D.A.;Siderovski,D.P.Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.Proc.Natl.Acad.Sci.USA.2008,105,6457-6462.
[20]Kimple,A.J.;Bosch,D.E.;Giguère,P.M.;Siderovski,D.P.Regulators of G-protein signaling and their Gαsubstrates:promises and challenges in their use as drug discovery targets.Pharmacol.Rev.2011,63,728-749.
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[22]Larminie,C.;Murdock,P.;Walhin,J.P.;Duckworth,M.;Blumer,K.J.;Scheideler,M.A.;Garnier,M.Selective expression of regulators of G-protein signaling(RGS)in the human central nervous system.Brain Res.Mol.Brain Res.2004,122,24-34.
[23]Garzón,J.;Rodríguez-Mu?oz,M.;Vicente-Sánchez,A.;Bailón,C.;Martínez-Murillo,R.;Sánchez-Blázquez,P.RGSZ2 binds to the neural nitric oxide synthase PDZ domain to regulate mu-opioid receptor-mediated potentiation of the N-methyl-D-aspartate receptor-calmodulindependent protein kinase II pathway.Antioxid.Redox Signal.2011,15,873-887.
[24]Mao,H.;Zhao,Q.S.;Daigle,M.;Ghahremani,M.H.;Chidiac,P.;Albert,P.R.RGS17/RGSZ2,a novel regulator of Gi/o,Gz,and Gq signaling.J.Biol.Chem.2004,279,26314-26322.
[25]Lafourcade,C.A.;Zhang,L.H.;Alger,B.E.Novel mGluR-and CB1R-independent suppression of GABA release caused by a contaminant of the group I metabotropic glutamate receptor agonist,DHPG.PLo S One.2009,4,e6122.
[26]Kohara,A.;Takahashi,M.;Yatsugi,S.;Tamura,S.;Shitaka,Y.;Hayashibe,S.;Kawabata,S.;Okada,M.Neuroprotective effects of the selective type 1 metabotropic glutamate receptor antagonist YM-202074 in rat stroke models.Brain Res.2008,1191,168-179.
[27]Rao,A.M.;Hatcher,J.F.;Dempsey,R.J.Neuroprotection by group I metabotropic glutamate receptor antagonists in forebrain ischemia of gerbil.Neurosci.Lett.2000,293,1-4.
[28]Liu,B.;Dong,Q.;Zhang,S.Y.;Su,D.Y.;Yang,Z.C.;Lv,M.;Zhang,S.Y.MGluR1,5 activation protects cortical astrocytes and GABAergic neurons from ischemia-induced impairment.Neurosci.Res.2013,75,160-166.
[29]Pai,M.Y.;Lomenick,B.;Hwang,H.;Schiestl,R.;Mc Bride,W.;Loo,J.A.;Huang,J.Drug affinity responsive target stability(DARTS)for small-molecule target identification.Methods Mol.Biol.2015,1263,287-298.
[30]Lomenick,B.;Jung,G.;Wohlschlegel,J.A.;Huang,J.Target identification using drug affinity responsive target stability(DARTS).Curr.Protoc.Chem.Biol.2011,3,163-180.
[2]Wang,Y.;Shen,J.G.;Wang,X.M.;Fu,D.L.;Chen,C.Y.;Lu,L.Y.;Lu,L.;Xie,C.L.;Fang,J.Q.;Zheng,G.Q.Scalp acupuncture for acute ischemic stroke:a meta-analysis of randomized controlled trials.Evid.Based.Complement.Alternat.Med.2012,2012,480950.
[3]R?ther,J.;Ford,G.A.;Thijs,V.N.Thrombolytics in acute ischaemic stroke:historical perspective and future opportunities.Cerebrovasc.Dis.2013,35,313-319.
[4]Goyal,M.;Menon,B.K.;van Zwam,W.H.;Dippel,D.W.J.;Mitchell,P.J.;Demchuk,A.M.;Dávalos,A.;Majoie,C.B.L.M.;van der Lugt,A.;de Miquel,M.A.;Donnan,G.A.;Roos,Y.B.W.E.M.;Bonafe,A.;Jahan,R.;Diener,H.C.;van den Berg,L.A.;Levy,E.I.;Berkhemer,O.A.;Pereira,V.M.;Rempel,J.;Millán,M.;Davis,S.M.;Roy,D.;Thornton,J.;Román,L.S.;Ribó,M.;Beumer,D.;Stouch,B.;Brown,S.;Campbell,B.C.V.;van Oostenbrugge,R.J.;Saver,J.L.;Hill,M.D.;Jovin,T.G.Endovascular thrombectomy after large-vessel ischaemic stroke:a meta-analysis of individual patient data from five randomised trials.Lancet.2016,387,1723-1731.
[5]Balami,J.S.;Chen,R.L.;Sutherland,B.A.;Buchan,A.M.Thrombolytic agents for acute ischaemic stroke treatment:the past,present and future.CNS Neurol.Disord.Drug Targets.2013,12,145-154.
[6]Balami,J.S.;Sutherland,B.A.;Edmunds,L.D.;Grunwald,I.Q.;Neuhaus,A.A.;Hadley,G.;Karbalai,H.;Metcalf,K.A.;DeLuca,G.C.;Buchan,A.M.A systematic review and meta-analysis of randomized controlled trials of endovascular thrombectomy compared with best medical treatment for acute ischemic stroke.Int.J.Stroke.2015,10,1168-1178.
[7]Zhu,Y.J.;Gao,Y.Q.;Zheng,D.P.;Shui,M.Y.;Yu,K.;Liu,X.Y.;Lin,Y.;Su,L.;Yang,W.X.;Wang,Y.Y.Design and evaluation of EphrinA1 mutants with cerebral protective effect.Sci.Rep.2017,7,1881.
[8]Bu,Q.X.;Liu,X.Y.;Zhu,Y.J.;Liu,Y.;Wang,Y.Y.W007B protects brain against ischemia-reperfusion injury in rats through inhibiting inflammation,apoptosis and autophagy.Brain Res.2014,1558,100-108.
[9]Wang,Y.;Liu,Y.;Zhang,P.;Tong,W.;Cai,Y.Magnolol derivatives,honokiol derivatives and their preparation method and application.China,2013.
[10]Ye,Z.A new compound W026B alleviates ischemic brain injury through inhibiting the production of inflammatory cytokines in pMCAO and tMCAO,and enhances the beneficial effect of tPA.J.Chin.Pharm.Sci.2018,27,675-685.
[11]Zhang,Z.J.;Zhang,G.X.;Sun,Y.W.;Szeto,S.S.W.;Law,H.C.H.;Quan,Q.;Li,G.H.;Yu,P.;Sho,E.;Siu,M.K.W.;Lee,S.M.Y.;Chu,I.K.;Wang,Y.Q.Tetramethylpyrazine nitrone,a multifunctional neuroprotective agent for ischemic stroke therapy.Sci.Rep.2016,6,37148.
[12]Llombart,V.;Trejo,S.A.;Bronsoms,S.;Morancho,A.;Ma,F.F.;Faura,J.;García-Berrocoso,T.;Simats,A.;Rosell,A.;Canals,F.;Hernández-Guillamón,M.;Montaner,J.Profiling and identification of new proteins involved in brain ischemia using MALDI-imaging-massspectrometry.J.Proteomics.2017,152,243-253.
[13]Shah,F.A.;Park,D.J.;Koh,P.O.Identification of proteins differentially expressed by quercetin treatment in a middle cerebral artery occlusion model:A proteomics approach.Neurochem.Res.2018,43,1608-1623.
[14]Shui,M.Y.;Liu,X.Y.;Zhu,Y.J.;Wang,Y.Y.Exogenous hydrogen sulfide attenuates cerebral ischemia-reperfusion injury by inhibiting autophagy in mice.Can.J.Physiol.Pharmacol.2016,94,1187-1192.
[15]Zhang,G.C.;Chen,L.;Yang,L.L.;Hua,X.D.;Zhou,B.Q.;Miao,Z.G.;Li,J.Z.;Hu,H.;Namaka,M.;Kong,J.M.;Xu,X.S.Combined use of spatial restraint stress and middle cerebral artery occlusion is a novel model of post-stroke depression in mice.Sci.Rep.2015,5,16751.
[16]Wi?niewski,J.R.;Zougman,A.;Nagaraj,N.;Mann,M.Universal sample preparation method for proteome analysis.Nat.Methods 2009,6,359-362.
[17]Hu,Z.Y.;Bian,X.L.;Liu,X.Y.;Zhu,Y.J.;Zhang,X.Y.;Chen,S.Z.;Wang,K.W.;Wang,Y.Y.Honokiol protects brain against ischemia-reperfusion injury in rats through disrupting PSD95-nNOS interaction.Brain Res.2013,1491,204-212.
[18]Zhang,H.P.;Wang,F.;Kranzler,H.R.;Anton,R.F.;Gelernter,J.Variation in regulator of G-protein signaling17 gene(RGS17)is associated with multiple substance dependence diagnoses.Behav.Brain Funct.2012,8,23.
[19]Soundararajan,M.;Willard,F.S.;Kimple,A.J.;Turnbull,A.P.;Ball,L.J.;Schoch,G.A.;Gileadi,C.;Fedorov,O.Y.;Dowler,E.F.;Higman,V.A.;Hutsell,S.Q.;Sundstr?m,M.;Doyle,D.A.;Siderovski,D.P.Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits.Proc.Natl.Acad.Sci.USA.2008,105,6457-6462.
[20]Kimple,A.J.;Bosch,D.E.;Giguère,P.M.;Siderovski,D.P.Regulators of G-protein signaling and their Gαsubstrates:promises and challenges in their use as drug discovery targets.Pharmacol.Rev.2011,63,728-749.
[21]Roman,D.L.;Traynor,J.R.Regulators of G protein signaling(RGS)proteins as drug targets:modulating G-protein-coupled receptor(GPCR)signal transduction.J.Med.Chem.2011,54,7433-7440.
[22]Larminie,C.;Murdock,P.;Walhin,J.P.;Duckworth,M.;Blumer,K.J.;Scheideler,M.A.;Garnier,M.Selective expression of regulators of G-protein signaling(RGS)in the human central nervous system.Brain Res.Mol.Brain Res.2004,122,24-34.
[23]Garzón,J.;Rodríguez-Mu?oz,M.;Vicente-Sánchez,A.;Bailón,C.;Martínez-Murillo,R.;Sánchez-Blázquez,P.RGSZ2 binds to the neural nitric oxide synthase PDZ domain to regulate mu-opioid receptor-mediated potentiation of the N-methyl-D-aspartate receptor-calmodulindependent protein kinase II pathway.Antioxid.Redox Signal.2011,15,873-887.
[24]Mao,H.;Zhao,Q.S.;Daigle,M.;Ghahremani,M.H.;Chidiac,P.;Albert,P.R.RGS17/RGSZ2,a novel regulator of Gi/o,Gz,and Gq signaling.J.Biol.Chem.2004,279,26314-26322.
[25]Lafourcade,C.A.;Zhang,L.H.;Alger,B.E.Novel mGluR-and CB1R-independent suppression of GABA release caused by a contaminant of the group I metabotropic glutamate receptor agonist,DHPG.PLo S One.2009,4,e6122.
[26]Kohara,A.;Takahashi,M.;Yatsugi,S.;Tamura,S.;Shitaka,Y.;Hayashibe,S.;Kawabata,S.;Okada,M.Neuroprotective effects of the selective type 1 metabotropic glutamate receptor antagonist YM-202074 in rat stroke models.Brain Res.2008,1191,168-179.
[27]Rao,A.M.;Hatcher,J.F.;Dempsey,R.J.Neuroprotection by group I metabotropic glutamate receptor antagonists in forebrain ischemia of gerbil.Neurosci.Lett.2000,293,1-4.
[28]Liu,B.;Dong,Q.;Zhang,S.Y.;Su,D.Y.;Yang,Z.C.;Lv,M.;Zhang,S.Y.MGluR1,5 activation protects cortical astrocytes and GABAergic neurons from ischemia-induced impairment.Neurosci.Res.2013,75,160-166.
[29]Pai,M.Y.;Lomenick,B.;Hwang,H.;Schiestl,R.;Mc Bride,W.;Loo,J.A.;Huang,J.Drug affinity responsive target stability(DARTS)for small-molecule target identification.Methods Mol.Biol.2015,1263,287-298.
[30]Lomenick,B.;Jung,G.;Wohlschlegel,J.A.;Huang,J.Target identification using drug affinity responsive target stability(DARTS).Curr.Protoc.Chem.Biol.2011,3,163-180.