基于磁性纳米颗粒载体的中药活性成分体内原位靶点鉴定方法学探索
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摘要
靶点鉴定是药物作用机制研究的重要前提。当前主流的药物靶点鉴定方法大多是利用体外培养的细胞模型开展的,然而,离体培养的细胞与体内细胞在生长微环境、营养代谢、生物学特性及功能等方面存在着巨大差异,因此,建立有效的药物体内靶点识别方法是一个具有挑战性的科学问题。在该研究中,作者制备了一种磁性纳米颗粒,该磁性纳米颗粒表面的环氧基团可以和天然药物分子松果菊苷(ECH)的羟基结构进行化学偶联,形成分布具有器官选择性又便于回收的纳米级靶点识别颗粒。进而将键合了ECH的磁性纳米颗粒静脉注射到大鼠体内,并通过体内分布到特定器官后,通过胞吞作用进入组织细胞内,通过磁性纳米颗粒表面键合的ECH原位捕获靶蛋白并进行鉴定。基于此方法,在大鼠脾脏中成功鉴定到ECH的潜在作用靶蛋白,初步阐明了ECH的免疫调节机制。因此,该研究创建了一种利用磁性纳米颗粒进行体内原位靶点鉴定的新方法,为今后的药物靶点确证和药理机制阐明提供了一种全新的技术策略。
Target identification is an important prerequisite for the study of medicine action mechanism. Currently,drug target identification is mostly based on various cell models in vitro. However,the growth microenvironment,nutrition metabolism,biological properties as well as functions are quite different between in vitro cell culture and physiological environment in vivo; wherefore,it is a challenging scientific issue to establish an effective method for identifying drug targets in vivo condition. In this study,we successfully prepared a kind of magnetic nanoparticles( MNPs) which can be chemically modified by the hydroxyl structure of natural bioactive compound echinacoside( ECH) via the epoxy group label on the surface of MNPs. Therefore,organ-selective and recoverable nanoscale target-recognizing particles were prepared. We then intravenously injected the ECH-binding MNPs into rats and distributed them to specific organs in vivo. After cell endocytosis,ECH-binding MNPs captured target proteins in situ for further analysis. Based on this method,we discovered several potential target proteins in the spleen lysates for ECH,and preliminarily clarified the immuno-regulation mechanism of ECH. Collectively,our strategy developed a proof-of-concept technology using nanoparticles for in vivo target identification,and also provided a feasible approach for drug target prediction and pharmacological mechanism exploration.
Target identification is an important prerequisite for the study of medicine action mechanism. Currently,drug target identification is mostly based on various cell models in vitro. However,the growth microenvironment,nutrition metabolism,biological properties as well as functions are quite different between in vitro cell culture and physiological environment in vivo; wherefore,it is a challenging scientific issue to establish an effective method for identifying drug targets in vivo condition. In this study,we successfully prepared a kind of magnetic nanoparticles( MNPs) which can be chemically modified by the hydroxyl structure of natural bioactive compound echinacoside( ECH) via the epoxy group label on the surface of MNPs. Therefore,organ-selective and recoverable nanoscale target-recognizing particles were prepared. We then intravenously injected the ECH-binding MNPs into rats and distributed them to specific organs in vivo. After cell endocytosis,ECH-binding MNPs captured target proteins in situ for further analysis. Based on this method,we discovered several potential target proteins in the spleen lysates for ECH,and preliminarily clarified the immuno-regulation mechanism of ECH. Collectively,our strategy developed a proof-of-concept technology using nanoparticles for in vivo target identification,and also provided a feasible approach for drug target prediction and pharmacological mechanism exploration.
引文
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[3] Ye X,He X,Lei Y,et al. One-pot synthesized Cu/Au/Pt trimetallic nanoparticles with enhanced catalytic and plasmonic properties as a universal platform for biosensing and cancer theranostics[J]. Chem Commun,2019,55(16):2321.
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[6] Ostroverkhov P V,Semkina A S,Naumenko V A,et al. Synthesis and characterization of bacteriochlorin loaded magnetic nanoparticles(MNP)for personalized MRI guided photosensitizers delivery to tumor[J]. J Colloid Interface Sci,2019,537:132.
[7] Ali E M M,Elashkar A A,El-Kassas H Y,et al. Methotrexate loaded on magnetite iron nanoparticles coated with chitosan:biosynthesis,characterization and impact on human breast cancer MCF-7 cell line[J]. Int J Biol Macromol,2018,120:1170.
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[9] Lu C W,Hsieh H L,Lin T Y,et al. Echinacoside,an active constituent of cistancheherba,exerts a neuroprotective effect in a kainic acid rat model by inhibiting inflammatory processes and activating the Akt/GSK3βpathway[J]. Biol Pharm Bull,2018,41:1685.
[10] Chen M,Wang X,Hu B,et al. Protective effects of echinacoside against anoxia/reperfusion injury in H9c2 cells via up-regulating p-AKT and SLC8A3[J]. Biomed Pharmacother,2018,104:52.
[11] Wang W,Luo J,Liang Y,et al. Echinacoside suppresses pancreatic adenocarcinoma cell growth by inducing apoptosis via the mitogen-activated protein kinase pathway[J]. Mol Med Rep,2016,13:2613.
[12]何文君,方泰惠,屠鹏飞.松果菊苷的药理研究进展[J].中国中药杂志,2009,34(4):476.
[13] Hanmer K L,Mavri-Damelin D. Peroxidasin is a novel target of the redox-sensitive transcription factor Nrf2[J]. Gene,2018,674:104.
[14] Soudia M,Zamockya M,Jakopitscha C,et al. Molecular evolution,structure,and function of peroxidasins[J]. Chem Biodiv,2012,9(9):1776.
[15] Ero-Tolliver I A,Hudson B G,Bhave G. The ancient immunoglobulin domains of peroxidasin are equired to form sulfilimine cross-links in collagenⅣ[J]. J Biol Chem,2015,290:21741.
[16]戴灵豪,沈钰明,伍义行,等.松果菊苷对乙肝病毒复制和抗原表达的影响[J].中国中药杂志,2015,40(15):3047.
[17] Tang F,Hao Y,Zhang X,et al. Effect of echinacoside on kidney fibrosis by inhibition of TGF-β1/Smads signaling pathway in the db/db mice model of diabetic nephropathy[J]. Drug Des Devel Ther,2017,11:2813.
[18] Osaki M,Oshimura M,Ito H. PI3K-Akt pathway:its functions and alterations in human cancer[J]. Apoptosis,2004,9(6):667.
[19] Maqbool M,Hoda N. GSK3 inhibitors in the therapeutic development of diabetes,cancer and neurodegeneration:past,present and future[J]. Curr Pharm Des,2017,23(29):4332.
[2] Saglam-Metiner P,Gulce-Iz S,Biray-Avci C. Bioengineering-inspired three-dimensional culture systems:organoids to create tumor microenvironment[J]. Gene,2019,686:203.
[3] Ye X,He X,Lei Y,et al. One-pot synthesized Cu/Au/Pt trimetallic nanoparticles with enhanced catalytic and plasmonic properties as a universal platform for biosensing and cancer theranostics[J]. Chem Commun,2019,55(16):2321.
[4] Cheng Q,Teng K X,Ding Y F,et al. Dual stimuli-responsive bispillar
[5]arene-based nanoparticles for precisely selective drug delivery in cancer cells[J]. Chem Commun,2019,55(16):2340.
[5] Pereira S A P,Passos M L C,Correia A,et al. Automatic methodologies to perform loading and release assays of anticancer drugs from mesoporous silicon nanoparticles[J]. Talanta,2019,196:227.
[6] Ostroverkhov P V,Semkina A S,Naumenko V A,et al. Synthesis and characterization of bacteriochlorin loaded magnetic nanoparticles(MNP)for personalized MRI guided photosensitizers delivery to tumor[J]. J Colloid Interface Sci,2019,537:132.
[7] Ali E M M,Elashkar A A,El-Kassas H Y,et al. Methotrexate loaded on magnetite iron nanoparticles coated with chitosan:biosynthesis,characterization and impact on human breast cancer MCF-7 cell line[J]. Int J Biol Macromol,2018,120:1170.
[8]曾克武,廖理曦,万彦军,等.基于靶点“钩钓”策略的肉苁蓉苯乙醇苷药理靶点鉴定及功效解析[J].中草药,2018,49(1):173.
[9] Lu C W,Hsieh H L,Lin T Y,et al. Echinacoside,an active constituent of cistancheherba,exerts a neuroprotective effect in a kainic acid rat model by inhibiting inflammatory processes and activating the Akt/GSK3βpathway[J]. Biol Pharm Bull,2018,41:1685.
[10] Chen M,Wang X,Hu B,et al. Protective effects of echinacoside against anoxia/reperfusion injury in H9c2 cells via up-regulating p-AKT and SLC8A3[J]. Biomed Pharmacother,2018,104:52.
[11] Wang W,Luo J,Liang Y,et al. Echinacoside suppresses pancreatic adenocarcinoma cell growth by inducing apoptosis via the mitogen-activated protein kinase pathway[J]. Mol Med Rep,2016,13:2613.
[12]何文君,方泰惠,屠鹏飞.松果菊苷的药理研究进展[J].中国中药杂志,2009,34(4):476.
[13] Hanmer K L,Mavri-Damelin D. Peroxidasin is a novel target of the redox-sensitive transcription factor Nrf2[J]. Gene,2018,674:104.
[14] Soudia M,Zamockya M,Jakopitscha C,et al. Molecular evolution,structure,and function of peroxidasins[J]. Chem Biodiv,2012,9(9):1776.
[15] Ero-Tolliver I A,Hudson B G,Bhave G. The ancient immunoglobulin domains of peroxidasin are equired to form sulfilimine cross-links in collagenⅣ[J]. J Biol Chem,2015,290:21741.
[16]戴灵豪,沈钰明,伍义行,等.松果菊苷对乙肝病毒复制和抗原表达的影响[J].中国中药杂志,2015,40(15):3047.
[17] Tang F,Hao Y,Zhang X,et al. Effect of echinacoside on kidney fibrosis by inhibition of TGF-β1/Smads signaling pathway in the db/db mice model of diabetic nephropathy[J]. Drug Des Devel Ther,2017,11:2813.
[18] Osaki M,Oshimura M,Ito H. PI3K-Akt pathway:its functions and alterations in human cancer[J]. Apoptosis,2004,9(6):667.
[19] Maqbool M,Hoda N. GSK3 inhibitors in the therapeutic development of diabetes,cancer and neurodegeneration:past,present and future[J]. Curr Pharm Des,2017,23(29):4332.