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大黄素对辣椒素诱导的TRPV1通道电流的抑制作用

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复旦学报（自然科学版） 2017年第3期56卷 366-373页

作者：胡益张迪 wolfgang schwarz 复旦大学航空航天系上海200433 上海市针灸机制与穴位功能重点实验室上海200433 上海市针灸经络研究中心上海201203 法兰克福大学生物物理学系法兰克福60438

本研究通过显微注射技术将TRPV1 cRNA(1ng/nL)注射至非洲爪蟾卵母细胞(20nL/个)体内,放置于G-ORi培养液中,并在(19±1)℃的恒温培养箱内表达.同时利用灌流技术将不同浓度梯度的大黄素、辣椒素按照设计顺序依次灌流进入流动腔,同时控制... 详细信息

本研究通过显微注射技术将TRPV1 cRNA(1ng/nL)注射至非洲爪蟾卵母细胞(20nL/个)体内,放置于G-ORi培养液中,并在(19±1)℃的恒温培养箱内表达.同时利用灌流技术将不同浓度梯度的大黄素、辣椒素按照设计顺序依次灌流进入流动腔,同时控制流速保证非洲爪蟾卵母细胞完全浸没于流动腔内.使用双电极电压钳技术记录0.1,1.0,10.0,50.0μmol/L浓度梯度的大黄素对500nmol/L浓度的辣椒素激活TRPV1电流的影响.得到大黄素作用辣椒素激活TRPV1通道的抑制作用表现出浓度依耐性和电压非依赖性,IC50=38μmol/L,Hill系数n=0.5.结果表明了大黄素对TRPV1位点的相互作用是负协同的,在天然药物里面是一类比较弱的拮抗剂,且大黄素在ORi溶液中开始析出沉淀的浓度在50~60μmol/L之间.我们首次发现了大黄素能够抑制TRPV1通道电流,这可能为开发新的镇痛药物提供理论基础.

关键词： TRPV1通道电流辣椒素大黄素镇痛机制双电极电压钳

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用于针刺穴位研究的结缔组织片模型

针刺研究 2009年第5期34卷 291-296页

作者：汪丽娜 wolfgang schwarz 顾全保丁光宏复旦大学力学与工程科学系上海市针灸经络研究中心上海200433 马克斯-普朗克生物物理所德国法兰克福60438

目的:先前研究发现,穴区功能与穴区结缔组织结构有密切关系,本文旨在建立基于穴区原位组织上的膜片钳研究方法。方法:急性分离出SD大鼠"后三里"穴皮下结缔组织,并安置在含有孵育液的浴槽内。组织片内肥大细胞可用甲苯胺蓝或中性红进行... 详细信息

目的:先前研究发现,穴区功能与穴区结缔组织结构有密切关系,本文旨在建立基于穴区原位组织上的膜片钳研究方法。方法:急性分离出SD大鼠"后三里"穴皮下结缔组织,并安置在含有孵育液的浴槽内。组织片内肥大细胞可用甲苯胺蓝或中性红进行标记。在膜片钳实验中,通过记录电极对组织片内原位肥大细胞施加-30、-60和-90 cmH2O的负压刺激,并记录细胞上机械激活性全细胞电流。结果:制备的皮下结缔组织片标本经甲苯胺蓝或中性红染色显示,肥大细胞散在分布于组织片中的基质中。肥大细胞脱颗粒现象也能通过染色清晰可见。膜片钳记录表明穴区组织片肥大细胞的全细胞电流随着机械负压的施加而增大。结论:大鼠穴区皮下结缔组织片原位肥大细胞全细胞电流能被机械刺激激活。结合显微技术和电生理技术,大鼠"后三里"穴区皮下结缔组织片可以做为研究针刺效应外周机制的一种模型,并提示穴位肥大细胞可能参与针刺过程中机械信号的转导。该模型也可为今后进一步在细胞水平上研究穴位中细胞、胶原纤维、蛋白多糖等各成分的特性及其之间的相互作用提供基础。

关键词：针刺结缔组织片肥大细胞机械激活性膜片钳

Heat induces adenosine triphosphate release from mast cells in vitro:a putative mechanism for moxibustion

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Journal of Traditional Chinese Medicine 2015年第3期35卷 323-328页

作者： Hu Lei Wang Lina Wei Jianzi Ryszard Grygorczyk Shen Xueyong wolfgang schwarz Acupuncture & Moxibustion and Tuina College Shanghai University of Traditional Chinese Medicine Cellular Electrophysiological Laboratory Shanghai Research Centre for Acupuncture and Meridians Research Centre Centre hospitalierde I'Universite de Montreal(CRCHUM)-Hotel-DieuQuebec H2W 1T8Canada Department of Medicine Universite de Montreal Institute for Biophysics Goethe-University Frankfurt

OBJECTIVE:To investigate the role of adenosine triphosphate(ATP) purinergic signaling in mast cells(MCs) modulated by heat to further understand the molecular mechanisms of ***:Skin temperatures induced by monkshood cake moxibustion were evaluated by measuring the Neiguan acupoint(PC 6) from 31 participants with a digital thermocouple *** of 43 ℃ and 52 ℃ were applied to cultured human leukemia mast cell line HMC-1 in *** fluorescence was applied to detect intracellular Ca^(2+)([Ca^(2+)]_i).Extracellular ATP contents were measured by luciferin-luciferase ***:Maximum skin temperatures mostly ranged from 40-45 ℃,but some reached up to50 ℃.Both 43 ℃,and 52 ℃ induced MC degranulation,which was accompanied by an increase in[Ca^(2+)]_i and ATP *** extracellular Ca^(2+) with 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid(EGTA) inhibited the noxious heat-induced elevation of[Ca^(2+)]_i and prevented the enhanced ATP secretion by those cells at 52℃ but not 43 ℃.CONCLUSION:Monkshood cake moxibustion can generate heat sufficient to trigger cellular events of MCs,including degranulation,[Ca^(2+)]_i elevation,and ATP release,suggesting that purinergic signals originating from MCs are possibly the initiating response of acupoints to moxibustion.

关键词： Fire toxin syndrome Mast cells Cell degranulation Calcium Adenosine triphosphate Moxibustion

Inhibition of the Na+/Ca2+Exchanger NCX1Expressed in XenopusOocyte by Glycyrrhizic Acid and Cyclophylin A

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Journal of Biosciences and Medicines 2017年第3期5卷 128-141页

作者： Jan Laudenbach Yu Wang Beibei Xing Silvia schwarz Yinfang Xu Quanbao Gu wolfgang schwarz Shanghai Research Center for Acupuncture & Meridians Shanghai China Max-Planck-Institute for Biophysics and Institute for Biophysics Frankfurt am Main Germany Institute for Biophysics Goethe University Frankfurt am Main Germany Shanghai Research Institute of Acupuncture & Moxibustion and Meridian Shanghai China Shanghai Key Laboratory for Acupuncture Mechanism and Acupoint Function Shanghai China

The Na+/Ca2+ exchanger plays an important role in regulation of airway smooth muscle contraction by regulating intracellular calcium, and is a potential target for treatment of asthma. To test modulation of exchanger activity, we used Xenopus oocytes as model system. Na+/Ca2+ exchanger was expressed in the cells by microinjection of cRNA of the exchanger isoform NCX1. The activity of NCX1 was determined as Ni2+-sensitive current under voltage clamp in low Cl– medium and in the presence of the Cl–-channel inhibitor niflumic acid. Only this composition of solution allowed determining NCX1-mediated current with sufficient accuracy. Among a few tested Chinese herbal drugs, glycyrrhizic acid turned out to be a potent inhibitor of NCX1 with an apparent IC50 value of 40 μM. Previous work had revealed elevated cyclophylin A concentration in serum of asthmatic rats after receiving acupuncture treatment. Extracellular incubation of the oocytes in cyclophylin A for one day led to significant inhibition with an apparent IC50 value of about 1 μM. We suggest that effects of acupuncture and application of glycyrrhizic acid as an active constituent of Chinese medicine for treatment of asthma symptoms may partially be attributed to inhibition of the reversed mode of NCX1 and that these compounds may stimulate the search for new anti-asthmatic drugs.

关键词： Sodium-Calcium Exchanger Asthma Voltage Clamp Glycyrrhizic Acid Cyclophylin A

TRPV2条件性基因剔除小鼠的建立及其基本表型分析

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中国细胞生物学学报 2013年第4期35卷 502-509页

作者：王瑶莉张梦杰 wolfgang schwarz 蔡蕾匡颖费俭同济大学生命科学与技术学院上海200092 上海针灸经络研究中心上海201203 上海南方模式生物研究中心上海201203

TRPV2是瞬时感受器离子通道蛋白亚家族成员之一,主要分布在感受伤害性热痛刺激和机械刺激的有髓Aδ神经和少部分C神经纤维中,在机体的热痛感觉、机械刺激感觉过程中发挥着重要的作用。此外,该基因在脑和多种非神经组织中也有表达,参与... 详细信息

TRPV2是瞬时感受器离子通道蛋白亚家族成员之一,主要分布在感受伤害性热痛刺激和机械刺激的有髓Aδ神经和少部分C神经纤维中,在机体的热痛感觉、机械刺激感觉过程中发挥着重要的作用。此外,该基因在脑和多种非神经组织中也有表达,参与维持体内Ca2+、Mg2+的离子平衡,暗示了其功能的多样性[1]。该研究建立了TRPV2基因条件性剔除小鼠模型,并研究了TRPV2基因全身性剔除小鼠在出生率、体重、血压、学习记忆、运动协调能力等方面的表型变化,结果显示,与野生型相比,纯合突变小鼠的出生率明显偏低,但成年雄鼠在上述指标方面并无显著差异。

关键词： TRPV2 条件性基因敲除表型分析发育体重行为

A FLUID MECHANICS MODEL OF TISSUE FLUID FLOW IN LIMB CONNECTIVE TISSUE——A MECHANISM OF ACUPUNCTURE SIGNAL TRANSMISSION

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Journal of Hydrodynamics 2009年第5期21卷 675-684页

作者： ZHANG Di YAO Wei DING Guang-hong YANG Jing schwarz wolfgang FEI Lun LIU Fang SHEN Xue-yong LAO Li-xing Shanghai Research Center of Acupuncture and Meridian Department of Mechanics and Engineering Science Fudan University Shanghai 200433 China Anatomy Department of the Second Medical University of PLA Shanghai 200433 China Acupuncture College Shanghai University of Traditional Chinese Medicine Shanghai 200032 China Center for Integrative Medicine University of Maryland School of Medicine Baltimore MD 21201 USA

This article explores the mechanisms of acupuncture meridians by determining characteristics of the tissue fluid flow in the connective tissue along meridians. Based on deep dissection of acupoints on the upper and lower limbs of the human body and micro and macro observation and measurement, a mathematical model of the flow of tissue fluid in interosseous membranes is constructed. It is shown that the signal transmission along acupuncture meridians may be determined by unique anatomical and physiological factors that govern the flow of tissue fluid in the connective tissue and involve mast cell degranulation. Our results provide a preliminary explanation of the phenomenon of the ＂de qi＂ sensation and the mechanism of acupuncture signal transmission along acupuncture meridians.

关键词： acupuncture meridian tissue-fluid flow mast cell

Inhibition of P2X_7 Receptor by Extracts of Chinese Medicine

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Journal of Acupuncture and Tuina Science 2008年第5期6卷 286-288页

作者： Andreas Spielmann 顾全保马春辉黄成钢宣利江 wolfgang schwarz Max-Planck-Institute for Biophysics Frankfurt/MainGernamy Shanghai Research Center of Acupuncture and Meridian Shanghai 201203R R.China Shanghai Institute of Materia Medica Chinese Academy of SciencesShanghai 201203P.R.China Max-Planck-Institute for Biophysics Frankfurt/MainGernamy Shanghai Research Center of Acupuncture and MeridianShanghai 201203E R.China

Objective： To investigate the influence of Acorus gramineus （Soland）, a crude extract, SCP01, and a purified component, SCP02, and of Rosmarinus officinalis L., X0728 on human mast cells （HMC-1 Cell Line）. Methods： Current-voltage of P2X7 receptors on human mast cell membrane activated by ATP was recorded by the whole-cell patch clamp technique. Results： The current at -100 mV mediated by P2X7 was inhibited by （27.6 ± 2.0） % in the presence of 40 μg/mL SCP01 and by （29.5 ± 2.2） % in the presence of 40 μg/mL SCP02, which was identified as α-asarone. 42 μg/mL of the commercially available α-asarone inhibited the P2X7-mediated current by （52.2 ± 2.0） %. In contrast to SCP01 and SCP02, 40 μg/mL X0728 provoked stimulation of the current by （28.6 ± 2.8） %. All effects were voltage- independent. Conclusion： The inhibition of P2X7 by α-asarone will inhibit intracellular calcium increase and this may account for the inhibition of reported excitotoxic cell death. The pharmacological function of P2X7 stimulation by X0728 needs further investigation.

关键词： Purinoceptor P2X1 Acorus Tatarinowii Asarone Rosmarinus Patch-Clamp Technique

Val 70, Phe 72 and the last seven amino acid residues of C-terminal are essential to the function of norepinephrine transporter

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Cell Research 1998年第4期8卷 311-315页

作者： LIU YAN HONG FANG HUANG JIAN FEI JIAN XING ZHAO QUANG BAO GU wolfgang schwarz LI HE GUO (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China)(Max-Plank Institute for Biophysik, Kennedy allee 70,60596 Frankfurt/M G Max-Plank Institute for Biophysik Kennedy allee 70 Frankfurt/M 60596 Germany

The norepinephrine transporter(NET) is a member of the Na+/Cl- dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. To delineate the critical amino acid residues and the function of C-terminal in regulating transport activity of NET, here we constructed two site mutants (V70F, F72V; V70I, F72V) and one C-terminal truncated mutant (△611-617). The wild type and mutants of NET were expressed in Xenopus oocytes by injection of their cRNA. We found that all of these mutants lost their transport activity. These results indicate that the amino acid residues of V70 and F72 3 and the last seven amino acids of C-terminal are essential to the transport activity of NET.

关键词： Norepinephrine transporter neurotransmitter uptake mutagenesis

Interaction of δ-opioid Receptor with Membrane Transporters: Possible Mechanisms in Pain Suppression by Acupuncture

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Journal of Acupuncture and Tuina Science 2008年第5期6卷 298-300页

作者：杨智杰鲍国斌邓海平杜慧明顾全保裴钢濮璐 wolfgang schwarz 夏鹏 Max-Planck-Institute for Biophysics Frankfurt/MainGermany Shanghai Research Center of Acupuncture and MeridianShanghai 201203P.R.China Max-Planck-Institute for Biophysics Frankfurt/MainGermany MPG Guest Laboratory at the Partner Institute of Computational BiologyShanghai Institutes of Biological Science Shanghai 200031R R.China Shanghai Research Center of Acupuncture and Meridian Shanghai 201203P.R.China MPG Guest Laboratory at the Partner Institute of Computational Biology Shanghai Institutes of Biological ScienceShanghai 200031R R.China Max-Planck-Institute for Btophysics Frankturt/MainGermany Shanghai Research Center of Acupuncture and MeridianShanghai 201203R R.China MPG Guest Laboratory at the Partner Institute of Computational BiologyShanghai Institutes of Biological Science Shanghai 200031R R.China

Objective： To investigate the possible mechanisms in acupuncture analgesia by interaction of δ-opioid receptor with neurotransmitter transport proteins or the Na^＋-K^＋ pump. Methods： Microinjection of respective heterologous cRNA into the Xenopus oocytes as a model system, and measurement of steady-state currents under two-electrode voltage clamp. Results： The co-expression of the 8-opioid receptor with GAT1, EAAC 1 or the sodium pump resulted in reducing activity of the respective transporter. Opioid receptor activation affected transporter activity in different ways： 1） GAT1 was further inhibited; 2） EAAC1 was stimulated; 3） Na^＋-K^＋ pump activity interfered with agonist sensitivity of DOR. Pump inhibition led to higher sensitivity for DPDPE. Conclusion： GABA transporter inhibition and glutamate transporter stimulation may counteract pain sensation by affecting the neurotransmitter concentration in the synaptic cleft and, therefore, may contribute synergistically to pain suppression by acupuncture. Sodium pump inhibition by endogenous ouabain may amplify these effects. These synergistic effects may be the molecular mechanism of inhibiting pain sense and/or acupuncture analgesia.

关键词： Acupuncture Analgesia Receptors, Opioid Neurotransmitter Transport Proteins Protein Interaction Domains and Motifs