桉叶素对大鼠脊髓胶状质神经元自发兴奋性传递的影响
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摘要
目的：TRP(transient receptor potential)通道是一类广泛存在神经系统的跨膜蛋白质，是一种非选择性阳离子通道。 TRP通道家族包含七种亚族，其中多种TRP通道参与疼痛信号的传递。脊髓背角胶状质（SG）含有丰富且经典的递质及其受体，是伤害性信息传入的第一站，是脊髓中神经结构和化学组成最复杂的区域，是疼痛调制的关键部位。尽管TRP通道在脊髓SG神经元痛觉传递的过程中发挥着重要的作用；但其作用尚不明确。TRP通道可被多种植物源性化学物质激活，如辣椒素和异硫氰酸盐等。桉叶素存在于多种植物精油中，最近研究报道它具有镇痛的作用，但其作用机制尚不清楚。本研究采用全细胞膜片钳技术，观察1,4-桉叶素和1,8-桉叶素对脊髓胶状质神经元谷氨酸能自发兴奋性传递的影响，探讨其作用机制是否与DRG神经元上的TRP通道有关。
方法：雄性SD大鼠(6-8周)，腹腔注射乌拉坦(1.2g/kg)麻醉后，行腰骶部椎板切除术。20min后，取出腰骶部脊髓(L1-S3)，利用振动切片机切取650-700μm厚的脊髓切片，放入记录槽内的尼龙网上。人工脑脊液(Krebs)灌流1h后，盲法全细胞膜片钳技术记录SG神经元的sEPSC。将1,4-桉叶素、1,8-桉叶素、(-)薄荷醇、辣椒素、TRPV1通道抑制剂抗辣椒素和TRPA1通道抑制剂HC-030031分别灌注记录槽。
结果：固定电位保持在-70mV，0.05-7mM1,8-桉叶素灌注3min，sEPSC的频率增加呈剂量依赖性，EC50为2.5mM (Hill系数为2.0)，波幅无明显改变。0.1-0.7mM1,4-桉叶素灌注3min，sEPSC的频率增加呈剂量依赖性，EC50为0.18mM (Hill系数为7.9)，伴随波幅轻度增加。0.5mM1,4-桉叶素（含有1,8-桉叶素植物中的一种微量提取物）灌注3min，sEPSC的频率增加程度较1,8-桉叶素高，伴随波幅轻微增加。桉叶素引起sEPSC频率增加的效果是可重复的，20min后再次用药有同化作用。桉叶素对自发兴奋性传递的促进作用不能被TRPV1通道抑制剂抗辣椒素阻断，也不影响辣椒素的作用。但是，桉叶素的作用可被TRPA1通道抑制剂HC-030031抑制。和(-)薄荷醇相比，1,8-桉叶素对sEPSC频率的增加率远大于预期的其通过激活TRPM8而产生的影响。在部分细胞中，桉叶素可引起内向电流。
结论：桉叶素通过激活神经元的TRPA1通道而不是TRPV1通道，引起脊髓SG神经元自发性左旋谷氨酸释放增加。1,4-桉叶素的作用强于1,8-桉叶素，该结论有助于了解作为疼痛和其他疾病治疗靶点的中枢TRPA1通道的特性。
Objective: The transient receptor potential (TRP) channel is a kind of transme-mbrane protein that exist in the nervous system widely, and a kind of nonselectivecation channel. There are seven subfamilies in TRP channels, and many of themregulate the sensation of pain. There are abundant of classical neurotransmitter and itsreceptors in substantia gelatinosa which is the important region to regulate thesensation of pain. Although TRP channels play a role in regulating nociceptivetransmission in spinal substantia gelatinosa (SG) neurons, the properties of the TRPchannels have not yet been examined fully. The central TRP channels are activated byvarious plant-derived chemicals such as capsaicin and isothiocyanate. In many kinds ofessential oil there is cineole which has analgesic effect, but the mechanism is not clearyet. The present study examined the effects of1,4-cineole and1,8-cineole onglutamatergic spontaneous excitatory postsynaptic currents (sEPSC) in the SG neuronsof spinal cord slices by using the whole-cell patch-clamp technique, and investigatewhether they can activate TRP channels.
Methods: Male adult Sprague-Dawley rats (6-8weeks old) were anesthetizedwith urethane (1.2g/kg, intraperitoneal), and then lumbosacral laminectomy wasperformed. The lumbosacral spinal cord (L1-S3) was removed and mounted on avibrating microslicer and then a650-700μm thick transverse slice was cut. The slicewas placed on nylon mesh in the recording chamber, superfused Krebs, one hour laterthe blind whole-cell patch-clamp technique was applied to the SG neurons of the slicesto record the sEPSC.1,4-cineole,1,8-cineole, menthol, capsaicin, capsazepine andHC-030031were applied by superfusion with a change in solutions in the recordingchamber.
Results: Bath-applied1,8-cineole for3min in a range of0.2-7mM increased thefrequency of sEPSC in a dose-dependent manner with a half-maximal effectiveconcentration value of2.5mM (Hill coefficient=2.0) with no significant increase in itsamplitude and unchanged holding currents at a holding potential of-70mV.1,4-Cineole, a minor component of plant extracts containing1,8-cineole, superfused for3min in a range of0.1-0.7mM increased sEPSC frequency (EC50=0.18mM, Hillcoefficient=7.9) effectively more than1,8-cineole with a small increase in itsamplitude. The sEPSC frequency increase produced by cineole was reversible, theeffect of twice was similar with a20min interval. The excitatory transmissionenhancement produced by cineole was unaffected by a TRPV1antagonist capsazepine;the cineole effect was not occluded by capsaicin. On the other hand, the facilitatoryeffect of cineole was inhibited by a TRPA1antagonist HC-030031. The sEPSCfrequency increase produced by1,8-cineole was much greater than that expected fromTRPM8activation by1,8-cineole compared to (-) menthol. Cineole produced aninward current in some of the neurons examined.
Conclusion: It is concluded that cineole enhances the spontaneous release ofL-glutamate onto SG neurons by activating TRPA1but not TRPV1channel; this actionof1,4-cineole is much larger than that of1,8-cineole. This result could serve to knowthe properties of the central TRPA1channels that are therapeutic targets for pain andother disorders.
方法：雄性SD大鼠(6-8周)，腹腔注射乌拉坦(1.2g/kg)麻醉后，行腰骶部椎板切除术。20min后，取出腰骶部脊髓(L1-S3)，利用振动切片机切取650-700μm厚的脊髓切片，放入记录槽内的尼龙网上。人工脑脊液(Krebs)灌流1h后，盲法全细胞膜片钳技术记录SG神经元的sEPSC。将1,4-桉叶素、1,8-桉叶素、(-)薄荷醇、辣椒素、TRPV1通道抑制剂抗辣椒素和TRPA1通道抑制剂HC-030031分别灌注记录槽。
结果：固定电位保持在-70mV，0.05-7mM1,8-桉叶素灌注3min，sEPSC的频率增加呈剂量依赖性，EC50为2.5mM (Hill系数为2.0)，波幅无明显改变。0.1-0.7mM1,4-桉叶素灌注3min，sEPSC的频率增加呈剂量依赖性，EC50为0.18mM (Hill系数为7.9)，伴随波幅轻度增加。0.5mM1,4-桉叶素（含有1,8-桉叶素植物中的一种微量提取物）灌注3min，sEPSC的频率增加程度较1,8-桉叶素高，伴随波幅轻微增加。桉叶素引起sEPSC频率增加的效果是可重复的，20min后再次用药有同化作用。桉叶素对自发兴奋性传递的促进作用不能被TRPV1通道抑制剂抗辣椒素阻断，也不影响辣椒素的作用。但是，桉叶素的作用可被TRPA1通道抑制剂HC-030031抑制。和(-)薄荷醇相比，1,8-桉叶素对sEPSC频率的增加率远大于预期的其通过激活TRPM8而产生的影响。在部分细胞中，桉叶素可引起内向电流。
结论：桉叶素通过激活神经元的TRPA1通道而不是TRPV1通道，引起脊髓SG神经元自发性左旋谷氨酸释放增加。1,4-桉叶素的作用强于1,8-桉叶素，该结论有助于了解作为疼痛和其他疾病治疗靶点的中枢TRPA1通道的特性。
Objective: The transient receptor potential (TRP) channel is a kind of transme-mbrane protein that exist in the nervous system widely, and a kind of nonselectivecation channel. There are seven subfamilies in TRP channels, and many of themregulate the sensation of pain. There are abundant of classical neurotransmitter and itsreceptors in substantia gelatinosa which is the important region to regulate thesensation of pain. Although TRP channels play a role in regulating nociceptivetransmission in spinal substantia gelatinosa (SG) neurons, the properties of the TRPchannels have not yet been examined fully. The central TRP channels are activated byvarious plant-derived chemicals such as capsaicin and isothiocyanate. In many kinds ofessential oil there is cineole which has analgesic effect, but the mechanism is not clearyet. The present study examined the effects of1,4-cineole and1,8-cineole onglutamatergic spontaneous excitatory postsynaptic currents (sEPSC) in the SG neuronsof spinal cord slices by using the whole-cell patch-clamp technique, and investigatewhether they can activate TRP channels.
Methods: Male adult Sprague-Dawley rats (6-8weeks old) were anesthetizedwith urethane (1.2g/kg, intraperitoneal), and then lumbosacral laminectomy wasperformed. The lumbosacral spinal cord (L1-S3) was removed and mounted on avibrating microslicer and then a650-700μm thick transverse slice was cut. The slicewas placed on nylon mesh in the recording chamber, superfused Krebs, one hour laterthe blind whole-cell patch-clamp technique was applied to the SG neurons of the slicesto record the sEPSC.1,4-cineole,1,8-cineole, menthol, capsaicin, capsazepine andHC-030031were applied by superfusion with a change in solutions in the recordingchamber.
Results: Bath-applied1,8-cineole for3min in a range of0.2-7mM increased thefrequency of sEPSC in a dose-dependent manner with a half-maximal effectiveconcentration value of2.5mM (Hill coefficient=2.0) with no significant increase in itsamplitude and unchanged holding currents at a holding potential of-70mV.1,4-Cineole, a minor component of plant extracts containing1,8-cineole, superfused for3min in a range of0.1-0.7mM increased sEPSC frequency (EC50=0.18mM, Hillcoefficient=7.9) effectively more than1,8-cineole with a small increase in itsamplitude. The sEPSC frequency increase produced by cineole was reversible, theeffect of twice was similar with a20min interval. The excitatory transmissionenhancement produced by cineole was unaffected by a TRPV1antagonist capsazepine;the cineole effect was not occluded by capsaicin. On the other hand, the facilitatoryeffect of cineole was inhibited by a TRPA1antagonist HC-030031. The sEPSCfrequency increase produced by1,8-cineole was much greater than that expected fromTRPM8activation by1,8-cineole compared to (-) menthol. Cineole produced aninward current in some of the neurons examined.
Conclusion: It is concluded that cineole enhances the spontaneous release ofL-glutamate onto SG neurons by activating TRPA1but not TRPV1channel; this actionof1,4-cineole is much larger than that of1,8-cineole. This result could serve to knowthe properties of the central TRPA1channels that are therapeutic targets for pain andother disorders.
引文
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