酸敏感离子通道与炎性痛中枢敏化机制的研究
发布时间：2018-08-02 11:18
【摘要】： 外周炎症和组织损伤都能导致病理痛的产生。作为伤害性信号传递的中枢第一站，脊髓是伤害性信息传递和调控的重要部位。已有的报道表明，脊髓的中枢敏化是病理痛形成的主要机制之一；然而，人们对这种敏化的机制却了解甚少。在炎性或神经病理性疼痛状态下，痛觉过敏产生的重要原因之一是由于脊髓背角神经元表达的离子通道和受体受到调控，从而使其持续过度兴奋。目前，有多种离子通道和受体被认为和疼痛的产生与维持相关。 酸敏感离子通道(ASICs)是一类由质子(H~+)激活的阳离子通道，属于ENaC／DEG家族，有四个编码的基因：ASIC1、ASIC2、ASIC3、ASIC4。它们编码六种亚基蛋白：ASIC1a、ASIC1b、ASIC2a、ASIC2b、ASIC3和ASIC4。酸敏感离子通道的N-端和C-端均在胞内，有两个跨膜区，胞外环上富含半胱氨酸。最新的结构生物学实验发现，体内的ASICs是由相同的或不同的亚基构成的三聚体。除了ASIC3和ASIC1b只在外周神经系统中特异表达外，ASICs其他亚基在外周和中枢神经系统都有存在。目前对ASICs的大部分研究主要集中在外周神经系统中，认为ASICs参与了痛觉、味觉以及机械感觉等的信息传递；而对中枢神经系统中的ASICs功能了解较少，尤其是对病理条件下ASICs的变化如何了解不多。 目前，背根神经节(DRG)的ASICs研究已有大量报道，并被认为与机械感受和痛觉相关。对于ASICs在中枢痛觉感受中的作用目前尚未见报道。已有报道提及ASICs在脊髓中有表达，另外在脊髓培养神经元中也观察到了酸诱导的电流。然而，对于脊髓背角神经元中酸诱导电流的分子基础、通道特性及其生理、病理功能却很不清楚。 因此，在本课题中，我们首先证明了脊髓背角神经元中主要表达Ca~(2+)通透性的ASIC1a通道。进一步，我们探讨了ASIC1a通道参与脊髓水平痛觉传递和调制的作用及其机制。主要实验结果如下： 1．脊髓背角神经元中酸敏感离子通道特性研究 运用电生理的方法，我们首先发现，对于急性分离的脊髓背角神经元，Psalmotoxin 1(PcTX1)，一种特异的ASIC1a同聚体通道抑制剂，能够非常明显的抑制酸性溶液(pH6.0)引发的内向电流。采用RNA干扰的手段，我们特异的抑制培养的脊髓背角神经元中ASIC1a的表达，随后的电生理实验发现，酸性溶液引发的内向电流也明显的降低。这提示我们ASIC1a同聚体通道是脊髓背角神经元中主要的质子感受器。进一步的钙离子成像实验表明，在脊髓背角神经元中，ASIC1a同聚体通道介导了酸性溶液引起的Ca~(2+)内流。 2．脊髓背角酸敏感离子通道与炎性痛觉超敏 运用完全弗氏佐剂(CFA)致炎模型，我们进一步研究了ASIC1a通道在炎症痛中的作用。行为学实验表明，大鼠脚底CFA致炎能够导致明显的热和机械超敏化，这种行为超敏化能够持续超过七天。然而，鞘内注射PcTX1能够明暴地削弱这种外周炎症引起的热和机械超敏化。同时，鞘内注射阿米洛利(anailoride)，一种非特异性的ASICs抑制剂，也产生同样的效果。进一步，我们使用一条特异性的抑制ASIC1a蛋白表达的反义寡核苷酸发现，与急性抑制ASIC1a功能相似，在抑制脊髓背角ASIC1a蛋白表达之后，外周炎症引起的热和机械超敏化也被明显削弱。与之相反的是，鞘内注射PcTX1并不影响正常大鼠的伤害性信号传递。 为了进一步证明脊髓背角的ASIC1a通道参与炎性痛觉超敏，我们采用福尔马林模型阐明脊髓背角和背根神经节中的ASIC1a通道的功能异同。给大鼠脚背注射福尔马林能够使其产生持续约一小时的伤害性反应。根据持续时间，这种反应被分为两相：第一相仅与背根神经节细胞超兴奋相关；第二相与脊髓背角神经元超兴奋关系密切。我们的实验表明，鞘内注射PcTX1仅抑制第二相持续反应，而不影响第一相反应。相反地，脚底注射PcTX1对伤害性传递没有作用。结合这两个实验，我们进一步证明脊髓背角ASIC1a通道参与伤害性信号传递。 3．脊髓背角酸敏感离子通道影响脊髓背角神经元的兴奋性和可塑性 脊髓背角ASIC1a通道是如何参与伤害性信号传递的呢?免疫组织化学实验表明，在外周致炎后，脊髓背角神经元中ASIC1a的蛋白表达量明显增加。与之相反，ASIC1a在背根神经节细胞的中枢端和胶质细胞中的表达却很少。进一步的在体电生理实验表明，电刺激大鼠足底神经激动C-纤维时，脊髓背角广动力域(WDR)神经元的兴奋性改变，其表现形式为自发放电增强(“wind-up”现象)。而在脊髓急性给予PcTX1能构有效的抑制“wind-up”现象。随后，向WDR神经元的外周感受野中心部位分别施加不同强度的刷(brushing)、压(pressing)和夹(pinching)等机械性刺激；而在外周致炎的条件下，WDR神经元对不同强度brushing、pressing和pinching等机械性刺激反应性明显增强，说明有机械性痛敏产生。进一步的实验表明，脊髓急性给予PcTX1，并不影响正常大鼠脊髓背角WDR神经元对机械性刺激的反应。然而，脊髓急性给予PcTX1能够明显抑制致炎大鼠的WDR神经元对伤害性机械刺激(包括pressing和pinching)的反应性，而对非伤害性机械刺激(brushing)引起的反应没有作用。 4．炎性痛中脊髓背角酸敏感离子通道上调机制初探 在炎症过程中，大量的神经递质(Glu)和调质[P物质(SP)、BDNF]等从DRG细胞突触终末释放，影响脊髓背角的突触传递。我们发现，慢性施加BDNF能够浓度依赖性的增加培养的脊髓背角神经元中ASIC1a表达。进一步，给大鼠鞘内长期注射BDNF受体-TrkB的抑制剂K252a，能够抑制外周炎症引起的脊髓背角ASIC1a的增加和痛行为。而且，在K252a慢性处理的大鼠脊髓中注射PcTX1，不能进一步的抑制痛行为。这提示我们可能是BDNF介导了炎性痛中ASIC1a的上调。另外，穿孔膜片钳实验表明，PI3K信号通路，而不是PKA和CaMKⅡ信号通路，介导了BDNF引起的ASIC1a上调。 综上所述，本文鉴定了Ca~(2+)通透性的ASIC1a同聚体通道是脊髓背角主要存在的ASIC；外周炎症导致脊髓背角神经元中ASIC1a的表达增加；过高表达的ASIC1a通道增加脊髓背角神经元的兴奋性和可塑性，并参与炎性痛觉敏化的过程。该研究结果揭示了生物体内痛觉诱发和维持的一种新机制，，并且提示脊髓背角的ASIC1a通道可能成为研究镇痛药物的新靶点。
[Abstract]:Peripheral inflammation and tissue damage can cause pathological pain. As the first station of nociceptive signal transmission, the spinal cord is an important part of the transmission and regulation of nociceptive information. It has been reported that the central sensitization of the spinal cord is one of the main mechanisms of the formation of pathological pain; however, few people know the mechanism of this sensitization. In the state of inflammatory or neuropathic pain, one of the important causes of hyperalgesia is the regulation of ion channels and receptors expressed in the dorsal horn neurons of the spinal cord, which makes it excessively excited. At present, a variety of ion channels and receptors are considered to be associated with the production and maintenance of pain.
Acid sensitive ion channel (ASICs) is a class of cationic channels activated by protons (H~+), belonging to the ENaC / DEG family. There are four encoded genes: ASIC1, ASIC2, ASIC3, and ASIC4., which encode six subunits: ASIC1a, ASIC1b, ASIC2a, ASIC2b, and two transmembrane regions. The outer ring is rich in cysteine. The latest structural biology experiments have found that the ASICs in the body is a trimer composed of the same or different subunits. In addition to the specific expression of ASIC3 and ASIC1b in the peripheral nervous system, the other subunits of ASICs are stored in the peripheral and central nervous systems. Most of the research on ASICs is mainly concentrated. In the peripheral nervous system, ASICs is considered to be involved in the transmission of information of pain, taste and mechanical sensation, but less on the ASICs function in the central nervous system, especially on how the changes of ASICs in the pathological conditions are not well understood.
Currently, the ASICs study of the dorsal root ganglion (DRG) has been widely reported and is considered to be associated with mechanical perception and pain. The role of ASICs in central pain perception has not yet been reported. It has been reported that ASICs has been expressed in the spinal cord, and acid induced current is also observed in spinal cultured neurons. The molecular basis, channel characteristics, physiological and pathological functions of acid induced currents in neurons of the dorsal horn of the spinal cord are unclear.
Therefore, in this subject, we first demonstrated the ASIC1a channel that mainly expressed Ca~ (2+) permeability in the spinal dorsal horn neurons. Further, we explored the role and mechanism of the ASIC1a channel in the spinal level of spinal level pain transmission and modulation. The main experimental results are as follows:
Characteristics of acid sensing ion channels in spinal dorsal horn neurons in 1.
Using electrophysiological methods, we first found that, for the acute isolated spinal dorsal horn neurons, Psalmotoxin 1 (PcTX1), a specific ASIC1a homopolymer inhibitor, can significantly inhibit the inward current induced by acidic solution (pH6.0). Using RNA interference, we specifically inhibit the cultured spinal dorsal horn neurons. The expression of ASIC1a, followed by electrophysiological experiments, found that the inward current caused by the acid solution was also significantly reduced. This suggests that our ASIC1a homopolymer channel is the main proton receptor in the spinal dorsal horn neurons. Further calcium imaging experiments showed that the ASIC1a homopolymer channel mediated acid dissolution in the dorsal horn of the spinal cord of the spinal cord. Ca~ (2+) internal flow caused by liquid.
2. acid sensitive ion channels in spinal dorsal horn and inflammatory hyperalgesia
Using the complete Freund adjuvant (CFA) induced inflammatory model, we further studied the role of the ASIC1a channel in inflammatory pain. Behavioral experiments showed that the CFA induced inflammation in the sole of the rat can lead to obvious thermal and mechanical hyper sensitization, and this behavioral hypersensitization can last over seven days. However, intrathecal PcTX1 can clearly weaken the peripheral inflammation. Thermal and mechanical hyper sensitization. Meanwhile, intrathecal amiloride (anailoride), a nonspecific ASICs inhibitor, also produced the same effect. Further, we use a specific antisense oligodeoligonucleotide to inhibit the expression of ASIC1a protein, similar to the acute inhibition of ASIC1a function, in inhibiting the ASIC1a protein table in the dorsal horn of the spinal cord. After that, the heat and mechanical hyper sensitization caused by peripheral inflammation were also significantly weakened. In contrast, intrathecal injection of PcTX1 did not affect the nociceptive signal transmission in normal rats.
To further demonstrate the involvement of the ASIC1a channel in the dorsal horn of the spinal cord in inflammatory pain hypersensitivity, we used the Faure Marin model to elucidate the functional differences in the ASIC1a channel in the dorsal horn of the spinal cord and the dorsal root ganglion. The injections of the dorsum of the rat to the dorsum of the rat were able to produce a persistent nociceptive response for about one hour. The first phase was related only to the superexcitability of the dorsal root ganglion cells; the second phase was closely related to the superexcitability of the dorsal horn neurons of the spinal cord. Our experiment showed that intrathecal injection of PcTX1 only inhibited the second phase, but did not affect the first phase reaction. On the contrary, the infoot injection of PcTX1 had no effect on nociceptive transmission. We further demonstrate that the ASIC1a channel in the dorsal horn of the spinal cord is involved in noxious signaling.
3. the acid sensitive ion channel of spinal dorsal horn affects the excitability and plasticity of neurons in spinal dorsal horn.
How does the spinal dorsal horn ASIC1a channel participate in nociceptive signal transduction? Immunohistochemical experiments show that the protein expression of ASIC1a in the dorsal horn neurons of the spinal cord increases obviously after the peripheral inflammation. On the contrary, the expression of ASIC1a in the central and glial cells of the dorsal root ganglion cells is few. The experimental results showed that the excitatory changes of the spinal dorsal horn (WDR) neurons were stimulated by electrical stimulation of the C- fibers in the plantar nerve of the rat, and the manifestation was the spontaneous discharge enhancement ("wind-up"). The acute administration of PcTX1 in the spinal cord could effectively inhibit the "wind-up" image. Then, the central part of the peripheral receptive field of the WDR neurons was divided. Do not apply mechanical stimuli such as brushing, pressing and pinching, and the mechanical irritation of WDR neurons to brushing, pressing, pinching and other mechanical stimuli in the peripheral inflammation, indicating that mechanical pain is produced. Further experiments suggest that PcTX1 is given acute spinal cord in the spinal cord. The response to mechanical stimulation of WDR neurons in the dorsal horn of the spinal cord of normal rats was not affected. However, acute spinal cord administration of the spinal cord in the spinal cord could significantly inhibit the reactivity of WDR neurons in the inflammatory rats to nociceptive mechanical stimulation (including pressing and pinching), but had no effect on the response to non nociceptive mechanical stimulation (brushing).
Mechanism of upregulated acid sensing ion channels in spinal dorsal horn in 4. inflammatory pain
During the process of inflammation, a large number of neurotransmitters (Glu) and substance [P (SP) and BDNF] are released from DRG cell synaptic terminals, affecting synaptic transmission in the dorsal horn of the spinal cord. We found that the chronic exertion of BDNF can increase the ASIC1a expression in the cultured spinal dorsal horn neurons in a concentration dependent manner. Further, the long-term injection of BDNF receptor -TrkB in the rat sheath K252a, a inhibitor, can inhibit the increase and pain behavior of the spinal dorsal horn ASIC1a caused by peripheral inflammation. Moreover, the injection of PcTX1 in the spinal cord of K252a chronic treated rats can not further inhibit the pain behavior. This suggests that we may mediate the up regulation of ASIC1a in inflammatory pain. Furthermore, the perforated patch clamp experiment indicates that the PI3K signal pathway is indicated by the perforated patch clamp test. Rather than PKA and CaMK II signaling pathways, BDNF induced ASIC1a upregulation.
In summary, the Ca~ (2+) permeability ASIC1a homopolymer channel is the main ASIC in the dorsal horn of the spinal cord; peripheral inflammation leads to an increase in the expression of ASIC1a in the dorsal horn neurons of the spinal cord; the exorbitant ASIC1a channel increases the excitability and plasticity of the spinal dorsal horn neurons and participates in the process of inflammatory pain sensitization. The results reveal a new mechanism of pain induction and maintenance in vivo, and suggest that ASC1a channel in spinal dorsal horn may be a new target for the study of analgesics.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363
本文编号：2159259
[Abstract]:Peripheral inflammation and tissue damage can cause pathological pain. As the first station of nociceptive signal transmission, the spinal cord is an important part of the transmission and regulation of nociceptive information. It has been reported that the central sensitization of the spinal cord is one of the main mechanisms of the formation of pathological pain; however, few people know the mechanism of this sensitization. In the state of inflammatory or neuropathic pain, one of the important causes of hyperalgesia is the regulation of ion channels and receptors expressed in the dorsal horn neurons of the spinal cord, which makes it excessively excited. At present, a variety of ion channels and receptors are considered to be associated with the production and maintenance of pain.
Acid sensitive ion channel (ASICs) is a class of cationic channels activated by protons (H~+), belonging to the ENaC / DEG family. There are four encoded genes: ASIC1, ASIC2, ASIC3, and ASIC4., which encode six subunits: ASIC1a, ASIC1b, ASIC2a, ASIC2b, and two transmembrane regions. The outer ring is rich in cysteine. The latest structural biology experiments have found that the ASICs in the body is a trimer composed of the same or different subunits. In addition to the specific expression of ASIC3 and ASIC1b in the peripheral nervous system, the other subunits of ASICs are stored in the peripheral and central nervous systems. Most of the research on ASICs is mainly concentrated. In the peripheral nervous system, ASICs is considered to be involved in the transmission of information of pain, taste and mechanical sensation, but less on the ASICs function in the central nervous system, especially on how the changes of ASICs in the pathological conditions are not well understood.
Currently, the ASICs study of the dorsal root ganglion (DRG) has been widely reported and is considered to be associated with mechanical perception and pain. The role of ASICs in central pain perception has not yet been reported. It has been reported that ASICs has been expressed in the spinal cord, and acid induced current is also observed in spinal cultured neurons. The molecular basis, channel characteristics, physiological and pathological functions of acid induced currents in neurons of the dorsal horn of the spinal cord are unclear.
Therefore, in this subject, we first demonstrated the ASIC1a channel that mainly expressed Ca~ (2+) permeability in the spinal dorsal horn neurons. Further, we explored the role and mechanism of the ASIC1a channel in the spinal level of spinal level pain transmission and modulation. The main experimental results are as follows:
Characteristics of acid sensing ion channels in spinal dorsal horn neurons in 1.
Using electrophysiological methods, we first found that, for the acute isolated spinal dorsal horn neurons, Psalmotoxin 1 (PcTX1), a specific ASIC1a homopolymer inhibitor, can significantly inhibit the inward current induced by acidic solution (pH6.0). Using RNA interference, we specifically inhibit the cultured spinal dorsal horn neurons. The expression of ASIC1a, followed by electrophysiological experiments, found that the inward current caused by the acid solution was also significantly reduced. This suggests that our ASIC1a homopolymer channel is the main proton receptor in the spinal dorsal horn neurons. Further calcium imaging experiments showed that the ASIC1a homopolymer channel mediated acid dissolution in the dorsal horn of the spinal cord of the spinal cord. Ca~ (2+) internal flow caused by liquid.
2. acid sensitive ion channels in spinal dorsal horn and inflammatory hyperalgesia
Using the complete Freund adjuvant (CFA) induced inflammatory model, we further studied the role of the ASIC1a channel in inflammatory pain. Behavioral experiments showed that the CFA induced inflammation in the sole of the rat can lead to obvious thermal and mechanical hyper sensitization, and this behavioral hypersensitization can last over seven days. However, intrathecal PcTX1 can clearly weaken the peripheral inflammation. Thermal and mechanical hyper sensitization. Meanwhile, intrathecal amiloride (anailoride), a nonspecific ASICs inhibitor, also produced the same effect. Further, we use a specific antisense oligodeoligonucleotide to inhibit the expression of ASIC1a protein, similar to the acute inhibition of ASIC1a function, in inhibiting the ASIC1a protein table in the dorsal horn of the spinal cord. After that, the heat and mechanical hyper sensitization caused by peripheral inflammation were also significantly weakened. In contrast, intrathecal injection of PcTX1 did not affect the nociceptive signal transmission in normal rats.
To further demonstrate the involvement of the ASIC1a channel in the dorsal horn of the spinal cord in inflammatory pain hypersensitivity, we used the Faure Marin model to elucidate the functional differences in the ASIC1a channel in the dorsal horn of the spinal cord and the dorsal root ganglion. The injections of the dorsum of the rat to the dorsum of the rat were able to produce a persistent nociceptive response for about one hour. The first phase was related only to the superexcitability of the dorsal root ganglion cells; the second phase was closely related to the superexcitability of the dorsal horn neurons of the spinal cord. Our experiment showed that intrathecal injection of PcTX1 only inhibited the second phase, but did not affect the first phase reaction. On the contrary, the infoot injection of PcTX1 had no effect on nociceptive transmission. We further demonstrate that the ASIC1a channel in the dorsal horn of the spinal cord is involved in noxious signaling.
3. the acid sensitive ion channel of spinal dorsal horn affects the excitability and plasticity of neurons in spinal dorsal horn.
How does the spinal dorsal horn ASIC1a channel participate in nociceptive signal transduction? Immunohistochemical experiments show that the protein expression of ASIC1a in the dorsal horn neurons of the spinal cord increases obviously after the peripheral inflammation. On the contrary, the expression of ASIC1a in the central and glial cells of the dorsal root ganglion cells is few. The experimental results showed that the excitatory changes of the spinal dorsal horn (WDR) neurons were stimulated by electrical stimulation of the C- fibers in the plantar nerve of the rat, and the manifestation was the spontaneous discharge enhancement ("wind-up"). The acute administration of PcTX1 in the spinal cord could effectively inhibit the "wind-up" image. Then, the central part of the peripheral receptive field of the WDR neurons was divided. Do not apply mechanical stimuli such as brushing, pressing and pinching, and the mechanical irritation of WDR neurons to brushing, pressing, pinching and other mechanical stimuli in the peripheral inflammation, indicating that mechanical pain is produced. Further experiments suggest that PcTX1 is given acute spinal cord in the spinal cord. The response to mechanical stimulation of WDR neurons in the dorsal horn of the spinal cord of normal rats was not affected. However, acute spinal cord administration of the spinal cord in the spinal cord could significantly inhibit the reactivity of WDR neurons in the inflammatory rats to nociceptive mechanical stimulation (including pressing and pinching), but had no effect on the response to non nociceptive mechanical stimulation (brushing).
Mechanism of upregulated acid sensing ion channels in spinal dorsal horn in 4. inflammatory pain
During the process of inflammation, a large number of neurotransmitters (Glu) and substance [P (SP) and BDNF] are released from DRG cell synaptic terminals, affecting synaptic transmission in the dorsal horn of the spinal cord. We found that the chronic exertion of BDNF can increase the ASIC1a expression in the cultured spinal dorsal horn neurons in a concentration dependent manner. Further, the long-term injection of BDNF receptor -TrkB in the rat sheath K252a, a inhibitor, can inhibit the increase and pain behavior of the spinal dorsal horn ASIC1a caused by peripheral inflammation. Moreover, the injection of PcTX1 in the spinal cord of K252a chronic treated rats can not further inhibit the pain behavior. This suggests that we may mediate the up regulation of ASIC1a in inflammatory pain. Furthermore, the perforated patch clamp experiment indicates that the PI3K signal pathway is indicated by the perforated patch clamp test. Rather than PKA and CaMK II signaling pathways, BDNF induced ASIC1a upregulation.
In summary, the Ca~ (2+) permeability ASIC1a homopolymer channel is the main ASIC in the dorsal horn of the spinal cord; peripheral inflammation leads to an increase in the expression of ASIC1a in the dorsal horn neurons of the spinal cord; the exorbitant ASIC1a channel increases the excitability and plasticity of the spinal dorsal horn neurons and participates in the process of inflammatory pain sensitization. The results reveal a new mechanism of pain induction and maintenance in vivo, and suggest that ASC1a channel in spinal dorsal horn may be a new target for the study of analgesics.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R363
【引证文献】
相关硕士学位论文 前1条
1 陆瑶;外周酸敏感离子通道在蜜蜂毒诱致的自发痛、痛敏和炎症中的作用[D];辽宁医学院;2012年
本文编号：2159259
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