趋化因子MCP-1对辣椒素受体（TRPV1）功能的易化作用及其机制研究
摘要
单核细胞趋化蛋白-1 (monocyte chemoattractant protein-1;MCP-1)属于CC类趋化因子。近年来的研究表明,在神经病理性疼痛、炎症或其他病理性变等情况下,趋化因子MCP-1及其受体CCR2在神经元和胶质细胞上表达上调,并能直接引起痛觉神经元兴奋性持续增高、伤害性感受器基因表达改变。MCP-1被认为是疼痛家族的一个新成员,是一个重要的导致疼痛发生的因子。
辣椒素受体TRPV1在背根神经节、三叉神经节等中小型神经元的C类无髓纤维或Aδ神经纤维等疼痛感受器上大量表达,是配体门控的非选择性阳离子通道。越来越多的研究表明,TRPV1在痛觉的产生及痛觉敏感性增强的病理过程中扮演着重要角色。
为了进一步研究MCP-1对TRPV1疼痛效应的调控机制,本课题在大鼠慢性压迫背根神经节(CCD)的模型上,应用Real-time PCR和免疫组织化学染色明确CCD背根神经节细胞上,MCP-1受体CCR2与TRPV1等的表达与分布;用Western blot的方法对CCD、CCI以及SNL三种神经损伤模型上CCR2在DRG中的表达进行了定量分析;应用行为学测试方法观察MCP-1是否易化CCD大鼠上TRPV1介导的痛觉行为,应用全细胞膜片钳及钙离子成像技术,观察了MCP-1对CCD大鼠DRG神经元上TRPV1通道的调控作用及可能的细胞内信号途径。主要研究结果如下;
1应用Real-time PCR方法显示,CCD后TRPV1的mRNA表达增加并应用免疫组化方法显示CCD后TRPV1表达增加,用Western blot的方法对CCD、CCI以及SNL三种神经损伤模型上CCR2在DRG中的表达进行了定量分析,结果表明,三种损伤方式都能引起CCR2的表达量增加,尤以CCD损伤后CCR2增加特别明显,在DRG组织切片和急性分离的DRG神经元上用免疫荧光方法显示CCR2和TRPV1共表达于CCD神经元,这就为MCP-1对TRPV1的调控作用提供形态学基础。
2动物自发痛反应行为测试结果显示,MCP-1增强Capsaicin诱发的疼痛行为,与单独给与MCP-1和Capsaicin有显著性差异。
3全细胞膜片钳记录显示,MCP-1增强在急性分离的CCD神经元上TRPV1受体激动剂Capsaicin诱导的膜电位去极化幅度及内向电流幅度。应用Ca~(2+)成像技术观察对急性分离的CCD神经元在MCP-1作用前后对Capsaicin诱发的细胞内游离钙离子的反应。结果表明,MCP-1(100 ng/ml)增强Capsaicin诱发的细胞内钙荧光强度
4在急性分离的CCD神经元上,应用Ca~(2+)成像技术观察PLC阻断剂U73122对MCP-1增强Capsaicin诱发的细胞内钙荧光强度的调控作用。结果显示U73122有效地阻断MCP-1对TRPV1的增强效应。动物自发痛反应行为测试结果显示,PLC阻断剂U73122有效地阻断MCP-1对Capsaicin引发的自发痛的易化作用。
综上所述,提示CCD损伤后DRG神经元上调表达的CCR2,在MCP-1作用激活后可通过增强TRPV1的功能,使痛觉神经元的兴奋性提高,从而参与痛觉过敏的产生。
MCP-1 (monocyte chemoattractant protein-1) is one of the CC chemokine subfamily members. The expression of MCP-1 was increased in neurons and glial cells after the nerve injuries,inflammation or other pathological changes. MCP-1 can directly enhance the excitability of neurons.As a new member of the family,MCP-1-1 is considered to be an important new mediator that produces pain.
TRPV1 is an thermal non-selective cation channel which mainly expresses in small or medium neurons and unmyelinated C fibers or Aδnerve fibers of the dorsal root ganglia, trigeminal ganglia. The accumulated evidences demonstrate that TRPV1 is a very important pain mediators mediating pain sensitivity.
The purpose of this research is to investigate the modulation of TRPV1 by MCP-1 in an animal pain model of the chronically compressed dorsal root ganglia (CCD). The main results are as follows:
1 As assessed by Real-time PCR, the mRNA expression of TRPV1 was increased in CCD-injured dorsal root ganglia (DRG). The co-expression of CCR2, the cognate receptor for MCP-1 TRPV1 was found DRG neurons in the DRG tissue slices by immunofluorescence method.
2. Spontaneous pain behavior test showed that MCP-1 enhanced capsaicin-induced flinching. The paw lifting time per minute (PLTPM) in CCD rats received a con-injection of MCP-1 with capsaicin is significantly higher than that received a single injection of capsaicin.
3. The whole-cell patch-clamp recordings demonstrated the enhancement by MCP-1 of membrane depolarization and inward current amplitude induced by capsaicin in acutely isolated CCD neurons. Also, the free intracellular calcium that entry through the TRPV1 channels from the extracellular fluid was increased by MCP-1 in the acutely isolated CCD neurons as assessed by the ratio Ca~(2+) imaging system.
4. The enhanced effects of MCP-1 on TRPV1 in CCD neurons was eliminated by U73122, an inhibitor of PLC. After the pretreatment of CCD neurons with U73122, The intracellular free calcium coming via the TRPV1 channels was not increased by MCP-1.
In conclusion, the enhancement by MCP-1 of TRPV1 function in CCD neurons, as revealed by a larger depolarization or inward current induced by capsaicin and a higher level of free intarcellular calcium via TRPV1 channels might contribute to hyperalgesia in CCD rats.
辣椒素受体TRPV1在背根神经节、三叉神经节等中小型神经元的C类无髓纤维或Aδ神经纤维等疼痛感受器上大量表达,是配体门控的非选择性阳离子通道。越来越多的研究表明,TRPV1在痛觉的产生及痛觉敏感性增强的病理过程中扮演着重要角色。
为了进一步研究MCP-1对TRPV1疼痛效应的调控机制,本课题在大鼠慢性压迫背根神经节(CCD)的模型上,应用Real-time PCR和免疫组织化学染色明确CCD背根神经节细胞上,MCP-1受体CCR2与TRPV1等的表达与分布;用Western blot的方法对CCD、CCI以及SNL三种神经损伤模型上CCR2在DRG中的表达进行了定量分析;应用行为学测试方法观察MCP-1是否易化CCD大鼠上TRPV1介导的痛觉行为,应用全细胞膜片钳及钙离子成像技术,观察了MCP-1对CCD大鼠DRG神经元上TRPV1通道的调控作用及可能的细胞内信号途径。主要研究结果如下;
1应用Real-time PCR方法显示,CCD后TRPV1的mRNA表达增加并应用免疫组化方法显示CCD后TRPV1表达增加,用Western blot的方法对CCD、CCI以及SNL三种神经损伤模型上CCR2在DRG中的表达进行了定量分析,结果表明,三种损伤方式都能引起CCR2的表达量增加,尤以CCD损伤后CCR2增加特别明显,在DRG组织切片和急性分离的DRG神经元上用免疫荧光方法显示CCR2和TRPV1共表达于CCD神经元,这就为MCP-1对TRPV1的调控作用提供形态学基础。
2动物自发痛反应行为测试结果显示,MCP-1增强Capsaicin诱发的疼痛行为,与单独给与MCP-1和Capsaicin有显著性差异。
3全细胞膜片钳记录显示,MCP-1增强在急性分离的CCD神经元上TRPV1受体激动剂Capsaicin诱导的膜电位去极化幅度及内向电流幅度。应用Ca~(2+)成像技术观察对急性分离的CCD神经元在MCP-1作用前后对Capsaicin诱发的细胞内游离钙离子的反应。结果表明,MCP-1(100 ng/ml)增强Capsaicin诱发的细胞内钙荧光强度
4在急性分离的CCD神经元上,应用Ca~(2+)成像技术观察PLC阻断剂U73122对MCP-1增强Capsaicin诱发的细胞内钙荧光强度的调控作用。结果显示U73122有效地阻断MCP-1对TRPV1的增强效应。动物自发痛反应行为测试结果显示,PLC阻断剂U73122有效地阻断MCP-1对Capsaicin引发的自发痛的易化作用。
综上所述,提示CCD损伤后DRG神经元上调表达的CCR2,在MCP-1作用激活后可通过增强TRPV1的功能,使痛觉神经元的兴奋性提高,从而参与痛觉过敏的产生。
MCP-1 (monocyte chemoattractant protein-1) is one of the CC chemokine subfamily members. The expression of MCP-1 was increased in neurons and glial cells after the nerve injuries,inflammation or other pathological changes. MCP-1 can directly enhance the excitability of neurons.As a new member of the family,MCP-1-1 is considered to be an important new mediator that produces pain.
TRPV1 is an thermal non-selective cation channel which mainly expresses in small or medium neurons and unmyelinated C fibers or Aδnerve fibers of the dorsal root ganglia, trigeminal ganglia. The accumulated evidences demonstrate that TRPV1 is a very important pain mediators mediating pain sensitivity.
The purpose of this research is to investigate the modulation of TRPV1 by MCP-1 in an animal pain model of the chronically compressed dorsal root ganglia (CCD). The main results are as follows:
1 As assessed by Real-time PCR, the mRNA expression of TRPV1 was increased in CCD-injured dorsal root ganglia (DRG). The co-expression of CCR2, the cognate receptor for MCP-1 TRPV1 was found DRG neurons in the DRG tissue slices by immunofluorescence method.
2. Spontaneous pain behavior test showed that MCP-1 enhanced capsaicin-induced flinching. The paw lifting time per minute (PLTPM) in CCD rats received a con-injection of MCP-1 with capsaicin is significantly higher than that received a single injection of capsaicin.
3. The whole-cell patch-clamp recordings demonstrated the enhancement by MCP-1 of membrane depolarization and inward current amplitude induced by capsaicin in acutely isolated CCD neurons. Also, the free intracellular calcium that entry through the TRPV1 channels from the extracellular fluid was increased by MCP-1 in the acutely isolated CCD neurons as assessed by the ratio Ca~(2+) imaging system.
4. The enhanced effects of MCP-1 on TRPV1 in CCD neurons was eliminated by U73122, an inhibitor of PLC. After the pretreatment of CCD neurons with U73122, The intracellular free calcium coming via the TRPV1 channels was not increased by MCP-1.
In conclusion, the enhancement by MCP-1 of TRPV1 function in CCD neurons, as revealed by a larger depolarization or inward current induced by capsaicin and a higher level of free intarcellular calcium via TRPV1 channels might contribute to hyperalgesia in CCD rats.
引文
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