15d-PGJ_2对TRPA1离子通道的作用以及对TRPA1介导的疼痛行为的抑制
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摘要
瞬时受体电位离子通道(Transient receptor potential ion channels, TRPs)是近年来发现存在于细胞膜或胞内细胞器膜上的非选择性阳离子通道。其中TRPA1亚型被认为介导生物对外界低温、刺激性化学物质和机械刺激的感受,TRPA1还参与炎症性和神经病理性疼痛的发病机制。
15-去氧-△-(12,14)前列腺素J2 (15d-PGJ2),是近年来发现的在组织损伤、炎症反应和氧化应激中释放的一种内源性物质。我们与其他研究小组以前的研究发现15d-PGJ2能够与TRPA1蛋白N-末端半胱氨酸共价结合而激动TRPA1离子通道,并引起TRPA1依赖性的急性疼痛行为。然而,最近的研究表明,在急性痛、炎症性疼痛和神经病理性疼痛模型中,15d-PGJ2还具有显著的镇痛作用。
本研究假设15d-PGJ2的镇痛作用是由于其对外周感觉神经元的激动以及随后的脱敏作用。为了证实该假设,本研究使用Fura-2AM钙离子成像技术首先比较了15d-PGJ2和AITC对TRPA1离子通道的激动作用,以及诱发的小鼠急性疼痛行为。随后,我们测试了不同浓度的15d-PGJ2和AITC预处理对TRPA1表达阳性的背根神经节神经元细胞的脱敏作用,以及足底预先注射15d-PGJ2或AITC对小鼠急性疼痛反应的作用。最后观察了15d-PGJ2对炎症性疼痛模型的镇痛作用,并使用TRPA1基因敲除小鼠,探讨了TRPA1离子通道在15d-PGJ2镇痛机制中的作用。主要实验结果如下：
1.15d-PGJ2和AITC剂量依赖性的激动TRPA1离子通道,引起小鼠的急性疼痛反应。
2.与同浓度AITC相比,25和50μM低浓度15d-PGJ2预处理能显著降低AITC反应阳性的DRG神经元数目和反应强度。100和200μM高浓度15d-PGJ2和AITC都能显著降低DRG神经元对AITC的反应性,但15d-PGJ2对辣椒素引起的神经元激动没有抑制作用。行为学实验也发现,足底预先注射15mM15d-PGJ2能显著降低随后15d-PGJ2或AITC注射引起的急性疼痛,但15d-PGJ2对辣椒素引起的急性疼痛没有抑制作用。任何浓度的AITC对小鼠急性疼痛行为都没有抑制作用。
3.足底注射15d-PGJ2能减轻CFA注射造成的炎症性机械痛敏感,这种镇痛作用在TRPA1基因敲除的小鼠中完全消失。
综上所述,本研究证实TRPA1激动剂15d-PGJ2能激动周围感觉神经元上的TRPA1离子通道,随后长时间脱敏离子通道,并产生由此介导的镇痛作用。这种作用是否是由于15d-PGJ2对TRPA1离子通道的特殊修饰作用仍有待进一步研究。
Transient receptor potential ion channels (TRPs) are recently founded non-selective cation channels which exist in plasmic membrane. The subtype TRPA1 was found mediating the sensation of cold, irritant chemicals and mechanical stimulus, it is also underlying the mechanism of inflammatory and neuropathic pain.
Newly found prostaglandin 15-deoxy-A 12,14-prostaglandin J2 (15d-PGJ2) is expressed when tissue is undergoing injury, inflammation or oxidative stress. We and others have shown that 15d-PGJ2can activate ion channel TRPA1 via covalent modification of N-terminal cysteines and causes channel-dependent nocifensive behavior. Paradoxically, recent studies indicate that 15d-PGJ2 is anti-nociceptive in several pain models.
We hypothesized that activation and subsequent desensitization of TRPA1 in peripheral sensory neurons might play a role in the anti-nociceptive property of 15d-PGJ2. To investigate this, we initially utilized Fura-2AM calcium imaging to test the activation of DRG neurons with different doses of 15d-PGJ2 and AITC, their nociceptive effect was observed as well. Then, compared to pre-exposure to allyl isothiocyanate (AITC, mustard oil), we tested whether pre-treatment with 15d-PGJ2 could desensitize TRPA1-expressing dorsal root ganglion (DRG) neurons and inhibit nociceptive behaviors to subsequent stimulation. Finally, we tested if intraplantar pre-injection of 15d-PGJ2 could reduce mechanical hypersensitivity in the Complete Freund's Adjuvant model, and whether TRPA1 was involved in the analgesic mechanism of 15d-PGJ2. The results were as followed:
1.15d-PGJ2 and AITC activated DRG neurons and caused nociceptive behaviors in a dose-dependent manner.15d-PGJ2 of a concentration higher than saturated one caused decresed nociceptive response.
2. We found that pre-exposure 15d-PGJ2 reduced the magnitude and number of subsequent responses to AITC, but not capsaicin (CAP). A series of analogous behavioral studies demonstrated that intraplantar pre-injection of 15d-PGJ2, in contrast to AITC, attenuated acute nocifensive responses to subsequent injections of 15d-PGJ2 and AITC, but not CAP.
3. Intraplantar 15d-PGJ2-administered after the induction of inflammation-also reduced mechanical hypersensitivity in the Complete Freund's Adjuvant (CFA) model for up to 2 hours post-injection. The 15d-PGJ2-mediated reduction in mechanical hypersensitivity was dependent on TRPA1, as this effect was absent in TRPA1 knockout mice.
In conclusion, our data supports the hypothesis that 15d-PGJ2 induces activation followed by persistent desensitization of TRPA1 channels expressed in peripheral sensory neurons both in vitro and in vivo. Whether this is a unique property of TRPA1 modulation by 15d-PGJ2 merits further investigation.
15-去氧-△-(12,14)前列腺素J2 (15d-PGJ2),是近年来发现的在组织损伤、炎症反应和氧化应激中释放的一种内源性物质。我们与其他研究小组以前的研究发现15d-PGJ2能够与TRPA1蛋白N-末端半胱氨酸共价结合而激动TRPA1离子通道,并引起TRPA1依赖性的急性疼痛行为。然而,最近的研究表明,在急性痛、炎症性疼痛和神经病理性疼痛模型中,15d-PGJ2还具有显著的镇痛作用。
本研究假设15d-PGJ2的镇痛作用是由于其对外周感觉神经元的激动以及随后的脱敏作用。为了证实该假设,本研究使用Fura-2AM钙离子成像技术首先比较了15d-PGJ2和AITC对TRPA1离子通道的激动作用,以及诱发的小鼠急性疼痛行为。随后,我们测试了不同浓度的15d-PGJ2和AITC预处理对TRPA1表达阳性的背根神经节神经元细胞的脱敏作用,以及足底预先注射15d-PGJ2或AITC对小鼠急性疼痛反应的作用。最后观察了15d-PGJ2对炎症性疼痛模型的镇痛作用,并使用TRPA1基因敲除小鼠,探讨了TRPA1离子通道在15d-PGJ2镇痛机制中的作用。主要实验结果如下：
1.15d-PGJ2和AITC剂量依赖性的激动TRPA1离子通道,引起小鼠的急性疼痛反应。
2.与同浓度AITC相比,25和50μM低浓度15d-PGJ2预处理能显著降低AITC反应阳性的DRG神经元数目和反应强度。100和200μM高浓度15d-PGJ2和AITC都能显著降低DRG神经元对AITC的反应性,但15d-PGJ2对辣椒素引起的神经元激动没有抑制作用。行为学实验也发现,足底预先注射15mM15d-PGJ2能显著降低随后15d-PGJ2或AITC注射引起的急性疼痛,但15d-PGJ2对辣椒素引起的急性疼痛没有抑制作用。任何浓度的AITC对小鼠急性疼痛行为都没有抑制作用。
3.足底注射15d-PGJ2能减轻CFA注射造成的炎症性机械痛敏感,这种镇痛作用在TRPA1基因敲除的小鼠中完全消失。
综上所述,本研究证实TRPA1激动剂15d-PGJ2能激动周围感觉神经元上的TRPA1离子通道,随后长时间脱敏离子通道,并产生由此介导的镇痛作用。这种作用是否是由于15d-PGJ2对TRPA1离子通道的特殊修饰作用仍有待进一步研究。
Transient receptor potential ion channels (TRPs) are recently founded non-selective cation channels which exist in plasmic membrane. The subtype TRPA1 was found mediating the sensation of cold, irritant chemicals and mechanical stimulus, it is also underlying the mechanism of inflammatory and neuropathic pain.
Newly found prostaglandin 15-deoxy-A 12,14-prostaglandin J2 (15d-PGJ2) is expressed when tissue is undergoing injury, inflammation or oxidative stress. We and others have shown that 15d-PGJ2can activate ion channel TRPA1 via covalent modification of N-terminal cysteines and causes channel-dependent nocifensive behavior. Paradoxically, recent studies indicate that 15d-PGJ2 is anti-nociceptive in several pain models.
We hypothesized that activation and subsequent desensitization of TRPA1 in peripheral sensory neurons might play a role in the anti-nociceptive property of 15d-PGJ2. To investigate this, we initially utilized Fura-2AM calcium imaging to test the activation of DRG neurons with different doses of 15d-PGJ2 and AITC, their nociceptive effect was observed as well. Then, compared to pre-exposure to allyl isothiocyanate (AITC, mustard oil), we tested whether pre-treatment with 15d-PGJ2 could desensitize TRPA1-expressing dorsal root ganglion (DRG) neurons and inhibit nociceptive behaviors to subsequent stimulation. Finally, we tested if intraplantar pre-injection of 15d-PGJ2 could reduce mechanical hypersensitivity in the Complete Freund's Adjuvant model, and whether TRPA1 was involved in the analgesic mechanism of 15d-PGJ2. The results were as followed:
1.15d-PGJ2 and AITC activated DRG neurons and caused nociceptive behaviors in a dose-dependent manner.15d-PGJ2 of a concentration higher than saturated one caused decresed nociceptive response.
2. We found that pre-exposure 15d-PGJ2 reduced the magnitude and number of subsequent responses to AITC, but not capsaicin (CAP). A series of analogous behavioral studies demonstrated that intraplantar pre-injection of 15d-PGJ2, in contrast to AITC, attenuated acute nocifensive responses to subsequent injections of 15d-PGJ2 and AITC, but not CAP.
3. Intraplantar 15d-PGJ2-administered after the induction of inflammation-also reduced mechanical hypersensitivity in the Complete Freund's Adjuvant (CFA) model for up to 2 hours post-injection. The 15d-PGJ2-mediated reduction in mechanical hypersensitivity was dependent on TRPA1, as this effect was absent in TRPA1 knockout mice.
In conclusion, our data supports the hypothesis that 15d-PGJ2 induces activation followed by persistent desensitization of TRPA1 channels expressed in peripheral sensory neurons both in vitro and in vivo. Whether this is a unique property of TRPA1 modulation by 15d-PGJ2 merits further investigation.
引文
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