脊髓MIF介导疼痛病理发生的分子机制研究
发布时间：2018-10-19 07:22
【摘要】：疾病的发生发展几乎总是引起不同程度的疼痛发生。尽管已有多种药物和方法可用来进行疼痛治疗,但其效果并不理想。因此寻求新的疼痛调节因子并进行干预治疗具有重要的临床意义。伤害性刺激或神经损伤诱导的炎症免疫反应与疼痛发生发展关系密切。MIF是参与几乎所有炎症性疾病过程的促炎性细胞因子,有研究提示MIF是神经损伤后再生与功能恢复的重要参与者。鉴于神经损伤与疼痛之间的因果联系,推测MIF可能在疼痛发生发展过程起着敏化神经及降低痛阈的作用。 本研究通过建立急性炎症性疼痛和慢性神经病理性疼痛模型,鞘内给药MIF互变异构酶小分子抑制剂ISO-1观察疼痛阈值变化与脊髓MIF水平之间的关系,进而分析MIF介导疼痛病理发生的可能信号活化机制。这为疼痛治疗潜在干预靶点提供线索,并为MIF相关疼痛药物的开发提供理论基础。整个研究分为如下三个部分： 一.MIF参与福尔马林诱导急性炎症性疼痛阈值的调节及其机制 通过福尔马林诱导炎症性疼痛模型大鼠鞘内给药不同剂量MIF小分子抑制剂ISO-1,发现福尔马林诱导的二相疼痛行为学改善呈现ISO-1剂量依赖性变化。伴随着福尔马林诱导疼痛行为学变化,大鼠脊髓背角MIF及其受体CD74蛋白表达呈现时间依赖性上调特征；同时脑脊液MIF水平在福尔马林注射后第二时相后期显著增加；而ISO-1的应用使得脊髓背角MIF及CD74表达下调,这说明脊髓MIF水平的变化参与福尔马林诱导炎症性疼痛的发生发展。在此基础上,应用蛋白印迹技术发现ISO-1可以显·著抑制ERK、p-p38 MAPK及NR2B表达；而ERK与p38 MAPK抑制剂又可显著下调NR2B蛋白表达,这说明ERK-p38 MAPK-NR2B信号通路的活化参与了MIF介导福尔马林诱导炎症性疼痛的病理发生过程。进一步地,通过免疫组化方法检测了脊髓背角MIF、CD11b、CD3的表达变化,结果发现MIF与CD11b共表达于脊髓背角,这说明福尔马林诱导炎症性疼痛大鼠脊髓背角MIF高表达来源于脊髓小胶质细胞。为了证明抑制MIF互变异构酶活性与抑制其生物活性等效,即鞘内ISO-1的应用是否有效抑制MIF生物活性,本研究以多巴铬甲酯为互变异构酶活性检测工具,进行了MIF互变异构酶活性与MIF生成之间的相关性观察,结果显示ISO-1抑制MIF互变异构酶活性的作用与抑制MIF生成的作用一致,从而证明选择MIF互变异构酶小分子抑制剂作为MIF相关性疼痛治疗与药物开发切实可行。 二.MIF参与CCI诱导神经病理性疼痛阂值变化的调节及其机制 通过建立CCI诱导的神经病理性疼痛小鼠模型,发现损伤神经同侧脊髓背角MIF高表达同时,机械刺激疼痛阈值下调程度与热源刺激疼痛潜伏期缩短水平呈现时间依赖性关系；并伴随产生刺激诱发脊髓放电振幅增加及刺激结束后脊髓放电趋于正常化时间延长；不仅如此,脑脊液MIF水平呈现时间依赖性增加特征,而鞘内应用不同剂量ISO-1后机械与热刺激疼痛行为学改善则呈现剂量依赖性关系,其效果与MIF Ab相似,这说明MIF参与并调节CCI诱导神经病理性疼痛阈值变化。进一步发现脊髓背角CD74表达与MIF呈相似变化；应用免疫荧光技术检测发现MIF与NeuN共表达,而CD74与CD11b共表达,这说明CD74在CCI诱导的疼痛病理过程中伴随MIF发生相应的表达变化,但MIF的产生与作用靶位不在同一细胞,从而使得MIF成为一个穿梭于神经元与小胶质细胞之间的疼痛敏化介质。在此基础上发现脊髓背角p-p44/42 MAPK及下游反应蛋白IL-8与NR2B表达及脑脊液PGE2水平在CCI小鼠显著升高,而鞘内ISO-1可有效降低p-p44/42 MAPK及其效应分子的水平；同时应用p44/42 MAPK抑制剂后小鼠机械性和热源性刺激疼痛阈值变化与ISO-1产生的效果相同,这提示p44/42 MAPK信号通路及其下游效应分子在MIF介导CCI疼痛模型小鼠中被活化,从而参与痛阂调节。为了进一步论证MIF在CCI疼痛模型小鼠痛阈变化中的作用,以MIF-1-小鼠建立CCI疼痛模型,发现MIF基因敲除后机械性和热源性疼痛阈值较野生型(Wild-type, WT)小鼠显著上移,其脊髓背角CD74、p-p44/42 MAPK、IL-8、NR2B表达显著下调,脑脊液PGE2水平显著降低,从而为MIF参与CCI诱导疼痛阈值调节提供了佐证。在此基础上正常小鼠鞘内给药rMIF后发现机械和热源刺激疼痛阈值下调,而p44/42 MAPK抑制剂可预防rMIF引起的痛阈变化,相应地脊髓背角IL-8及NR2B表达表现出与疼痛变化一致的改变,从而论证了CCI诱导小鼠痛阈的下调与脑脊液MIF的增加相关,说明脑脊液MIF与脊髓背角MIF在CCI诱导的疼痛发生发展过程中起着联合协同的作用。最后,通过培养的小胶质细胞观察了ISO-1抑制MIF互变异构酶活性的作用及rMIF的互变异构酶活性。 三.MIF活化培养的离体脊髓小胶质细胞 通过离体脊髓小胶质细胞培养,脂多糖(Lipopolysaccharide, LPS)刺激作为炎症发生及相应炎性介质释放的参照,rMIF刺激培养小胶质细胞发现rMIF诱导环氧化酶2 (Cyclooxygenase 2, COX 2)及微粒体前列腺素E2合酶-1(Microsomal prostaglandin E2 synthase, mPGES-1)活化,从而引起PGE2合成增加,而且呈现rMIF剂量依赖性变化；这种rMIF引起的炎症级联反应可以被ISO-1所抑制。在此基础上,发现p38与p44/42 MAPK信号传导通路活化参与调控rMIF引起COX 2/mPGES-1/PGE2级联反应。这说明外源性MIF自身即可活化小胶质细胞产生类似LPS诱导的炎症反应。 综上所述,脊髓MIF在急性炎症性与慢性神经病理性疼痛病理发生过程中通过不同的信号活化通路起着疼痛阂值调节的作用,这种作用的发挥与脊髓背角小胶质细胞炎症级联反应的发生密切相关。通过鞘内给药MIF互变异构酶小分子抑制剂,论证了应用MIF酶活性抑制剂进行其生物活性抑制的可行性,进而为MIF相关疼痛干预的实施提供了基础数据。
[Abstract]:The development of disease almost always causes different levels of pain. Although many drugs and methods have been used to treat pain, the effect is not ideal. Therefore, it is important to seek new pain regulation factor and intervene treatment. The inflammatory immune response induced by nociceptive stimulation or nerve injury is closely related to the development of pain. MIF is a pro-inflammatory cytokine involved in almost all inflammatory disease processes, and studies suggest MIF is an important participant in post-nerve regeneration and functional recovery. Given the causal link between nerve injury and pain, it is estimated that MIF may play a role in sensitizing nerve and reducing pain threshold during the development of pain. In this study, the relationship between the change of pain threshold and MIF level was observed through the establishment of a model of acute inflammatory pain and chronic neuropathic pain, and the relationship between the change of pain threshold and MIF level was observed by MIIF tautomeric small molecule inhibitor ISO-1, and the possible signal activity of MIF-mediated pain pathology was analyzed. Mechanism. This provides clues to the potential intervention targets for pain therapy and provides the development of MIF-related pain medications. On the basis, the whole study is divided into the following three parts: a part of the department Subdivision: I. MIF participates in formalin-induced acute inflammatory pain threshold The adjustment of the value and its mechanism induced inflammatory pain model in formalin-induced inflammatory pain model rats with different doses of MIF small molecular inhibitor ISO-1, and found that formalin-induced two-phase pain behavior improved presentation I. Changes in the dose-dependent manner of SO-1. The expression of MIF and its receptor CD74 protein in spinal dorsal horn of rats presented a time-dependent upregulation with formalin-induced pain behavior; CSF MIF levels were injected in formalin at the same time. The post-second phase was significantly increased; while ISO-1 was used to downregulate the expression of MIF and CD74 in the dorsal horn of the spinal cord, indicating that the change of MIF level in the spinal cord was induced in formalin. On the basis of this, it was found that ISO-1 could significantly inhibit the expression of CD44v6, p-p38 MAPK and NR2B by Western blot. The expression of NR2B protein could be significantly reduced by p38 MAPK-NR2B signaling pathway, which indicated that activation of p38 MAPK-NR2B signaling pathway was involved in MIF-mediated formalin-induced inflammation. The expression of MIF, CD11b and CD3 in dorsal horn of spinal cord was detected by immunohistochemistry. The results showed that MIF and MIF were co-expressed in dorsal horn of spinal cord, which indicated that MIF was higher than that in spinal dorsal horn of rats with inflammatory pain induced by formalin. To demonstrate the effective inhibition of MIF bioactivity by inhibiting MIF tautomeric activity and inhibition of MIF bioactivity, this study carried out MIF tautomeric enzyme activity and MI by using DOPA as a tautomeric enzyme activity detection tool. Correlation between F-generation showed that the effect of ISO-1 inhibition of MIF tautomeric activity was consistent with inhibition of MIF generation, thus demonstrating the choice of MIF tautomeric small molecule inhibitor as MIF-related pain. Treatment It's feasible to develop drugs with drugs. II.MIF is involved in CCI-induced nerves. Modulation of pathological pain and its mechanism by establishing CCI-induced neuropathic pain mouse model, hair At the same time, the reduction degree of mechanical stimulation pain threshold and the latency of heat source stimulation pain shorten the time-dependent relationship, and the stimulation-induced increase of the amplitude of spinal cord discharge and the end of stimulation were observed. The post-spinal cord discharge tended to normalize time. Furthermore, the MIF level presented a time-dependent increase in the CSF MIF level, whereas a dose-dependent relationship was presented by the use of different doses of ISO-1 post-mechanical and heat-stimulated pain behavior in the brain. The effect was similar to MIF Ab, indicating MIF involvement. It was found that the expression of CD74 in dorsal horn of spinal cord was similar to MIF. The expression of CD74 in dorsal horn of spinal cord was similar to MIF, and the co-expression of MIF with MLIF was detected by immunofluorescence technique, and CD74 was co-expressed with CD11b, indicating the pain induced by CD74 in CCI. There was a corresponding change in the expression of MIF in the course of pathology, but the production of MIF and the target site were not in the same cell, so MIF became a shuttle. On this basis, we found that the expression of p-p44/ 42MAPK and the downstream reactive protein IL-8 and NR2B in the dorsal horn of the spinal cord and the levels of IL-8 and NR2B were significantly increased in CCI mice. The expression of p44/ 42 MAPK and its effector molecule was similar to that produced by ISO-1 after application of p44/ 42 MAPK inhibitor, suggesting that p44/ 42 MAPK signaling pathway and its downstream effector molecule mediate CCI in MIF. In order to further demonstrate the effect of MIF in the pain threshold change of CCI pain model mice, the CCI pain model was established by MIF-1-mouse, and the mechanical and heat source pain thresholds were found to be significantly higher than that of wild type (Wild-type, WT) mice. 4. The expression of p-p44/ 42MAPK, IL-8 and NR2B was down-regulated, and the level of cerebral spinal fluid level decreased significantly. F is involved in the regulation of CCI-induced pain threshold. On the basis of this, we found that mechanical and heat source stimulation pain thresholds were down-regulated after administration of rMIF in normal mice, while p44/ 42 MAPK inhibitor could prevent the change of pain threshold caused by rMIF, and the corresponding spinal dorsal horn IL-8. The expression of NR2B showed a consistent change in the pain threshold, which demonstrated that the reduction of pain threshold in CCI-induced mice was associated with an increase in MIF, suggesting that MIF and spinal dorsal horn MIF were induced by CCI. The effect of joint synergy in the development of pain occurs. Finally, the inhibition of MIF tautomer by ISO-1 was observed by cultured microglial cells. enzyme activity The Role of Sex and the tautomeric Enzyme Activity of rMIF.. MIF activated cultured oligodendrocytes were cultured by microglia cell culture, lipopolysaccharide (LPS) stimulation as a reference for inflammation and corresponding inflammatory mediators release, and rMIF stimulated the culture of microglial cells. rMIF-induced cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) have been activated, resulting in an increase in protein synthesis, and the presence of rMIF dose-dependent changes. The inflammatory cascade response induced by this rMIF could be inhibited by ISO-1. On the basis of this, it was found that p38 and p44/ 42 MAPK signal transduction pathways were involved in the regulation and control. rMIF causes a cascade reaction of COX 2/ mPGES-1/ gal. This means that exogenous M IF itself can activate microglia to produce similar LPS-induced inflammatory responses. In conclusion, spinal cord MIF plays a role in the regulation of pain relief by different signal activation pathways in the pathogenesis of acute inflammatory and chronic neuropathic pain. The role of MIF is closely related to the occurrence of inflammatory cascade in the dorsal horn of spinal cord.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
本文编号：2280521
[Abstract]:The development of disease almost always causes different levels of pain. Although many drugs and methods have been used to treat pain, the effect is not ideal. Therefore, it is important to seek new pain regulation factor and intervene treatment. The inflammatory immune response induced by nociceptive stimulation or nerve injury is closely related to the development of pain. MIF is a pro-inflammatory cytokine involved in almost all inflammatory disease processes, and studies suggest MIF is an important participant in post-nerve regeneration and functional recovery. Given the causal link between nerve injury and pain, it is estimated that MIF may play a role in sensitizing nerve and reducing pain threshold during the development of pain. In this study, the relationship between the change of pain threshold and MIF level was observed through the establishment of a model of acute inflammatory pain and chronic neuropathic pain, and the relationship between the change of pain threshold and MIF level was observed by MIIF tautomeric small molecule inhibitor ISO-1, and the possible signal activity of MIF-mediated pain pathology was analyzed. Mechanism. This provides clues to the potential intervention targets for pain therapy and provides the development of MIF-related pain medications. On the basis, the whole study is divided into the following three parts: a part of the department Subdivision: I. MIF participates in formalin-induced acute inflammatory pain threshold The adjustment of the value and its mechanism induced inflammatory pain model in formalin-induced inflammatory pain model rats with different doses of MIF small molecular inhibitor ISO-1, and found that formalin-induced two-phase pain behavior improved presentation I. Changes in the dose-dependent manner of SO-1. The expression of MIF and its receptor CD74 protein in spinal dorsal horn of rats presented a time-dependent upregulation with formalin-induced pain behavior; CSF MIF levels were injected in formalin at the same time. The post-second phase was significantly increased; while ISO-1 was used to downregulate the expression of MIF and CD74 in the dorsal horn of the spinal cord, indicating that the change of MIF level in the spinal cord was induced in formalin. On the basis of this, it was found that ISO-1 could significantly inhibit the expression of CD44v6, p-p38 MAPK and NR2B by Western blot. The expression of NR2B protein could be significantly reduced by p38 MAPK-NR2B signaling pathway, which indicated that activation of p38 MAPK-NR2B signaling pathway was involved in MIF-mediated formalin-induced inflammation. The expression of MIF, CD11b and CD3 in dorsal horn of spinal cord was detected by immunohistochemistry. The results showed that MIF and MIF were co-expressed in dorsal horn of spinal cord, which indicated that MIF was higher than that in spinal dorsal horn of rats with inflammatory pain induced by formalin. To demonstrate the effective inhibition of MIF bioactivity by inhibiting MIF tautomeric activity and inhibition of MIF bioactivity, this study carried out MIF tautomeric enzyme activity and MI by using DOPA as a tautomeric enzyme activity detection tool. Correlation between F-generation showed that the effect of ISO-1 inhibition of MIF tautomeric activity was consistent with inhibition of MIF generation, thus demonstrating the choice of MIF tautomeric small molecule inhibitor as MIF-related pain. Treatment It's feasible to develop drugs with drugs. II.MIF is involved in CCI-induced nerves. Modulation of pathological pain and its mechanism by establishing CCI-induced neuropathic pain mouse model, hair At the same time, the reduction degree of mechanical stimulation pain threshold and the latency of heat source stimulation pain shorten the time-dependent relationship, and the stimulation-induced increase of the amplitude of spinal cord discharge and the end of stimulation were observed. The post-spinal cord discharge tended to normalize time. Furthermore, the MIF level presented a time-dependent increase in the CSF MIF level, whereas a dose-dependent relationship was presented by the use of different doses of ISO-1 post-mechanical and heat-stimulated pain behavior in the brain. The effect was similar to MIF Ab, indicating MIF involvement. It was found that the expression of CD74 in dorsal horn of spinal cord was similar to MIF. The expression of CD74 in dorsal horn of spinal cord was similar to MIF, and the co-expression of MIF with MLIF was detected by immunofluorescence technique, and CD74 was co-expressed with CD11b, indicating the pain induced by CD74 in CCI. There was a corresponding change in the expression of MIF in the course of pathology, but the production of MIF and the target site were not in the same cell, so MIF became a shuttle. On this basis, we found that the expression of p-p44/ 42MAPK and the downstream reactive protein IL-8 and NR2B in the dorsal horn of the spinal cord and the levels of IL-8 and NR2B were significantly increased in CCI mice. The expression of p44/ 42 MAPK and its effector molecule was similar to that produced by ISO-1 after application of p44/ 42 MAPK inhibitor, suggesting that p44/ 42 MAPK signaling pathway and its downstream effector molecule mediate CCI in MIF. In order to further demonstrate the effect of MIF in the pain threshold change of CCI pain model mice, the CCI pain model was established by MIF-1-mouse, and the mechanical and heat source pain thresholds were found to be significantly higher than that of wild type (Wild-type, WT) mice. 4. The expression of p-p44/ 42MAPK, IL-8 and NR2B was down-regulated, and the level of cerebral spinal fluid level decreased significantly. F is involved in the regulation of CCI-induced pain threshold. On the basis of this, we found that mechanical and heat source stimulation pain thresholds were down-regulated after administration of rMIF in normal mice, while p44/ 42 MAPK inhibitor could prevent the change of pain threshold caused by rMIF, and the corresponding spinal dorsal horn IL-8. The expression of NR2B showed a consistent change in the pain threshold, which demonstrated that the reduction of pain threshold in CCI-induced mice was associated with an increase in MIF, suggesting that MIF and spinal dorsal horn MIF were induced by CCI. The effect of joint synergy in the development of pain occurs. Finally, the inhibition of MIF tautomer by ISO-1 was observed by cultured microglial cells. enzyme activity The Role of Sex and the tautomeric Enzyme Activity of rMIF.. MIF activated cultured oligodendrocytes were cultured by microglia cell culture, lipopolysaccharide (LPS) stimulation as a reference for inflammation and corresponding inflammatory mediators release, and rMIF stimulated the culture of microglial cells. rMIF-induced cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) have been activated, resulting in an increase in protein synthesis, and the presence of rMIF dose-dependent changes. The inflammatory cascade response induced by this rMIF could be inhibited by ISO-1. On the basis of this, it was found that p38 and p44/ 42 MAPK signal transduction pathways were involved in the regulation and control. rMIF causes a cascade reaction of COX 2/ mPGES-1/ gal. This means that exogenous M IF itself can activate microglia to produce similar LPS-induced inflammatory responses. In conclusion, spinal cord MIF plays a role in the regulation of pain relief by different signal activation pathways in the pathogenesis of acute inflammatory and chronic neuropathic pain. The role of MIF is closely related to the occurrence of inflammatory cascade in the dorsal horn of spinal cord.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363
【参考文献】
相关期刊论文 前3条
1 ;Activation of ERK1/2 in spinal cord contributes to the development of acute cystic pain in rabbits[J];Neuroscience Bulletin;2006年04期
2 ;Macrophage migration inhibitory factor regulates proliferation of gastric cancer cells via the PI3K/Akt pathway[J];World Journal of Gastroenterology;2009年44期
3 Nitsan Maharshak;Sivan Cohen;Frida Lantner;Gili Hart;Richard Bucala;Idit Shachar;;CD74 is a survival receptor on colon epithelial cells[J];World Journal of Gastroenterology;2010年26期
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