脊髓MIF介導(dǎo)疼痛病理發(fā)生的分子機(jī)制研究
發(fā)布時(shí)間：2018-10-19 07:22
【摘要】：疾病的發(fā)生發(fā)展幾乎總是引起不同程度的疼痛發(fā)生。盡管已有多種藥物和方法可用來進(jìn)行疼痛治療,但其效果并不理想。因此尋求新的疼痛調(diào)節(jié)因子并進(jìn)行干預(yù)治療具有重要的臨床意義。傷害性刺激或神經(jīng)損傷誘導(dǎo)的炎癥免疫反應(yīng)與疼痛發(fā)生發(fā)展關(guān)系密切。MIF是參與幾乎所有炎癥性疾病過程的促炎性細(xì)胞因子,有研究提示MIF是神經(jīng)損傷后再生與功能恢復(fù)的重要參與者。鑒于神經(jīng)損傷與疼痛之間的因果聯(lián)系,推測(cè)MIF可能在疼痛發(fā)生發(fā)展過程起著敏化神經(jīng)及降低痛閾的作用。 本研究通過建立急性炎癥性疼痛和慢性神經(jīng)病理性疼痛模型,鞘內(nèi)給藥MIF互變異構(gòu)酶小分子抑制劑ISO-1觀察疼痛閾值變化與脊髓MIF水平之間的關(guān)系,進(jìn)而分析MIF介導(dǎo)疼痛病理發(fā)生的可能信號(hào)活化機(jī)制。這為疼痛治療潛在干預(yù)靶點(diǎn)提供線索,并為MIF相關(guān)疼痛藥物的開發(fā)提供理論基礎(chǔ)。整個(gè)研究分為如下三個(gè)部分： 一.MIF參與福爾馬林誘導(dǎo)急性炎癥性疼痛閾值的調(diào)節(jié)及其機(jī)制 通過福爾馬林誘導(dǎo)炎癥性疼痛模型大鼠鞘內(nèi)給藥不同劑量MIF小分子抑制劑ISO-1,發(fā)現(xiàn)福爾馬林誘導(dǎo)的二相疼痛行為學(xué)改善呈現(xiàn)ISO-1劑量依賴性變化。伴隨著福爾馬林誘導(dǎo)疼痛行為學(xué)變化,大鼠脊髓背角MIF及其受體CD74蛋白表達(dá)呈現(xiàn)時(shí)間依賴性上調(diào)特征；同時(shí)腦脊液MIF水平在福爾馬林注射后第二時(shí)相后期顯著增加；而ISO-1的應(yīng)用使得脊髓背角MIF及CD74表達(dá)下調(diào),這說明脊髓MIF水平的變化參與福爾馬林誘導(dǎo)炎癥性疼痛的發(fā)生發(fā)展。在此基礎(chǔ)上,應(yīng)用蛋白印跡技術(shù)發(fā)現(xiàn)ISO-1可以顯·著抑制ERK、p-p38 MAPK及NR2B表達(dá)；而ERK與p38 MAPK抑制劑又可顯著下調(diào)NR2B蛋白表達(dá),這說明ERK-p38 MAPK-NR2B信號(hào)通路的活化參與了MIF介導(dǎo)福爾馬林誘導(dǎo)炎癥性疼痛的病理發(fā)生過程。進(jìn)一步地,通過免疫組化方法檢測(cè)了脊髓背角MIF、CD11b、CD3的表達(dá)變化,結(jié)果發(fā)現(xiàn)MIF與CD11b共表達(dá)于脊髓背角,這說明福爾馬林誘導(dǎo)炎癥性疼痛大鼠脊髓背角MIF高表達(dá)來源于脊髓小膠質(zhì)細(xì)胞。為了證明抑制MIF互變異構(gòu)酶活性與抑制其生物活性等效,即鞘內(nèi)ISO-1的應(yīng)用是否有效抑制MIF生物活性,本研究以多巴鉻甲酯為互變異構(gòu)酶活性檢測(cè)工具,進(jìn)行了MIF互變異構(gòu)酶活性與MIF生成之間的相關(guān)性觀察,結(jié)果顯示ISO-1抑制MIF互變異構(gòu)酶活性的作用與抑制MIF生成的作用一致,從而證明選擇MIF互變異構(gòu)酶小分子抑制劑作為MIF相關(guān)性疼痛治療與藥物開發(fā)切實(shí)可行。 二.MIF參與CCI誘導(dǎo)神經(jīng)病理性疼痛閡值變化的調(diào)節(jié)及其機(jī)制 通過建立CCI誘導(dǎo)的神經(jīng)病理性疼痛小鼠模型,發(fā)現(xiàn)損傷神經(jīng)同側(cè)脊髓背角MIF高表達(dá)同時(shí),機(jī)械刺激疼痛閾值下調(diào)程度與熱源刺激疼痛潛伏期縮短水平呈現(xiàn)時(shí)間依賴性關(guān)系；并伴隨產(chǎn)生刺激誘發(fā)脊髓放電振幅增加及刺激結(jié)束后脊髓放電趨于正�；瘯r(shí)間延長(zhǎng)；不僅如此,腦脊液MIF水平呈現(xiàn)時(shí)間依賴性增加特征,而鞘內(nèi)應(yīng)用不同劑量ISO-1后機(jī)械與熱刺激疼痛行為學(xué)改善則呈現(xiàn)劑量依賴性關(guān)系,其效果與MIF Ab相似,這說明MIF參與并調(diào)節(jié)CCI誘導(dǎo)神經(jīng)病理性疼痛閾值變化。進(jìn)一步發(fā)現(xiàn)脊髓背角CD74表達(dá)與MIF呈相似變化；應(yīng)用免疫熒光技術(shù)檢測(cè)發(fā)現(xiàn)MIF與NeuN共表達(dá),而CD74與CD11b共表達(dá),這說明CD74在CCI誘導(dǎo)的疼痛病理過程中伴隨MIF發(fā)生相應(yīng)的表達(dá)變化,但MIF的產(chǎn)生與作用靶位不在同一細(xì)胞,從而使得MIF成為一個(gè)穿梭于神經(jīng)元與小膠質(zhì)細(xì)胞之間的疼痛敏化介質(zhì)。在此基礎(chǔ)上發(fā)現(xiàn)脊髓背角p-p44/42 MAPK及下游反應(yīng)蛋白IL-8與NR2B表達(dá)及腦脊液PGE2水平在CCI小鼠顯著升高,而鞘內(nèi)ISO-1可有效降低p-p44/42 MAPK及其效應(yīng)分子的水平；同時(shí)應(yīng)用p44/42 MAPK抑制劑后小鼠機(jī)械性和熱源性刺激疼痛閾值變化與ISO-1產(chǎn)生的效果相同,這提示p44/42 MAPK信號(hào)通路及其下游效應(yīng)分子在MIF介導(dǎo)CCI疼痛模型小鼠中被活化,從而參與痛閡調(diào)節(jié)。為了進(jìn)一步論證MIF在CCI疼痛模型小鼠痛閾變化中的作用,以MIF-1-小鼠建立CCI疼痛模型,發(fā)現(xiàn)MIF基因敲除后機(jī)械性和熱源性疼痛閾值較野生型(Wild-type, WT)小鼠顯著上移,其脊髓背角CD74、p-p44/42 MAPK、IL-8、NR2B表達(dá)顯著下調(diào),腦脊液PGE2水平顯著降低,從而為MIF參與CCI誘導(dǎo)疼痛閾值調(diào)節(jié)提供了佐證。在此基礎(chǔ)上正常小鼠鞘內(nèi)給藥rMIF后發(fā)現(xiàn)機(jī)械和熱源刺激疼痛閾值下調(diào),而p44/42 MAPK抑制劑可預(yù)防rMIF引起的痛閾變化,相應(yīng)地脊髓背角IL-8及NR2B表達(dá)表現(xiàn)出與疼痛變化一致的改變,從而論證了CCI誘導(dǎo)小鼠痛閾的下調(diào)與腦脊液MIF的增加相關(guān),說明腦脊液MIF與脊髓背角MIF在CCI誘導(dǎo)的疼痛發(fā)生發(fā)展過程中起著聯(lián)合協(xié)同的作用。最后,通過培養(yǎng)的小膠質(zhì)細(xì)胞觀察了ISO-1抑制MIF互變異構(gòu)酶活性的作用及rMIF的互變異構(gòu)酶活性。 三.MIF活化培養(yǎng)的離體脊髓小膠質(zhì)細(xì)胞 通過離體脊髓小膠質(zhì)細(xì)胞培養(yǎng),脂多糖(Lipopolysaccharide, LPS)刺激作為炎癥發(fā)生及相應(yīng)炎性介質(zhì)釋放的參照,rMIF刺激培養(yǎng)小膠質(zhì)細(xì)胞發(fā)現(xiàn)rMIF誘導(dǎo)環(huán)氧化酶2 (Cyclooxygenase 2, COX 2)及微粒體前列腺素E2合酶-1(Microsomal prostaglandin E2 synthase, mPGES-1)活化,從而引起PGE2合成增加,而且呈現(xiàn)rMIF劑量依賴性變化；這種rMIF引起的炎癥級(jí)聯(lián)反應(yīng)可以被ISO-1所抑制。在此基礎(chǔ)上,發(fā)現(xiàn)p38與p44/42 MAPK信號(hào)傳導(dǎo)通路活化參與調(diào)控rMIF引起COX 2/mPGES-1/PGE2級(jí)聯(lián)反應(yīng)。這說明外源性MIF自身即可活化小膠質(zhì)細(xì)胞產(chǎn)生類似LPS誘導(dǎo)的炎癥反應(yīng)。 綜上所述,脊髓MIF在急性炎癥性與慢性神經(jīng)病理性疼痛病理發(fā)生過程中通過不同的信號(hào)活化通路起著疼痛閡值調(diào)節(jié)的作用,這種作用的發(fā)揮與脊髓背角小膠質(zhì)細(xì)胞炎癥級(jí)聯(lián)反應(yīng)的發(fā)生密切相關(guān)。通過鞘內(nèi)給藥MIF互變異構(gòu)酶小分子抑制劑,論證了應(yīng)用MIF酶活性抑制劑進(jìn)行其生物活性抑制的可行性,進(jìn)而為MIF相關(guān)疼痛干預(yù)的實(shí)施提供了基礎(chǔ)數(shù)據(jù)。
[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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
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
，本文編號(hào)：2280521
本文鏈接：http://sikaile.net/xiyixuelunwen/2280521.html
最近更新
教材專著
