HMGB1在吗啡耐受形成过程中的表达调节及介导吗啡耐受的信号途径

发布时间：2018-06-17 04:20

本文选题：吗啡耐受 + HMGB1　；参考：《郑州大学》2017年硕士论文

【摘要】：研究背景阿片类药物吗啡由于其强大、可靠的镇痛效能,常被作为临床治疗顽固性疼痛的金标准。但吗啡等镇痛药在多次反复应用后,往往会产生镇痛作用的下降,即产生了所谓的镇痛耐受。虽然加大剂量可一定程度恢复其镇痛的效能,但其副作用常使病人难以忍受。因此,吗啡耐受严重限制其在临床上的应用。目前认为反复应用吗啡后引起的阿片受体功能的变化、以及中枢痛易化神经环路的激活是吗啡耐受产生的主要原因。大量实验证明吗啡可以引起脊髓胶质细胞的激活,进而促使其表达趋化因子、细胞因子等致痛物质,这些致痛物质在吗啡耐受的形成、发展和维持过程中起着至关重要的作用。HMGB1高迁移率族蛋白1(High Mobility Group Box-1 protein HMGB1)属于HMGB家族,是一种与细胞因子类似的炎症介质。近年来的研究发现,脊髓中的HMGB1在神经病理性疼痛和骨癌痛中发挥了重要作用,但HMGB1在吗啡耐受形成过程中的作用及其机制目前仍无报道。根据HMGB1促进细胞因子的释放以及自身所具有的细胞因子样作用,我们推测HMGB1可能参与吗啡耐受的形成。据此,我们实验室开展了系列研究,初步证明了脊髓HMGB1上调参与了吗啡耐受的形成和维持。但吗啡引起的脊髓HMGB1表达分泌的机制以及HMGB1介导吗啡耐受的细胞分子原理仍需进一步研究。研究目的本实验观察吗啡耐受及吗啡停用后,大鼠脊髓(Spinal Cord SC)和背根神经节(Dorsal Root Ganglion DRG)中HMGB1的表达变化,并且观察HMGB1参与吗啡耐受及其引起的痛觉过敏可能的信号通路中相关分子的表达变化,以确定HMGB1在吗啡耐受形成过程中的表达调节,并确定HMGB1介导吗啡耐受形成的信号途径。确定HMGB1在吗啡耐受的形成过程中分子机制。实验结果1.鞘内微量注射吗啡对脊髓HMGB1及其受体、磷酸化NF-κB p65、TNF-α和IL-1β表达的影响连续6天进行大鼠鞘内注射10μg/10μl的吗啡,每天两次,第7天给予诱导剂量。在给药的第1、3天吗啡的镇痛效率在90%-100%,给药的第5天吗啡的镇痛效率只有20%左右(***P0.001),第7天吗啡的镇痛效率则几乎为0(***P0.001)。在吗啡停药后,引起了大鼠双侧后足机械痛觉超敏和热痛觉过敏(*P0.05***P0.001是与第8天的control组相比,###P0.001是与自身baseline相比)。分别在给药的第1、3、5、7天以及停药后第3天进行大鼠脊髓的取材,具体为大鼠L4-L5的脊髓,之后进行western blot检测。Western Blot结果显示在吗啡注射的第1、3、5、7天,脊髓HMGB1及其受体RAGE和TLR4、磷酸化NF-κB p65以及致炎因子TNF-α、IL-1β的表达量维持在较高水平(*P0.05**P0.01),第三天达到峰值,停药后其水平逐渐恢复,且在停药后的第3天其表达量恢复至正常水平。由此说明鞘内微量注射吗啡可以引起脊髓HMGB1及其受体、磷酸化NF-κB p65、TNF-α和IL-1β表达量升高。2.吗啡对离体培养大鼠胎鼠脊髓神经元HMGB1及其下游致炎因子TNF-α和IL-1β的表达的影响离体的脊髓神经元培养10天细胞生长状态稳定,向培养基中加入1 mg/ml的吗啡30μl,使其终浓度为20μM/L,分别作用0 h、3 h、6 h、12 h、24 h和48 h后提取蛋白进行western blot检测,结果显示与吗啡作用0 h组相比其他组的HMGB1和TNF-α、IL-1β的表达量显著升高(*P0.05**P0.01***P0.001),作用6 h时三者的表达量达到峰值,且三者的表达变化趋势一致。与上述相同,在细胞生长稳定后向培养基中分别加入saline、1 mg/ml的吗啡15μl、30μl、60μl、120μl和240μl使其终浓度分别为0、10、20、40、80和160μM/L,待吗啡作用6 h后提取蛋白进行western blot检测,结果显示与saline组相比,吗啡组的HMGB1的表达量明显升高(***P0.001),且吗啡浓度为20μM/L时达到峰值。由以上结果说明吗啡可以对离体的大鼠脊髓神经元产生作用,可以导致神经元中HMGB1和TNF-α、IL-1β表达的增加,且对吗啡具有一定的时间和剂量依赖性。3.TLR4和μ受体阻断剂对离体培养大鼠胎鼠脊髓神经元HMGB1和致炎因子TNF-α表达的影响离体的大鼠脊髓神经元培养10 d后,首先分别向培养基中加入TLR4阻断剂TAK-242(10μM/L)、μ-受体阻断剂CTOP(10μM/L)预先作用30 min后,加入吗啡,使其终浓度为20μM/L,作用6 h后收取细胞,提取蛋白进行western blot检测,结果显示与单独加入吗啡相比,加入Mor+TAK-242组的HMGB1、TNF-α的表达量明显降低(*P0.05),并且单独加入TAK-242组两者的表达量与溶剂组、吗啡加TAK-242组的相比没有统计学意义。而Mor+CTOP组与吗啡加溶剂组相比,HMGB1的表达量没有改变,且这两组的HMGB1的表达量明显高于溶剂组和CTOP组。据此我们得出结论阻断TLR4可以抑制脊髓神经元HMGB1、TNF-α的表达,而阻断μ受体不能抑制两者的表达,由此说明TLR4参与了HMGB1介导的吗啡耐受的形成过程,而μ受体则没有参与该过程。4.鞘内注射TLR4阻断剂对脊髓和背根神经节HMGB1、磷酸化NF-κB p65表达及吗啡耐受形成的影响实验组大鼠吗啡注射前30 min进行鞘内注射TLR4阻断剂TAK-242(剂量为5,10,20μg/10μl),一天一次,吗啡一天两次,连续六天;对照组为吗啡+溶剂鞘内注射,一天两次,连续六天。行为学结果显示鞘内预注射TAK-242组与鞘内预注射溶剂组相比较在注射吗啡第5天(***P0.001)和第7天(***P0.001*P0.05)吗啡的最大镇痛效率有明显的升高。在吗啡注射后第8天(即吗啡停止注射后第1天),TAK-242组与溶剂组相比较PWMT和PWHL显著增高(**P0.01),但与自身的BL相比较无显著差异,显著的缓解了连续使用吗啡后引起的痛觉过敏现象,并且具有一定的药物剂量依赖性。在停药后的第二天进行western blot的取材。Western Blot结果显示鞘内预注射吗啡+TAK-242组的脊髓背角和DRG中HMGB1、p-p65的表达量与吗啡+溶剂组比较明显减少(**P0.01)。以上结果说明阻断TLR4可以缓解吗啡耐受的形成,抑制脊髓和背根神经节HMGB1、p-p65的表达释放。5.鞘内注射HMGB1 siRNA对吗啡耐受形成的细胞内信号途径的影响实验组吗啡注射前30 min注射HMGB1 siRNA、Scramble si RNA和Transfection regent,一天一次,吗啡一天两次,连续六天;对照组单独吗啡注射,一天两次,连续六天。在停药后的第二天进行western blot的取材。Western Blot结果显示鞘内预注射吗啡+HMGB1 siRNA组脊髓背角和DRG中HMGB1、p-p65、p-JNK的表达量与其他三组相比明显减少(*P0.05**P0.01***P0.001)。说明鞘内预注射HMGB1 siRNA可以缓解吗啡耐受的形成,抑制脊髓和背根神经节HMGB1、p-p65和p-JNK的表达释放。结果提示鞘内吗啡诱导的脊髓HMGB1上调可能是通过NF-κB和JNK信号通路的激活介导吗啡耐受的形成。结论吗啡通过TLR4-NF-κB信号通路的激活介导了SC和DRG中HMGB1的表达;脊髓HMGB1通过激活NF-κB、p-JNK信号通路,调控细胞因子的表达介导了吗啡耐受的形成。
[Abstract]:Background opioid morphine is often used as the gold standard for clinical treatment of intractable pain because of its strong and reliable analgesic efficacy. However, morphine and other analgesics often produce a decrease in analgesic effect after repeated use of morphine, which produces a so-called analgesic tolerance. But the side effects often make the patient unbearable. Therefore, morphine tolerance severely restricts its clinical application. The changes in the function of opioid receptor caused by repeated use of morphine, and the activation of the central pain prone neural loop are the main causes of morphine tolerance. A large number of experiments have shown that morphine can cause spinal glial cells. Activation, thus promoting the expression of chemokines, cytokines, and other pain causing substances, which play a vital role in the formation, development and maintenance of morphine tolerance,.HMGB1 high mobility group protein 1 (High Mobility Group Box-1 protein HMGB1) belongs to the HMGB family, which is an inflammatory mediator similar to the cytokine. Recent studies have found that HMGB1 plays an important role in neuropathic and bone cancer pain in the spinal cord, but the role and mechanism of HMGB1 in the formation of morphine tolerance are still not reported. Do we speculate that HMGB1 may be involved in promoting the release of cytokines and the cytokine like effects of HMGB1 The formation of endorphin, according to this, our laboratory has conducted a series of studies that preliminarily demonstrated that the HMGB1 up regulation of the spinal cord participates in the formation and maintenance of morphine tolerance. However, the mechanism of HMGB1 induced secretion of morphine and the molecular principle of HMGB1 mediated morphine tolerance are still needed to be further studied. Changes in the expression of HMGB1 in the spinal cord (Spinal Cord SC) and the dorsal root ganglion (Dorsal Root Ganglion DRG) after morphine discontinuation, and to observe the changes in the expression of the related molecules in the signaling pathway of the morphine tolerance and its induced hyperalgesia in order to determine the expression of HMGB1 in the formation of morphine tolerance and to determine HMGB. 1 to guide the signaling pathway of morphine tolerance formation. Determine the molecular mechanism of HMGB1 in the formation of morphine tolerance. Results the effects of microinjection of morphine on the HMGB1 and its receptor in the spinal cord in the 1. sheath, the effects of the expression of phosphorylated NF- kappa B p65, TNF- A and IL-1 beta in the rat sheath for 6 days for 6 days in the rat sheath were injected with 10 u g/10 Mu morphine, two times a day, and seventh days of induction. The analgesic efficiency of morphine on day 1,3 of the administration was 90%-100%. The analgesic efficiency of morphine was only about 20% (***P0.001) on the fifth day of administration, and the analgesic efficiency of morphine was almost 0 (***P0.001) in seventh days. After the withdrawal of morphine, the mechanical pain hypersensitivity and hyperalgesia of bilateral hind feet of the rats were induced (*P0.05***P0.001 and eighth days cont). Compared with the rol group, ###P0.001 was compared with its own baseline). The spinal cord of rats was taken on day 1,3,5,7 of the drug delivery and third days after the withdrawal of the drug, specifically the spinal cord of the rat L4-L5. Then the Western blot detection of.Western Blot showed that the.Western Blot result was on the day of the morphine injection. The expression of p65 and inflammation factor TNF- a, IL-1 beta was maintained at a high level (*P0.05**P0.01) and reached a peak at third days. After stopping the drug, its level gradually resumed and its expression recovered to the normal level on the third day after withdrawal. Thus, the intrathecal microinjection of morphine could cause HMGB1 and its receptor, phosphorylated NF- kappa B p65, TNF- A and IL-. 1 beta expression increased the expression of.2. morphine on the HMGB1 of spinal cord and the expression of TNF- A and IL-1 beta in the spinal cord of the rat fetal rat in vitro. The growth state of the cells in the cultured spinal neurons was stable for 10 days, and 1 mg/ml of morphine 30 u l were added to the medium, and the final concentration was 20 mu M/L, which acted as 0 h, 3 h, 6 h, 12 h, 24 h and 48. After h, the extracted protein was detected by Western blot. The results showed that the expression of IL-1 beta was significantly increased (*P0.05**P0.01***P0.001) and the expression amount of the three in the other group was significantly higher than that of the other groups, compared with the 0 h group of morphine, and the expression of the three was at the peak value at 6 h, and the trend of expression of the three were the same. After adding saline, 1 mg/ml morphine 15 l, 30 mu L, 60 mu L, 120 mu L and 240 Mu L, the final concentration was 0,10,20,40,80 and 160 micron respectively. The protein extracted from morphine 6 h was detected by Western blot. The results showed that the expression of morphine was significantly higher than that of the morphine group, and the concentration of morphine was 20 mu. The results showed that morphine could produce the effect of morphine on the isolated rat spinal cord neurons, which could lead to the increase of the expression of HMGB1 and TNF- a, IL-1 beta in the neurons, and the expression of HMGB1 and the expression of inflammation factor TNF- alpha in the cultured rat spinal cord neurons in vitro by a certain dose dependent.3.TLR4 and the dose dependent.3.TLR4 receptor blocker. After 10 d of the isolated rat spinal cord neurons were cultured, the TLR4 blocker TAK-242 (10 M/L) was first added to the culture medium, and the micron receptor blocker CTOP (10 mu M/L) was pre acted on 30 min, and the final concentration was 20 u M/L, and the cells were collected after the action of 6 h, and the extracted protein was detected for Western blot. The results showed that the morphine was added to the morphine alone. The results showed that morphine was added separately to the morphine. The result showed that morphine was added to the morphine alone. The expression of HMGB1, TNF- alpha in the Mor+TAK-242 group was significantly lower (*P0.05), and the expression of the two groups in the TAK-242 group was not statistically significant compared with the solvent group and the morphine plus TAK-242 group. The expression of HMGB1 in the Mor+CTOP group was not changed compared with the morphine plus solvent group, and the expression of HMGB1 in these two groups was significantly higher. In the solvent group and the CTOP group, we concluded that blocking TLR4 could inhibit the expression of HMGB1 and TNF- alpha in the spinal cord neurons, while blocking the micron receptor did not inhibit the expression of the two. Thus, TLR4 was involved in the formation process of HMGB1 mediated morphine tolerance, while the micron receptor did not participate in the.4. sheath of the TLR4 blocker to the spinal cord and dorsal root. The effect of HMGB1, phosphorylated NF- kappa B p65 expression and morphine tolerance formation in the experimental group was 30 min before injection of morphine TLR4 blocker TAK-242 (dose 5,10,20 u g/10 micron L), once a day, two times a day for six days, and the control group was injected with morphine + Solution for six days, two times a day for six days. Compared with the preintrathecal PREINJECTED TAK-242 group and the intrathecal PREINJECTED solvent group, the maximum analgesic efficiency of morphine was significantly higher than that of morphine fifth days (***P0.001) and seventh days (***P0.001*P0.05). Eighth days after morphine injection (first days after morphine stopped injection), group TAK-242 was significantly higher than that of PWMT and PWHL (**P0.01) compared with that of the solvent group (**P0.01). There was no significant difference in the BL phase of its own, significantly relieving the hyperalgesia caused by the continuous use of morphine, and having a dose dependence. The.Western Blot results of Western blot in the second day after the withdrawal showed the spinal dorsal horn of the morphine +TAK-242 group and the HMGB1 in DRG, and the expression of p-p65. Compared with the morphine + solvent group (**P0.01), the results showed that blocking the TLR4 could alleviate the formation of morphine tolerance, inhibit the HMGB1 of the spinal cord and dorsal root ganglia, and the expression of p-p65 released the.5. intrathecal HMGB1 siRNA for the intracellular signaling pathway of morphine tolerance in the experimental group, the 30 Min injection HMGB1 siRNA, Scr before morphine injection. Amble Si RNA and Transfection regent, once a day, morphine two times a day for six days. The control group was injected with morphine alone, two times a day for six days. The.Western Blot results of Western blot in the second day after the withdrawal of the drug showed the intrathecal preinjection of morphine +HMGB1 siRNA group of spinal dorsal horn and DRG. The other three groups were significantly reduced (*P0.05**P0.01***P0.001). It was suggested that intrathecal pre injection of HMGB1 siRNA could alleviate the formation of morphine tolerance and inhibit the release of HMGB1, p-p65 and p-JNK in the spinal and dorsal root ganglia. The results suggest that intrathecal morphine induced up-regulated spinal HMGB1 may be mediated by the activation of NF- kappa B and JNK signaling pathway to mediate morphine tolerance. It is concluded that the activation of morphine through the TLR4-NF- kappa B signaling pathway mediates the expression of HMGB1 in SC and DRG, and spinal HMGB1 mediates the formation of morphine tolerance by activating NF- kappa B, p-JNK signaling pathway, and regulating the expression of cytokines.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

【相似文献】