HMGB1在吗啡耐受中的作用

发布时间：2018-06-26 02:41

本文选题：吗啡耐受 + 痛觉过敏　；参考：《郑州大学》2016年硕士论文

【摘要】：实验背景与目的吗啡是阿片类受体激动剂,是应用于临床镇痛的金标准,但是在多次反复使用后,会产生镇痛耐受以及停药后的痛觉过敏现象。对吗啡耐受虽然已经进行了大量研究,但其产生机制迄今仍不十分清楚。最近研究发现,反复大量使用吗啡后,在神经系统会引起一些适应性变化,包括阿片类受体的脱敏,内化,下调以及磷酸化现象或者与其他受体异源二聚化。慢性阿片类物质的接触会引起神经胶质细胞以及谷氨酸受体的活化和促炎细胞因子的释放等。其中细胞因子的释放可能是引起吗啡镇痛耐受和痛觉过敏产生的主要原因。高迁移率族蛋白1(High mobility group box protein 1,HMGB1)是一种类似于细胞因子的炎性介质,属于HMGB家族。HMGB1广泛存在于哺乳动物的细胞中,在接受刺激时可以从免疫和非免疫细胞当中释放到细胞外环境,从而引起各种炎症反应。近年来的研究发现,周围神经损伤可引起HMGB1在背根神经节(Dorsal Root Ganglia,DRG)和脊髓背角的表达变化,阻断HMGB1的作用可减轻大鼠神经病理性疼痛。目前,多数国内外学者认为神经病理性痛和吗啡耐受有部分相似的发生机制。虽然HMGB1在病理性疼痛中的作用已经取得了明确的实验结果,但HMGB1在吗啡耐受中的作用和具体的分子机制迄今未见报道。本研究的主要目的就是通过动物实验证明HMGB1在吗啡耐受形成过程的作用。为此本实验首先采用多次(每天2次,共6天)鞘内微量注射吗啡(10μg/10μl)建立大鼠吗啡镇痛耐受模型,通过检测大鼠甩尾潜伏期(Tail-flick la tency)观测吗啡镇痛作用的变化,通过测试大鼠机械刺激撤足阈值(Paw With drawal Threshold,PWT)和热刺激撤足潜伏期(Paw Withdrawal Latency,PWL)评估停用吗啡后大鼠的痛行为学变化。实验过程通过在吗啡耐受形成的不同时段(吗啡耐受形成前和吗啡耐受形成后)鞘内注射Glycyrrhizin(GL)(HMGB1的小分子抑制剂)或HMGB1 si RNA,结合以上行为学测试,以观察阻断HM GB1的作用或抑制HMGB1的合成对吗啡耐受形成的影响。实验还通过Western Blot法进一步观察了在吗啡耐受形成过程HMGB1在DRG和脊髓背角的表达变化以及鞘内注射HMGB1 si RNA对HMGB1表达的影响;通过免疫荧光双染色法观察了吗啡诱导的HMGB1在大鼠DRG和脊髓背角表达的细胞类型。实验结果将对进一步阐明吗啡耐受形成的机制提供理论支持,同时对临床防治吗啡耐受的产生有一定实际参考价值。实验结果1.吗啡耐受形成过程HMGB1在大鼠DRG和脊髓背角的表达变化以及表达的细胞类型大鼠鞘内注射吗啡10μg/10μl一天两次,连续6天注射。在给药的第5天吗啡的镇痛效率在20%左右(**P0.01),在第7天吗啡的镇痛效率几乎为0(与注射生理盐水组比较,***P0.001)。吗啡停药后第8天会引起大鼠的双侧机械痛觉超敏和热痛觉过敏(与注射生理盐水组比较,***P0.001)。West ern Blot检测发现鞘内注射吗啡引起了HMGB1在脊髓背角和DRG中的表达量的明显升高(与注射生理盐水组比较,**P0.001),通过免疫荧光双染发现H MGB1在脊髓背角中与神经元、星形胶质细胞以及小胶质细胞均有共定位,在背根神经节中HMGB1与A类C类神经元以及卫星样胶质细胞共定位。2.鞘内预注射HMGB1小分子抑制剂Glycyrrhizin部分抑制了吗啡耐受的形成鞘内注射GL组和注射溶剂组(Vehicle,Veh)相比较,在吗啡注射的第5天(***P0.001)和第7天(**P0.01,***P0.001)吗啡的镇痛效率有了明显的提升。停药后第8天大鼠的PWT和PWL和溶剂组相比较均有一定程度的提高(*P0.05,**P0.01)。结果提示鞘内注射GL延缓了吗啡耐受的形成,同时部分减轻了吗啡停药后所引起的痛觉过敏,作用具有一定的时间和剂量依赖性。3.吗啡耐受模型建立后鞘内预注射GL能够部分逆转吗啡耐受症状大鼠吗啡耐受模型建立后第7天,在鞘内注射吗啡的同时,鞘内注射GL(提前30 min,50μg/10μl,每天一次,共5天)。结果发现在从第7天开始到第11天鞘内注射Gl组和溶剂组相比较,鞘内注射GL组的吗啡镇痛效率逐渐升高(**P0.01,***P0.001)。在第12天停止给药后大鼠的PWT和PWL和溶剂组相比较均有明显的升高(**P0.01,***P0.001)。结果表明鞘内注射GL可部分逆转已经形成的吗啡镇痛耐受。4.鞘内注射HMGB1 si RNA可以延缓吗啡耐受的形成鞘内注射吗啡+HMGB1 si RNA组和吗啡+生理盐水组、吗啡+HMGB1 Scra mble si RNA组以及吗啡+Transfection Regant组相比较,在吗啡注射的第5天(***P0.001)和第7天(**P0.01)吗啡的镇痛效率有了明显的提升。停药后第8天大鼠的PWT和PWl和溶剂组相比较均有一定程度的提高(*P0.05,**P0.01)。HMGB1 Scramble si RNA组和Transfection Regant组行为学结果和吗啡+生理盐水组相比较无统计学差异。Western Blot结果显示,吗啡+HMGB1 si RN A组与生理盐水组、吗啡+HMGB1 Scramble si RNA组以及吗啡+Transfection R egant组相比较,脊髓背角HMGB1的表达量最低(**P0.01),表明鞘内注射HMGB1 si RNA有效抑制了HMGB1在大鼠脊髓背角的合成。5.运动功能检测发现鞘内连续注射吗啡、GL以及HMGB1 si RNA对大鼠的运动功能没有影响大鼠完成机械撤足阈值和热撤足潜伏期测试后,进行运动功能检测,通过放置反射、抓握反射、翻正反射三种测试方式,与对照组比较,给药组大鼠没有产生运动功能障碍。实验结论抑制大鼠脊髓HMGB1的表达或阻断HMGB1的作用,可以有效延缓吗啡引起的镇痛耐受以及停用吗啡后的机械刺激痛觉超敏和热刺激痛觉过敏症状。
[Abstract]:Background and objective morphine is opioid receptor agonist, which is the gold standard for clinical analgesia, but it will produce analgesic tolerance and hyperalgesia after repeated use. Although many studies have been done on morphine tolerance, the production mechanism is still not very clear. After the use of morphine, some adaptive changes are caused in the nervous system, including desensitization, internalization, downregulation and phosphorylation of opioid receptors, or heterogenous fusion with other receptors. The contact of chronic opioids may cause the activation of neuroglial cells and the activation of glutamate receptors and the release of proinflammatory cytokines. The release of cytokines may be the main cause of morphine tolerance and hyperalgesia. High mobility group protein 1 (High mobility group box protein 1, HMGB1) is an inflammatory medium similar to cytokine, which belongs to the HMGB family.HMGB1 widely existed in mammalian cells. Non immune cells are released into the extracellular environment and cause various inflammatory responses. Recent studies have found that peripheral nerve damage can cause changes in the expression of HMGB1 in the dorsal root ganglion (Dorsal Root Ganglia, DRG) and the dorsal horn of the spinal cord. Blocking the action of HMGB1 can reduce the neuropathic pain in rats. Most domestic and foreign scholars believe that the neuropathic pain can be reduced. Neuropathic pain and morphine tolerance are partly similar to the mechanism of morphine tolerance. Although the role of HMGB1 in pathological pain has been made clear, the role of HMGB1 in morphine tolerance and specific molecular mechanisms have not been reported. The main purpose of this study was to demonstrate that HMGB1 was in morphine tolerance through animal experiments. In this experiment, a rat model of morphine analgesic tolerance was established by microinjection of morphine (10 mu g/10 Mu L) in the sheath for 6 days (2 times a day for a total of 6 days). The changes in the analgesic effect of morphine were measured by the test of rat tail flick latency (Tail-flick La tency), and the threshold of the withdrawal of morphine (Paw With drawal Thresh) was tested. Old, PWT) and thermal stimulation of the foot latency (Paw Withdrawal Latency, PWL) assessment of the changes in the pain behavior of rats after the discontinuation of morphine. The experimental process was combined with the intrathecal injection of Glycyrrhizin (GL) (HMGB1's small molecule inhibitor) or HMGB1 Si RNA, in the different periods of morphine tolerance formation (morphine tolerance formation before and after morphine tolerance formation). The effects of blocking HM GB1 or inhibiting the synthesis of HMGB1 on the formation of morphine tolerance were observed. The effects of HMGB1 on the expression of HMGB1 in DRG and spinal dorsal horn in morphine tolerance formation and the effect of HMGB1 Si RNA on HMGB1 expression in the sheath were further observed by Western Blot; immunofluorescence double staining was used. The expression of morphine induced HMGB1 in rat DRG and dorsal horn of spinal cord was observed. The results of the experiment will provide theoretical support for further elucidate the mechanism of morphine tolerance formation, and have some practical reference value for the production of morphine tolerance in clinical. 1. the experimental results of morphine tolerance formation in rat DRG and spinal cord dorsal The changes in the expression of the angle and the expression of the cell type in the rat sheath were injected with morphine 10 g/10 Mu l a day two times a day for 6 days. The analgesic efficiency of morphine was about 20% (**P0.01) on the fifth day of the administration, and the analgesic efficiency of morphine was almost 0 in seventh days (compared with that of the injected saline group, ***P0.001). The morphine was stopped at eighth days after the withdrawal of morphine. The lateral mechanical pain hypersensitivity and hyperalgesia (compared with the injected physiological saline group, ***P0.001).West ERN Blot detected that intrathecal morphine induced a significant increase in HMGB1 expression in the dorsal horn and DRG of the spinal cord (compared with the injected physiological saline group, **P0.001), and found that H MGB1 was in the dorsal horn of the spinal cord and neurons by immunofluorescence double staining. Astrocytes and microglia have co localization. In the dorsal root ganglia, HMGB1, a class a C neuron and the satellite like glial cells are Co located in the.2. sheath for the pre injection of the HMGB1 small molecule inhibitor Glycyrrhizin, which inhibits the formation of morphine tolerance in the intrathecal GL group and the injection solvent group (Vehicle, Veh), in morphine injection. The analgesic efficiency of morphine, fifth days (***P0.001) and seventh days (**P0.01, ***P0.001), was significantly improved. The PWT and PWL in the rats were improved to a certain extent on the eighth day after the withdrawal (*P0.05, **P0.01). The results suggested that intrathecal GL delayed the formation of morphine tolerance and partly alleviated morphine induced withdrawal. Hyperalgesia, the effect has a certain time and dose dependent.3. morphine tolerance model after the establishment of intrathecal preinjection of GL can partially reverse morphine tolerance model rats after the establishment of morphine tolerance model seventh days after intrathecal injection of morphine, intrathecal injection of GL (early 30 min, 50 mu g/10 L, a total of 5 days a day). Results found in from seventh The analgesic efficiency of morphine in the intrathecal GL group was increased gradually (**P0.01, ***P0.001) in the intrathecal injection of Gl and the solvent group at the beginning of the day to eleventh days. The PWT and PWL and the PWL in the rats were significantly increased (**P0.01, ***P0.001) after the twelfth day of withdrawal (**P0.01, ***P0.001). The results showed that intrathecal GL partly reversed the morphine that had been formed. The intrathecal injection of HMGB1 Si RNA for analgesic tolerance to.4. could delay morphine tolerance in the formation of morphine +HMGB1 Si RNA group and morphine + physiological saline group. Compared with morphine +HMGB1 Scra mble Si RNA group and morphine group, the analgesic efficiency of morphine fifth days and seventh days of morphine was obvious. On the eighth day after stopping the drug, the PWT and PWl and the solvent group had a certain degree of improvement (*P0.05, **P0.01).HMGB1 Scramble Si RNA group and Transfection Regant group, and there was no statistical difference between the morphine + physiological saline group and the morphine + physiological saline group. Compared with the +HMGB1 Scramble Si RNA group and the morphine +Transfection R egant group, the expression of HMGB1 in the dorsal horn of the spinal cord was the lowest (**P0.01). It showed that the intrathecal HMGB1 Si RNA effectively inhibited the synthesis of the spinal dorsal horn of the rat. After the rats completed the test of the threshold of mechanical withdrawal and the incubation period of the heat withdrawal, the exercise function test was carried out, and three kinds of test methods were carried out by placing reflection, grasping reflex and turning positive reflex. Compared with the control group, the rats did not produce motor dysfunction. The experimental conclusion inhibited the expression of HMGB1 in the spinal cord of rats or blocked the effect of HMGB1. The analgesic effect of morphine is delayed, and the symptoms of hyperalgesia and heat induced hyperalgesia after morphine withdrawal are delayed.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R402

【相似文献】