川芎嗪调节星形胶质细胞紊乱改善慢性疼痛的实验研究

发布时间：2018-05-03 06:24

本文选题：川芎嗪 + 星形胶质胶质细胞　；参考：《南京医科大学》2017年硕士论文

【摘要】：背景:慢性疼痛是危害人类健康的严重问题。数据显示,全球成人慢性疼痛的平均发病率约为30%,由于其病因复杂,治疗非常困难。目前临床上现有治疗神经痛的药物,只能暂时性缓解疼痛,并且有许多副作用,因此当务之急是了解神经病理性疼痛的发生发展机制,并且寻找安全有效副作用少的治疗药物。越来越多的研究表明,脊髓胶质细胞在神经病理性疼痛过程中,发挥着重要的作用。胶质细胞的调控逐渐成为治疗神经病理疼痛的新靶点。本实验室的前期研究结果与神经科学领域的前沿研究均提示:川芎嗪是一个具有较大镇痛潜能的天然产物。川芎是我国的一味传统生物碱类中药,作为慢性痛的治疗药物已经有超过一百多年的历史,具有活血化瘀的作用,同时又可理气、疏风止痛。川芎嗪被认为是川芎发挥生物活性的主要物质,本实验室以往实验结发现在CCI诱导的神经病理性疼痛模型中,川芎嗪可以缓解疼痛。但川芎嗪缓解神经病理性疼痛的具体机制并不是很清楚。因此本文拟深入探讨川芎嗪对神经病理性疼痛进程中的星形胶质细胞功能紊乱的影响,并进一步探索它的镇痛机制。方法:(1)建立大鼠坐骨神经慢性压迫损伤(CCI)模型,利用行为学考察单次、多次给予川芎嗪对大鼠机械性缩足反射阈值影响。(2)利用Western blot和免疫荧光方法检测川芎嗪对神经病理性疼痛发生后脊髓星形胶质细胞活化的影响。(3)利用明胶酶谱评估体内川芎嗪对CCI造模的大鼠脊髓水平基质金属蛋白酶-9和2的作用。(4)利用免疫印迹(Western blot)法检测单次、多次给予川芎嗪对CCI造模的大鼠脊髓水平MAPK家族蛋白p-P38、p-JNK、p-ERK的影响。免疫印迹(Western blot)法检测单次、多次给予川芎嗪对CCI造模大鼠脊髓水平 p-PKCγ、p-NR1 和 IL-1β的影响。(5)为了进一步探索川芎嗪潜在的镇痛机制,利用分子模拟技术,对接JNK的上游分子TAK1。(6)利用CCI模型,检测TAK1和JNK抑制剂对大鼠机械性缩足反射阈值影响;使用TAK1和JNK抑制剂考察其对MMP-9和2的影响;利用免疫印迹(Western blot)法,在整体和具有星形胶质细胞特性的C8细胞中检测川芎嗪对p-TAK1和p-JNK的影响。结果:(1)单次川芎嗪腹腔给药能够剂量依赖性地抑制CCI诱导的机械超敏;连续五天给予川芎嗪(30mg/kg)能够发挥持续的镇痛效果;川芎嗪(30mg/kg)多次给药能够显著延缓神经病理性疼痛的发生;川芎嗪腹腔给药能够剂量依赖性的选择性抑制脊髓的JNK的磷酸化水平。(2)多次川芎嗪腹腔给药显著抑制CCI诱发的脊髓背角星形胶质细胞标记物GFAP表达上调。(3)体内明胶酶谱实验结果表明单次或多次腹腔给予川芎嗪,能够显著抑制CCI引起的大鼠脊髓水平MMP-9/2的激活。(4)川芎嗪能显著抑制CCI诱导的大鼠脊髓MAPK家族蛋白p-JNK水平的升高和IL-1β活化以及PKCγ和NR1的磷酸化。(5)CCI可引起p-TAK1水平显著上升。给予川芎嗪后,脊髓p-TAK1水平显著降低。(6)鞘内注射TAK1抑制剂和JNK抑制剂可以抑制CCI诱导的大鼠疼痛超敏的发生发展。(7)给予川芎嗪可以显著抑制IL-1β引起的p-TAK1,p-JNK和MMP-9和2的升高。同时,体外培养星形胶质细胞C8细胞,IL-1β刺激后诱导C8细胞中TAK1、JNK的磷酸化,以及MMP-9/2的升高,而给予川芎嗪可以明显降低CaMKII、TAK1、JNK的磷酸化,以及MMP-9/2的升高。结论:川芎嗪能够显著抑制CCI诱导的脊髓星形胶质细胞的激活,通过抑制神经炎症产生镇痛作用,我们的结果进一步暗示JNK-MMP-2/9作为神经性疼痛减轻的新靶标。
[Abstract]:Background: chronic pain is a serious problem that endangers human health. Data shows that the average incidence of chronic pain in adults around the world is about 30%, and the treatment is very difficult because of its complicated causes. Currently, the current clinical drugs for neuralgia can only temporarily relieve pain and have many side effects, so the urgent task is to understand neuropathy. More and more studies have shown that the spinal glial cells play an important role in the process of neuropathic pain. The regulation of glial cells has gradually become a new target for the treatment of neuropathic pain. The frontier research in the field of neuroscience has suggested that Ligustrazine is a natural product with greater analgesic potential. Ligusticum chuanxiong is a traditional Chinese traditional Chinese medicine. As a chronic pain treatment drug, it has more than more than 100 years history. It has the function of activating blood and removing stasis, and it can also be rational and relieving pain. Ligustrazine is considered to be Ligusticum chuanxiong plays a major bioactive substance. In our laboratory, ligustrazine can relieve pain in the current CCI induced neuropathic pain model, but the specific mechanism of Ligustrazine to relieve neuropathic pain is not very clear. Therefore, this article is to explore the star of ligustrazine in the process of neuropathic pain. The effect of glial cell dysfunction and further explore its analgesic mechanism. Methods: (1) establish the rat sciatic nerve chronic compression injury (CCI) model, use the behavior study to examine the single time, and give the effect of Ligustrazine on the mechanical contraction reflex threshold of rats many times. (2) Western blot and immunofluorescence were used to detect the neuropathology of Ligustrazine. The effect of astrocyte activation after the occurrence of sexual pain. (3) the effect of Tetramethylpyrazine on the spinal cord level matrix metalloproteinase -9 and 2 in CCI model rats was evaluated using gelatinase spectrum. (4) using the immunoblotting (Western blot) method to detect the single time, and the MAPK family protein p-P38, p- of the rat spinal cord with Ligustrazine on CCI The influence of JNK, p-ERK. Immunoblotting (Western blot) method was used to detect the effects of Ligustrazine on the level of p-PKC gamma, p-NR1 and IL-1 beta in the spinal cord of the rat model of CCI. (5) in order to further explore the potential analgesic mechanism of Ligustrazine, using molecular simulation technology, the upstream molecule TAK1. (6) of JNK was used for CCI model to detect TAK1 and inhibition. Effect of the agent on the reflex threshold of mechanical contraction in rats; the effects of TAK1 and JNK inhibitors on MMP-9 and 2; the effects of Tetramethylpyrazine on p-TAK1 and p-JNK were detected in the whole and astrocyte specific C8 cells by Western blot (Western blot). Results: (1) the single tetramethylpyrazine intraperitoneal administration could be dose-dependent. The mechanical hypersensitivity induced by CCI was inhibited; tetramethylpyrazine (30mg/kg) was given a continuous analgesic effect for five days; Ligustrazine (30mg/kg) could significantly delay the occurrence of neuropathic pain; tetramethylpyrazine intraperitoneal administration could selectively inhibit the phosphorylation of JNK in the spinal cord. (2) multiple Ligustrazine intraperitoneal administration The drug significantly inhibited the up-regulated expression of the astrocyte marker GFAP in the dorsal horn of the spinal cord induced by CCI. (3) the results of the gelatinase test in vivo showed that a single or multiple intraperitoneal administration of Ligustrazine could significantly inhibit the activation of MMP-9/2 in the spinal cord of rats induced by CCI. (4) tetramethylpyrazine could significantly inhibit the p-JNK water of MAPK family protein in spinal cord induced by CCI. Level elevation and IL-1 beta activation, and phosphorylation of PKC gamma and NR1. (5) CCI could cause a significant increase in p-TAK1 levels. After Ligustrazine, the level of p-TAK1 in the spinal cord decreased significantly. (6) intrathecal TAK1 inhibitors and JNK inhibitors could inhibit the occurrence of CCI induced pain hypersensitivity in rats. (7) tetramethylpyrazine could significantly inhibit the IL-1 beta. P-TAK1, p-JNK and MMP-9 and 2 increased. At the same time, cultured astrocyte C8 cells in vitro, IL-1 beta stimulated TAK1, JNK phosphorylation and MMP-9/2 increase in C8 cells, and tetramethylpyrazine could significantly reduce CaMKII, TAK1, JNK phosphorylation and increase. Conclusion: Ligustrazine can significantly inhibit the induced spinal star. Activation of the glial cells produces analgesic effects by inhibiting neuritis. Our results further imply that JNK-MMP-2/9 is a new target for neuropathic pain relief.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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