背根神经节P2X7受体在术后急慢性痛转化中的作用
发布时间：2018-08-22 09:19
【摘要】：背景和目的国际疼痛协会(IASP)将术后慢性疼痛(CPSP)定义为发生在手术后,与手术相关的,排除其他原因且持续至少2个月的慢性疼痛。CPSP的发病率居高不下,最近一项国内大样本研究结果显示,3110名纳入对象术后6月CPSP发生率达29.6%,其中30.3%的患者合并焦虑症状,24.4%的患者存在抑郁表现。P2X7受体是腺苷三磷酸(ATP)门控的离子通道,属于嘌呤能P2受体家族,参与细胞信号传导、细胞因子的分泌等多种生理功能。研究表明,P2X7受体不仅在免疫细胞中能够引起细胞凋亡,且能影响到细胞许多复杂的生理生化功能。在神经病理性疼痛和炎性疼痛模型大鼠,P2X7受体在脊髓背角水平,通过调控炎性因子如肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)等的分化和释放,参与神经元和胶质细胞的对话,在中枢敏感化的发生发展中扮演重要角色。我们前期研究已证实P2X7受体在脊髓背角可通过调控小胶质细胞的激活及炎性因子的释放从而参与到术后急慢性痛的转化过程中。抑制脊髓背角P2X7受体激活可下调肿瘤坏死因子-α的释放并显著改善SMIR术后慢性疼痛。背根神经(Dorsal root ganglion,DRG)位于脊神经后根,是外周神经与中枢神经的传递中枢。DRG上神经元胞体为假单极神经元,神经节上每个神经元胞体会发出一个轴突进而形成两个分支,分布在外周各处(皮肤、内脏等)接收外周刺激信号并传递到中枢神经系统。DRG上神经元传递伤害性感受,与痛觉传导机制密切相关,是外周痛觉信号逐级传入至大脑皮层的第一级神经元。当机体受到外周刺激时,首先由DRG感知这一变化,并激活DRG神经元,引起外周敏化,进而传递到脊髓背角,引起脊髓背角投射神经元兴奋性异常增高,诱发脊髓背角C纤维LTP产生并引起中枢敏化,传递到大脑最终导致病理性疼痛的产生。虽然我们前期的结果已经表明脊髓背角P2X7受体的激活是术后疼痛慢性化的重要中枢机制,但关于CPSP产生的外周机制我们并不清楚。基于以上,我们把研究焦点转移到背根神经节,期望从DRG层面来探究术后疼痛慢性化形成的外周机制。本研究通过SMIR模型模拟术后急性疼痛转化为术后慢性疼痛,探究背根神经节中P2X7受体在术后慢性疼痛形成中的作用机制,并深入探讨大鼠背根神经节中P2X7受体介导的ERK/MAPK信号通路的激活在术后急慢性痛转化中的作用和地位。方法选取体重180~220 g的SD雄性成年大鼠,将其随机分为2组:模型组(SMIR组)和假手术组(Sham组)。SMIR组采用Flatter的下肢皮肤肌肉切开牵拉法(skin/muscle incision and retraction,SMIR)来建立大鼠手术后慢性疼痛模型。Sham采用假手术作为对照。首先分别测定术前一天及术后各时间点(1、3、7、12、22及32天)各组大鼠的机械缩足反应阈值(PWT)(n=14)。上述各时间点痛阈测试完毕后,分时间点(0、1、3、7、12、22及32天)取大鼠手术侧L2-4 DRGs采用免疫印迹试验(Western Blot)及免疫双标染色法的方法对大鼠背根神经节P2X7受体、ERK/MAPK信号通路及TNF-α各时间点蛋白表达量变化趋势及细胞定位进行观察(n=6)。然后经鞘内给予ERK抑制剂SCH772984进行干预,观察ERK抑制剂SCH772984对SMIR术后大鼠的行为学的影响,采用免疫印迹试验检测术后第7天各组手术侧L2-4 DRGs P2X7受体、ERK/MAPK信号通路及TNF-α表达量的变化。另外再经腹腔注射P2X7受体拮抗剂BBG进行干预,观察BBG对SMIR术后大鼠机械缩足反应阈值的影响,并采用Western Blot方法观察P2X7受体拮抗剂BBG对SMIR术后大鼠术侧L2-4 DRGs P2X7受体、ERK/MAPK信号通路及TNF-α表达量的影响。以进一步确认P2X7受体、ERK/MAPK信号通路及TNF-α三者之间的关系。明确背根神经节P2X7受体在术后急慢性痛转化中的作用。结果1.SMIR术后大鼠术侧缩足反应阈值明显降低通过对各时间点大鼠机械缩足反应阈值进行统计分析,结果显示:两组大鼠术前的PWT基础值无统计学差异(P0.05)。SMIR组手术侧痛阈术后1d即发生降低并持续至术后32天后才恢复至术前水平,SMIR组大鼠手术侧PWT值术后第1d与术前基础值相比即发生降低(P0.01),持续降低至术后第22d(P0.001),术后第12d达到最低峰值(P0.001),疼痛持续至术后32d才基本恢复正常(P0.05)。SMIR组大鼠手术对侧足底PWT值与基础值相比变化无统计学差异(P0.05),Sham组大鼠双侧足底术后的PWT值与基础值相比均无统计学差异(P0.05)。2.SMIR模型组大鼠术侧背根神经节P2X7受体表达增多且特异性表达在卫星胶质细胞上。免疫印迹试验方法结果显示SMIR模型组大鼠L2-4背根神经节P2X7受体表达量在术后第一天开始上调(P0.05),术后7d达峰值(P0.001),随后逐渐减少,直至术后第32d恢复术前水平(P0.05)。将术后7d大鼠手术侧L3 DRG进行免疫荧光双标染色,分别将P2X7受体与神经元特异性标记物NF200、CGRP、Ib4及卫星胶质细胞特异性标记物GFAP进行双染,结果显示,P2X7受体与GFAP大量共标,而与NF200、CGRP、Ib4未见共标。提示,在SMIR模型大鼠背根神经节,P2X7受体完全特异性表达在卫星胶质细胞(Satellite glial cells,SGCs)上。3.SMIR模型组大鼠背根神经节ERK/MAPK信号通路被激活SMIR模型制备后第1d可见大鼠背根神经节p-c Raf、p-MEK及p-ERK蛋白表达水平显著性增高(P0.001),并持续增高至SMIR术后第12d达峰值(p0.001),术后第32d恢复至术前水平(P0.05)。SMIR术后各时间点p-c Raf、p-MEK及p-ERK蛋白表达水平与SMIR术后各时间对应点的PWT值变化的相关性分析显示,p-c Raf、p-MEK及p-ERK都与术后PWT值的变化具有显著相关性,p-c-Raf(Pearson correlation;r=-0.965,P=0.002),p-MEK(Pearson correlation;r=-0.906,P=0.013),p-ERK(Pearson correlation;r=-0.887,P=0.018)。术后7d SMIR模型组大鼠手术侧L3 DRG的P2X7受体和p-ERK的免疫双标染色显示P2X7受体与p-ERK大量共标。以上提示SMIR术后ERK/MAPK信号通路被激活,且ERK/MAPK信号通路的激活可能与P2X7受体有关。4.SMIR模型组大鼠手术侧脊髓背根神经节TNF-α释放水平上调SMIR模型组大鼠L2-4 DRGs各时间点TNF-α的Western blot结果分析显示,模型组大鼠手术侧L2-4 DRGs TNF-α在术后第1d即上调,手术后第12d达到峰值(P0.001),之后逐渐下调直至术后第32d才恢复至术前水平(P0.05),SMIR术后大鼠背根神经节TNF-α的表达变化与SMIR术后PWT值变化具有相关性(Pearson correlation;r=-0.871,P=0.011)。5.ERK的特异性拮抗剂SCH772984可缓解SMIR术后大鼠疼痛,下调背根神经节TNF-α释放水平,但不影响SMIR术后P2X7受体的激活结果显示,鞘内注射ERK特异性拮抗剂SCH772984可显著改善大鼠SMIR术后的痛觉过敏。而单纯注射SCH772984对正常大鼠的PWT值不产生影响(P0.05)。SMIR术后第1d至术后第22d各时间点,给药组和对照组两组组间PWT值结果比较显示:给药组PWT值均显著增加(P0.001)。这表明ERK的特异性拮抗剂SCH772984可缓解SMIR术后大鼠疼痛。术后第7d给药组(SCH+SMIR)和对照组(DMSO+SMIR)两组大鼠L2-4DRGs的TNF-α表达量的结果分析显示,与Sham相比,给药组TNF-α表达水平发生下降(P0.01),与Sham相比,对照组TNF-α表达水平发生显著性上调(P0.001),而与对照组相比,给药组TNF-α表达水平显著性降低(P0.001)。手术后第7d两组大鼠L2-4 DRGs的P2X7受体的Western blot结果显示,与Sham组相比,给药组和对照组P2X7受体表达水平均发生显著性上调(P0.001),与对照组相比,给药组P2X7受体表达水平没有明显改变(P0.05)。这表明ERK的特异性拮抗剂SCH772984可下调SMIR术后大鼠背根神经节TNF-α释放水平,但不影响SMIR术后P2X7受体的表达。6.P2X7受体特异性拮抗剂BBG可上调SMIR模型组大鼠痛阈值并下调ERK/MAPK信号通路及TNF-α表达水平结果显示,腹腔注射BBG的给药组(BBG+SMIR)大鼠机械缩足反应阈值与腹腔注射生理盐水的对照组(NS+SMIR)相比有明显改善。给药组PWT值大鼠与Sham组相比无明显统计学差异(P0.05)。而术后第1d至术后第22d,给药组与对照组相比,对照组PWT值均显著性增加(P0.001),表明给予BBG抑制P2X7受体的激活可显著改善大鼠SMIR术后疼痛。手术后第7d两组大鼠术侧L2-4 DRGs的ERK/MAPK信号通路和TNF-α表达量的Western blot结果显示,与Sham组相比,对照组p-c Raf、p-MEK、p-ERK和TNF-α表达水平均发生显著上调(P0.001);与Sham组相比,给药组p-c Raf、p-MEK、p-ERK和TNF-α表达水平均发生下调(P0.05);与对照组相比,给药组p-c Raf、p-MEK、p-ERK和TNF-α表达水平均减少(P0.001)。表明P2X7受体拮抗剂BBG可抑制SMIR术后大鼠背根神经节ERK/MAPK信号通路的激活及下调TNF-α的表达。结论本研究通过大鼠皮肤/肌肉切口牵拉(SMIR)模型模拟术后急慢性疼痛转化过程,发现:1、SMIR术后大鼠术侧背根神经节卫星胶质细胞上P2X7受体表达上调,ERK/MAPK信号通路被激活,背根神经节神经元上TNF-α释放增多;2、鞘内给予ERK的特异性拮抗剂SCH772984可缓解SMIR诱发的痛觉过敏,下调背根神经节TNF-α的释放,但对不影响SMIR术后P2X7受体的上调;3、腹腔注射P2X7受体拮抗剂BBG不仅能明显预防SMIR术后疼痛的发生,而且能抑制SMIR术后ERK/MAPK信号通路的激活并下调TNF-α的释放。故而,术后急慢性疼痛的转化可能是由脊髓背根神经节卫星胶质细胞P2X7受体上调,进而介导ERK/MAPK信号通路激活,引起DRG神经元炎性反应增加所导致。
[Abstract]:BACKGROUND AND OBJECTIVE The International Association for Pain (IASP) defines postoperative chronic pain (CPSP) as postoperative, surgery-related, chronic pain lasting at least two months, excluding other causes. The incidence of CPSP remains high. A recent large-sample study in China showed that the incidence of CPSP was 29.6% in 3110 subjects 6 months after surgery. Among them, 30.3% had anxiety symptoms and 24.4% had depression. P2X7 receptor is an ATP-gated ion channel, belonging to the purinergic P2 receptor family, which participates in many physiological functions such as cell signal transduction and cytokine secretion. In neuropathic pain and inflammatory pain model rats, P2X7 receptor is involved in the dialogue between neurons and glial cells at the level of spinal dorsal horn and is sensitive in the central nervous system by regulating the differentiation and release of inflammatory factors such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1bet). Inhibition of P2X7 receptor activation in the spinal dorsal horn can down-regulate the release of tumor necrosis factor-alpha and significantly improve SMIR. Postoperative chronic pain. The dorsal root ganglion (DRG) is located in the posterior root of the spinal nerve and is the transmission center of the peripheral and central nerves. The neurons on DRG transmit nociceptive sensation, which is closely related to the mechanism of pain conduction. It is the first stage of peripheral nociceptive signal that passes into the cerebral cortex step by step. The activation of P2X7 receptors in the dorsal horn of the spinal cord has been shown to be an important central mechanism of postoperative pain chronicity. The peripheral mechanism of CPSP is unclear. Based on the above, we shifted our focus to the DRG level to explore the peripheral mechanism of chronic pain after surgery. Methods Male SD rats weighing 180-220 g were randomly divided into two groups: model group (SMIR group) and sham operation group (Sham group). Sham used sham operation as control. Firstly, the mechanical shrinkage thresholds (PWT) (n=14) of the rats in each group were measured at 1, 3, 7, 12, 22 and 32 days before and after operation. L2-4 DRGs were harvested at different time points (0,1,3,7,12,22 and 32 days) after the time point pain threshold test. The expression of P2X7 receptor, ERK/MAPK signaling pathway and TNF-alpha protein in dorsal root ganglion of rats were observed by Western Blot and immunostaining (n=6). The effects of ERK inhibitor SCH772984 on the behavior of rats after SMIR were observed. The changes of L2-4 DRGs P2X7 receptor, ERK/MAPK signaling pathway and TNF-alpha expression were detected by Western blotting on the 7th day after SMIR. Intervention was conducted to observe the effect of BBG on the threshold of mechanical foot contraction in rats after SMIR. Western Blot method was used to observe the effects of BBG, a P2X7 receptor antagonist, on L2-4 DRGs P2X7 receptor, ERK/MAPK signaling pathway and TNF-alpha expression on the operative side of rats after SMIR. Results 1. After SMIR, the threshold of contraction reaction was significantly reduced. By statistical analysis of the threshold of mechanical contraction reaction at each time point, the results showed that there was no significant difference in the baseline value of PWT between the two groups (P 0.05). In SMIR group, the PWT value on the operative side decreased at the first day after operation (P 0.01), and continued to decrease until the 22nd day after operation (P 0.001), and reached the lowest peak at the 12th day after operation (P 0.001). The pain did not return to normal until 32 days after operation (P 0.05). There was no significant difference in PWT value between the contralateral plantar ganglia and the baseline values (P 0.05). There was no significant difference in PWT value between the sham group and the baseline values (P 0.05). 2. The expression of P2X7 receptor in the dorsal root ganglia of the SMIR model group increased and expressed specifically on the satellite glia. The results showed that the expression of P2X7 receptor in the L2-4 dorsal root ganglion of SMIR model rats was up-regulated at the first day after operation (P 0.05), peaked at 7 days after operation (P 0.001), then decreased gradually until the preoperative level was restored at 32 days after operation (P 0.05). The results showed that P2X7 receptor was highly co-labeled with GFAP, but not with NF200, CGRP and Ib4. It was suggested that P2X7 receptor was completely and specifically expressed on the satellite glial cells (SGCs) in the dorsal root ganglion of SMIR rats. The expression of P-C Raf, p-MEK and p-ERK protein in rat dorsal root ganglion was significantly increased on the first day after SMIR (P 0.001), and reached the peak at the 12th day after SMIR (P 0.001), and returned to the preoperative level at the 32nd day after SMIR (P 0.05). Correlation analysis showed that P-C Raf, p-MEK and p-ERK were significantly correlated with the changes of PWT after SMIR, P-C Raf (Pearson correlation; r=-0.965, P=0.002), p-MEK (Pearson correlation; r=-0.906, P=0.013), p-ERK (Pearson correlation; r=-0.887, P=0.018). Immunodouble-labeled staining of P2X7 receptor and p-ERK in L3 DRG of SMIR rats on the 7th day revealed a large number of P2X7 receptor and p-ERK co-labeled. These results suggest that ERK/MAPK signaling pathway is activated after SMIR, and the activation of ERK/MAPK signaling pathway may be related to P2X7 receptor. 4. TNF-alpha release level in dorsal root ganglion of spinal cord in SMIR rats Western blot analysis showed that the expression of TNF-alpha in the dorsal root ganglion of SMIR rats was up-regulated on the first day after operation, peaked at the 12th day after operation (P 0.001), and then gradually decreased until the 32nd day after operation (P 0.05). The expression of TNF-alpha in the dorsal root ganglion of SMIR rats was restored to the preoperative level (P 0.05). The changes were correlated with the changes of PWT after SMIR (Pearson correlation; r = - 0.871, P = 0.011). 5. SCH772984, a specific antagonist of ERK, could alleviate the pain after SMIR in rats, and down-regulate the release of TNF-a in dorsal root ganglion, but did not affect the activation of P2X7 receptor after SMIR. SCH772984, a specific antagonist of ERK, could be significantly injected intrathecally. Compared with the control group, the PWT values of the two groups were significantly increased (P 0.001). This indicated that the specific antagonist of ERK, SCH772984, could be slowed down. The expression of TNF-alpha in L2-4DRGs in SCH+SMIR group and DMSO+SMIR group was significantly higher than that in Sham group (P 0.01) and control group (P 0.001). The expression of P2X7 receptor in L2-4 DRGs was significantly increased in both groups on the 7th day after operation (P 0.001). Compared with Sham group, the expression of P2X7 receptor was significantly increased in both groups (P 0.001). Compared with the control group, the expression of P2X7 receptor was not significantly changed (P 0.05). ERK-specific antagonist SCH772984 could down-regulate the release of TNF-a in the dorsal root ganglion of rats after SMIR, but did not affect the expression of P2X7 receptor after SMIR. 6. P2X7 receptor-specific antagonist BBG could up-regulate the pain threshold and down-regulate the expression of ERK/MAPK signaling pathway and TNF-a in the SMIR model rats. The results showed that the intraperitoneal injection of BBG could up-regulate the pain threshold and down-regulate the expression of TNF-a Compared with the control group (NS + SMIR), there was no significant difference in PWT between the two groups (P 0.05). However, from the 1st day to the 22nd day after operation, the PWT of the control group was significantly increased (P 0.001), indicating that BBG inhibited P2X7. The ERK/MAPK signaling pathway and the expression of TNF-alpha in the L2-4 DRGs were significantly increased in the control group (P 0.001) and the Sham group (P 0.001) on the 7th day after SMIR. The expression levels of p-ERK and TNF-alpha were down-regulated (P 0.05). Compared with the control group, the expression levels of P-C Raf, p-MEK, p-ERK and TNF-alpha were decreased in the treatment group (P 0.001). It indicated that BBG, a P2X7 receptor antagonist, could inhibit the activation of ERK/MAPK signaling pathway and down-regulate the expression of TNF-alpha in the dorsal root ganglion of SMIR rats. The incision traction (SMIR) model simulated the process of acute and chronic pain transformation after SMIR. It was found that: 1. P2X7 receptor expression was up-regulated, ERK/MAPK signaling pathway was activated and TNF-a release was increased in dorsal root ganglion neurons after SMIR; 2. SCH772984, a specific antagonist of ERK, could alleviate SMIR-induced pain. Hyperalgesia can down-regulate the release of TNF-a in dorsal root ganglion, but does not affect the up-regulation of P2X7 receptor after SMIR. 3. Intraperitoneal injection of BBG, a P2X7 receptor antagonist, can not only significantly prevent pain after SMIR, but also inhibit the activation of ERK/MAPK signaling pathway and down-regulate the release of TNF-a after SMIR. P2X7 receptor is up-regulated by the satellite glial cell P2X7 receptor in the dorsal root ganglion of the spinal cord, and then mediates the activation of ERK/MAPK signaling pathway, resulting in an increased inflammatory response of DRG neurons.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R402
本文编号：2196666
[Abstract]:BACKGROUND AND OBJECTIVE The International Association for Pain (IASP) defines postoperative chronic pain (CPSP) as postoperative, surgery-related, chronic pain lasting at least two months, excluding other causes. The incidence of CPSP remains high. A recent large-sample study in China showed that the incidence of CPSP was 29.6% in 3110 subjects 6 months after surgery. Among them, 30.3% had anxiety symptoms and 24.4% had depression. P2X7 receptor is an ATP-gated ion channel, belonging to the purinergic P2 receptor family, which participates in many physiological functions such as cell signal transduction and cytokine secretion. In neuropathic pain and inflammatory pain model rats, P2X7 receptor is involved in the dialogue between neurons and glial cells at the level of spinal dorsal horn and is sensitive in the central nervous system by regulating the differentiation and release of inflammatory factors such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1bet). Inhibition of P2X7 receptor activation in the spinal dorsal horn can down-regulate the release of tumor necrosis factor-alpha and significantly improve SMIR. Postoperative chronic pain. The dorsal root ganglion (DRG) is located in the posterior root of the spinal nerve and is the transmission center of the peripheral and central nerves. The neurons on DRG transmit nociceptive sensation, which is closely related to the mechanism of pain conduction. It is the first stage of peripheral nociceptive signal that passes into the cerebral cortex step by step. The activation of P2X7 receptors in the dorsal horn of the spinal cord has been shown to be an important central mechanism of postoperative pain chronicity. The peripheral mechanism of CPSP is unclear. Based on the above, we shifted our focus to the DRG level to explore the peripheral mechanism of chronic pain after surgery. Methods Male SD rats weighing 180-220 g were randomly divided into two groups: model group (SMIR group) and sham operation group (Sham group). Sham used sham operation as control. Firstly, the mechanical shrinkage thresholds (PWT) (n=14) of the rats in each group were measured at 1, 3, 7, 12, 22 and 32 days before and after operation. L2-4 DRGs were harvested at different time points (0,1,3,7,12,22 and 32 days) after the time point pain threshold test. The expression of P2X7 receptor, ERK/MAPK signaling pathway and TNF-alpha protein in dorsal root ganglion of rats were observed by Western Blot and immunostaining (n=6). The effects of ERK inhibitor SCH772984 on the behavior of rats after SMIR were observed. The changes of L2-4 DRGs P2X7 receptor, ERK/MAPK signaling pathway and TNF-alpha expression were detected by Western blotting on the 7th day after SMIR. Intervention was conducted to observe the effect of BBG on the threshold of mechanical foot contraction in rats after SMIR. Western Blot method was used to observe the effects of BBG, a P2X7 receptor antagonist, on L2-4 DRGs P2X7 receptor, ERK/MAPK signaling pathway and TNF-alpha expression on the operative side of rats after SMIR. Results 1. After SMIR, the threshold of contraction reaction was significantly reduced. By statistical analysis of the threshold of mechanical contraction reaction at each time point, the results showed that there was no significant difference in the baseline value of PWT between the two groups (P 0.05). In SMIR group, the PWT value on the operative side decreased at the first day after operation (P 0.01), and continued to decrease until the 22nd day after operation (P 0.001), and reached the lowest peak at the 12th day after operation (P 0.001). The pain did not return to normal until 32 days after operation (P 0.05). There was no significant difference in PWT value between the contralateral plantar ganglia and the baseline values (P 0.05). There was no significant difference in PWT value between the sham group and the baseline values (P 0.05). 2. The expression of P2X7 receptor in the dorsal root ganglia of the SMIR model group increased and expressed specifically on the satellite glia. The results showed that the expression of P2X7 receptor in the L2-4 dorsal root ganglion of SMIR model rats was up-regulated at the first day after operation (P 0.05), peaked at 7 days after operation (P 0.001), then decreased gradually until the preoperative level was restored at 32 days after operation (P 0.05). The results showed that P2X7 receptor was highly co-labeled with GFAP, but not with NF200, CGRP and Ib4. It was suggested that P2X7 receptor was completely and specifically expressed on the satellite glial cells (SGCs) in the dorsal root ganglion of SMIR rats. The expression of P-C Raf, p-MEK and p-ERK protein in rat dorsal root ganglion was significantly increased on the first day after SMIR (P 0.001), and reached the peak at the 12th day after SMIR (P 0.001), and returned to the preoperative level at the 32nd day after SMIR (P 0.05). Correlation analysis showed that P-C Raf, p-MEK and p-ERK were significantly correlated with the changes of PWT after SMIR, P-C Raf (Pearson correlation; r=-0.965, P=0.002), p-MEK (Pearson correlation; r=-0.906, P=0.013), p-ERK (Pearson correlation; r=-0.887, P=0.018). Immunodouble-labeled staining of P2X7 receptor and p-ERK in L3 DRG of SMIR rats on the 7th day revealed a large number of P2X7 receptor and p-ERK co-labeled. These results suggest that ERK/MAPK signaling pathway is activated after SMIR, and the activation of ERK/MAPK signaling pathway may be related to P2X7 receptor. 4. TNF-alpha release level in dorsal root ganglion of spinal cord in SMIR rats Western blot analysis showed that the expression of TNF-alpha in the dorsal root ganglion of SMIR rats was up-regulated on the first day after operation, peaked at the 12th day after operation (P 0.001), and then gradually decreased until the 32nd day after operation (P 0.05). The expression of TNF-alpha in the dorsal root ganglion of SMIR rats was restored to the preoperative level (P 0.05). The changes were correlated with the changes of PWT after SMIR (Pearson correlation; r = - 0.871, P = 0.011). 5. SCH772984, a specific antagonist of ERK, could alleviate the pain after SMIR in rats, and down-regulate the release of TNF-a in dorsal root ganglion, but did not affect the activation of P2X7 receptor after SMIR. SCH772984, a specific antagonist of ERK, could be significantly injected intrathecally. Compared with the control group, the PWT values of the two groups were significantly increased (P 0.001). This indicated that the specific antagonist of ERK, SCH772984, could be slowed down. The expression of TNF-alpha in L2-4DRGs in SCH+SMIR group and DMSO+SMIR group was significantly higher than that in Sham group (P 0.01) and control group (P 0.001). The expression of P2X7 receptor in L2-4 DRGs was significantly increased in both groups on the 7th day after operation (P 0.001). Compared with Sham group, the expression of P2X7 receptor was significantly increased in both groups (P 0.001). Compared with the control group, the expression of P2X7 receptor was not significantly changed (P 0.05). ERK-specific antagonist SCH772984 could down-regulate the release of TNF-a in the dorsal root ganglion of rats after SMIR, but did not affect the expression of P2X7 receptor after SMIR. 6. P2X7 receptor-specific antagonist BBG could up-regulate the pain threshold and down-regulate the expression of ERK/MAPK signaling pathway and TNF-a in the SMIR model rats. The results showed that the intraperitoneal injection of BBG could up-regulate the pain threshold and down-regulate the expression of TNF-a Compared with the control group (NS + SMIR), there was no significant difference in PWT between the two groups (P 0.05). However, from the 1st day to the 22nd day after operation, the PWT of the control group was significantly increased (P 0.001), indicating that BBG inhibited P2X7. The ERK/MAPK signaling pathway and the expression of TNF-alpha in the L2-4 DRGs were significantly increased in the control group (P 0.001) and the Sham group (P 0.001) on the 7th day after SMIR. The expression levels of p-ERK and TNF-alpha were down-regulated (P 0.05). Compared with the control group, the expression levels of P-C Raf, p-MEK, p-ERK and TNF-alpha were decreased in the treatment group (P 0.001). It indicated that BBG, a P2X7 receptor antagonist, could inhibit the activation of ERK/MAPK signaling pathway and down-regulate the expression of TNF-alpha in the dorsal root ganglion of SMIR rats. The incision traction (SMIR) model simulated the process of acute and chronic pain transformation after SMIR. It was found that: 1. P2X7 receptor expression was up-regulated, ERK/MAPK signaling pathway was activated and TNF-a release was increased in dorsal root ganglion neurons after SMIR; 2. SCH772984, a specific antagonist of ERK, could alleviate SMIR-induced pain. Hyperalgesia can down-regulate the release of TNF-a in dorsal root ganglion, but does not affect the up-regulation of P2X7 receptor after SMIR. 3. Intraperitoneal injection of BBG, a P2X7 receptor antagonist, can not only significantly prevent pain after SMIR, but also inhibit the activation of ERK/MAPK signaling pathway and down-regulate the release of TNF-a after SMIR. P2X7 receptor is up-regulated by the satellite glial cell P2X7 receptor in the dorsal root ganglion of the spinal cord, and then mediates the activation of ERK/MAPK signaling pathway, resulting in an increased inflammatory response of DRG neurons.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R402
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