针刺扶突穴等对颈部切口痛大鼠脊髓SP、NK-1R、COX-1及5-HT1AR、5-HT2AR表达的影响
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摘要
研究背景
痛症是针灸治疗的第一适应大症,在针刺镇痛(针刺麻醉)的适应症中,首选之一为甲状腺手术。但是,针刺镇痛行甲状腺手术的神经生物学机制尚未进行过系统研究。术后痛是急性疼痛的一种表现形式,临床上,针刺可明显缓解口腔手术术后痛、阑尾切除术后痛、骨科术后痛、甲状腺术后切口痛、四肢及妇科手术后切口痛等。动物实验也证明,电针可减轻足底切口痛行为反应,电生理学实验证明电针可以抑制足底切口痛大鼠Aδ纤维和C类纤维的放电。但是,针刺缓解切口痛的作用机制还不完全清楚,研究还不多；特别是针刺减轻颈部切口痛还没有任何报道。因此,本实验在大鼠甲状腺区切口痛的模型上,探讨电针双侧扶突穴对动物疼痛反应的影响及脊髓背角SP及其NK1受体与COX-1、5-HT1A、5-HT2A的表达变化,探讨电针缓解术后疼痛的脊髓机制,为临床针麻行甲状腺手术提供实验依据。
材料与方法
Wistar雄性大鼠120只,随机分为正常对照组(n=24),模型组(n=24),扶突穴组(n=24),足三里-阳陵泉穴组(n=24),合谷-内关穴组(n=24)。异氟醚麻醉下,在大鼠颈部做纵行切口,造成切口痛模型,分别电针双侧扶突穴,双侧足三里、阳陵泉穴,双侧合谷、内关穴(2／15 Hz,15min,1mA；15min,2 mA),用辐射热分别在术前、术后、针后照射大鼠颈部／切口引起的躲避潜伏期作为痛敏分数衡量动物痛反应(每组10例),在麻醉状态下,一部分(每组8例)动物常规灌流取颈段脊髓(颈1-颈4),连续冰冻切片,用免疫组织化学方法检测颈段脊髓背角SP、NK-1、COX-1和5-HT1A、5-HT2A的表达；另一部分动物(每组6例)取颈段脊髓(颈1-颈4)组织液氮研磨提取RNA和蛋白,分别用定量PCR和Western blot方法来检测脊髓中SP、COX-1和5-HT2A的表达的变化。
结果
1)电针对颈部切口痛大鼠痛行为的影响
与颈部切口术前(模型组15.94±4.21,扶突穴组14.48±2.76,合谷内关组13.72±2.54,足三里-阳陵泉组13.04±3.20)比较,各组动物的热辐射躲避潜伏期(模型组10.10±3.21,扶突穴组8.99±2.99,内关组8.33±1.33,足三里-阳陵泉组8.27±1.67)明显缩短(P<0.05)；电针扶突穴后动物痛阈值(15.98±5.04)与本组术后4h(8.99±2.99)比较、电针合谷、内关穴后动物痛阈值(12.56±3.05)与本组术后4h(8.33±1.33)比较,其躲避潜伏期均显著延长(P<0.05),电针扶突穴后动物的痛阈值明显高于电针合谷、内关动物(P<0.05)。电针足三里-阳陵泉穴后痛阈值(9.54±2.34)与本组术后(8.27±1.67)比较差异无统计学意义(P>0.05)。
2)电针对颈部切口痛大鼠脊髓背角背角SP、NK-1R、COX-1及5-HT1A,5-HT2A影响的免疫组织化学研究
颈部切口术后,模型组动物脊髓背角浅层SP(76.76±12.51)、NK-1(71.02±7.08)、COX-1(60.84±8.02)的灰度值均明显低于正常对照组(93.74±14.01,110.60±0.97,95.16±8.73)(P<0.05),即表达显著升高。与模型组比较,扶突穴组SP(94.90±7.32)、NK-1(91.69±1.28)、COX-1(105.60±13.84)的灰度值显著升高,表达明显下调(P<0.05))；足三里-阳陵泉穴组SP灰度值(73.72±11.06)、NK-1灰度值(67.81±15.23)、COX-1灰度值(76.59±8.07),与模型组比较(76.76±12.51,71.02±7.08,60.84±8.02)无显著差异(P>0.05)。
术后,与正常组(113.9±6.76,134.33±8.06)比较,模型组5-HT1A受体灰度值(95.13±4.56)明显降低(P<0.05),5-HT2A灰度值稍有降低(129.4515.62)。与模型组比较,扶突穴组5-HT1A(78.81±4.57)、5-HT2A(110.89±8.33)灰度值明显下降(P<0.05),而足三里-阳陵泉组5-HT1AR灰度值(94.36±4.20)5-HT2AR灰度值(130.43±14.70)与之没有明显差异(P>0.05)。
3)电针对颈部切口痛大鼠脊髓背角NK-1R mRNA、COX-1mRNA及5-HT1AR mRNA、5-HT2ARmRNA及蛋白水平表达的影响
与正常动物组NK-1R mRNA的表达水平(1.393±0.430)相比,模型组动物颈部手术后,颈段脊髓(C1-C4)的NK-1R mRNA的表达水平(6.608±0.689)明显增高(P<0.05),与模型组比较(6.608±0.689),电针双侧扶突穴后颈段脊髓(C1-C4)NK-1受体的表达(0.339±0.074)显著降低(P<0.05),电针双侧的合谷-内关穴后颈段脊髓(C1-C4) NK-1受体的表达(1.089±0.797)与模型组比较(6.608±0.689)也显著降低(P<0.05),而电针双侧扶突穴组mRNA水平与电针双侧合谷、内关穴组比较没有差别。足三里-阳陵泉组颈段脊髓(C1-C4)NK-1受体的表达(4.513±1.556)明显高于扶突穴组(0.339±0.074)与合谷-内关组(1.089±0.797),足三里-阳陵泉组腰段脊髓(L4-S3)mRNA的表达(2.3708±0.988)低于本组颈段脊髓,但没有统计学差异。
与正常动物组(1.227±0.158)相比,模型组动物颈部手术后,颈段脊髓(C1-C4)的COX-1mRNA的表达水平(4.519±1.211)明显增高(P<0.05),与模型组比较(4.519±1.211),电针双侧扶突穴后颈段脊髓(C1-C4)COX-1 mRNA的表达(1.171±0.249)、电针双侧的合谷、内关穴后颈段脊髓(C1-C4) COX-1mRNA的表达(1.321±0.160)都显著降低(P<0.05),而电针双侧扶突穴组mRNA水平与电针双侧合谷-内关穴组比较没有差别。足三里-阳陵泉组颈段脊髓(C1-C4) COX-1mRNA的表达(5.812±1.734)明显高于扶突穴组(1.171±0.249)与合谷-内关组(1.321±0.160),足三里-阳陵泉组腰段脊髓(L4-S3)COX-1mRNA的表达(2.3708±0.988)明显低于本组颈段脊髓表达水平(2.685±0.921)(P<0.05)。
与正常动物组(1.109±0.154)相比,模型组动物颈部手术后,颈段脊髓(C1-C4)的5-HT1ARmRNA的表达水平(6.439±1.770)明显增高(P<0.05),与模型组比较(6.439±1.770),电针双侧扶突穴后颈段脊髓(C1-C4) 5-HT1AR mRNA的表达(1.676±0.684)、电针双侧的合谷-内关穴后颈段脊髓(C1-C4)5-HT1AR mRNA的表达(2.320±1.043)都显著降低(P<0.05),而电针双侧扶突穴组mRNA水平与电针双侧合谷、内关穴组比较没有显著差别。足三里-阳陵泉组腰段脊髓(L4-S3)5-HTR mRNA的表达(20.647±0.238)明显低于本组颈段脊髓表达水平(3.057±1.757)(P<0.05)。
Western blot结果显示,所有标本的内参都显示清晰的条带,经统计,模型组5-HT2AR蛋白的表达(0.882±0.139)明显高于正常组(0.448±0.106)(P<0.05),电针双侧扶突穴后5-HT2AR的表达(1.871±0.264)较模型组(0.882±0.139)明显升高(P<0.05),电针双侧合谷-内关穴后5-HT2A的表达(1.430±0.507)也明显高于模型组(0.882±0.139)(P<0.05)。并且电针双侧扶突穴后5-HT2A受体的表达(1.871±0.264)水平显著高于电针双侧合谷-内关穴后5-HT2A蛋白的表达(1.430±0.507)水平(P<0.05)
结论
1)大鼠颈部切口可引起的明显的痛反应,上调颈段脊髓SP、NK-1R、COX-1表达增高,说明手术造成的组织损伤引起了外周及中枢的敏化。
2)电针双侧“扶突穴”与合谷-内关穴均可缓解颈部切口可引起的明显的痛反应,并且扶突穴产生的对痛敏的抑制效应优于针刺双侧合谷-内关穴。针刺双侧“扶突穴”或“合谷”-“内关”穴可显著下调颈部切口痛引起的脊髓增加的NK-1 mRNA、COX-1mRNA、5-HT1ARmRNA的表达和加强5-HT2AR蛋白的表达水平的上调,说明颈部脊髓内5-HT1AR和5-HT2AR参与电针缓解颈部切口痛行为反应。
3)针刺双侧足三里-阳陵泉穴对颈部切口痛反应及SP、NK-1R、COX-1、5-HT1AR、5-HT2AR的表达没有明显作用,而可下调腰段脊髓的NK-1R mRNA、COX-1mRNA、5-HT1AR mRNA,说明电针足三里-阳陵泉的镇痛作用有神经节段性差异。
Effects of Electroacupuncture at Futu(LI18), etc. on the Expression of SP, NK-1R,COX-1 and 5-HT1A and 5-HT2AR in the Spinal Cord in Rats with Neck Incision Pain
Background
It has been well documented that acupuncture therapy is effective for relieving pain in clinical practice. Thyroid surgery is one of the best indications for acupuncture anesthesia. The dorsal horn of the spinal cord is the first station where the nociceptive information enter the central nervous system and the key link where the nocicetptive information transmitted from the primary peripheral afferents is processed and controlled. Following traumatic injury of the skin and muscles and nerves, the receptors in the injured tissues and the primary afferent fibers will be activated, resulting in an increase of the the release of a lot of substances, such as glutamate (Glu),aspartic acid, substance P (SP), calcitonin gene-related protein (CGRP), etc. from the endings of the afferent fibers, followed by activation of the secondary neurons of the spinal cord, and leading to neuropathic pain or hyperalgesia after the nociceptive inputs reaching the cerebral cortex. It has been proved that it is effective for inflammatory pain and neuropathic pain to be treated by electroacupuncture. Scientific researchers have conducted abundant experimental studies about the mechanisms of hyeralgesia and allodynia at different levels of the central nervous system.Postoperative pain induced by oral surgery, appendectomy surgery, thyroidectomy, abdominal surgery, etc. is one type of acute pain, and can be relieved effectively by acupuncture. It has been proved from animal experiments that acupuncture can raise the pain threshold in rats and mice undergoing plantar incision, and could inhibit the afferent discharges of the afferent A-delta and C fibers. But the underlying mechanisms of acupuncture in relieving pain after surgical incision in both man and animals remain unclear up to now. For this reason, we conducted a series of experiments in rats experiencing neck incision around the bilateral thyroid gland regions for observing the effects of electroacupuncture (EA) of Futu (LI18), Hegu (LI4)-Neiguan (PC6), Zusanli(ST36)-Yanglingquan (GB34) on the expressions of SP, NK-1 receptor (R), cyclooxygenase (COX)-1, serotonin (5-HT)1AR and 5-HT2AR mRNA and proteins in spinal cord by using immunohistochemistry, quantitative real-time polymerase chain reaction (RT-PCR) and western blot techniques, so as to provide scientific evidence for clinical application of acupuncture analgesia to thyroidectomy.
Materials and Methods
One hundred and twenty male Wistar rats(250-300g) were randomized into normal control, model, EA-"Futu"(LI18), EA"Hegu-Neiguan"(LI4-PC6) group, EA-Zusanli-Yanglingquan (ST36-GB34) groups (n=24/group). Among the 24 rats in each group, 10 were used for observing the behavioral changes,8 for immunohistochemistry, and 6 for Real-Time PCR and Western blotting. Rats were given with a longitudinal incision in the neck around the thyroid gland region under anesthesia (isoflurane) except the normal group, and were treated by EA at bilateral Futu (LI18), Hegu (LI4)-Neiguan (PC6), Zusanli(ST36)-Yanglingquan (GB34) (2/15Hz, 1-2mA,30min), respectively. The changes of behavior was detected by heat radiation before and after surgery, and post-acupuncture treatment. When the treatment course was over, rats for immunohistochemistry were anesthetized and perfused with 4% paraformaldehyde (PFA), for removing the neck segment of the spinal cord (C1-C4) and cutting into sections (with a freezing microtome). The changes of the immunoreactivity of SP, NK-1 R, COX-1 and 5-HT1AR and 5-HT2AR of the dorsal horn of the spinal cord were determined by immunohistochmistry, and fresh spinal NK-1 R mRNA, COX-1 mRNA,5-HT1AR mRNA and 5-HT2AR mRNA, and 5-HT2AR protein expression were detected by RT-PCR and western blot.
Results
1) Effects of EA on pain threshold
Compared with the pain threshold of pre-operation (model group 15.94±4.21, Futu group 14.48±2.76, Hegu-Neiguan group 13.72±2.54, Zusanli-Yanglignquan group 13.04±3.20), the pain threshold (PT) of the rats decreased significantly after neck incision (10.10±3.21,8.99±2.99,8.33±1.33,8.27±1.67) (P<0.05). Compared with that of model group, the PTs of EA-Futu (LI18) group (15.98±5.04) and EA-Neiguan-Hegu(LI4-PC6) group (12.56±3.05) increased markedly after EA treatment (P<0.05), and the PT of EA Futu(LI18) group was significantly higher than that of EA "Neiguan-Hegu" (LI4-PC6) group (P<0.05). While the PT of EA "Zusanli-Yanglingquan" (EA-ST36-GB34) group (9.54±2.34) had no apparent changes in comparison with that of model group.
2) Effects of EA on immunoreactivity of SP, NK-1R, COX-1,5-HT1AR and 5-HT2AR in the spinal dorsal horn
Result of immunohistochemistry showed that in the superficial dorsal horn the expressions of SP(76.76±12.51 integral grey value), NK-1 (71.02±7.08), COX-1 (60.84±8.02) in model group were significantly lower than those in normal group (SP93.74±14.01, NK-1110.60±0.97, COX-195.16±8.73) (P<0.05), meaning upregulation of the 3 indexes after neck incision. Compared with model group, the expressions of SP(94.90±7.32), NK-1 (91.69±1.28), COX-1 (105.60±13.84) in Futu(LI18) group were significantly higher than those in model group(SP93.74±14.01, NK-1110.60±0.97, COX-195.16±8.73) (P<0.05), while there were no apparent changes in "Zusanli-Yanglingquan" (ST36-GB34)group in expression of SP (73.72±11.06)、NK-1 (67.81±15.23)、COX-1 (76.59±8.07) compared with model group SP (76.76±12.51, NK-171.02±7.08, COX-160.84±8.02) (P>0.05)。
Compared with normal group (113.9±6.76,134.33±8.06),the expression of 5-HT1AR in model group (95.13±4.56) was significantly lower (P<0.05).In "Futu" (LI18)group,the expression of 5-HT1AR (78.81±4.57)、5-HT2A (110.89±8.33) were clearly lower than those of model group (5-HT1A95.13±4.56) (5-HT2A 129.4515.62) (P<0.05).While there were no difference between the expression of 5-HT1AR (94.36±4.20)、5-HT2AR (130.43±14.70) and the model group (P>0.05)
3)Effect of EA on expression of NK-1RmRNA、COX-1mRNA、5-HT1ARmRNA and 5-HT2AR protein in the spinal cord
Compared with normal group(1.393±0.430 Relative expression valve),the expression of NK-1RmRNA in cervical spinal cord of model group(6.608±0.689)was significantly increased(P<0.05),and the relative expression value in "Futu" (LI18) group(0.339±0.074)and "Hegu-Neiguan" (LI4-PC6) group(1.089±0.797) were clearly decresade in comparision with that of model group (6.608±0.689)(P<0.05),and there was no difference in expression of NK-1R mRNA between those of "Futu" (LI18)group and "Hegu-Neiguan" group.While the expression of NK-1RmRNA in cervical spinal cord in "Zusanli-Yanglingquan"(ST36-GB34) group (4.513±1.556) was significently higher than those of Futu group and "Hegu-Neiguan" (LI4-PC6)group (P<0.05), and the NK-1RmRNA expression in lumbar spinal cord (2.3708±0.988) of "Zusanli-Yanglingquan" (ST36-GB34) group was much lower than that in cervical spinal cord (P<0.05)
Compared with normal group(1.227±0.158 Relative expression valve),the expression of COX-1 RmRNA in cervical spinal cord of model group(4.519±1.211)was significantly increased(P<0.05),and the relative expression value in "Futu" (LI 18) group (1.171±0.249) and "Hegu-Neiguan" (LI4-PC6)group (1.321±0.160) were clearly decresade in comparision with that of model group (4.519±1.211) (P<0.05).and there was no difference in expression of COX-1R mRNA between those of Futu group and "Hegu-Neiguan" (LI4-PC6) group. While the expression of COX-1 RmRNA in cervical spinal cord in Zusanli-Yanglingquan group (5.812±1.734) was significantly higher than those of "Futu" (LI18) group and "Hegu-Neiguan" (LI4-PC6) group (P<0.05), and the COX-1 RmRNA expression in lumbar spinal cord (2.3708±0.988) of "Zusanli-Yanglingquan" (ST36-GB34) group was much lower than that in cervical spinal cord (P<0.05)
Compared with normal group (1.109±0.154 Relative expression valve), the expression of 5-HT1ARmRNA in cervical spinal cord of model group (6.439±1.770) was significantly increased (P<0.05), and the relative expression value in "Futu" (LI18) group (1.676±0.684) and "Hegu-Neiguan" (LI4-PC6) group (2.320±1.043) were clearly decreased in comparison with that of model group (6.439±1.770) (P<0.05). and there was no significant difference in the expression of 5-HT1AR mRNA between Futu (LI18) group and "Hegu-Neiguan" (LI4-PC6) group. While the expression of 5-HT1ARmRNA in cervical spinal cord in Zusanli-Yanglingquan group (3.057±1.757) was significantly higher than those of "Futu" (LI18) and "Hegu-Neiguan" (LI4-PC6) group (P<0.05), and 5-HT1ARmRNA expression in the lumbar spinal cord (2.3708±0.988) of "Zusanli-Yanglingquan"(ST36-GB34) group was much lower than that in cervical spinal cord (20.647±0.238) (P<0.05)
Result of Western blotting showed that the expression of spinal 5-HT2A protein in model group (0.882±0.139) was significantly higher than that in normal group (0.448±0.106) (P<0.05), and the expressions were significantly increased in "Futu" (LI18) (1.871±0.264) and "Hegu-Neiguan" (LI4-PC6) group(1.430±0.507)compared with that of model group(0.882±0.139) (P<0.05), and there was an obvious difference between the expressions of spinal 5-HT2A protein in "Futu" (LI18) and "Hegu-Neiguan" (LI4-PC6) group (P<0.05)
Conclusions
1)There are remarkable changes in behavior in rats undergoing incision surgery, and the expressions of SP, NK-1R and COX-1 in the dorsal horn of the cervical spinal cord, indicating a hyperalgesia after neck incision.
2) EA of "Futu"(LI18) and Hegu-Neiguan (LI4-PC6) acupoint areas can relieve the pain behavior reaction, and the effect of EA "Futu"(LI18) was superior to that of EA "Hegu-Neiguan"(LI4-PC6). EA of Futu(LI18) and Hegu-Neiguan (LI4-PC6) can effectively suppress incision-pain induced upregulation of SP, NK-1R, COX-1 and 5-HT1AR immunoactivity in the dorsal horn of the cervical spinal cord, and those of spinal NK-1R mRNA, COX-1 mRNA and 5-HT1AR mRNA are inhibited after EA of Futu(LI18) and Hegu-Neiguan (LI4-PC6), and further potentiate incision pain induced upregulation of 5-HT2AR mRNA.
3) EA at "Zusanli-Yanglingquan"(ST36-G34) have no remarkable effects on neck-incision pain reaction, and on SP, NK-1R, COX-1,5-HT1AR,5-HT2AR immunoactivity, NK-1R mRNA, COX-1 mRNA,5-HT1AR mRNA expressions in the cervical spinal cord, but can down-regulate neck-incision induced upregulation of NK-1R mRNA, COX-1 mRNA and 5-HT1AR mRNA in the lumbar spinal cord, suggesting a relative specificity of the effects of acupoints at different nerve segments.
痛症是针灸治疗的第一适应大症,在针刺镇痛(针刺麻醉)的适应症中,首选之一为甲状腺手术。但是,针刺镇痛行甲状腺手术的神经生物学机制尚未进行过系统研究。术后痛是急性疼痛的一种表现形式,临床上,针刺可明显缓解口腔手术术后痛、阑尾切除术后痛、骨科术后痛、甲状腺术后切口痛、四肢及妇科手术后切口痛等。动物实验也证明,电针可减轻足底切口痛行为反应,电生理学实验证明电针可以抑制足底切口痛大鼠Aδ纤维和C类纤维的放电。但是,针刺缓解切口痛的作用机制还不完全清楚,研究还不多；特别是针刺减轻颈部切口痛还没有任何报道。因此,本实验在大鼠甲状腺区切口痛的模型上,探讨电针双侧扶突穴对动物疼痛反应的影响及脊髓背角SP及其NK1受体与COX-1、5-HT1A、5-HT2A的表达变化,探讨电针缓解术后疼痛的脊髓机制,为临床针麻行甲状腺手术提供实验依据。
材料与方法
Wistar雄性大鼠120只,随机分为正常对照组(n=24),模型组(n=24),扶突穴组(n=24),足三里-阳陵泉穴组(n=24),合谷-内关穴组(n=24)。异氟醚麻醉下,在大鼠颈部做纵行切口,造成切口痛模型,分别电针双侧扶突穴,双侧足三里、阳陵泉穴,双侧合谷、内关穴(2／15 Hz,15min,1mA；15min,2 mA),用辐射热分别在术前、术后、针后照射大鼠颈部／切口引起的躲避潜伏期作为痛敏分数衡量动物痛反应(每组10例),在麻醉状态下,一部分(每组8例)动物常规灌流取颈段脊髓(颈1-颈4),连续冰冻切片,用免疫组织化学方法检测颈段脊髓背角SP、NK-1、COX-1和5-HT1A、5-HT2A的表达；另一部分动物(每组6例)取颈段脊髓(颈1-颈4)组织液氮研磨提取RNA和蛋白,分别用定量PCR和Western blot方法来检测脊髓中SP、COX-1和5-HT2A的表达的变化。
结果
1)电针对颈部切口痛大鼠痛行为的影响
与颈部切口术前(模型组15.94±4.21,扶突穴组14.48±2.76,合谷内关组13.72±2.54,足三里-阳陵泉组13.04±3.20)比较,各组动物的热辐射躲避潜伏期(模型组10.10±3.21,扶突穴组8.99±2.99,内关组8.33±1.33,足三里-阳陵泉组8.27±1.67)明显缩短(P<0.05)；电针扶突穴后动物痛阈值(15.98±5.04)与本组术后4h(8.99±2.99)比较、电针合谷、内关穴后动物痛阈值(12.56±3.05)与本组术后4h(8.33±1.33)比较,其躲避潜伏期均显著延长(P<0.05),电针扶突穴后动物的痛阈值明显高于电针合谷、内关动物(P<0.05)。电针足三里-阳陵泉穴后痛阈值(9.54±2.34)与本组术后(8.27±1.67)比较差异无统计学意义(P>0.05)。
2)电针对颈部切口痛大鼠脊髓背角背角SP、NK-1R、COX-1及5-HT1A,5-HT2A影响的免疫组织化学研究
颈部切口术后,模型组动物脊髓背角浅层SP(76.76±12.51)、NK-1(71.02±7.08)、COX-1(60.84±8.02)的灰度值均明显低于正常对照组(93.74±14.01,110.60±0.97,95.16±8.73)(P<0.05),即表达显著升高。与模型组比较,扶突穴组SP(94.90±7.32)、NK-1(91.69±1.28)、COX-1(105.60±13.84)的灰度值显著升高,表达明显下调(P<0.05))；足三里-阳陵泉穴组SP灰度值(73.72±11.06)、NK-1灰度值(67.81±15.23)、COX-1灰度值(76.59±8.07),与模型组比较(76.76±12.51,71.02±7.08,60.84±8.02)无显著差异(P>0.05)。
术后,与正常组(113.9±6.76,134.33±8.06)比较,模型组5-HT1A受体灰度值(95.13±4.56)明显降低(P<0.05),5-HT2A灰度值稍有降低(129.4515.62)。与模型组比较,扶突穴组5-HT1A(78.81±4.57)、5-HT2A(110.89±8.33)灰度值明显下降(P<0.05),而足三里-阳陵泉组5-HT1AR灰度值(94.36±4.20)5-HT2AR灰度值(130.43±14.70)与之没有明显差异(P>0.05)。
3)电针对颈部切口痛大鼠脊髓背角NK-1R mRNA、COX-1mRNA及5-HT1AR mRNA、5-HT2ARmRNA及蛋白水平表达的影响
与正常动物组NK-1R mRNA的表达水平(1.393±0.430)相比,模型组动物颈部手术后,颈段脊髓(C1-C4)的NK-1R mRNA的表达水平(6.608±0.689)明显增高(P<0.05),与模型组比较(6.608±0.689),电针双侧扶突穴后颈段脊髓(C1-C4)NK-1受体的表达(0.339±0.074)显著降低(P<0.05),电针双侧的合谷-内关穴后颈段脊髓(C1-C4) NK-1受体的表达(1.089±0.797)与模型组比较(6.608±0.689)也显著降低(P<0.05),而电针双侧扶突穴组mRNA水平与电针双侧合谷、内关穴组比较没有差别。足三里-阳陵泉组颈段脊髓(C1-C4)NK-1受体的表达(4.513±1.556)明显高于扶突穴组(0.339±0.074)与合谷-内关组(1.089±0.797),足三里-阳陵泉组腰段脊髓(L4-S3)mRNA的表达(2.3708±0.988)低于本组颈段脊髓,但没有统计学差异。
与正常动物组(1.227±0.158)相比,模型组动物颈部手术后,颈段脊髓(C1-C4)的COX-1mRNA的表达水平(4.519±1.211)明显增高(P<0.05),与模型组比较(4.519±1.211),电针双侧扶突穴后颈段脊髓(C1-C4)COX-1 mRNA的表达(1.171±0.249)、电针双侧的合谷、内关穴后颈段脊髓(C1-C4) COX-1mRNA的表达(1.321±0.160)都显著降低(P<0.05),而电针双侧扶突穴组mRNA水平与电针双侧合谷-内关穴组比较没有差别。足三里-阳陵泉组颈段脊髓(C1-C4) COX-1mRNA的表达(5.812±1.734)明显高于扶突穴组(1.171±0.249)与合谷-内关组(1.321±0.160),足三里-阳陵泉组腰段脊髓(L4-S3)COX-1mRNA的表达(2.3708±0.988)明显低于本组颈段脊髓表达水平(2.685±0.921)(P<0.05)。
与正常动物组(1.109±0.154)相比,模型组动物颈部手术后,颈段脊髓(C1-C4)的5-HT1ARmRNA的表达水平(6.439±1.770)明显增高(P<0.05),与模型组比较(6.439±1.770),电针双侧扶突穴后颈段脊髓(C1-C4) 5-HT1AR mRNA的表达(1.676±0.684)、电针双侧的合谷-内关穴后颈段脊髓(C1-C4)5-HT1AR mRNA的表达(2.320±1.043)都显著降低(P<0.05),而电针双侧扶突穴组mRNA水平与电针双侧合谷、内关穴组比较没有显著差别。足三里-阳陵泉组腰段脊髓(L4-S3)5-HTR mRNA的表达(20.647±0.238)明显低于本组颈段脊髓表达水平(3.057±1.757)(P<0.05)。
Western blot结果显示,所有标本的内参都显示清晰的条带,经统计,模型组5-HT2AR蛋白的表达(0.882±0.139)明显高于正常组(0.448±0.106)(P<0.05),电针双侧扶突穴后5-HT2AR的表达(1.871±0.264)较模型组(0.882±0.139)明显升高(P<0.05),电针双侧合谷-内关穴后5-HT2A的表达(1.430±0.507)也明显高于模型组(0.882±0.139)(P<0.05)。并且电针双侧扶突穴后5-HT2A受体的表达(1.871±0.264)水平显著高于电针双侧合谷-内关穴后5-HT2A蛋白的表达(1.430±0.507)水平(P<0.05)
结论
1)大鼠颈部切口可引起的明显的痛反应,上调颈段脊髓SP、NK-1R、COX-1表达增高,说明手术造成的组织损伤引起了外周及中枢的敏化。
2)电针双侧“扶突穴”与合谷-内关穴均可缓解颈部切口可引起的明显的痛反应,并且扶突穴产生的对痛敏的抑制效应优于针刺双侧合谷-内关穴。针刺双侧“扶突穴”或“合谷”-“内关”穴可显著下调颈部切口痛引起的脊髓增加的NK-1 mRNA、COX-1mRNA、5-HT1ARmRNA的表达和加强5-HT2AR蛋白的表达水平的上调,说明颈部脊髓内5-HT1AR和5-HT2AR参与电针缓解颈部切口痛行为反应。
3)针刺双侧足三里-阳陵泉穴对颈部切口痛反应及SP、NK-1R、COX-1、5-HT1AR、5-HT2AR的表达没有明显作用,而可下调腰段脊髓的NK-1R mRNA、COX-1mRNA、5-HT1AR mRNA,说明电针足三里-阳陵泉的镇痛作用有神经节段性差异。
Effects of Electroacupuncture at Futu(LI18), etc. on the Expression of SP, NK-1R,COX-1 and 5-HT1A and 5-HT2AR in the Spinal Cord in Rats with Neck Incision Pain
Background
It has been well documented that acupuncture therapy is effective for relieving pain in clinical practice. Thyroid surgery is one of the best indications for acupuncture anesthesia. The dorsal horn of the spinal cord is the first station where the nociceptive information enter the central nervous system and the key link where the nocicetptive information transmitted from the primary peripheral afferents is processed and controlled. Following traumatic injury of the skin and muscles and nerves, the receptors in the injured tissues and the primary afferent fibers will be activated, resulting in an increase of the the release of a lot of substances, such as glutamate (Glu),aspartic acid, substance P (SP), calcitonin gene-related protein (CGRP), etc. from the endings of the afferent fibers, followed by activation of the secondary neurons of the spinal cord, and leading to neuropathic pain or hyperalgesia after the nociceptive inputs reaching the cerebral cortex. It has been proved that it is effective for inflammatory pain and neuropathic pain to be treated by electroacupuncture. Scientific researchers have conducted abundant experimental studies about the mechanisms of hyeralgesia and allodynia at different levels of the central nervous system.Postoperative pain induced by oral surgery, appendectomy surgery, thyroidectomy, abdominal surgery, etc. is one type of acute pain, and can be relieved effectively by acupuncture. It has been proved from animal experiments that acupuncture can raise the pain threshold in rats and mice undergoing plantar incision, and could inhibit the afferent discharges of the afferent A-delta and C fibers. But the underlying mechanisms of acupuncture in relieving pain after surgical incision in both man and animals remain unclear up to now. For this reason, we conducted a series of experiments in rats experiencing neck incision around the bilateral thyroid gland regions for observing the effects of electroacupuncture (EA) of Futu (LI18), Hegu (LI4)-Neiguan (PC6), Zusanli(ST36)-Yanglingquan (GB34) on the expressions of SP, NK-1 receptor (R), cyclooxygenase (COX)-1, serotonin (5-HT)1AR and 5-HT2AR mRNA and proteins in spinal cord by using immunohistochemistry, quantitative real-time polymerase chain reaction (RT-PCR) and western blot techniques, so as to provide scientific evidence for clinical application of acupuncture analgesia to thyroidectomy.
Materials and Methods
One hundred and twenty male Wistar rats(250-300g) were randomized into normal control, model, EA-"Futu"(LI18), EA"Hegu-Neiguan"(LI4-PC6) group, EA-Zusanli-Yanglingquan (ST36-GB34) groups (n=24/group). Among the 24 rats in each group, 10 were used for observing the behavioral changes,8 for immunohistochemistry, and 6 for Real-Time PCR and Western blotting. Rats were given with a longitudinal incision in the neck around the thyroid gland region under anesthesia (isoflurane) except the normal group, and were treated by EA at bilateral Futu (LI18), Hegu (LI4)-Neiguan (PC6), Zusanli(ST36)-Yanglingquan (GB34) (2/15Hz, 1-2mA,30min), respectively. The changes of behavior was detected by heat radiation before and after surgery, and post-acupuncture treatment. When the treatment course was over, rats for immunohistochemistry were anesthetized and perfused with 4% paraformaldehyde (PFA), for removing the neck segment of the spinal cord (C1-C4) and cutting into sections (with a freezing microtome). The changes of the immunoreactivity of SP, NK-1 R, COX-1 and 5-HT1AR and 5-HT2AR of the dorsal horn of the spinal cord were determined by immunohistochmistry, and fresh spinal NK-1 R mRNA, COX-1 mRNA,5-HT1AR mRNA and 5-HT2AR mRNA, and 5-HT2AR protein expression were detected by RT-PCR and western blot.
Results
1) Effects of EA on pain threshold
Compared with the pain threshold of pre-operation (model group 15.94±4.21, Futu group 14.48±2.76, Hegu-Neiguan group 13.72±2.54, Zusanli-Yanglignquan group 13.04±3.20), the pain threshold (PT) of the rats decreased significantly after neck incision (10.10±3.21,8.99±2.99,8.33±1.33,8.27±1.67) (P<0.05). Compared with that of model group, the PTs of EA-Futu (LI18) group (15.98±5.04) and EA-Neiguan-Hegu(LI4-PC6) group (12.56±3.05) increased markedly after EA treatment (P<0.05), and the PT of EA Futu(LI18) group was significantly higher than that of EA "Neiguan-Hegu" (LI4-PC6) group (P<0.05). While the PT of EA "Zusanli-Yanglingquan" (EA-ST36-GB34) group (9.54±2.34) had no apparent changes in comparison with that of model group.
2) Effects of EA on immunoreactivity of SP, NK-1R, COX-1,5-HT1AR and 5-HT2AR in the spinal dorsal horn
Result of immunohistochemistry showed that in the superficial dorsal horn the expressions of SP(76.76±12.51 integral grey value), NK-1 (71.02±7.08), COX-1 (60.84±8.02) in model group were significantly lower than those in normal group (SP93.74±14.01, NK-1110.60±0.97, COX-195.16±8.73) (P<0.05), meaning upregulation of the 3 indexes after neck incision. Compared with model group, the expressions of SP(94.90±7.32), NK-1 (91.69±1.28), COX-1 (105.60±13.84) in Futu(LI18) group were significantly higher than those in model group(SP93.74±14.01, NK-1110.60±0.97, COX-195.16±8.73) (P<0.05), while there were no apparent changes in "Zusanli-Yanglingquan" (ST36-GB34)group in expression of SP (73.72±11.06)、NK-1 (67.81±15.23)、COX-1 (76.59±8.07) compared with model group SP (76.76±12.51, NK-171.02±7.08, COX-160.84±8.02) (P>0.05)。
Compared with normal group (113.9±6.76,134.33±8.06),the expression of 5-HT1AR in model group (95.13±4.56) was significantly lower (P<0.05).In "Futu" (LI18)group,the expression of 5-HT1AR (78.81±4.57)、5-HT2A (110.89±8.33) were clearly lower than those of model group (5-HT1A95.13±4.56) (5-HT2A 129.4515.62) (P<0.05).While there were no difference between the expression of 5-HT1AR (94.36±4.20)、5-HT2AR (130.43±14.70) and the model group (P>0.05)
3)Effect of EA on expression of NK-1RmRNA、COX-1mRNA、5-HT1ARmRNA and 5-HT2AR protein in the spinal cord
Compared with normal group(1.393±0.430 Relative expression valve),the expression of NK-1RmRNA in cervical spinal cord of model group(6.608±0.689)was significantly increased(P<0.05),and the relative expression value in "Futu" (LI18) group(0.339±0.074)and "Hegu-Neiguan" (LI4-PC6) group(1.089±0.797) were clearly decresade in comparision with that of model group (6.608±0.689)(P<0.05),and there was no difference in expression of NK-1R mRNA between those of "Futu" (LI18)group and "Hegu-Neiguan" group.While the expression of NK-1RmRNA in cervical spinal cord in "Zusanli-Yanglingquan"(ST36-GB34) group (4.513±1.556) was significently higher than those of Futu group and "Hegu-Neiguan" (LI4-PC6)group (P<0.05), and the NK-1RmRNA expression in lumbar spinal cord (2.3708±0.988) of "Zusanli-Yanglingquan" (ST36-GB34) group was much lower than that in cervical spinal cord (P<0.05)
Compared with normal group(1.227±0.158 Relative expression valve),the expression of COX-1 RmRNA in cervical spinal cord of model group(4.519±1.211)was significantly increased(P<0.05),and the relative expression value in "Futu" (LI 18) group (1.171±0.249) and "Hegu-Neiguan" (LI4-PC6)group (1.321±0.160) were clearly decresade in comparision with that of model group (4.519±1.211) (P<0.05).and there was no difference in expression of COX-1R mRNA between those of Futu group and "Hegu-Neiguan" (LI4-PC6) group. While the expression of COX-1 RmRNA in cervical spinal cord in Zusanli-Yanglingquan group (5.812±1.734) was significantly higher than those of "Futu" (LI18) group and "Hegu-Neiguan" (LI4-PC6) group (P<0.05), and the COX-1 RmRNA expression in lumbar spinal cord (2.3708±0.988) of "Zusanli-Yanglingquan" (ST36-GB34) group was much lower than that in cervical spinal cord (P<0.05)
Compared with normal group (1.109±0.154 Relative expression valve), the expression of 5-HT1ARmRNA in cervical spinal cord of model group (6.439±1.770) was significantly increased (P<0.05), and the relative expression value in "Futu" (LI18) group (1.676±0.684) and "Hegu-Neiguan" (LI4-PC6) group (2.320±1.043) were clearly decreased in comparison with that of model group (6.439±1.770) (P<0.05). and there was no significant difference in the expression of 5-HT1AR mRNA between Futu (LI18) group and "Hegu-Neiguan" (LI4-PC6) group. While the expression of 5-HT1ARmRNA in cervical spinal cord in Zusanli-Yanglingquan group (3.057±1.757) was significantly higher than those of "Futu" (LI18) and "Hegu-Neiguan" (LI4-PC6) group (P<0.05), and 5-HT1ARmRNA expression in the lumbar spinal cord (2.3708±0.988) of "Zusanli-Yanglingquan"(ST36-GB34) group was much lower than that in cervical spinal cord (20.647±0.238) (P<0.05)
Result of Western blotting showed that the expression of spinal 5-HT2A protein in model group (0.882±0.139) was significantly higher than that in normal group (0.448±0.106) (P<0.05), and the expressions were significantly increased in "Futu" (LI18) (1.871±0.264) and "Hegu-Neiguan" (LI4-PC6) group(1.430±0.507)compared with that of model group(0.882±0.139) (P<0.05), and there was an obvious difference between the expressions of spinal 5-HT2A protein in "Futu" (LI18) and "Hegu-Neiguan" (LI4-PC6) group (P<0.05)
Conclusions
1)There are remarkable changes in behavior in rats undergoing incision surgery, and the expressions of SP, NK-1R and COX-1 in the dorsal horn of the cervical spinal cord, indicating a hyperalgesia after neck incision.
2) EA of "Futu"(LI18) and Hegu-Neiguan (LI4-PC6) acupoint areas can relieve the pain behavior reaction, and the effect of EA "Futu"(LI18) was superior to that of EA "Hegu-Neiguan"(LI4-PC6). EA of Futu(LI18) and Hegu-Neiguan (LI4-PC6) can effectively suppress incision-pain induced upregulation of SP, NK-1R, COX-1 and 5-HT1AR immunoactivity in the dorsal horn of the cervical spinal cord, and those of spinal NK-1R mRNA, COX-1 mRNA and 5-HT1AR mRNA are inhibited after EA of Futu(LI18) and Hegu-Neiguan (LI4-PC6), and further potentiate incision pain induced upregulation of 5-HT2AR mRNA.
3) EA at "Zusanli-Yanglingquan"(ST36-G34) have no remarkable effects on neck-incision pain reaction, and on SP, NK-1R, COX-1,5-HT1AR,5-HT2AR immunoactivity, NK-1R mRNA, COX-1 mRNA,5-HT1AR mRNA expressions in the cervical spinal cord, but can down-regulate neck-incision induced upregulation of NK-1R mRNA, COX-1 mRNA and 5-HT1AR mRNA in the lumbar spinal cord, suggesting a relative specificity of the effects of acupoints at different nerve segments.
引文
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