甲醛炎性痛诱导大鼠脊髓HO-1蛋白表达增加及其促痛作用研究

发布时间：2018-01-16 11:31

本文关键词：甲醛炎性痛诱导大鼠脊髓HO-1蛋白表达增加及其促痛作用研究　出处：《河北医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:内源性一氧化碳(Carbon monoxide,CO)作为一种新型神经递质已经被大家所公认。血红素氧合酶(Heme oxygenase,HO)是生成CO的限速酶,广泛分布于中枢神经系统,其催化亚铁血红素Heme产生CO、游离铁和胆绿素,胆绿素在胆绿素还原酶的作用下进一步生成胆红素。HO-1为HO的一种亚型,正常时在神经系统表达较少,但可被多种因素所诱导表达。我室已往的研究已证实,甲醛炎性痛时脊髓后角一氧化氮合酶(Nitricoxide synthase,NOS)活性增加,一氧化氮(Nitrogen monoxidum,NO)产生增多。研究结果证实,NOS/NO和HO/CO两个系统之间存在密切的联系。有关HO/CO系统在脊髓伤害性信息传递和痛觉过敏方面作用的研究已有一些报道,研究发现,结扎坐骨神经引起的神经损伤可诱导脊髓HO表达增多,CO产生增多,但甲醛炎性痛引起的伤害性信息传入是否会诱导脊髓HO-1表达发生改变尚未见相关报道。有研究证实,鞘内给予HO抑制剂可剂量依赖性地减轻甲醛炎性痛大鼠自发痛行为,表明脊髓后角产生的CO在伤害性信息传递和痛感觉方面发挥重要作用,但有关HO和CO在热痛觉过敏和机械痛敏形成中作用尚无定论,有待进一步的研究加以确定。因此,本实验采用免疫组织化学方法,观察大鼠足底注射甲醛引起的炎性痛过程中,脊髓后角HO-1蛋白表达改变及其时间特征。通过甲醛炎性痛大鼠鞘内注射HO的抑制剂锌原卟啉(zinc protoporphyrin,Znpp),观察其对甲醛炎性痛大鼠的自发痛行为以及对热和机械痛觉过敏的影响;通过正常大鼠鞘内注射HO激动剂氯化血红素(Hemin),观察其对正常大鼠热和机械痛阈的影响,探讨HO/CO在痛觉过敏形成中的作用。 1甲醛炎性痛诱导脊髓HO-1蛋白表达改变 42只雄性Sprague-Dawley大鼠,体重250±20克,随机分为7组,分别为正常对照组(control组)、甲醛6h组、甲醛12h组、甲醛1d组、甲醛2d组、甲醛3d组、甲醛7d组,每组6只动物。Control组不做任何处理,直接断头取材。甲醛各组动物均于足底注射甲醛后行痛反应评分,然后于相应时间点断头取腰5脊髓节段(L5),石蜡切片上行HO-1免疫组化染色。结果发现:大鼠右后足底注射甲醛后,即刻出现舔、摇、和抬高注射足等自发痛行为,且注射部位出现红、肿等明显的炎症反应,注射足出现典型的双向性自发缩足反应,持续1 h以上。免疫组化检测结果发现,Control组大鼠脊髓HO-1免疫反应阳性细胞在脊髓后角及中央管周围仅有极少量分布。足底注射甲醛后,双侧L5脊髓后角均可观察到HO-1免疫反应阳性细胞数目即有所增多,双侧无差别。足底注射甲醛后6h,注射足同侧的L5脊髓后角可观察到HO-1免疫反应阳性细胞数目即有所增多,阳性面积增大,而灰度值未见明显降低,中央管周围阳性细胞数目也明显增多;足底注射甲醛后12h时,脊髓后角免疫反应阳性细胞数目进一步升高和阳性面积进一步增大,中央管周围阳性细胞数进一步增多;1d时脊髓后角及中央管周围HO-1阳性细胞数、阳性面积及灰度值均达到峰值,7d时仍高于正常对照组水平。 2 HO/CO在甲醛诱导的自发痛及痛觉过敏中的作用 45只雄性Sprague-Dawley大鼠,体重250±20克,随机分为两个大组。第一组又分为5个亚组,分别为正常对照组(control组)、甲醛组(F24h组)、甲醛+溶剂DMSO组(DMSO组)、甲醛+Znpp50μg组(ZnPP50μg组)、甲醛+Znpp100μg组(ZnPP100μg组)、甲醛+Znpp300μg组(ZnPP300μg组),每组5只动物。甲醛+DMSO组和甲醛+各剂量Znpp组预先鞘内注射DMSO或HO抑制剂Znpp,30分钟后足底注射甲醛并行痛反应评分,24h后再次鞘内注射DMSO或Znpp,30分钟后测定热辐射缩足反射潜伏期和机械缩足反射阈值。第二组又分为4个亚组,分别为正常对照组(control组)、溶剂NaOH组、Hemin187.5μg组、Hemin375μg组,每组5只动物。溶剂NaOH组和Hemin各组于正常大鼠鞘内注射溶剂NaOH和HO激动剂Hemin,30分钟后测定热辐射缩足反射潜伏期和机械缩足反射阈值。结果发现:甲醛炎性痛大鼠鞘内注射HO抑制剂溶剂DMSO后,甲醛诱导的大鼠的痛行为及热和机械痛觉过敏程度均较甲醛组无明显改变。而鞘内注射HO抑制剂Znpp后,大鼠疼痛反应评分较甲醛组明显降低,且随Znpp剂量的增大,其对大鼠痛反应的抑制作用越明显。与正常对照组比较,甲醛组大鼠注射足热辐射缩足潜伏期明显延长,机械刺激缩足反射阈值也明显升高,而非注射足热辐射缩足潜伏期明显缩短,机械刺激缩足反射阈值也明显降低;甲醛+Znpp各剂量组与甲醛组相比,注射足热辐射缩足潜伏期和机械刺激缩足反射阈值均无明显变化;而非注射足热辐射缩足潜伏期和机械刺激缩足反射阈值均明显升高,且随着Znpp剂量的增加,非注射足热辐射缩足潜伏期和机械刺激缩足反射阈值明显高于正常对照组水平。与Control组相比,正常大鼠鞘内注射HO激动剂溶剂NaOH后,左右两侧足热辐射缩足潜伏期和机械刺激缩足反射阈值均无明显差别,正常大鼠鞘内注射HO的激动剂Hemin后,双侧足热辐射缩足潜伏期缩短,并且机械刺激缩足反射阈值也明显降低,两足比较无明显差别。结论:大鼠足底注射甲醛引起的炎性痛诱导了脊髓后角和中央管周围HO-1表达增多,以注射甲醛后1d增多最为明显。鞘内给予HO抑制剂可明显抑制甲醛诱导的痛行为反应及热和机械性痛觉过敏程度;正常大鼠鞘内给于HO激动剂则可诱发热和机械性痛觉过敏的产生。这些结果表明,HO/CO系统参与伤害性信息的传导和痛觉过敏的产生过程。
[Abstract]:Objective: endogenous carbon monoxide (Carbon monoxide, CO) as a new neurotransmitter has been recognized. Heme oxygenase (Heme oxygenase HO) is the rate limiting enzyme of CO generation, are widely distributed in the CNS, the catalytic heme Heme CO, free iron and biliverdin, biliverdin on biliverdin reductase under the action of further generated bilirubin.HO-1 is a subtype of HO, less expression in the nervous system is normal, but can be induced by various factors.
Our previous studies demonstrated that formaldehyde inflammatory pain in spinal dorsal horn of nitric oxide synthase (Nitricoxide synthase, NOS) activity increased, nitric oxide (Nitrogen monoxidum, NO) have increased. The results confirmed that between NOS/NO and HO/CO two systems are closely linked. In the study of spinal cord nociceptive transmission and pain the role of allergy on the HO/CO system has been reported, the study found that ligation of sciatic nerve injury caused by spinal cord can induce the increased expression of HO, CO increased, but the formaldehyde inflammatory pain caused by nociceptive input will induce spinal HO-1 expression changes have not been reported.
Studies have demonstrated that intrathecal HO inhibitor dose dependently reduced formalin induced inflammatory pain pain behavior, that play an important role in the spinal dorsal horn of CO in nociceptive transmission and pain, but the HO and CO effect on the formation of inconclusive in thermal hyperalgesia and mechanical allodynia. Further research is to be determined.
Therefore, this experiment using immunohistochemical method, the process of inflammatory pain rats caused by injection of formalin, angle changes in the HO-1 protein and the time characteristics. The expression of spinal cord after ZnPP formalininflammatorypain intrathecal injection of HO (zinc protoporphyrin Znpp), the observation of spontaneous pain behavior rats of formalin inflammatory pain and the influence of thermal and mechanical pain hypersensitivity; normal rats by intrathecal injection of HO agonist hemin (Hemin), to observe its effect on normal rat heat and mechanical pain threshold, to investigate the effects of HO/CO on hyperalgesia formation.
Changes of expression of HO-1 protein in spinal cord induced by 1 inflammatory pain in the spinal cord
42 male Sprague-Dawley rats, weighing 250 - 20 g, were randomly divided into 7 groups: normal control group (control group), 6h group, 12h group, formaldehyde, formaldehyde, formaldehyde, 1D group, 2D group, 3D group, formaldehyde, formaldehyde, formaldehyde, 7d group, 6 rats in each animal group.Control without any treatment. Were directly sacrificed. Each animal was formaldehyde injection of formalin after pain response score, and then take the waist at the corresponding time points in 5 spinal segments (L5), paraffin sections upstream HO-1 immunohistochemical staining.
Results: right after injection of formalin in rats after immediate licking, shake, and raise the injection foot pain behavior, and the injection site redness, inflammation swelling obviously, injection foot typical two-way spontaneous flinching response, for more than 1 h.
The immunohistochemistry results showed that spinal HO-1 immunoreactivity in rats of group Control positive cells in the spinal dorsal horn and around the central canal. Only a very small amount of the distribution by formalin injection, L5 bilateral posterior horn of the spinal cord was observed HO-1 immunoreactive cell number is increased, the bilateral difference. Injection of formalin after 6H injection. The foot on the same side of L5 spinal cord were observed HO-1 immunoreactive cell number is increased, the positive area increases, and the gray value was not obviously decreased, the number of positive cells around the central canal was significantly increased; 12h after injection of formalin, angle immunoreactive cell number increased after spinal cord and the positive area further increases around the central canal, the number of positive cells increased further; 1D spinal dorsal horn and around the central canal of HO-1 positive cells and positive gray value reached the peak area, 7d is still higher than the The level of the normal control group.
2 HO/CO in formaldehyde induced spontaneous pain and hyperalgesia in action
45 male Sprague-Dawley rats, with a weight of 250 + 20 grams, were randomly divided into two groups.
The first group was divided into 5 subgroups, respectively as the normal control group (control group), formaldehyde group (F24h group), formaldehyde + solvent DMSO group (DMSO group), G group, +Znpp50 formaldehyde (ZnPP50 G group), G group, +Znpp100 formaldehyde (ZnPP100 G group), formaldehyde +Znpp300 G group (ZnPP300 G group), 5 rats in each group. +DMSO group and animal formaldehyde formaldehyde + each dose group Znpp intrathecal injection of DMSO or HO inhibitor Znpp, 30 minutes after the injection of formalin parallel pain response score, 24h again after intrathecal injection of DMSO or Znpp, 30 minutes after the determination of the thermal radiation flinching reflex the latency and mechanical withdrawal threshold.
The second group was divided into 4 subgroups, respectively as the normal control group (group control), solvent group NaOH, Hemin187.5 G group, Hemin375 G group, 5 rats in each animal. NaOH group and Hemin group in solvent injection solvent NaOH and normal HO rats with intrathecal agonist Hemin, 30 minutes after the determination thermal paw withdrawal latency and mechanical withdrawal threshold.
Results: Intrathecal formalin inflammatory pain in the injection of HO inhibitor DMSO solvent after formalin induced rat pain behavior and thermal and mechanical hyperalgesia was higher than the formaldehyde group had no obvious change. But the intrathecal injection of HO inhibitor Znpp after rat pain group was significantly lower than that of formaldehyde, and with the increase of the dose of Znpp, inhibition of the reaction of pain in rats was more obvious. Compared with normal control group, formalin group rats were injected with enough heat radiation paw withdrawal latency was prolonged and the mechanical withdrawal threshold was significantly increased, while the non thermal radiation injection foot paw withdrawal latency was significantly shorter, mechanical withdrawal the threshold is significantly lower than +Znpp; formaldehyde groups with formaldehyde group, heat radiation injected paw withdrawal latency and mechanical withdrawal thresholds were not significantly changed; and the non thermal radiation injected paw withdrawal latency and mechanical withdrawal threshold The values were all significantly increased. With the increase of Znpp dose, the latency and the reflex threshold of mechanical stimulation were significantly higher than those of the normal control group.
Compared with the Control group, normal rats, intrathecal injection of HO agonist NaOH solvent, the left and right sides of the foot thermal withdrawal latency and mechanical withdrawal threshold were no significant difference between normal rats, intrathecal injection of HO agonist Hemin, bilateral foot withdrawal latency shortened heat radiation, and mechanical stimuli foot reflex threshold was significantly decreased, there is no obvious difference between two.
Conclusion: rat intraplantar injection of formalin induced inflammatory pain induced by spinal dorsal horn and around the central canal increased expression of HO-1 in 1D after injection of formaldehyde increased obviously. Intrathecal HO inhibitor can inhibit the reaction of formaldehyde induced pain behavior and thermal and mechanical hyperalgesia; normal rats with intrathecal production HO agonists can induce heat and mechanical hyperalgesia. These results show that HO/CO system plays an important role in nociceptive information transmission and hyperalgesia.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

【参考文献】