本文關鍵詞：甲醛炎性痛誘導大鼠脊髓HO-1蛋白表達增加及其促痛作用研究　出處：《河北醫(yī)科大學》2008年碩士論文　論文類型：學位論文

  更多相關文章： 甲醛炎性痛 痛覺過敏 HO-1 Znpp Hemin 脊髓 大鼠

【摘要】： 目的:內(nèi)源性一氧化碳(Carbon monoxide,CO)作為一種新型神經(jīng)遞質(zhì)已經(jīng)被大家所公認。血紅素氧合酶(Heme oxygenase,HO)是生成CO的限速酶,廣泛分布于中樞神經(jīng)系統(tǒng),其催化亞鐵血紅素Heme產(chǎn)生CO、游離鐵和膽綠素,膽綠素在膽綠素還原酶的作用下進一步生成膽紅素。HO-1為HO的一種亞型,正常時在神經(jīng)系統(tǒng)表達較少,但可被多種因素所誘導表達。 我室已往的研究已證實,甲醛炎性痛時脊髓后角一氧化氮合酶(Nitricoxide synthase,NOS)活性增加,一氧化氮(Nitrogen monoxidum,NO)產(chǎn)生增多。研究結(jié)果證實,NOS/NO和HO/CO兩個系統(tǒng)之間存在密切的聯(lián)系。有關HO/CO系統(tǒng)在脊髓傷害性信息傳遞和痛覺過敏方面作用的研究已有一些報道,研究發(fā)現(xiàn),結(jié)扎坐骨神經(jīng)引起的神經(jīng)損傷可誘導脊髓HO表達增多,CO產(chǎn)生增多,但甲醛炎性痛引起的傷害性信息傳入是否會誘導脊髓HO-1表達發(fā)生改變尚未見相關報道。 有研究證實,鞘內(nèi)給予HO抑制劑可劑量依賴性地減輕甲醛炎性痛大鼠自發(fā)痛行為,表明脊髓后角產(chǎn)生的CO在傷害性信息傳遞和痛感覺方面發(fā)揮重要作用,但有關HO和CO在熱痛覺過敏和機械痛敏形成中作用尚無定論,有待進一步的研究加以確定。 因此,本實驗采用免疫組織化學方法,觀察大鼠足底注射甲醛引起的炎性痛過程中,脊髓后角HO-1蛋白表達改變及其時間特征。通過甲醛炎性痛大鼠鞘內(nèi)注射HO的抑制劑鋅原卟啉(zinc protoporphyrin,Znpp),觀察其對甲醛炎性痛大鼠的自發(fā)痛行為以及對熱和機械痛覺過敏的影響;通過正常大鼠鞘內(nèi)注射HO激動劑氯化血紅素(Hemin),觀察其對正常大鼠熱和機械痛閾的影響,探討HO/CO在痛覺過敏形成中的作用。 1甲醛炎性痛誘導脊髓HO-1蛋白表達改變 42只雄性Sprague-Dawley大鼠,體重250±20克,隨機分為7組,分別為正常對照組(control組)、甲醛6h組、甲醛12h組、甲醛1d組、甲醛2d組、甲醛3d組、甲醛7d組,每組6只動物。Control組不做任何處理,直接斷頭取材。甲醛各組動物均于足底注射甲醛后行痛反應評分,然后于相應時間點斷頭取腰5脊髓節(jié)段(L5),石蠟切片上行HO-1免疫組化染色。 結(jié)果發(fā)現(xiàn):大鼠右后足底注射甲醛后,即刻出現(xiàn)舔、搖、和抬高注射足等自發(fā)痛行為,且注射部位出現(xiàn)紅、腫等明顯的炎癥反應,注射足出現(xiàn)典型的雙向性自發(fā)縮足反應,持續(xù)1 h以上。 免疫組化檢測結(jié)果發(fā)現(xiàn),Control組大鼠脊髓HO-1免疫反應陽性細胞在脊髓后角及中央管周圍僅有極少量分布。足底注射甲醛后,雙側(cè)L5脊髓后角均可觀察到HO-1免疫反應陽性細胞數(shù)目即有所增多,雙側(cè)無差別。足底注射甲醛后6h,注射足同側(cè)的L5脊髓后角可觀察到HO-1免疫反應陽性細胞數(shù)目即有所增多,陽性面積增大,而灰度值未見明顯降低,中央管周圍陽性細胞數(shù)目也明顯增多;足底注射甲醛后12h時,脊髓后角免疫反應陽性細胞數(shù)目進一步升高和陽性面積進一步增大,中央管周圍陽性細胞數(shù)進一步增多;1d時脊髓后角及中央管周圍HO-1陽性細胞數(shù)、陽性面積及灰度值均達到峰值,7d時仍高于正常對照組水平。 2 HO/CO在甲醛誘導的自發(fā)痛及痛覺過敏中的作用 45只雄性Sprague-Dawley大鼠,體重250±20克,隨機分為兩個大組。 第一組又分為5個亞組,分別為正常對照組(control組)、甲醛組(F24h組)、甲醛+溶劑DMSO組(DMSO組)、甲醛+Znpp50μg組(ZnPP50μg組)、甲醛+Znpp100μg組(ZnPP100μg組)、甲醛+Znpp300μg組(ZnPP300μg組),每組5只動物。甲醛+DMSO組和甲醛+各劑量Znpp組預先鞘內(nèi)注射DMSO或HO抑制劑Znpp,30分鐘后足底注射甲醛并行痛反應評分,24h后再次鞘內(nèi)注射DMSO或Znpp,30分鐘后測定熱輻射縮足反射潛伏期和機械縮足反射閾值。 第二組又分為4個亞組,分別為正常對照組(control組)、溶劑NaOH組、Hemin187.5μg組、Hemin375μg組,每組5只動物。溶劑NaOH組和Hemin各組于正常大鼠鞘內(nèi)注射溶劑NaOH和HO激動劑Hemin,30分鐘后測定熱輻射縮足反射潛伏期和機械縮足反射閾值。 結(jié)果發(fā)現(xiàn):甲醛炎性痛大鼠鞘內(nèi)注射HO抑制劑溶劑DMSO后,甲醛誘導的大鼠的痛行為及熱和機械痛覺過敏程度均較甲醛組無明顯改變。而鞘內(nèi)注射HO抑制劑Znpp后,大鼠疼痛反應評分較甲醛組明顯降低,且隨Znpp劑量的增大,其對大鼠痛反應的抑制作用越明顯。與正常對照組比較,甲醛組大鼠注射足熱輻射縮足潛伏期明顯延長,機械刺激縮足反射閾值也明顯升高,而非注射足熱輻射縮足潛伏期明顯縮短,機械刺激縮足反射閾值也明顯降低;甲醛+Znpp各劑量組與甲醛組相比,注射足熱輻射縮足潛伏期和機械刺激縮足反射閾值均無明顯變化;而非注射足熱輻射縮足潛伏期和機械刺激縮足反射閾值均明顯升高,且隨著Znpp劑量的增加,非注射足熱輻射縮足潛伏期和機械刺激縮足反射閾值明顯高于正常對照組水平。 與Control組相比,正常大鼠鞘內(nèi)注射HO激動劑溶劑NaOH后,左右兩側(cè)足熱輻射縮足潛伏期和機械刺激縮足反射閾值均無明顯差別,正常大鼠鞘內(nèi)注射HO的激動劑Hemin后,雙側(cè)足熱輻射縮足潛伏期縮短,并且機械刺激縮足反射閾值也明顯降低,兩足比較無明顯差別。 結(jié)論:大鼠足底注射甲醛引起的炎性痛誘導了脊髓后角和中央管周圍HO-1表達增多,以注射甲醛后1d增多最為明顯。鞘內(nèi)給予HO抑制劑可明顯抑制甲醛誘導的痛行為反應及熱和機械性痛覺過敏程度;正常大鼠鞘內(nèi)給于HO激動劑則可誘發(fā)熱和機械性痛覺過敏的產(chǎn)生。這些結(jié)果表明,HO/CO系統(tǒng)參與傷害性信息的傳導和痛覺過敏的產(chǎn)生過程。
[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.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R363

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相關期刊論文 前1條

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本文編號：1432873