脊髓神经细胞自噬在氢气治疗神经病理性疼痛中作用机制研究

发布时间：2018-06-21 01:27

本文选题：神经痛 + 氢　；参考：《天津医科大学》2017年博士论文

【摘要】：神经病理性疼痛是由于躯体感觉神经系统的损伤或疾病引起的一种长期慢性疼痛。其形成的机制复杂,是当今医学界较难治疗的一类疾病,常常迁延不愈,给患者造成极大痛苦,生活质量极其低下。大量研究表明,细胞自噬功能障碍参与神经病理性疼痛形成和发展,适当的诱导细胞自噬功能成为神经病理性疼痛治疗突破口。近年众多研究表明,低浓度氢分子(4%)和富氢液具有选择性抗氧化、抗炎、抗凋亡作用,可应用于多种疾病的研究和治疗。因此,越来越多的学者认为氢气与富氢液是一种新型的医学气体分子。一些研究已发现腹腔注射富氢液可改善作用神经病理性疼痛大鼠行为学,对神经病理性疼痛有治疗作用,具体的机制仍在探讨中。缺氧诱导因子-1(HIF-1)是缺氧条件下调节细胞自噬的关键因子,也是细胞适应低氧环境的关键调节因子,研究证实其在神经病理性疼痛的模型初级传导通路中表达增多。本课题通过建立神经病理性疼痛大鼠模型腹腔注射富氢液和带状疱疹神经痛患者雾化吸入氢气,探讨氢气对神经病理性疼痛变保护作用的可能机制,为临床治疗奠定基础。实验一、富氢液激活脊髓神经细胞自噬在神经病理性疼痛中作用目的:细胞自噬功能障碍是神经病理性疼痛形成机制之一,通过诱导细胞自噬反应,成为目前治疗神经病理性疼痛的新靶点。富氢液对神经病理性疼痛有一定的治疗作用,具体的机制尚不明确。本实验拟利用建立神经病理性疼痛大鼠模型(CCI),探讨富氢液对神经病理性疼痛大鼠脊髓细胞自噬的影响。方法:鞘内置管成功的雄性SD大鼠60只,采用随机数字表法共分为5组(n=12):假手术组(S组),神经病理性疼痛模型组(C组),神经病理性疼痛模型组+富氢液组(C+H组),神经病理性疼痛模型组+3-甲基腺嘌呤组(C+M组),神经病理性疼痛模型+3-甲基腺嘌呤+富氢液组(C+H+M组),神经病理性疼痛模型采用慢性坐骨神经结扎模型CCI。C+H组和C+H+M组于术后1h开始腹腔注射富氢液(0.6 mmol/L)5ml/kg Bid,C+M组和C+H+M组术后1h开始鞘内注射自噬抑制剂3-甲基腺嘌呤10μl/kg Bid,其他组腹腔/鞘内给予等量生理盐水Bid。分别于术前1天(-1d),术后1天(1d),3天(3d),7天(7d),10天(10d),14天(14d)测大鼠机械刺激缩足阈值和冷、热刺激缩足潜伏期,术后第14天测定痛阈后心脏灌注取脊髓L4-6节段,采用Western blot和Real-time PCR法测定脊髓的自噬相关基因和蛋白LC3Ⅱ,Beclin-1,P62的表达;采用电镜观察各组大鼠脊髓背角自噬小体的形态和数量;应用酶联免疫法测定各组大鼠血清炎症因子(IL-6,TNF-α)表达,脊髓超氧化物歧化酶(SOD)和丙二醛(MDA)。结果:与S组比较,C组3d后MWT降低,TWL缩短,冷痛阈降低,大鼠脊髓组织Beclin-1、LC3-II基因及蛋白表达上调,p62表达下调,自噬小体数量增多,SOD活力降低,MDA、IL-6和TNF-α表达水平上调(P0.05);与C组比较,富氢液注射逆转了这些变化C+H组的MWT升高,TWL延长,冷痛阈升高,大鼠脊髓组织Beclin-1、LC3-II表达上调,p62基因及蛋白下调,自噬小体增多,SOD活力增高,MDA,IL-6和TNF-α表达水平水平下调(P0.05);与C组比较,应用3-MA的C+M组3d后MWT降低,TWL缩短,冷痛阈降低,痛觉过敏行为加剧,Beclin-1、LC3-II表达下调,p62基因及蛋白上调,自噬小体减少,SOD活力下降,MDA,IL-6和TNF-α表达水平上调(P0.05)。结论:富氢液减轻大鼠神经病理性痛的机制与诱导脊髓神经细胞自噬,降低氧化应激,抑制炎症反应有关。实验二HIF-1α介导细胞自噬在富氢液治疗神经病理性疼痛中的作用目的:缺血缺氧的微环境是神经病理性疼痛的重要形成因素,在缺氧条件下激活一系列适应性反应,低氧诱导因子HIF-1α在其中发挥中心作用,通过转录各种帮助细胞因子适应低氧环境,研究证实它及下游通路是低氧环境下调节自噬经典通路。本实验拟利用HIF-1α激动剂和抑制剂,探讨HIF-1α在富氢液对神经病理性疼痛大鼠脊髓细胞自噬的作用。方法:健康雄性SD大鼠96只,采用随机数字表法分为8组(n=12):假手术组(S组),假手术+富氢液组(S+H组),神经病理性疼痛模型组(C组),神经病理性疼痛+富氢液组(C+H组),神经病理性疼痛+2-甲氧基雌二醇组(C+2Me2组),CCI+H2组+2-甲氧基雌二醇(C+H+2Me2组),CCI+二羟基苯甲酸乙酯组(C+EDHB组),CCI+H2+二羟基苯甲酸乙酯组(C+H+EDHB组)。富氢液给药方式同实验一。HIF-1α抑制剂2ME2溶解在0.5%的二甲基亚砜,于术后30分钟(10mg/kg)腹腔注射Qd,HIF-1α拮抗剂EDHB在CCI术后30分钟以100 mg/kg腹腔注射Qd。分别于术前1天(-1d),术后1天(1d),3天(3d),7天(7d),10天(10d),14天(14d)测大鼠行为学变化,术后第14天(T5)测定痛阈后心脏灌注取脊髓L4-6节段,采用Western blot和Real-time PCR法测定脊髓的自噬相关蛋白和基因Beclin-1,HIF-1α,BNIP3的表达;应用酶联免疫法测定各组大鼠血清IL-6,TNF-α,脊髓SOD和MDA的表达水平。结果:在术后14天,与S组相比,Beclin-1,HIF-1α,BNIP3在C组,C+H组,C+H+2Me2组,C+EDHB组和C+H+EDHB组表达是显著增多,MWT降低,TWL缩短,SOD活力降低,MDA、IL-6和TNF-α表达水平上调(P0.05);与C组相比,在C+H组、C+EDHB及C+H+EDHB组的Beclin-1,HIF-1α,和BNIP3在脊髓表达上调,逆转了患肢的痛觉过敏现象MWT升高,TWL延长,SOD活力降低,MDA、炎性因子IL-6和TNF-α表达水平下调;在C+2Me2组Beclin-1,HIF-1α和BNIP3在脊髓表达下调,患肢的痛觉过敏加剧,MWT降低,TWL缩短,SOD活力降低,MDA、IL-6和TNF-α表达水平上调(P0.05);与C+H组相比,C+H+EDHB组的Beclin-1,HIF-1α和BNIP3在脊髓表达上调,MWT降低,TWL缩短,SOD活力升高,MDA、IL-6和TNF-α表达水平下调;在C+H+2Me2组Beclin-1,HIF-1α和BNIP3在脊髓表达下调,MWT降低,TWL缩短,SOD活力降低,MDA、IL-6和TNF-α含量上调(P0.05)。结论:富氢液通过HIF-1α介导的细胞自噬对神经病理性疼痛大鼠起到治疗作用。实验三、氢气雾化吸入对带状疱疹后神经痛患者的随机对照临床研究目的:带状疱疹后神经痛(PHN)为由水痘-带状疱疹病毒(varicella zoster virus,VZV)引起的带状疱疹的皮疹治愈后3月仍遗留的持续性疼痛。它属于神经病理性疼痛,PHN的治疗可谓世界性难题,寻找安全有效的治疗方法至关重要。本研究拟采用随机对照研究方法,明确雾化吸入氢气对PHN的治疗安全性及有效性,探讨氢气对神经病理性疼痛的治疗机制。方法:2016年01月-2016年06月在天津医科大学第二医院连续就诊的带状疱疹后遗神经痛患者60例,男31例,女29例,年龄45~79岁。采用随机数字法分为3组(n=20):对照组(S组),低频率组(H1组),高频率组(H2组)。S组为基础药物+100%氧气以3L/min速度吸入30分钟,Qd,H1组为基础药物+混合气体(67%H2+33%O2)吸入30分钟Qd,H2组基础药物+混合气体(67%H2+33%O2)吸入30分钟Bid,均连续治疗7天。分别于治疗前1天(0d),治疗后1天(1d),3天(3d),5天(5d),7天(7d),1月(1M),3月(3M),6月(6M)进行门诊评估疼痛程度和静脉采血。主要评价指标:视觉模拟评分(visual analogue scale,VAS)和次要评价指标:1)简化的Mc Gill疼痛问卷评分;2)睡眠评分;3)镇痛和抗癫痫药物用量。治疗过程每日晨起8点检测生命体征。静脉采实验室检测血常规、肝肾功能评估其安全性。治疗后7天采用酶联免疫吸附法(ELISA)测定IL-6,TNF-α,LC3Ⅱ,Beclin-1。结果:三组患者生命体征平稳,血常规,肝肾功能未见异常。与0d比较,S组各时间点差异无统计学意义(P0.05),H1组,H2组的VAS、SF-MPQ、SIS评分、曲马多和加巴喷丁用量在治疗后3d后开始时明显降低,差异有统计学意义(P0.05);与S组比较,H1组在5d后VAS、SF-MPQ、SIS评分、曲马多和加巴喷丁用量明显降低,H2组在3d后VAS、SF-MPQ、SIS评分、曲马多和加巴喷丁用量明显降低差异有统计学意义(P0.05);与H1组比较,H2组VAS、SF-MPQ、SIS评分、曲马多和加巴喷丁用量在5d后明显降低,差异有统计学意义(P0.05)。在治疗后7d,与S组比较,H1和H2组SOD,LC3Ⅱ,Beclin-1水平升高,MDA,IL-6,TNF-α表达减少(P0.05),与H1组比较,H2组患者SOD、LC3Ⅱ、Beclin-1水平升高,MDA,IL-6,TNF-α表达减少(P0.05)。结论:吸入氢气对带状疱疹神经痛患者起到治疗作用,可能与减少氧化应激,降低炎性反应,激活细胞自噬有关。
[Abstract]:Neuropathic pain is a chronic and chronic pain caused by the injury or disease of the somatosensory nervous system. The mechanism of its formation is complex. It is a kind of disease which is difficult to treat in the medical field today. It often causes great pain and low quality of life for the patients. A large number of studies show that the cell autophagy dysfunction is involved. Neuropathic pain is formed and developed, and proper induction of autophagic function has become a breakthrough in the treatment of neuropathic pain. In recent years, a number of studies have shown that low concentration hydrogen molecules (4%) and hydrogen rich fluids have selective antioxidant, anti-inflammatory and anti apoptosis effects, which can be applied to the research and treatment of various diseases. Hydrogen and hydrogen rich liquid are a new kind of medical gas molecules. Some studies have found that intraperitoneal injection of hydrogen rich liquid can improve the behavior of neuropathic pain rats, and it has a therapeutic effect on neuropathic pain. The specific mechanism is still discussed. Hypoxia inducible factor -1 (HIF-1) is the key factor to regulate autophagy in the condition of hypoxia. It is also the key regulator of the cell adaptation to the hypoxic environment. The study confirms that it is more expressed in the primary pathway of neuropathic pain. This topic is to explore the neuropathic pain of the neuropathic pain in the neuropathic pain rat model by intraperitoneal injection of hydrogen rich fluid and herpes zoster neuralgia. The possible mechanism of protective action is the basis for clinical treatment. Experiment 1, hydrogen rich fluid activates the role of autophagy in neuropathic pain of spinal nerve cells: autophagic dysfunction is one of the mechanisms of neuropathic pain formation. By inducing autophagy reaction, it is a new target for the treatment of neuropathic pain. The hydrogen solution has a certain therapeutic effect on neuropathic pain. The specific mechanism is not clear. This experiment is to establish a neuropathic pain rat model (CCI) to explore the effect of hydrogen rich solution on the autophagy of spinal cord cells in neuropathic pain rats. Methods: 60 male SD rats with a successful sheath in the sheath were divided into a random digital table. 5 groups (n=12): sham operation group (group S), neuropathic pain model group (group C), neuropathic pain model group + rich hydrogen group (group C+H), neuropathic pain model group +3- methyl adenine group (C+M group), neuropathic pain model +3- methyladenopurinopterin + rich hydrogen group (C+H+M group), neuropathic pain model using chronic sciatic God After the ligation of the CCI.C+H group and the C+H+M group, the peritoneal injection of hydrogen rich liquid (0.6 mmol/L) 5ml/kg Bid was injected into the abdominal cavity after the operation, and the C+M group and the C+H+M group were injected with the autophagic inhibitor, 3- methyl adenine 3-, 3- methyl adenine l/kg Bid. The other groups were given equal amount of saline in the abdominal cavity / sheath for 1 days before the operation, 1 days after the operation, 3 days, 7 days, 10 days. 10d), 14 days (14d) measured the threshold of mechanical stimulation of the foot and cold, the incubation period of heat stimulation, and the L4-6 segment of the spinal cord after the fourteenth day after the operation. The autophagy related genes and LC3 II, Beclin-1, P62 expression of the spinal cord were measured by Western blot and Real-time PCR, and the autophagy of the spinal dorsal horn of the rats was observed by electron microscopy. The expression of serum inflammatory factors (IL-6, TNF- a), superoxide dismutase (SOD) and malondialdehyde (MDA) in spinal cord of rats were measured by enzyme linked immunosorbent assay. Results: compared with group S, MWT decreased, TWL shortened, cold pain threshold decreased, Beclin-1, LC3-II gene and protein expression of rat spinal cord was up, p62 expression was down, down regulation of p62 expression, and down regulation of p62 expression. The number of phagocytic bodies increased, the activity of SOD decreased, and the expression level of MDA, IL-6 and TNF- alpha was up (P0.05). Compared with the C group, the injection of hydrogen rich solution reversed these changes in the MWT of the C+H group, the TWL lengthening, the increase of the cold pain threshold, the Beclin-1 of the spinal cord and the LC3-II expression in the rat spinal cord, the downregulation of the p62 genes and proteins, the increase of autophagic bodies and the increase of vitality. Expression level decreased (P0.05); compared with group C, MWT decreased after 3D in group C+M of 3-MA, TWL shortened, cold pain threshold decreased, hyperalgesia increased, Beclin-1, LC3-II downregulation, p62 gene and protein up-regulated, autophagic corpuscle decreased, SOD vitality decreased. Conclusion: rich hydrogen solution alleviated neuropathy in rats. The mechanism of rational pain is associated with the induction of autophagy in the spinal cord, reducing oxidative stress and inhibiting the inflammatory response. Experiment two HIF-1 alpha mediates the role of autophagy in the treatment of neuropathic pain in the hydrogen rich solution. The microenvironment of ischemic anoxia is an important form of neuropathic pain and activates a series of adaptations under the condition of hypoxia. Hypoxic inducible factor HIF-1 alpha plays a central role in it. By transcription of various help cytokines to adapt to the hypoxic environment, the study confirms that the downstream pathway is a classical pathway to regulate autophagy in low oxygen environments. This experiment is to use HIF-1 alpha agonists and inhibitors to explore the spinal cord of HIF-1 alpha in the neuropathic pain rats with hydrogen rich fluid Methods: the function of autophagy. Methods: 96 healthy male SD rats were divided into 8 groups (n=12): sham operation group (group S), sham operation + hydrogen rich group (group S+H), neuropathic pain model group (group C), neuropathic pain + hydrogen rich group (C+H group), neuropathic pain +2- methoxy estradiol group (C+2Me2 group), CCI+H2 group +2- a Oxygen based estradiol (group C+H+2Me2), CCI+ two hydroxy benzoate group (group C+EDHB), CCI+H2+ two hydroxy benzoate group (C+H+EDHB group). The way of hydrogen rich solution was dissolved in 0.5% of two methyl sulfoxide with experimental one.HIF-1 alpha inhibitor 2ME2, Qd in 30 minutes after operation (10mg/kg), HIF-1 alpha antagonist EDHB in 30 minutes after CCI. Intraperitoneal injection of Qd. was performed 1 days (-1d), 1 days (1D), 3 days (3D), 7 days (7D), 10 days (10d), 14 days (14d) to measure the behavioral changes of rats, and the L4-6 segment of the spinal cord was perfused after fourteenth days (T5) for the determination of the pain threshold. The expression of autophagy related protein and gene of spinal cord was measured by Western blot and Real-time. The expression level of serum IL-6, TNF- alpha and spinal cord SOD and MDA were measured by ELISA. Results: at the 14 day after the operation, the expression of Beclin-1, HIF-1 a, BNIP3 in C, C+H, C+H+2Me2, C+EDHB and other groups were increased significantly. In group C+H, in group C+H, Beclin-1, HIF-1 a, and BNIP3 in the group of C+EDHB and C+H+EDHB were up-regulated in the spinal cord, reversing the hyperalgesia phenomenon of the affected limbs, MWT increased, TWL prolonged, SOD activity decreased, MDA, inflammatory factor IL-6 and TNF- alpha expression down regulated. Decreased TWL, decreased SOD activity, MDA, IL-6 and TNF- alpha expression level up up (P0.05). Compared with group C+H, Beclin-1, HIF-1 A and BNIP3 were up regulation of the spinal cord expression, MWT decreased, decreased activity and decreased expression level. Shorten the activity of SOD and increase the content of MDA, IL-6 and TNF- alpha (P0.05). Conclusion: hydrogen rich solution can play a therapeutic role in neuropathic pain rats through HIF-1 alpha mediated autophagy. Experiment three, a randomized controlled clinical study of hydrogen atomization inhalation for patients with post herpetic neuralgia: postherpetic neuralgia (PHN) from varicella to band The rash of herpes zoster caused by herpes zoster virus (varicella zoster virus, VZV) remains persistent pain in March. It belongs to neuropathic pain. The treatment of PHN is a worldwide problem. It is very important to find a safe and effective treatment method. This study is to use a randomized controlled study to clarify the atomization inhalation of hydrogen to PHN The therapeutic mechanism of hydrogen on neuropathic pain was discussed. Methods: 60 cases of herpes zoster sequela neuralgia in Second Hospital Affiliated to Tianjin Medical University in 01 months of 2016 -2016, 31 men, 29 women, age 45~79 years, were divided into 3 groups (n=20): the control group (group S), low frequency rate. Group (group H1), group.S of high frequency (group H2) group.S as base drug +100% oxygen inhaled at 3L/min rate for 30 minutes, Qd, H1 group as basic drug + gas mixture (67%H2+33%O2) inhaled Qd, H2 group basic drugs + mixture gas (67%H2+33%O2) inhalation 30 minutes Bid, 7 days, respectively, 1 days before treatment, 1 days after treatment, 3 days 3 days, 5 days ), 7 days (7D), January (1M), March (3M), June (6M) to evaluate the degree of pain and venous blood collection. Major evaluation indexes: visual analogue scale (visual analogue scale, VAS) and secondary evaluation index: 1) simplified Mc Gill pain questionnaire score; 2) sleep and sleep score; 3) dosage of analgesic and antiepileptic drugs. The treatment process was tested at 8 points of life on the morning of the morning. Test blood routine, liver and kidney function to assess the safety of the blood routine test. 7 days after treatment, IL-6, TNF- a, LC3 II, Beclin-1. results were measured by enzyme linked immunosorbent assay (ELISA). The results of the three groups were stable, blood routine, liver and kidney function was not abnormal. Compared with 0d, there was no statistical significance (P0.05), H1 group, H2 group VA, compared with 0d. S, SF-MPQ, SIS score, the dosage of tramadol and gabapentin decreased significantly after the treatment after 3D, and the difference was statistically significant (P0.05). Compared with the S group, the H1 group was significantly reduced in VAS, SF-MPQ, SIS score after 5D, and the dosage of tramadol and gabapentin significantly decreased after the H2 group, and the dosage of tramadol and gabapentin significantly decreased the difference. Compared with group H1, H2 group VAS, SF-MPQ, SIS score, the dosage of tramadol and Gaba Martin decreased significantly after 5D, and the difference was statistically significant (P0.05). Flat MDA, IL-6, TNF- increased and decreased expression of alpha (P0.05). Conclusion: hydrogen inhalation on patients with herpes zoster neuralgia plays a role in the treatment, and may reduce oxidative stress, reduce inflammatory reaction, activation of autophagy.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R402

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