脊髓TLR9在大鼠胫骨癌痛中的作用及电针作用观察
摘要
由外周神经损伤、炎症和肿瘤引起的神经病理性疼痛、炎症痛和癌痛是最常见的三类慢性痛。现今癌症已成为威胁人类生存的重要疾病之一。众所周知,肿瘤引起的疼痛严重破坏了患者的生活质量,其中以由乳腺癌、前列腺癌、肺癌和卵巢癌骨转移引起的骨癌痛最为常见,也最为严重。有研究认为,癌痛可能有神经痛和炎症痛两种成分存在,但与炎症痛和神经痛又不完全相同。在癌痛早期,由于肿瘤与周围组织释放炎性因子而表现出与炎症痛相似的症状；在癌痛晚期由于肿瘤细胞的生长浸润引起神经压迫和损伤,从而表现出与神经痛类似的症状。这提示癌痛机理有其独特性和复杂性。因此将癌痛与神经痛、炎症痛做比较性研究对揭示癌痛独特的机理有重要的理论和临床意义。
Toll样受体(Toll like receptor, TLRs)是一个模式识别受体家族,属于Ⅰ型跨膜受体蛋白,已经发现其13个家族成员。它们主要通过识别保守的病原相关分子模式(Pathogen-Associated Molecular Pattern, PAMP),感知病原微生物的存在,对其产生免疫炎症反应。最近发现TLRs还能识别受损细胞或坏死细胞来源的内源性危险信号。这些内源性危险信号和PAMP类似,在外周可以促进抗原呈递细胞(antigen-presenting cells, APC)的成熟进而激发T细胞免疫应答。热休克蛋白(Heat shock protein, HSP)是TLRs常见的内源性激动剂,HSP与TLRs结合后,能通过髓样分化因子(myeloid differentiation factor88, MyD88)激活下游信号通路。已有研究发现中枢神经系统内的TLR2和TLR4在炎症痛与神经痛的发生发展中有重要的作用,我实验室前期的工作也发现TLR4在癌痛的发生与维持中发挥着关键性作用。TLR9是TLRs家族中重要一员,近来研究发现TLR9不仅在外周免疫细胞中表达,在中枢神经系统内也有广泛表达,并能调节下游的IL-1β、 TNF-α等炎性细胞因子,这些炎性因子在痛觉敏化的过程中有重要的作用。那么脊髓TLR9在癌痛的发生与发展中作用如何昵?因此本研究深入地观察了TLR9在癌痛中的作用。
祖国医学对疼痛有着深刻的认识,《黄帝内经》认为疼痛产生的重要原因是“经脉流行不止,环周不休,泣而不行,脉中则气不通,故卒然而痛。”,此外“风、寒、湿三气杂至合而为痹”。因此“不通则痛”,淤血阻滞,痰凝积结,湿邪内阻,毒火结聚等阻滞经络,引起疼痛。针刺是祖国医学的精华之一,具有作用广,毒副作用小,经济简便等优点。针刺能够疏通经络,调节气血。大量的临床实践证明,针刺疗法对急、慢性疼痛均有良好的治疗效果,但是针刺对骨癌痛的镇痛作用尚存在争议,针刺镇痛的机理也不是十分清楚。因此本研究观察了电针对不同性质疼痛的镇痛作用,以期进一步阐述骨癌痛的独特机理并探索针刺镇痛新的机理。
综上所述,本论文拟开展下列研究：
1.建立癌痛、炎症痛和神经痛模型并比较其行为学差异
(1)参照本研究组毛应启梁等之前的报道,建立大鼠胫骨癌痛模型。采用机械性痛觉超敏与热痛觉过敏等指标检验其行为学特征。
(2)建立新的大鼠胫骨炎症痛模型。采用病理学、血常规分析、免疫荧光、机械性痛觉超敏与热痛觉过敏等方法对其进行评价并检验其行为学特征。
(3)参照Woolf等建立的SNI (spared nerve injury)模型,并进行改进,新建大鼠胫神经病理性疼痛模型,并采用机械性痛觉超敏与热痛觉过敏等指标检验其行为学特征。
(4)通过给予三种疼痛模型大鼠抗炎镇痛药塞来昔布灌胃治疗,比较三种疼痛模型大鼠对塞来昔布的镇痛敏感性。
2.比较脊髓TLR9在三种疼痛模型中的作用及其机制
(1)运用RT-PCR方法在mRNA水平比较TLR9在三种疼痛模型中表达变化。
(2)运用Western Blot方法在蛋白水平比较TLR9在三种疼痛模型中表达变化。
(3)采用免疫荧光双标的方法检测TLR9在胫骨癌痛大鼠脊髓内细胞定位。
(4)通过鞘内注射TLR9的拮抗剂ODN2088,比较其对三种疼痛模型大鼠机械性痛阂的影响。
(5)通过鞘内注射TLR9内源性配体HSP90的抑制剂,17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride),比较其对三种疼痛模型大鼠机械性痛闽的影响。
(6)通过Bio-Plex悬液芯片方法检测TLR9通过下游的细胞因子IL-1β、IL-4、 IL-6、 IL-10与TNF-a的表达变化。
3.比较电针对不同慢性痛的镇痛作用及其对TLR9表达的影响
(1)观察多次电针对大鼠胫骨癌痛的镇痛作用及脊髓TLR9表达的影响。
(2)观察多次电针对大鼠胫骨炎症痛的镇痛作用及脊髓TLR9表达的影响。
(3)观察多次电针对大鼠胫神经痛的镇痛作用及脊髓TLR9表达的影响。
实验结果如下：
1.建立胫骨癌痛、胫骨炎症痛和胫神经痛模型并通过比较其行为学差异
(1)大鼠胫骨癌痛模型
参照毛应启梁等报道的大鼠胫骨癌痛模型,采用微量进样器将Walker256乳腺癌细胞注入大鼠胫骨骨髓腔内。随着肿瘤细胞的生长,大鼠后肢无明显热痛觉过敏,但是产生明显的机械性痛觉超敏,与之前的报道表现一致,表明该模型建立成功。
(2)大鼠胫骨炎症痛模型
与胫骨癌痛模型类似,采用微量进样器将金黄色葡萄球菌(S.aureus)注入大鼠胫骨骨髓腔内。随着细菌的生长,4天后模型大鼠胫骨骨髓腔内产生急性炎症,8天后发展为亚急性炎症,12天后进入慢性炎症阶段,16天后炎症开始恢复。血常规结果表现出一致的变化,在第4天与第8天白细胞数量显著增高,12天后与注射纤维蛋白胶对照组大鼠无显著差异,血常规的其它指标未发现显著性改变。模型大鼠从术后2天开始产生明显的机械性痛觉超敏和热痛觉过敏。有趣的是模型大鼠双后肢产生明显的机械性痛觉超敏,但是只有患肢表现出显著的热痛觉过敏。此外,免疫荧光结果显示脊髓背角内星形胶质细胞和小胶质细胞表达显著增加。表明该炎症痛模型的建立是成功的、可靠的。
(3)大鼠胫神经痛模型
参照Woolf等建立的SNI (spared nerve injury)神经痛模型,我们在此只结扎大鼠的胫神经,而保留腓肠神经和腓总神经的完整。从手术后第2天开始,大鼠手术侧后肢表现出明显的机械性痛觉超敏和热痛觉过敏,一直到16天还保持稳定的痛觉敏化状态,表明该神经痛模型是成功的。
(4)三种疼痛模型对塞来昔布的敏感性比较
从术后第6天给予塞来昔布灌胃治疗,持续到第10天,20mg/kg.day,早晚各一次。在术后第8天,血药浓度稳定后,观察不同疼痛模型大鼠对塞来昔布的敏感性。结果显示胫骨炎症痛大鼠对塞来昔布有很好的敏感性,而胫骨癌痛与胫神经痛大鼠对塞来昔布的敏感性比较弱。
小结：通过本部分的研究,我们建立了大鼠胫骨炎症痛、胫神经痛模型作为大鼠胫骨癌痛模型的研究对照模型,并系统的对比了三种模型机械性痛觉超敏、热痛觉过敏等疼痛行为的差异,此外我们还比较了三种模型大鼠对抗炎镇痛药塞来昔布的敏感性。我们的结果提示,大鼠胫骨癌痛与胫骨炎症痛、胫神经痛行为上既有相似之处,又有不同的地方,提示了骨癌痛可能有神经痛与炎症痛两种成份共同参与,但又有其独特的机理。采用胫骨炎症痛与胫神经模型作为对照模型研究胫骨癌痛的机理有重要的临床意义,为深入开展大鼠胫骨癌痛的机理研究提供了有效的方法。
2.观察脊髓TLR9在三种疼痛模型中的作用及其机制
(1)通过RT-PCR的方法检测TLR9mRNA在三种模型上的表达变化。结果显示,手术后12天,与正常组大鼠相比,胫骨炎症痛与胫骨癌痛大鼠脊髓内TLR9mRNA的表达显著升高,而胫神经痛大鼠则无明显的改变。各模型组大鼠手术同侧和对侧变化趋势一致。
(2)通过Western-Blot方法检测TLR9蛋白在三种疼痛模型上的表达变化,结果显示,手术后12天,与各自模型对照组大鼠相比,胫骨癌痛与胫骨炎症痛大鼠脊髓背角内TLR9的表达显著升高,而胫神经痛大鼠TLR9的表达未发生显著性的改变。
(3)为了明确胫骨癌痛时TLR9在脊髓背角内的表达定位,我们采用免疫双标的方法进行检测。结果表明,TLR9与神经元标记物NeuN存在大量共标,但是与星形胶质细胞标记物GFAP和小胶质细胞标记物CDllb几乎无共存。表明在胫骨癌痛时,神经元内的TLR9大量表达。
(4)为了观察TLR9在疼痛中的作用,我们通过鞘内注射的方法给予三种模型大鼠TLR9的拮抗剂ODN2088,并观察其对三种疼痛模型大鼠的镇痛作用。结果显示,ODN2088在给药后1小时与2小时均能显著性提高胫骨癌痛、胫骨炎症痛与胫神经痛大鼠的机械性痛阈,4小时后作用消失。但是产生相同程度的镇痛作用,胫骨癌痛大鼠所需要的剂量远远高于胫骨炎症痛和胫神经痛大鼠。
(5)为了观察TLR9的内源性配体HSP90在疼痛中的作用,我们通过鞘内注射的方法给予三种模型大鼠HSP90的抑制剂17-DMAG,并观察其对三种疼痛模型大鼠的镇痛作用。结果显示,17-DMAG在给药后1小时与2小时均能显著性提高胫骨癌痛、胫骨炎症痛与胫神经痛大鼠的机械性痛阈,4小时后作用消失。但是产生相同程度的镇痛作用,胫骨癌痛大鼠所需要的剂量远远高于胫骨炎症痛和胫神经痛。
(6)TLR9信号通路下游的细胞因子的表达变化
为了观察TLR9在疼痛中的作用机制,我们采用Bio-Plex悬液芯片法检测了细胞因子IL-1βp、 IL-4、 IL-6、 IL-10与TNF-a在三种疼痛模型中的表达变化以及ODN2088与17-DMAG对这些细胞因子的表达影响。结果显示,胫骨癌痛大鼠脊髓内IL-1β的表达显著升高,鞘内给予ODN2088与17-DMAG均能有效下调IL-1β的表达。胫骨炎症痛与胫神经痛大鼠脊髓内IL-1p没有显著变化。此外,IL-4、 IL-6、 IL-10与TNF-a在三种疼痛模型大鼠的表达均没有发生显著性改变。
小结：综上所述,造模12天后,胫骨癌痛大鼠脊髓背角内TLR9表达显著升高,鞘内给予TLR9的拮抗剂ODN2088能显著性的提高胫骨癌痛大鼠机械性痛阂,鞘内给予HSP90抑制剂17-DMAG对胫骨癌痛大鼠也有显著的镇痛作用。胫骨癌痛大鼠脊髓内炎性细胞因子IL-1β表达显著升高,ODN2088与17-DMAG均能显著性下调IL-1β的表达。虽然ODN2088与17-DMAG对胫骨炎症痛与胫神经痛大鼠也有明显的镇痛作用,但是我们所检测的炎性细胞细胞因子在胫骨炎症痛与胫神经痛中没有发生显著性改变,提示可能通过其它的机制发挥作用,有待进一步的研究。综上,这提示HSP90-TLR9-IL-1βp信号通路可能是胫骨癌痛所特有的,对此信号通路进行深入的研究有可能为临床治疗骨癌痛提供新的突破点。
3.电针对模型大鼠的镇痛作用
为了验证电针对不同疼痛模型大鼠的镇痛作用,我们对三种模型大鼠分别给予多次电针治疗,针刺穴位为“足三里”与“昆仑”,隔天检测机械性痛阈,并进一步观察电针对TLR9表达的影响。
(1)电针对胫骨癌痛大鼠的作用
结果显示,给予胫骨癌痛大鼠多次电针治疗,对其机械性痛阈没有显著性改变,对模型大鼠脊髓内TLR9表达无明显的改变。
(2)电针对胫骨炎症痛大鼠的作用
结果显示,给予胫骨炎症痛大鼠多次电针治疗,能显著提高其机械性痛阈,并能显著性降低模型大鼠脊髓内TLR9的表达。
(3)电针对胫神经痛大鼠的作用
结果显示,给予胫神经痛模型大鼠多次电针治疗,能显著提高其机械性痛阈,但是对模型大鼠脊髓内TLR9的表达无显著性改变。
小结：电针对胫骨癌痛大鼠TLR9的表达无显著性改变,对其机械性痛阈也没有显著性的影响。而电针能显著性下调胫骨炎症痛大鼠TLR9的表达,并且能显著性提高其机械性痛阈。电针对胫神经痛大鼠TLR9的表达没有明显改变,但是能显著提高胫神经痛大鼠的机械性痛阂。由于胫神经痛大鼠本身TLR9的表达不明显,综合之前的研究表明电针通过其它的机制参与胫神经痛的镇痛。电针对胫骨癌痛与胫骨炎症痛的作用表明,TLR9在针刺镇痛中可能发挥着重要的作用。
综上所述,本研究通过对大鼠胫骨癌痛、胫骨炎症痛、胫神经痛三种不同慢性痛的对比研究,表明胫骨癌痛与胫骨炎症痛、神经痛既有相似之处,又有其独特性。
1.在行为学表现方面,胫骨癌痛时机械性痛觉超敏显著而热痛觉过敏不显著,提示癌痛有其独特性。
2.HSP90-TLR9-IL-1β信号通路在大鼠胫骨癌痛的形成与维持中可能发挥着重要的作用,是癌痛不同于炎症痛、神经痛的重要机理之一。
3.电针对癌痛的作用不同于胫骨炎症痛与胫神经痛,TLR9在电针镇痛过程中可能发挥一定的作用。
Pain is a major symptom of bone cancer, and decreased the patient's quality of life greatly. It has been reported that70-90%of patients with advanced cancer experience intractable pain. Metastatic bone tumors are thought to be the most common cause of cancer-related pain and are particularly prevalent in patients with breast, prostate, lung and ovarian cancer. The cancer pain may be involved in the gredits ingredients including inflammatory pain and neuropathic pain, however, the cancer pain has its unique mechanisms refer to neurochemical changes that occur in the spinal cord and primary afferent neurons. At its earlier phase, an inflammatory response is inevitable, while in its later phase, nerve infiltration and compression by tumor, cancer pain may also be characterized as neuropathic pain. Therefore, comparison the cancer pain, inflammatory pain and cancer pain to reveal the unique mechanism of cancer pain may be of clinical benefit.
Toll-like receptors (TLRs) are closely related type I transmembrane proteins, which form the major pattern-recognition receptors (PRRs) to transduce the signals in response to microbial intruders such as protozoa, bacteria, fungi and viruses. In the rat inflammatory pain and neuropathic pain model, TLR2and TLR4transcripts are up-regulated in the spinal cord upon spinal nerve injury. It has also been reported that TLR4is required for the maximum induction of pain hypersensitivity upon spinal nerve transection. TLR9is one member of the TLRs family. It is expressed not only in immune celles, but also in CNS. TLR9involved in the development of many inflammatory, and regulate the expression of inflammatory factory, such as IL-1, TNF-a and IL-6. Recent research discoveried that in trigeminal tissues, stimulation of TLR9with CpG ODN can up-regulate of the expression of TRPV1, and can enhance the calcium flux in TRPV1-expressing DRG neurons, indicating the envolvement TLR9in nociception. Here we investigated the role of TLR9in the bone cancer pain.
Acupuncture has been a healing art in traditional Chinese medicine for more than2000years. Various diseases including pain can be cured effectively by inserting the needles into acupoints on the skin. It is accepted widely with the advantages of simple application, lost cost and minimal side effects. Among acupuncture therapies, the acupuncture-induced analgesic effect has been used widely and researched by scientists from all over the world; however, the mechanisms of acupuncture analgesia remain unclear. In the present research, the effects of acupuncture on different kinds of pain including bone inflammatory pain, neuropathic pain and bone cancer pain were observed and the mechanisms of acupuncture analgesia were also investigated.
Based on these, the present research was done as following:
1. The established models of bone cancer pain, inflammatory pain and neuropathic pain, and compared the behaviors differences among these models to reveal the character of the bone cancer pain.
Ⅰ. According to bone cancer model reported by Mao-ying, Walker256cancer cells were injected into the tibial cavity directly. As the growing of cancer cells, the rats injected with cancer cells displayed a profound decrease in mechanical threshold to the von Frey filaments stimulation, not only on the ipsilateral hind paws, but also on the contralateral hind paws. In contrast, there were no significant thermal hyperalgesia in rats injected with cancer cells. Consists with prior reported and suggests it a successful model for bone cancer pain research.
Ⅱ. The same surgical procedure as bone cancer pain, Staphylo coccus aureus was injected into the tibial cavity directly. Four days after an intra-tibial bacteria injection, acute inflammation was seen in the bone marrow, and this inflammation developed into chronic inflammation12days after the operation. The results from the blood analysis further confirmed the existence of bone inflammation. A pain related behavior analysis showed that significant mechanical allodynia and thermal hyperalgesia were developed on both sides of the hind limbs shortly after the unilateral bacteria injection. Moreover, this osteomyelitis-induced pain behavior was reversed by Celecoxib, a nonsteroidal antiinflammatory drug. Furthermore, significant hypertrophy of both microglia and astrocytes, which had been demonstrated to play an important role in many chronic pain conditions, was observed in the spinal cord. Our results suggested that osteomyelitis-induced rats displayed pain related behaviors and related neurochemical changes. This study thus provides a novel practical rat model of bone inflammation induced pain for bone cancer pain research.
Ⅲ. According to model of SNI reported by Woolf, modified tibial ligation was carefully performed. In this model, only the tibial nerve was ligated, and left the sural nerve and common peroneal nerve intact to minimize damage. After operation, the model rats displayed profound mechanical allodynia and thermal hyperalgesia.
IV. The Celecoxib was delivered from day6after surgery through day10. The bacteria-injected rats displayed higher sensitivity in mechanical threshold than bone cancer pain and neuropathic pain after treatment with Celecoxib (20mg/kg per day) on8days after surgery.
Summury:In this part, we established models of bone cancer pain, bone inflammatory pain and neuropathic pain successfully, and compared the bone cancer pain with bone inflammatory pain and neuropathic pain in mechanical allodynia and thermal hyperalgesia. Moreover, the sensitivity of Celecoxib on different pain models has been compared. The results indicated that there is a fundamental difference in the behaviors among bone cancer pain, bone inflammatory pain, and neuropathic pain. Compared the bone cancer pain with bone inflammatory pain and neuropathic pain may be useful for elucidating the complex mechanisms involved in bone cancer pain.
2. The expression of TLR9in bone cancer pain, bone inflammatory pain and neuropathic pain and the unique mechanism involved in bone cancer pain.
I. The RT-PCR was used to detect the expression of TLR9mRNA. The results indicated that12day after the operation, compared with normal rats, the mRNA of TLR9in bone cancer pain and bone inflammatory pain obviously increased in spinal cord. However, there was no significant change in the neuropathic pain rats. The both sides of spinal cord showed similar changes.
Ⅱ. The WB was used to detect the expression of TLR9. The results indicated that12day after the operation, compared with normal rats, the protein of TLR9in bone cancer pain and bone inflammatory pain obviously increased in spinal cord. However, there was no significant change in the neuropathic pain rats. The both sides of spinal cord showed similar changes.
Ⅱ. To definite the subcellular localization of TLR9, double immunostaining was used. In bone cancer pain rats, TLR9-immunoreactive cells was positive for NeuN, a neuron marker, and rarely for glia marker GFAP and CD1lb, showing that TLR9was mainly expressed in neuron.
IV. To observe the role of TLR9in the pain, ODN2088, a TLR9inhibitor, was intrathecal injected. Result indicated ODN2088can reduce pain effectively in bone cancer pain, bone inflammatory pain and neuropathic pain. However, the dose needed in cancer pain was higer than the other two kinds of pain condition.
V. HSP90is an endogenous agonist for TLR9.17-DMAG, a HSP90inhibitor, was intrathecal injected.17-DMAG can reduce pain effectively in bone cancer pain, bone inflammatory pain and neuropathic pain. However, the dose needed in bone cancer pain was higer than the other two kinds of pain condition.
VI. To observe the change of cytokines downstream of TLR9, Bio-Plex was used to decect the expressiom of IL-1β\IL-4, IL-6, IL-10and TNF-a. Result indicated that IL-1β was significantly increased in the spinal cord of bone cancer pain rats. Intrathecal injection of ODN2088and17-DMAG significantly inhibited the expression of IL-1β\However, there was no change for the expression of IL-1β in the bone inflammatory pain and neuropathic pain. The expressiom of IL-4, IL-6, IL-10and TNF-a did not change in all of three pain models.
Summury:12days after operation, the expression of TLR9in bone cancer pain rats increased significantly. And intrathecal injection of ODN2088and17-DMAG increased the mechanical threshold of bone cancer pain. Moreover, the expression of IL-1β increased only in the bone cancer pain, and can be inhibited by ODN2088and17-DMAG. The results indicated that the HSP90-TLR9-IL-1β signal pathway may be of vital involved in the bone cancer pain.
3. The anti-allodynic effect of electroacupuncture on bone cancer pain, inflammatory pain and neuropathic pain
I. In order to investigate the analgesic effects of electroacupuncture on pain, electroacupuncture was applied to the rat model every other day. The results showed the electroacupuncture can reduce bone inflammatory pain and neuropathic pain effectively while there was no significant analgesia effect on bone cancer pain.
Ⅱ. Electroacupuncture can decrease the expression of TLR9in inflammatory pain rats, and there was no effect on TLR9expression in bone cancer pain rats and neuropathic pain rats.
In summary:the electroacupuncture showed no significant analgesia effect on bone cancer pain and the expression of TLR9. However, electroacupuncture can alleviate pain effectively in the bone inflammatory pain. In neuropathic pain model, the electroacupuncture can reduce pain effectively while no effect on the expression of TLR9. All above indicated that TLR9is involved in the pain, and bone cancer pain has its own characters and mechanisms.
Toll样受体(Toll like receptor, TLRs)是一个模式识别受体家族,属于Ⅰ型跨膜受体蛋白,已经发现其13个家族成员。它们主要通过识别保守的病原相关分子模式(Pathogen-Associated Molecular Pattern, PAMP),感知病原微生物的存在,对其产生免疫炎症反应。最近发现TLRs还能识别受损细胞或坏死细胞来源的内源性危险信号。这些内源性危险信号和PAMP类似,在外周可以促进抗原呈递细胞(antigen-presenting cells, APC)的成熟进而激发T细胞免疫应答。热休克蛋白(Heat shock protein, HSP)是TLRs常见的内源性激动剂,HSP与TLRs结合后,能通过髓样分化因子(myeloid differentiation factor88, MyD88)激活下游信号通路。已有研究发现中枢神经系统内的TLR2和TLR4在炎症痛与神经痛的发生发展中有重要的作用,我实验室前期的工作也发现TLR4在癌痛的发生与维持中发挥着关键性作用。TLR9是TLRs家族中重要一员,近来研究发现TLR9不仅在外周免疫细胞中表达,在中枢神经系统内也有广泛表达,并能调节下游的IL-1β、 TNF-α等炎性细胞因子,这些炎性因子在痛觉敏化的过程中有重要的作用。那么脊髓TLR9在癌痛的发生与发展中作用如何昵?因此本研究深入地观察了TLR9在癌痛中的作用。
祖国医学对疼痛有着深刻的认识,《黄帝内经》认为疼痛产生的重要原因是“经脉流行不止,环周不休,泣而不行,脉中则气不通,故卒然而痛。”,此外“风、寒、湿三气杂至合而为痹”。因此“不通则痛”,淤血阻滞,痰凝积结,湿邪内阻,毒火结聚等阻滞经络,引起疼痛。针刺是祖国医学的精华之一,具有作用广,毒副作用小,经济简便等优点。针刺能够疏通经络,调节气血。大量的临床实践证明,针刺疗法对急、慢性疼痛均有良好的治疗效果,但是针刺对骨癌痛的镇痛作用尚存在争议,针刺镇痛的机理也不是十分清楚。因此本研究观察了电针对不同性质疼痛的镇痛作用,以期进一步阐述骨癌痛的独特机理并探索针刺镇痛新的机理。
综上所述,本论文拟开展下列研究：
1.建立癌痛、炎症痛和神经痛模型并比较其行为学差异
(1)参照本研究组毛应启梁等之前的报道,建立大鼠胫骨癌痛模型。采用机械性痛觉超敏与热痛觉过敏等指标检验其行为学特征。
(2)建立新的大鼠胫骨炎症痛模型。采用病理学、血常规分析、免疫荧光、机械性痛觉超敏与热痛觉过敏等方法对其进行评价并检验其行为学特征。
(3)参照Woolf等建立的SNI (spared nerve injury)模型,并进行改进,新建大鼠胫神经病理性疼痛模型,并采用机械性痛觉超敏与热痛觉过敏等指标检验其行为学特征。
(4)通过给予三种疼痛模型大鼠抗炎镇痛药塞来昔布灌胃治疗,比较三种疼痛模型大鼠对塞来昔布的镇痛敏感性。
2.比较脊髓TLR9在三种疼痛模型中的作用及其机制
(1)运用RT-PCR方法在mRNA水平比较TLR9在三种疼痛模型中表达变化。
(2)运用Western Blot方法在蛋白水平比较TLR9在三种疼痛模型中表达变化。
(3)采用免疫荧光双标的方法检测TLR9在胫骨癌痛大鼠脊髓内细胞定位。
(4)通过鞘内注射TLR9的拮抗剂ODN2088,比较其对三种疼痛模型大鼠机械性痛阂的影响。
(5)通过鞘内注射TLR9内源性配体HSP90的抑制剂,17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride),比较其对三种疼痛模型大鼠机械性痛闽的影响。
(6)通过Bio-Plex悬液芯片方法检测TLR9通过下游的细胞因子IL-1β、IL-4、 IL-6、 IL-10与TNF-a的表达变化。
3.比较电针对不同慢性痛的镇痛作用及其对TLR9表达的影响
(1)观察多次电针对大鼠胫骨癌痛的镇痛作用及脊髓TLR9表达的影响。
(2)观察多次电针对大鼠胫骨炎症痛的镇痛作用及脊髓TLR9表达的影响。
(3)观察多次电针对大鼠胫神经痛的镇痛作用及脊髓TLR9表达的影响。
实验结果如下：
1.建立胫骨癌痛、胫骨炎症痛和胫神经痛模型并通过比较其行为学差异
(1)大鼠胫骨癌痛模型
参照毛应启梁等报道的大鼠胫骨癌痛模型,采用微量进样器将Walker256乳腺癌细胞注入大鼠胫骨骨髓腔内。随着肿瘤细胞的生长,大鼠后肢无明显热痛觉过敏,但是产生明显的机械性痛觉超敏,与之前的报道表现一致,表明该模型建立成功。
(2)大鼠胫骨炎症痛模型
与胫骨癌痛模型类似,采用微量进样器将金黄色葡萄球菌(S.aureus)注入大鼠胫骨骨髓腔内。随着细菌的生长,4天后模型大鼠胫骨骨髓腔内产生急性炎症,8天后发展为亚急性炎症,12天后进入慢性炎症阶段,16天后炎症开始恢复。血常规结果表现出一致的变化,在第4天与第8天白细胞数量显著增高,12天后与注射纤维蛋白胶对照组大鼠无显著差异,血常规的其它指标未发现显著性改变。模型大鼠从术后2天开始产生明显的机械性痛觉超敏和热痛觉过敏。有趣的是模型大鼠双后肢产生明显的机械性痛觉超敏,但是只有患肢表现出显著的热痛觉过敏。此外,免疫荧光结果显示脊髓背角内星形胶质细胞和小胶质细胞表达显著增加。表明该炎症痛模型的建立是成功的、可靠的。
(3)大鼠胫神经痛模型
参照Woolf等建立的SNI (spared nerve injury)神经痛模型,我们在此只结扎大鼠的胫神经,而保留腓肠神经和腓总神经的完整。从手术后第2天开始,大鼠手术侧后肢表现出明显的机械性痛觉超敏和热痛觉过敏,一直到16天还保持稳定的痛觉敏化状态,表明该神经痛模型是成功的。
(4)三种疼痛模型对塞来昔布的敏感性比较
从术后第6天给予塞来昔布灌胃治疗,持续到第10天,20mg/kg.day,早晚各一次。在术后第8天,血药浓度稳定后,观察不同疼痛模型大鼠对塞来昔布的敏感性。结果显示胫骨炎症痛大鼠对塞来昔布有很好的敏感性,而胫骨癌痛与胫神经痛大鼠对塞来昔布的敏感性比较弱。
小结：通过本部分的研究,我们建立了大鼠胫骨炎症痛、胫神经痛模型作为大鼠胫骨癌痛模型的研究对照模型,并系统的对比了三种模型机械性痛觉超敏、热痛觉过敏等疼痛行为的差异,此外我们还比较了三种模型大鼠对抗炎镇痛药塞来昔布的敏感性。我们的结果提示,大鼠胫骨癌痛与胫骨炎症痛、胫神经痛行为上既有相似之处,又有不同的地方,提示了骨癌痛可能有神经痛与炎症痛两种成份共同参与,但又有其独特的机理。采用胫骨炎症痛与胫神经模型作为对照模型研究胫骨癌痛的机理有重要的临床意义,为深入开展大鼠胫骨癌痛的机理研究提供了有效的方法。
2.观察脊髓TLR9在三种疼痛模型中的作用及其机制
(1)通过RT-PCR的方法检测TLR9mRNA在三种模型上的表达变化。结果显示,手术后12天,与正常组大鼠相比,胫骨炎症痛与胫骨癌痛大鼠脊髓内TLR9mRNA的表达显著升高,而胫神经痛大鼠则无明显的改变。各模型组大鼠手术同侧和对侧变化趋势一致。
(2)通过Western-Blot方法检测TLR9蛋白在三种疼痛模型上的表达变化,结果显示,手术后12天,与各自模型对照组大鼠相比,胫骨癌痛与胫骨炎症痛大鼠脊髓背角内TLR9的表达显著升高,而胫神经痛大鼠TLR9的表达未发生显著性的改变。
(3)为了明确胫骨癌痛时TLR9在脊髓背角内的表达定位,我们采用免疫双标的方法进行检测。结果表明,TLR9与神经元标记物NeuN存在大量共标,但是与星形胶质细胞标记物GFAP和小胶质细胞标记物CDllb几乎无共存。表明在胫骨癌痛时,神经元内的TLR9大量表达。
(4)为了观察TLR9在疼痛中的作用,我们通过鞘内注射的方法给予三种模型大鼠TLR9的拮抗剂ODN2088,并观察其对三种疼痛模型大鼠的镇痛作用。结果显示,ODN2088在给药后1小时与2小时均能显著性提高胫骨癌痛、胫骨炎症痛与胫神经痛大鼠的机械性痛阈,4小时后作用消失。但是产生相同程度的镇痛作用,胫骨癌痛大鼠所需要的剂量远远高于胫骨炎症痛和胫神经痛大鼠。
(5)为了观察TLR9的内源性配体HSP90在疼痛中的作用,我们通过鞘内注射的方法给予三种模型大鼠HSP90的抑制剂17-DMAG,并观察其对三种疼痛模型大鼠的镇痛作用。结果显示,17-DMAG在给药后1小时与2小时均能显著性提高胫骨癌痛、胫骨炎症痛与胫神经痛大鼠的机械性痛阈,4小时后作用消失。但是产生相同程度的镇痛作用,胫骨癌痛大鼠所需要的剂量远远高于胫骨炎症痛和胫神经痛。
(6)TLR9信号通路下游的细胞因子的表达变化
为了观察TLR9在疼痛中的作用机制,我们采用Bio-Plex悬液芯片法检测了细胞因子IL-1βp、 IL-4、 IL-6、 IL-10与TNF-a在三种疼痛模型中的表达变化以及ODN2088与17-DMAG对这些细胞因子的表达影响。结果显示,胫骨癌痛大鼠脊髓内IL-1β的表达显著升高,鞘内给予ODN2088与17-DMAG均能有效下调IL-1β的表达。胫骨炎症痛与胫神经痛大鼠脊髓内IL-1p没有显著变化。此外,IL-4、 IL-6、 IL-10与TNF-a在三种疼痛模型大鼠的表达均没有发生显著性改变。
小结：综上所述,造模12天后,胫骨癌痛大鼠脊髓背角内TLR9表达显著升高,鞘内给予TLR9的拮抗剂ODN2088能显著性的提高胫骨癌痛大鼠机械性痛阂,鞘内给予HSP90抑制剂17-DMAG对胫骨癌痛大鼠也有显著的镇痛作用。胫骨癌痛大鼠脊髓内炎性细胞因子IL-1β表达显著升高,ODN2088与17-DMAG均能显著性下调IL-1β的表达。虽然ODN2088与17-DMAG对胫骨炎症痛与胫神经痛大鼠也有明显的镇痛作用,但是我们所检测的炎性细胞细胞因子在胫骨炎症痛与胫神经痛中没有发生显著性改变,提示可能通过其它的机制发挥作用,有待进一步的研究。综上,这提示HSP90-TLR9-IL-1βp信号通路可能是胫骨癌痛所特有的,对此信号通路进行深入的研究有可能为临床治疗骨癌痛提供新的突破点。
3.电针对模型大鼠的镇痛作用
为了验证电针对不同疼痛模型大鼠的镇痛作用,我们对三种模型大鼠分别给予多次电针治疗,针刺穴位为“足三里”与“昆仑”,隔天检测机械性痛阈,并进一步观察电针对TLR9表达的影响。
(1)电针对胫骨癌痛大鼠的作用
结果显示,给予胫骨癌痛大鼠多次电针治疗,对其机械性痛阈没有显著性改变,对模型大鼠脊髓内TLR9表达无明显的改变。
(2)电针对胫骨炎症痛大鼠的作用
结果显示,给予胫骨炎症痛大鼠多次电针治疗,能显著提高其机械性痛阈,并能显著性降低模型大鼠脊髓内TLR9的表达。
(3)电针对胫神经痛大鼠的作用
结果显示,给予胫神经痛模型大鼠多次电针治疗,能显著提高其机械性痛阈,但是对模型大鼠脊髓内TLR9的表达无显著性改变。
小结：电针对胫骨癌痛大鼠TLR9的表达无显著性改变,对其机械性痛阈也没有显著性的影响。而电针能显著性下调胫骨炎症痛大鼠TLR9的表达,并且能显著性提高其机械性痛阈。电针对胫神经痛大鼠TLR9的表达没有明显改变,但是能显著提高胫神经痛大鼠的机械性痛阂。由于胫神经痛大鼠本身TLR9的表达不明显,综合之前的研究表明电针通过其它的机制参与胫神经痛的镇痛。电针对胫骨癌痛与胫骨炎症痛的作用表明,TLR9在针刺镇痛中可能发挥着重要的作用。
综上所述,本研究通过对大鼠胫骨癌痛、胫骨炎症痛、胫神经痛三种不同慢性痛的对比研究,表明胫骨癌痛与胫骨炎症痛、神经痛既有相似之处,又有其独特性。
1.在行为学表现方面,胫骨癌痛时机械性痛觉超敏显著而热痛觉过敏不显著,提示癌痛有其独特性。
2.HSP90-TLR9-IL-1β信号通路在大鼠胫骨癌痛的形成与维持中可能发挥着重要的作用,是癌痛不同于炎症痛、神经痛的重要机理之一。
3.电针对癌痛的作用不同于胫骨炎症痛与胫神经痛,TLR9在电针镇痛过程中可能发挥一定的作用。
Pain is a major symptom of bone cancer, and decreased the patient's quality of life greatly. It has been reported that70-90%of patients with advanced cancer experience intractable pain. Metastatic bone tumors are thought to be the most common cause of cancer-related pain and are particularly prevalent in patients with breast, prostate, lung and ovarian cancer. The cancer pain may be involved in the gredits ingredients including inflammatory pain and neuropathic pain, however, the cancer pain has its unique mechanisms refer to neurochemical changes that occur in the spinal cord and primary afferent neurons. At its earlier phase, an inflammatory response is inevitable, while in its later phase, nerve infiltration and compression by tumor, cancer pain may also be characterized as neuropathic pain. Therefore, comparison the cancer pain, inflammatory pain and cancer pain to reveal the unique mechanism of cancer pain may be of clinical benefit.
Toll-like receptors (TLRs) are closely related type I transmembrane proteins, which form the major pattern-recognition receptors (PRRs) to transduce the signals in response to microbial intruders such as protozoa, bacteria, fungi and viruses. In the rat inflammatory pain and neuropathic pain model, TLR2and TLR4transcripts are up-regulated in the spinal cord upon spinal nerve injury. It has also been reported that TLR4is required for the maximum induction of pain hypersensitivity upon spinal nerve transection. TLR9is one member of the TLRs family. It is expressed not only in immune celles, but also in CNS. TLR9involved in the development of many inflammatory, and regulate the expression of inflammatory factory, such as IL-1, TNF-a and IL-6. Recent research discoveried that in trigeminal tissues, stimulation of TLR9with CpG ODN can up-regulate of the expression of TRPV1, and can enhance the calcium flux in TRPV1-expressing DRG neurons, indicating the envolvement TLR9in nociception. Here we investigated the role of TLR9in the bone cancer pain.
Acupuncture has been a healing art in traditional Chinese medicine for more than2000years. Various diseases including pain can be cured effectively by inserting the needles into acupoints on the skin. It is accepted widely with the advantages of simple application, lost cost and minimal side effects. Among acupuncture therapies, the acupuncture-induced analgesic effect has been used widely and researched by scientists from all over the world; however, the mechanisms of acupuncture analgesia remain unclear. In the present research, the effects of acupuncture on different kinds of pain including bone inflammatory pain, neuropathic pain and bone cancer pain were observed and the mechanisms of acupuncture analgesia were also investigated.
Based on these, the present research was done as following:
1. The established models of bone cancer pain, inflammatory pain and neuropathic pain, and compared the behaviors differences among these models to reveal the character of the bone cancer pain.
Ⅰ. According to bone cancer model reported by Mao-ying, Walker256cancer cells were injected into the tibial cavity directly. As the growing of cancer cells, the rats injected with cancer cells displayed a profound decrease in mechanical threshold to the von Frey filaments stimulation, not only on the ipsilateral hind paws, but also on the contralateral hind paws. In contrast, there were no significant thermal hyperalgesia in rats injected with cancer cells. Consists with prior reported and suggests it a successful model for bone cancer pain research.
Ⅱ. The same surgical procedure as bone cancer pain, Staphylo coccus aureus was injected into the tibial cavity directly. Four days after an intra-tibial bacteria injection, acute inflammation was seen in the bone marrow, and this inflammation developed into chronic inflammation12days after the operation. The results from the blood analysis further confirmed the existence of bone inflammation. A pain related behavior analysis showed that significant mechanical allodynia and thermal hyperalgesia were developed on both sides of the hind limbs shortly after the unilateral bacteria injection. Moreover, this osteomyelitis-induced pain behavior was reversed by Celecoxib, a nonsteroidal antiinflammatory drug. Furthermore, significant hypertrophy of both microglia and astrocytes, which had been demonstrated to play an important role in many chronic pain conditions, was observed in the spinal cord. Our results suggested that osteomyelitis-induced rats displayed pain related behaviors and related neurochemical changes. This study thus provides a novel practical rat model of bone inflammation induced pain for bone cancer pain research.
Ⅲ. According to model of SNI reported by Woolf, modified tibial ligation was carefully performed. In this model, only the tibial nerve was ligated, and left the sural nerve and common peroneal nerve intact to minimize damage. After operation, the model rats displayed profound mechanical allodynia and thermal hyperalgesia.
IV. The Celecoxib was delivered from day6after surgery through day10. The bacteria-injected rats displayed higher sensitivity in mechanical threshold than bone cancer pain and neuropathic pain after treatment with Celecoxib (20mg/kg per day) on8days after surgery.
Summury:In this part, we established models of bone cancer pain, bone inflammatory pain and neuropathic pain successfully, and compared the bone cancer pain with bone inflammatory pain and neuropathic pain in mechanical allodynia and thermal hyperalgesia. Moreover, the sensitivity of Celecoxib on different pain models has been compared. The results indicated that there is a fundamental difference in the behaviors among bone cancer pain, bone inflammatory pain, and neuropathic pain. Compared the bone cancer pain with bone inflammatory pain and neuropathic pain may be useful for elucidating the complex mechanisms involved in bone cancer pain.
2. The expression of TLR9in bone cancer pain, bone inflammatory pain and neuropathic pain and the unique mechanism involved in bone cancer pain.
I. The RT-PCR was used to detect the expression of TLR9mRNA. The results indicated that12day after the operation, compared with normal rats, the mRNA of TLR9in bone cancer pain and bone inflammatory pain obviously increased in spinal cord. However, there was no significant change in the neuropathic pain rats. The both sides of spinal cord showed similar changes.
Ⅱ. The WB was used to detect the expression of TLR9. The results indicated that12day after the operation, compared with normal rats, the protein of TLR9in bone cancer pain and bone inflammatory pain obviously increased in spinal cord. However, there was no significant change in the neuropathic pain rats. The both sides of spinal cord showed similar changes.
Ⅱ. To definite the subcellular localization of TLR9, double immunostaining was used. In bone cancer pain rats, TLR9-immunoreactive cells was positive for NeuN, a neuron marker, and rarely for glia marker GFAP and CD1lb, showing that TLR9was mainly expressed in neuron.
IV. To observe the role of TLR9in the pain, ODN2088, a TLR9inhibitor, was intrathecal injected. Result indicated ODN2088can reduce pain effectively in bone cancer pain, bone inflammatory pain and neuropathic pain. However, the dose needed in cancer pain was higer than the other two kinds of pain condition.
V. HSP90is an endogenous agonist for TLR9.17-DMAG, a HSP90inhibitor, was intrathecal injected.17-DMAG can reduce pain effectively in bone cancer pain, bone inflammatory pain and neuropathic pain. However, the dose needed in bone cancer pain was higer than the other two kinds of pain condition.
VI. To observe the change of cytokines downstream of TLR9, Bio-Plex was used to decect the expressiom of IL-1β\IL-4, IL-6, IL-10and TNF-a. Result indicated that IL-1β was significantly increased in the spinal cord of bone cancer pain rats. Intrathecal injection of ODN2088and17-DMAG significantly inhibited the expression of IL-1β\However, there was no change for the expression of IL-1β in the bone inflammatory pain and neuropathic pain. The expressiom of IL-4, IL-6, IL-10and TNF-a did not change in all of three pain models.
Summury:12days after operation, the expression of TLR9in bone cancer pain rats increased significantly. And intrathecal injection of ODN2088and17-DMAG increased the mechanical threshold of bone cancer pain. Moreover, the expression of IL-1β increased only in the bone cancer pain, and can be inhibited by ODN2088and17-DMAG. The results indicated that the HSP90-TLR9-IL-1β signal pathway may be of vital involved in the bone cancer pain.
3. The anti-allodynic effect of electroacupuncture on bone cancer pain, inflammatory pain and neuropathic pain
I. In order to investigate the analgesic effects of electroacupuncture on pain, electroacupuncture was applied to the rat model every other day. The results showed the electroacupuncture can reduce bone inflammatory pain and neuropathic pain effectively while there was no significant analgesia effect on bone cancer pain.
Ⅱ. Electroacupuncture can decrease the expression of TLR9in inflammatory pain rats, and there was no effect on TLR9expression in bone cancer pain rats and neuropathic pain rats.
In summary:the electroacupuncture showed no significant analgesia effect on bone cancer pain and the expression of TLR9. However, electroacupuncture can alleviate pain effectively in the bone inflammatory pain. In neuropathic pain model, the electroacupuncture can reduce pain effectively while no effect on the expression of TLR9. All above indicated that TLR9is involved in the pain, and bone cancer pain has its own characters and mechanisms.
引文
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