脊髓小胶质细胞调控氯离子稳态在慢性胰腺炎痛觉过敏的机制研究

Recurrent abdominal pain is considered as one of the most prominent clinical features of chronic pancreatitis（CP）. One hot topic for CP pain mechanisms is the sensitization of visceral sensory conduction. A study had shown that spinal cord microglia may play an essential role in the development and maintenance of pain hypersensitivity of CP. However, the specific mechanism for spinal microglia contributing to chronic pain of CP has not been elucidated. Recently, by using a rat model of CP induced by trinitrobenzene sulfonic acid（TNBS） infusion, we have found that TNBS treatment might lead to decreased expression of the K--Cl- cotransporter isoform 2（KCC2）in the thoracic spinal cord. As the major protein responsible for neuronal chloride homeostasis regulation, KCC2 also serves as an important target for microglia-neuron interaction, and many studies have demonstrated that altered chloride homeostasis mediated by KCC2 downregulation plays a critical role in somatic pain hypersensitivity following peripheral nerve injury. Thus, we hypothesize that the spinal microglia may contribute to CP pain hypersensitivity by downregulating KCC2 expression in the spinal dorsal horn. To verify the hypothesis, we first investigate the role of KCC2 downregulation mediated chloride homeostasis impairment in the central sensitization of CP chronic pain by using electrophysiological, molecular biological and behavioral techniques. Next, by using intrathecal injection for in vivo intervention, we explore the specific mechanisms for spinal microglia regulating KCC2 expression and pain sensitivity in the CP rat model. The goal of our project is to provide some new therapeutic direction and target for CP chronic pain intervention.

反复发作的腹痛是慢性胰腺炎（CP）最显著的临床特点之一，而内脏感觉传导的敏化机制是目前CP疼痛研究的热点。有研究发现脊髓小胶质细胞在CP痛觉过敏中起重要作用，但具体机制尚不明确。近来检测CP模型大鼠胸段脊髓钾氯共同转运体亚型2（KCC2）蛋白水平明显下降，而KCC2作为影响神经元氯离子稳态的重要调控蛋白，是小胶质细胞-神经元间交互作用的靶点之一，且有研究证实KCC2下调所导致的氯离子稳态失衡在神经性疼痛等躯体痛中起重要作用，推测脊髓小胶质细胞可能通过调控KCC2蛋白表达进一步介导CP痛觉过敏的产生。围绕以上科学假设，本课题拟首先通过膜片钳电生理技术，结合分子生物学、行为学检测，探讨KCC2下调介导的脊髓氯离子稳态失衡在CP中枢敏化中的作用；接下来，通过鞘内注射在体干预的方式，探讨脊髓小胶质细胞调控KCC2表达及CP痛觉行为的相关机制，为CP内脏痛临床治疗提供新的方向及靶点。

内脏痛觉高敏感是慢性胰腺炎（CP）腹痛的重要病理生理学特点，而近年来研究显示脊髓小胶质细胞介导的中枢敏化在CP痛觉过敏中起重要作用，其具体机制仍不明了。本研究旨在探讨脊髓小胶质细胞介导CP痛觉过敏的具体机制。通过胰管内逆行灌注TNBS 构建CP 大鼠模型，利用实时定量PCR、Western免疫印记及免疫荧光技术发现CP大鼠其胸腰段脊髓背角氯离子稳态相关蛋白钾氯共同转运体亚型2（KCC2）表达显著下降，而钠钾氯共同转运体亚型1（NKCC1）的表达没有明显改变。通过制备成年大鼠的脊髓薄片，利用全细胞膜片钳技术发现CP大鼠脊髓Ⅰ板层神经元由于KCC2下调而导致氯离子稳态失衡（表现为氯离子平衡电位的去极化方向偏移），其结果可导致脊髓水平GABA/甘氨酸能突触抑制效率的降低，即去抑制化。通过腹腔注射的方式给予KCC2增强剂CLP257后可部分逆转模型组神经元的氯离子稳态失衡，并降低CP的内脏高敏感。以上结果提示脊髓KCC2蛋白下调导致的氯离子稳态失衡可能是CP内脏高敏感的重要机制。接下来，通过鞘内注射的方式给予小胶质细胞抑制剂米诺环素可上调CP大鼠脊髓KCC2表达、逆转脊髓背角神经元氯离子稳态失衡并降低CP大鼠痛觉敏感性，提示激活的小胶质细胞可能通过调控脊髓KCC2表达及氯离子稳态介导CP痛觉过敏。进一步的研究发现，CP大鼠脊髓背角BDNF含量升高，而通过鞘内给予BDNF-TrkB 通路阻断剂TrkB-Fc可上调CP大鼠脊髓KCC2表达、逆转脊髓背角神经元氯离子稳态失衡并降低CP大鼠痛觉敏感性，提示BDNF-TrkB-KCC2通路在CP痛觉过敏中起重要作用。本课题应用分子生物学、电生理及行为学等多项技术从体外和体内不同角度探讨了氯离子稳态及相关调控机制在CP内脏高敏感中的可能作用机制，为临床干预提供新的方向及靶点。