Calpain 上调 TNF-α 参与运动神经损伤后慢性痛诱导的作用及机制

Our previous work has shown that selective injury of motor fibers, but leaving sensory neurons intact by L5 ventral root transection (L5-VRT), which leads to neuropathic pain, up-regulates tumor necrosis factor-alpha (TNF-α) and TNF receptor 1 (TNFR1) in uninjured dorsal root ganglia (DRG) and that the increased TNF-α is responsible for the upregulation of voltage-gated sodium channels Nav1.3 and Nav1.8 in uninjured DRG neurons. However, what factors contribute to the upregulation of TNF-α in primary sensory afferents following motor fiber injury is still unknown. A large body of studies has demonstrated that the activation of nucleus factor-kappaB (NF-κB) is required for the development of neuropathic pain, which regulates the expression of TNF-α. Calpain is an ubiquitous calcium-sensitive protease, the activation of which may be an early event in a proteolytic cascade that is initiated by axonal injury and culminates with axonal degeneration. Several groups have shown that calpain can activate IκBα degradation and NF-κB translocation into the neucleus. In our preliminary experiment, administration of the recombinant rat m-calpain (rm-calpain) onto the surface of L5 spinal root induced the decrease of paw withdrawal threshold and up-regulates the expression of TNF-α protein in bilateral DRGs and spinal cord. Based on our present works, we will further investigate ① whether motor nerve injury by L5-VRT activates μ- and m-calpain in DRGs and spinal cord, and whether the intervention of calpain inhibits the induction of chronic pain? ② whether calpain is involved in the long-term potentiation (LTP) of C-fiber-evoked field potentials in spinal dorsal horn,and whether calpain affects the neuronal action potential threshold and spinal NMDA/AMPA receptor current and dendritic morphosis? ③ whether the upregulation of TNF-α after L5-VRT was triggered by calpain via IκBα/ NF-κB pathway? The present project aims to reveal the roles and mechanisms of calpain in neuropathic pain, and to provide a new target for clinical therapy.

我们曾报道切断腰5运动前根（L5-VRT）引起背根神经节内肿瘤坏死因子α（TNF-α）上调；后者通过促使钠通道过表达导致慢性痛产生。但L5-VRT上调TNF-α的机制尚不清楚。研究表明，核转录因子NF-κB参与TNF-α的基因调控；而钙依赖性蛋白酶calpain又可促使NF-κB活化入核。预实验显示腰5神经根局部敷予calpain诱导痛阈下降和TNF-α上调。我们推测,运动神经损伤激活的calpain可能通过NF-κB上调TNF-α，诱导慢性痛产生。本项目拟进一步明确： Calpain在运动神经损伤后慢性痛诱导和维持中的作用？ Calpain是否通过促使感觉传入异常兴奋、增强脊髓背角突触传递效率参与痛诱导？探讨L5-VRT 后由"Calpain→NF-κB→TNF-α→Calpain"环路介导的外周/中枢敏感化机制？本项目将揭示calpain在慢性痛中的作用及机制，为防治慢性痛提供新靶点。

脊髓和背根神经节（DRG）内肿瘤坏死因子α（TNF-α）、白介素6（IL-6）的上调在运动神经损伤（切断腰5运动前根，L5-VRT）引起的慢性痛中扮演重要角色。核转录因子NF-κB参与TNF-α、IL-6的基因调控，钙依赖性蛋白酶calpain促使NF-κB活化入核，可能是TNF-α、IL-6的上游调控分子。本项目研究结果表明L5-VRT后半小时脊髓背角、前角以及DRG内calpain酶活性增强、calpain-2表达增加，在时间上远远先于VRT诱导的机械性痛过敏的产生；双免疫荧光染色发现calpain-2的增强主要表达于神经元内，胶质细胞中无表达；L5-VRT诱导TNF-α、IL-6在双侧脊髓和DRG内表达增强，其时程与calpain的激活时程紧密相关。免疫荧光双染显示 L5-VRT后在脊髓背角和前角以及DRG内阳性表达的calpain-2神经元也同时表达TNF-α、IL-6；术前腹腔或鞘内给予calpain抑制剂MDL28170可显著缓解由VRT神经损伤诱导的大鼠机械性痛阈值的下降，并有效阻断TNF-α、IL-6在脊髓和DRG内的表达增加；通过鞘内给予calpain2 RNA干扰病毒（PAVT1389）选择性干预calpain2的基因表达，在阻断脊髓背角calpain2异常表达的同时也阻断了L5-VRT诱导的IL-6蛋白的异常分泌并缓解了大鼠机械性痛过敏的诱导；不损伤任何神经，腰5 DRG表面给予外源性calpain-2在诱导大鼠短时程痛过敏的同时，也直接促使脊髓和DRG内TNF-α、IL-6表达增加；此外，L5-VRT后半小时，双侧脊髓背角和脊髓前角都有Iba1标记的小胶质细胞蛋白表达增强，ED1标记的巨噬细胞信号也显著增强，但GFAP标记的星型胶质细胞蛋白几乎无改变。使用选择性的小胶质细胞抑制剂Mac-1-saporin，发现术前阻断小胶质细胞的激活可阻断运动神经损伤诱发的大鼠后肢慢性痛以及脊髓内IL-6的异常表达。这一结果充分证实了脊髓小胶质细胞激活也参与了细胞因子的异常表达。通过本项目的研究，我们已经对运动神经损伤诱导的慢性痛形成的早期机制有了较充分的认识，calpain-2可作为临床干预慢性神经病理性痛的早期治疗靶点。