Department of Anesthesiology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, P. R. China.
Department of Anesthesiology, Cardiovascular Institute, Nanning, Guangxi, P. R. China.
J Neurochem. 2019 Oct;151(2):166-184. doi: 10.1111/jnc.14827. Epub 2019 Sep 9.
The specific mechanisms underlying cyclin-dependent kinase 5 (Cdk5)-mediated neuropathic pain at the spinal cord level remain elusive. The aim of the present study was to explore the role of crosstalk between Cdk5/p35 and extracellular signal-regulated kinase 1/2 (ERK1/2) signalling in mediating spinal astrocyte activity via the PPARγ pathway in a rat model of chronic constriction injury (CCI). Here, we quantified pain behaviour after CCI; detected the localization of p35, Cdk5, phosphorylated ERK1/2 (pERK1/2), phosphorylated peroxisome proliferator-activated receptor γ (pPPARγ), neuronal nuclei (a neuronal marker), glial fibrillary acidic protein (GFAP, an activated astrocyte marker) and ionized calcium binding adaptor molecule 1 (a microglial marker) in the dorsal horn using immunofluorescence; measured the protein levels of Cdk5, p35, pERK1/2, pPPARγ and GFAP using western blot analysis; and gauged the enzyme activity of Cdk5/p35 kinase using a Cdk5/p35 kinase activity assay kit. Tumour necrosis factor-α, interleukin (IL)-1β and IL-6 levels were measured using enzyme-linked immunosorbent assay (ELISA). Ligation of the right sciatic nerve induced mechanical allodynia; thermal hyperalgesia; and the time-dependent upregulation of p35, pERK1/2 and GFAP and downregulation of pPPARγ. p35 colocalized with Cdk5, pERK1/2, pPPARγ, neurons and astrocytes but not microglia. Meanwhile, intrathecal injection of the Cdk5 inhibitor roscovitine, the mitogen-activated ERK kinase (MEK) inhibitor U0126 and the PPARγ agonist pioglitazone prevented or reversed behavioural allodynia, increased pPPARγ expression, inhibited astrocyte activation and alleviated proinflammatory cytokine (tumour necrosis factor-α, IL-1β, and IL-6) release from activated astrocytes. Furthermore, crosstalk between the Cdk5/p35 and ERK1/2 pathways was observed with CCI. Blockade of either Cdk5/p35 or ERK1/2 inhibited Cdk5 activity. These findings indicate that spinal crosstalk between the Cdk5/p35 and ERK1/2 pathways mediates astrocyte activity via the PPARγ pathway in CCI rats and that targeting this crosstalk could be an effective strategy to attenuate CCI and astrocyte-derived neuroinflammation.
在脊髓水平，细胞周期蛋白依赖性激酶 5（Cdk5）介导的神经病理性疼痛的具体机制仍不清楚。本研究旨在探讨 Cdk5/p35 与细胞外信号调节激酶 1/2（ERK1/2）信号通路之间的串扰在慢性缩窄性损伤（CCI）大鼠模型中通过过氧化物酶体增殖物激活受体 γ（PPARγ）通路介导脊髓星形胶质细胞活性中的作用。在这里，我们通过免疫荧光法检测 CCI 后疼痛行为；检测背角中 p35、Cdk5、磷酸化 ERK1/2（pERK1/2）、磷酸化过氧化物酶体增殖物激活受体 γ（pPPARγ）、神经元核（神经元标志物）、胶质纤维酸性蛋白（GFAP，激活的星形胶质细胞标志物）和钙结合蛋白 1（一种小胶质细胞标志物）的定位；使用 Western blot 分析测量 Cdk5、p35、pERK1/2、pPPARγ 和 GFAP 的蛋白水平；并使用 Cdk5/p35 激酶活性测定试剂盒测量 Cdk5/p35 激酶的酶活性。使用酶联免疫吸附测定（ELISA）测量肿瘤坏死因子-α、白细胞介素（IL）-1β 和 IL-6 的水平。结扎右侧坐骨神经诱导机械性痛觉过敏；热痛觉过敏；以及 p35、pERK1/2 和 GFAP 的时间依赖性上调和 pPPARγ 的下调。p35 与 Cdk5、pERK1/2、pPPARγ、神经元和星形胶质细胞共定位，但与小胶质细胞不共定位。同时，鞘内注射 Cdk5 抑制剂罗西维林、丝裂原激活的 ERK 激酶（MEK）抑制剂 U0126 和 PPARγ 激动剂吡格列酮可预防或逆转行为性痛觉过敏，增加 pPPARγ 表达，抑制星形胶质细胞激活，并减轻激活星形胶质细胞中促炎细胞因子（肿瘤坏死因子-α、IL-1β 和 IL-6）的释放。此外，CCI 中观察到 Cdk5/p35 和 ERK1/2 通路之间的串扰。阻断 Cdk5/p35 或 ERK1/2 均可抑制 Cdk5 活性。这些发现表明，CCI 大鼠脊髓中 Cdk5/p35 和 ERK1/2 通路之间的串扰通过 PPARγ 通路介导星形胶质细胞活性，靶向这种串扰可能是减轻 CCI 和星形胶质细胞源性神经炎症的有效策略。
