Department of Anesthesiology, Ningbo Medical Treatment Center Lihuili Hospital, China.
FEBS Open Bio. 2021 Oct;11(10):2858-2865. doi: 10.1002/2211-5463.13258. Epub 2021 Aug 24.
Neuropathic pain (NP) is a clinical symptom that accompanies many diseases. We investigated the effect of receptor-interacting protein kinase 3 (RIP3)-regulated necroptosis on NP and explored its relationship with microglia, in order to provide a theoretical basis for further research and provide new insights into the treatment of NP. In this study, the spared nerve injury (SNI) model was used along with intervention with necrostatin and the inhibitor of necroptosis necrostatin-1 (Nec-1). Pain behavior tests were performed 1 and 3 days before the nerve injury (or sham) operation, and on days 1, 3, 5, 7, 10, and 14 after the operation. The spinal cord tissues were collected for detection of RIP3 expression and distribution, changes in the number of microglia cells, activation of necroptosis, and the level of proinflammatory factors. Collected spinal cord tissues were analyzed using western blot, immunohistochemistry, immunofluorescence, immunoprecipitation assays, and ELISA, respectively. We found that, compared with the sham group, the expression of RIP3 protein in the spinal cord of rats in the SNI group increased from 3 to 14 days after surgery. Immunofluorescence staining showed that RIP3 was coexpressed with the microglia and the number of microglia increased significantly in the SNI model group. The results of immunoprecipitation assays suggested that a RIP3-mediated necroptosis pathway promotes NP. After treatment with Nec-1, the expression of RIP3 protein and the number of microglia were significantly reduced, and the expression levels of TNF-α, IL-1β, and IL-6 in spinal dorsal horns were significantly decreased. These results indicate that RIP3 promotes necroptosis to increase the occurrence of NP via microglia.
神经病理性疼痛（NP）是伴随许多疾病的一种临床症状。我们研究了受体相互作用蛋白激酶 3（RIP3）调控的坏死性凋亡对 NP 的影响，并探讨了其与小胶质细胞的关系，以期为进一步的研究提供理论依据，并为 NP 的治疗提供新的思路。在这项研究中，我们使用 spared nerve injury（SNI）模型，结合坏死性凋亡抑制剂 necrostatin 和 necrostatin-1（Nec-1）抑制剂进行干预。在神经损伤（或假手术）操作前 1 天和 3 天，以及手术后第 1 天、3 天、5 天、7 天、10 天和 14 天进行疼痛行为测试。收集脊髓组织，检测 RIP3 表达和分布、小胶质细胞数量变化、坏死性凋亡激活以及促炎因子水平。分别采用 Western blot、免疫组化、免疫荧光、免疫沉淀和 ELISA 分析收集的脊髓组织。我们发现，与假手术组相比，SNI 组大鼠脊髓中 RIP3 蛋白的表达在手术后 3 天至 14 天逐渐增加。免疫荧光染色显示，RIP3 与小胶质细胞共表达，且在 SNI 模型组中，小胶质细胞的数量显著增加。免疫沉淀实验结果提示，RIP3 介导的坏死性凋亡通路促进了 NP 的发生。用 Nec-1 处理后，RIP3 蛋白表达和小胶质细胞数量明显减少，脊髓背角 TNF-α、IL-1β 和 IL-6 的表达水平也明显降低。这些结果表明，RIP3 通过小胶质细胞促进坏死性凋亡，增加 NP 的发生。
