Department of Anatomy and Neurobiology, Institute of Epilepsy Research, College of Medicine, Hallym University, Kangwon-Do, Chuncheon, 24252, South Korea.
J Neuroinflammation. 2021 Jan 6;18(1):14. doi: 10.1186/s12974-020-02058-y.
P2X7 receptor (P2X7R) is an ATP-gated nonselective cationic channel playing important roles in a variety of physiological functions, including inflammation, and apoptotic or necrotic cell death. An extracellular domain has ten cysteine residues forming five intrasubunit disulfide bonds, which are needed for the P2X7R trafficking to the cell surface and the recognition of surface epitopes of apoptotic cells and bacteria. However, the underlying mechanisms of redox/S-nitrosylation of cysteine residues on P2X7R and its role in P2X7R-mediated post-status epilepticus (SE, a prolonged seizure activity) events remain to be answered.
Rats were given pilocarpine (380 mg/kg i.p.) to induce SE. Animals were intracerebroventricularly infused N-nitro-L-arginine methyl ester hydrochloride (L-NAME, a NOS inhibitor) 3 days before SE, or protein disulfide isomerase (PDI) siRNA 1 day after SE using an osmotic pump. Thereafter, we performed Western blot, co-immunoprecipitation, membrane fraction, measurement of S-nitrosylated (SNO)-thiol and total thiol, Fluoro-Jade B staining, immunohistochemistry, and TUNEL staining.
SE increased S-nitrosylation ratio of P2X7R and the PDI-P2X7R bindings, which were abolished by L-NAME and PDI knockdown. In addition, both L-NAME and PDI siRNA attenuated SE-induced microglial activation and astroglial apoptosis. L-NAME and PDI siRNA also ameliorated the increased P2X7R surface expression induced by SE.
These findings suggest that PDI-mediated redox/S-nitrosylation may facilitate the trafficking of P2X7R, which promotes microglial activation and astroglial apoptosis following SE. Therefore, our findings suggest that PDI-mediated regulations of dynamic redox status and S-nitrosylation of P2X7R may be a critical mechanism in the neuroinflammation and astroglial death following SE.
P2X7 受体（P2X7R）是一种 ATP 门控非选择性阳离子通道，在多种生理功能中发挥重要作用，包括炎症和细胞凋亡或坏死。细胞外结构域有十个半胱氨酸残基形成五个亚基内二硫键，这对于 P2X7R 向细胞表面转运以及识别凋亡细胞和细菌的表面表位是必要的。然而，P2X7R 上半胱氨酸残基的氧化还原/S-亚硝基化的潜在机制及其在 P2X7R 介导的 post-status epilepticus（SE，一种延长的癫痫发作活动）事件中的作用仍有待回答。
大鼠腹腔注射匹鲁卡品（380mg/kg）诱导 SE。动物在 SE 前 3 天通过脑室内输注 N-硝基-L-精氨酸甲酯盐酸盐（L-NAME，一种 NOS 抑制剂），或在 SE 后 1 天通过渗透泵输注蛋白二硫键异构酶（PDI）siRNA。此后，我们进行 Western blot、共免疫沉淀、膜部分、S-亚硝基化（SNO）-巯基和总巯基的测定、Fluoro-Jade B 染色、免疫组织化学和 TUNEL 染色。
SE 增加了 P2X7R 的 S-亚硝基化比率和 PDI-P2X7R 结合，这两种作用均被 L-NAME 和 PDI 敲低所消除。此外，L-NAME 和 PDI siRNA 均减轻了 SE 诱导的小胶质细胞激活和星形胶质细胞凋亡。L-NAME 和 PDI siRNA 也改善了 SE 诱导的 P2X7R 表面表达增加。
这些发现表明，PDI 介导的氧化还原/S-亚硝基化可能有助于 P2X7R 的转运，从而促进 SE 后小胶质细胞激活和星形胶质细胞凋亡。因此，我们的研究结果表明，PDI 介导的 P2X7R 动态氧化还原状态和 S-亚硝基化的调节可能是 SE 后神经炎症和星形胶质细胞死亡的关键机制。
