Tang Min, Li Xia, Liu Ping, Wang Jianwen, He Fangping, Zhu Xiongchao
Department of Neurology, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou, Zhejiang Province, 310003, China.
Curr Neurovasc Res. 2018;15(2):138-144. doi: 10.2174/1567202615666180528073141.
Kinins are pro-inflammatory peptides that mediate numerous vascular and pain responses in tissue injury. Kinins exert their biological functions via two G-protein-coupled receptors: Bradykinin 1 Receptor (B1R) and Bradykinin 2 Receptor (B2R). We previously demonstrated the up-regulation of B2R after Hypoxia/Reoxygenation (H/R) injury in primary cultured cortical neurons. However, the role of B2R in inflammasome-induced pyroptosis remains unknown.
We induced H/R neuronal injury in primary cultured cortical neurons harvested from embryonic day 17 brains. Next, we examined the neuroprotective function of B2R in H/R-induced neuronal apoptosis or necrosis using an annexin V FITC/Propidium Iodide (PI) double-staining technique. The pyroptosis signaling cascade, including caspase-1, IL-1β and IL-18 levels and Cleaved Gasdermin D (GSDMD) expression was examined by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting to explore the underlying molecular mechanism.
H/R injury significantly increased B2R protein expression (P<0.05) as well as the percentage of early apoptotic and necrotic or late apoptotic neurons as verified by the annexin V FITC/PI flow cytometric analysis. Bradykinin (BK), a specific B2R agonist, caused a significant decrease in apoptotic neuronal death after H/R injury, while HOE140, a specific B2R antagonist, markedly reduced the neuroprotective effect of B2R. Following H/R injury, BK downregulated the caspase-1, IL-1β and IL-18 levels (P<0.01). In contrast, pretreatment with HOE140 significantly increased caspase-1, IL-1β, and IL-18 levels (P<0.01). Further analysis revealed that GSDMD, a key executioner of pyroptosis, is a target for B2R-mediated inhibition of neuronal pyroptosis. Cleaved GSDMD expression was significantly inhibited by BK pretreatment and significantly enhanced by HOE140 pretreatment (P<0.01).
These results indicate that activation of B2R plays an important role in pyroptosis mediated by H/R injury.
激肽是促炎肽，在组织损伤中介导多种血管和疼痛反应。激肽通过两种G蛋白偶联受体发挥其生物学功能：缓激肽1受体（B1R）和缓激肽2受体（B2R）。我们之前证明了原代培养的皮质神经元在缺氧/复氧（H/R）损伤后B2R上调。然而，B2R在炎性小体诱导的细胞焦亡中的作用尚不清楚。
我们在从胚胎第17天大脑收获的原代培养皮质神经元中诱导H/R神经元损伤。接下来，我们使用膜联蛋白V FITC/碘化丙啶（PI）双染技术检测B2R在H/R诱导的神经元凋亡或坏死中的神经保护功能。通过实时定量逆转录聚合酶链反应（RT-qPCR）和蛋白质印迹法检测细胞焦亡信号级联反应，包括半胱天冬酶-1、白细胞介素-1β和白细胞介素-18水平以及裂解的Gasdermin D（GSDMD）表达，以探索潜在的分子机制。
H/R损伤显著增加B2R蛋白表达（P<0.05）以及早期凋亡和坏死或晚期凋亡神经元的百分比，膜联蛋白V FITC/PI流式细胞术分析证实了这一点。缓激肽（BK），一种特异性B2R激动剂，导致H/R损伤后凋亡性神经元死亡显著减少，而HOE140，一种特异性B2R拮抗剂，显著降低了B2R的神经保护作用。H/R损伤后，BK下调了半胱天冬酶-1、白细胞介素-1β和白细胞介素-18水平（P<0.01）。相反，用HOE140预处理显著增加了半胱天冬酶-1、白细胞介素-1β和白细胞介素-18水平（P<0.01）。进一步分析表明，GSDMD是细胞焦亡的关键执行者，是B2R介导的抑制神经元细胞焦亡的靶点。BK预处理显著抑制了裂解的GSDMD表达，而HOE140预处理显著增强了其表达（P<0.01）。
这些结果表明，B2R的激活在H/R损伤介导的细胞焦亡中起重要作用。
