Yu Yunling, Liao Xinglan, Xing Kehui, Xie Ziyu, Xie Ningyuan, He Yinwen, Huang Zhihua, Tang Xiaolu, Liu Ruizhen
Key Laboratory of Prevention and treatment of cardiovascular and cerebrovascular diseases of Ministry of Education, Gannan Medical University, Ganzhou, 341000, China.
Department of Clinical Laboratory, Chinese Medicine Hospital, Tongnan District, Chongqing, 400000, China.
Metab Brain Dis. 2025 Jan 14;40(1):99. doi: 10.1007/s11011-025-01530-z.
Cerebral ischemia-induced pyroptosis contributes to the dissemination of neuroinflammation, and Nod-like receptor protein-3 (NLRP3) inflammasome plays a key role in this process. Previous studies have indicated that Genistein-3'-sodiumsulfonate (GSS) can inhibit neuroinflammation caused by cerebral ischemia, exert cerebroprotective effects, but its specific mechanism has not been comprehensively understood. The aim of this study was to explore the effect of GSS on ischemic stroke-induced cell pyroptosis. SD rats were randomly assigned to Sham group, transient middle cerebral artery occlusion (tMCAO) group, and tMCAO + GSS group. The open field test (OFT) was utilized to assess animals' spontaneous movement and anxiety-like behavior. Immunofluorescence was adopted to observe nod-like receptor pyrin domain containing 3 (NLRP3)/neuronal nuclei (NeuN) double-positive cells in the ischemic penumbra of each group. Western blot (WB) was conducted to detect levels of NLRP3 inflammasomes and pyroptosis-related proteins in the ischemic cortex tissue. Furthermore, the G protein-coupled estrogen receptor 1 (GPER1) inhibitor G15 was administered to monitor tMCAO rats' motor function, emotional state, and NLRP3 inflammasome activation. Compared with the Sham group, rats in the tMCAO group exhibited significant motor dysfunction and anxiety, increased NLRP3/NeuN co-expressing cells in the ischemic penumbra, and elevated levels of NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), pro-cysteinyl aspartate specific proteinase-1 (pro-caspase-1), cleaved-cysteinyl aspartate specific proteinase-1 (cleaved-caspase-1), gasdermin D (GSDMD), GSDMD-N-terminal domain (GSDMD-N), interleukin (IL)-1β, and IL-18 in the ischemic cortex. Treatment with GSS reversed these trends. Additionally, post G15 treatment, the therapeutic effects of GSS were reversed. GSS may inhibit NLRP3 inflammasome activation via GPER1, reducing membrane perforation and pro-inflammatory cytokine secretion, suppressing cell pyroptosis, and mitigating neuroinflammation, thereby improving chronic motor dysfunction and anxiety in tMCAO rats. Our study uncovers a potential novel mechanism for GSS treatment in ischemic stroke and provides new ideas for the treatment of ischemic stroke.
脑缺血诱导的细胞焦亡促进神经炎症的扩散，而NOD样受体蛋白3（NLRP3）炎性小体在这一过程中起关键作用。先前的研究表明，染料木黄酮-3'-磺酸钠（GSS）可抑制脑缺血引起的神经炎症，发挥脑保护作用，但其具体机制尚未完全明确。本研究旨在探讨GSS对缺血性脑卒中诱导的细胞焦亡的影响。将SD大鼠随机分为假手术组、短暂大脑中动脉闭塞（tMCAO）组和tMCAO+GSS组。采用旷场试验（OFT）评估动物的自发活动和焦虑样行为。采用免疫荧光法观察各组缺血半暗带中含NOD样受体吡咯结构域3（NLRP3）/神经元细胞核（NeuN）双阳性细胞。采用蛋白质免疫印迹法（WB）检测缺血皮质组织中NLRP3炎性小体和细胞焦亡相关蛋白的水平。此外，给予G蛋白偶联雌激素受体1（GPER1）抑制剂G15，以监测tMCAO大鼠的运动功能、情绪状态和NLRP3炎性小体激活情况。与假手术组相比，tMCAO组大鼠表现出明显的运动功能障碍和焦虑，缺血半暗带中NLRP3/NeuN共表达细胞增加，缺血皮质中NLRP3、含半胱天冬酶募集结构域的凋亡相关斑点样蛋白（ASC）、前半胱天冬酶-1（pro-caspase-1）、裂解的半胱天冬酶-1（cleaved-caspase-1）、gasdermin D（GSDMD）、GSDMD-N端结构域（GSDMD-N）、白细胞介素（IL）-1β和IL-18水平升高。GSS治疗可逆转这些趋势。此外，给予G15后，GSS的治疗效果被逆转。GSS可能通过GPER1抑制NLRP3炎性小体激活，减少膜穿孔和促炎细胞因子分泌，抑制细胞焦亡，减轻神经炎症，从而改善tMCAO大鼠的慢性运动功能障碍和焦虑。我们的研究揭示了GSS治疗缺血性脑卒中的潜在新机制，为缺血性脑卒中的治疗提供了新思路。
