大豆异黄酮通过抑制NLRP3炎症小体介导的神经元焦亡改善脑缺血再灌注损伤

Amelioration of cerebral ischemia-reperfusion injury by soy isoflavones via inhibiting NLRP3 inflammasome-mediated neuronal pyroptosis

摘要

目的：探究大豆异黄酮（soy isoflavones，SI）抑制NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor thermal protein domain associated protein 3，NLRP3）炎症小体介导的神经元焦亡对脑缺血再灌注损伤（cerebral ischemia-reperfusion injury，CIRI）的神经保护作用及其机制。方法：将60只健康SD大鼠随机分为假手术组、模型组、SI组和SI+尼日利亚菌素（Nigericin）组。SI组连续灌胃21 d，1次/d（120 mg/kg）。采用线栓法建立大鼠大脑中动脉缺血/再灌注（middle cerebral artery occlusion/reperfusion，MCAO/R）模型，缺血2 h后拔除线栓进行再灌注；假手术组仅分离血管，不插入线栓。再灌注前5 min，SI+Nigericin组经尾静脉注射Nigericin（1 mg/kg）。再灌注24 h后，通过神经功能缺损评分、干湿重法测定脑组织含水量、2,3,5-三苯基氯化四氮唑（2,3,5-triphenyltetrazolium chloride，TTC）染色及苏木精-伊红（hematoxylin-eosin，HE）染色法评估脑损伤程度。采用免疫荧光和western blotting法检测焦亡相关蛋白表达，酶联免疫吸附试验（enzyme-linked immunosorbent assay，ELISA）法检测血清白细胞介素（interleukin，IL）-1β、IL-18含量及乳酸脱氢酶（lactate dehydrogenase，LDH）活性。结果：与模型组相比，SI组大鼠神经功能缺损评分、脑组织含水量及脑梗死面积均显著降低（P＜0.001），皮层病理学损伤明显减轻。SI组NLRP3、消皮素D（Gasdermin D，GSDMD）、GSDMD N端结构域（GSDMD N-terminal domain，GSDMD-N）、caspase-1、cleaved caspase-1、IL-18、IL-1β及IL-1β p17蛋白表达下调，血清IL-1β、IL-18含量及LDH活性降低（均 P＜0.05），脑组织中NLRP3和GSDMD-N荧光信号明显减弱。Nigericin可部分逆转SI的上述保护效应（P＜0.05）。结论：SI通过抑制NLRP3炎症小体活化减少神经元焦亡，进而有效缓解CIRI，实现神经保护的生物学效应。

Abstract

Objective: To explore the neuroprotective effect and mechanism of soy isoflavones (SI) on cerebral ischemia-reperfusion injury (CIRI) by inhibiting NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome-mediated neuronal pyroptosis. Methods: Sixty healthy Sprague-Dawley (SD) rats were randomly divided into sham group, model group, SI group and SI+Nigericin group. Rats in the SI group were pretreated with intragastric administration of SI (120 mg/kg) once daily for 21 consecutive days. The middle cerebral artery occlusion (MCAO) rat model was established by the suture-occluded method, and reperfusion was induced by withdrawing the suture after 2 hours of ischemia; the sham group only underwent vascular separation without suture insertion. Five minutes before reperfusion, rats in the SI+Nigericin group were injected with Nigericin (1 mg/kg) via the tail vein. Twenty-four hours after reperfusion, the severity of brain injury was assessed by neurological deficit score, brain water content (dry-wet weight method), 2,3,5-triphenyltetrazolium chloride (TTC) staining, and hematoxylin-eosin (HE) staining. The expression of pyroptosis-related proteins was measured by immunofluorescence staining and western blotting. The serum levels of interleukin (IL)-1β, IL-18 and the activity of lactate dehydrogenase (LDH) were measured by enzyme-linked immunosorbent assay (ELISA). Results: Compared with the model group, the SI group exhibited significantly reduced neurological deficit scores, brain water content, cerebral infarct volume (P＜0.001), as well as obviously alleviated cortical pathological injury. In the SI group, the protein expression levels of NLRP3, Gasdermin D (GSDMD), GSDMD N-terminal domain (GSDMD-N), caspase-1, cleaved caspase-1, IL-18, IL-1β and IL-1β p17 were downregulated; serum IL-1β and IL-18 contents as well as LDH activity were reduced (all P＜0.05), and the fluorescence signals of NLRP3 and GSDMD-N in brain tissues were obviously weakened. Nigericin could partially reverse the above protective effects of SI (P＜0.05). Conclusion: SI reduces neuronal pyroptosis by inhibiting NLRP3 inflammasome activation, thereby effectively alleviating CIRI and exerting neuroprotective effects.

关键词

大豆异黄酮 / 脑缺血再灌注损伤 / 缺血性脑卒中 / 炎症 / 小体 / NOD样受体热蛋白结构域相关蛋白3 / 细胞焦亡 / GSDMD N端结构域 / 神经保护

Key words

soy isoflavones / cerebral ischemia-reperfusion injury / inflammasome / NOD-like receptor thermal protein domain associated protein 3 / pyroptosis / GSDMD N-terminal domain / neuroprotection

参考文献

[1] Mao Z G, Tian L Y, Liu J, et al. Ligustilide ameliorates hippocampal neuronal injury after cerebral-ischemia reperfusion through activating PINK1/Parkin-dependent mitophagy[J]. Phytomedicine, 2022, 101: 154111.

[2] Zeng X, Zhang Y D, Ma R Y, et al. Activated Drp1 regulates p62-mediated autophagic flux and aggravates inflammation in cerebral ischemia-reperfusion via the ROS-RIP1/RIP3-exosome axis[J]. Military Medical Research, 2022, 9(1): 25.

[3] Wan J C M, White J R, Diaz L A Jr. "Hey CIRI, what's my prognosis?"[J]. Cell, 2019, 178(3): 518-520.

[4] Shi S Y, Zhang C Y, Liu J X . TIMP2 facilitates CIRI through activating NLRP3-mediated pyroptosis[J]. Aging, 2023, 15(9): 3635-3643.

[5] Zhang Y G, Ye P, Zhu H, et al. Neutral polysaccharide from Gastrodia elata alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis-mediated neuroinflammation via the NRF2/HO-1 signaling pathway[J]. CNS Neuroscience & Therapeutics, 2024, 30(3): e14456.

[6] Duan W L, Wang X J, Ma Y P, et al. Therapeutic strategies targeting the NLRP3-mediated inflammatory response and pyroptosis in cerebral ischemia/reperfusion injury (review)[J]. Molecular Medicine Reports, 2024, 29(3): 46.

[7] Liu Y, Jiang X, Zhang Y F, et al. miR-135a-5p alleviates cerebral ischemia-reperfusion injury by inhibiting pyroptosis mediated through the DDX3X/NLRP3 pathway[J]. Experimental Neurology, 2025, 385: 115127.

[8] Cheng Z F, Shao W, Wei C Q, et al. Cascade-type microglial pyroptosis inhibitors for enhanced treatment of cerebral ischemia-reperfusion injury[J]. ACS Nano, 2025, 19(10): 10529-10548.

[9] 徐建国, 谷荣辉, 李晒, 等. 大豆异黄酮对大鼠脑缺血再灌注损伤的保护作用[J]. 中国医药科学, 2023, 13(9): 30-33, 50.

[10] Xu J G, Gu R H, Li S, et al. Protective effect of soy isoflavones on rats after cerebral ischemia-reperfusion injury[J]. China Medicine and Pharmacy, 2023, 13(9): 30-33, 50.

[11] St Clair R, Anthony M . Atherosclerosis: Diet and Drugs[M]. Berlin, Heidelberg: Springer, 2005: 301-323.

[12] Gil-Izquierdo A, Penalvo J L, Gil J I, et al. Soy isoflavones and cardiovascular disease epidemiological, clinical and-omics perspectives[J]. Current Pharmaceutical Biotechnology, 2012, 13(5): 624-631.

[13] 李丽, 汪梦哲, 刘赛赛, 等. 大豆异黄酮通过抑制Wnt/Ca²⁺信号通路减轻大鼠脑缺血再灌注引起的钙超载 [J]. 南方医科大学学报, 2024, 44(6): 1048-1058.

[14] Li L, Wang M Z, Liu S S, et al. Soy isoflavones alleviates calcium overload in rats with cerebral ischemia-reperfusion by inhibiting the Wnt/Ca²⁺ signaling pathway [J]. Journal of Southern Medical University, 2024, 44(6): 1048-1058.

[15] 赵士弟, 陈耀, 董银凤, 等. 大豆异黄酮对全脑缺血/再灌注大鼠海马神经细胞凋亡的影响[J]. 上海交通大学学报(医学版), 2015, 35(4): 521-529.

[16] Zhao S D, Chen Y, Dong Y F, et al. Effects of soybean isoflavones on apoptosis of rat hippocampal neurons after global cerebral ischemia/reperfusion[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2015, 35(4): 521-529.

[17] Xue H M, Feng Z, Jin C, et al. Soy isoflavones protects against stroke by inhibiting Keap1/NQO1/Nrf2/HO-1 signaling pathway: network pharmacology analysis combined with the experimental validation[J]. Pharmaceuticals, 2025, 18(4): 548.

[18] Sharif H, Wang L, Wang W L, et al. Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome[J]. Nature, 2019, 570(7761): 338-343.

[19] 卢小叶, 吕倩忆, 李棋龙, 等. Zea-longa评分与改良Garcia评分应用于针刺治疗CIRI大鼠神经功能缺损评估的研究[J]. 湖南中医药大学学报, 2021, 41(9): 1356-1360.

[20] Lu X Y, Lü Q Y, Li Q L, et al. Evaluation research of Zea-longa score and modified Garcia score applied to acupuncture treatment of neurological deficits in CIRI rats[J]. Journal of Traditional Chinese Medicine University of Hunan, 2021, 41(9): 1356-1360.

[21] Liu X Y, Xie C M, Wang Y, et al. Ferritinophagy and ferroptosis in cerebral ischemia reperfusion injury[J]. Neurochemical Research, 2024, 49(8): 1965-1979.

[22] Wang Z, Li Y N, Ye Y Z, et al. NLRP3 inflammasome deficiency attenuates cerebral ischemia-reperfusion injury by inhibiting ferroptosis[J]. Brain Research Bulletin, 2023, 193: 37-46.

[23] 杨溢伟, 孙晓伟. 治疗脑缺血/再灌注损伤中NLRP3炎性小体介导的细胞焦亡的研究进展[J]. 基础医学与临床, 2026, 46(3): 435-439.

[24] Yang Y W, Sun X W. Research progress in the treatment of NLRP3 inflammasome-mediated pyroptosis in cerebral ischemia-reperfusion injury[J]. Basic & Clinical Medicine, 2026, 46(3): 435-439.

[25] Wei C, Jiang W, Wang R Y, et al. Brain endothelial GSDMD activation mediates inflammatory BBB breakdown[J]. Nature, 2024, 629(8013): 893-900.

[26] Shi M, Chen J, Liu T X, et al. Protective effects of remimazolam on cerebral ischemia/reperfusion injury in rats by inhibiting of NLRP3 inflammasome-dependent pyroptosis[J]. Drug Design, Development and Therapy, 2022, 16: 413-423.

[27] 陈露露, 苏凯奇, 陈渤霏, 等. 电针通过调控海马神经元线粒体分裂/融合改善大鼠脑缺血再灌注后的学习记忆障碍[J]. 针刺研究, 2026, 51(1): 11-20.

[28] Chen L L, Su K Q, Chen B F, et al. Electroacupuncture improves learning-memory ability following cerebral ischemia-reperfusion in rats by regulating mitochondrial fission/fusion of hippocampal neurons[J]. Acupuncture Research, 2026, 51(1): 11-20.

[29] Mayo B, Vázquez L, Flórez A B . Equol: a bacterial metabolite from the daidzein isoflavone and its presumed beneficial health effects[J]. Nutrients, 2019, 11(9): 2231.

[30] Song S H, Jun S, Joung H, et al. Dietary intakes of soy and isoflavones and risk of mortality in a population with high soy consumption[J]. The Journal of Nutrition, 2025, 155(10): 3517-3525.

[31] 薛靖, 李军涛, 贾林伟. 大豆异黄酮对大鼠局灶性脑缺血再灌注损伤的保护作用及其机制研究[J]. 中药药理与临床, 2015, 31(4): 110-114.

[32] Xue J, Li J T, Jia L W. The protective effects and mechanism of soybean isoflavone on focal cerebral ischemia-reperfusion injury in rats[J]. Pharmacology and Clinics of Chinese Materia Medica, 2015, 31(4): 110-114.

[33] Li J J, Xu P F, Hong Y, et al. Lipocalin-2-mediated astrocyte pyroptosis promotes neuroinflammatory injury via NLRP3 inflammasome activation in cerebral ischemia/reperfusion injury[J]. Journal of Neuroinflammation, 2023, 20(1): 148.

[34] 曹雪芬. 川芎嗪抑制NLRP3炎性小体活化减轻肠缺血再灌注损伤细胞焦亡的研究[D]. 兰州: 兰州大学, 2024.

[35] Cao X F. Tetramethylpyrazine protected against intestinal ischemia-reperfusion injury induced pyroptosis by inhibiting NLRP3 inflammasome activation[D]. Lanzhou: Lanzhou University, 2024.

基金资助

国家自然科学基金资助项目(82060226)