Table_1_Didymin Suppresses Microglia Pyroptosis and Neuroinflammation Through the Asc/Caspase-1/GSDMD Pathway Following Experimental Intracerebral Hemorrhage.doc

Neuroinflammation has been proven to exert an important effect on brain injury after intracerebral hemorrhage (ICH). Previous studies reported that Didymin possessed anti-inflammatory properties after acute hepatic injury, hyperglycemia-induced endothelial dysfunction, and death. However, the role of Didymin in microglial pyroptosis and neuroinflammation after ICH is unclear. The current study aimed to investigate the effect of Didymin on neuroinflammation mediated by microglial pyroptosis in mouse models of ICH and shed some light on the underlying mechanisms. In this study, we observed that Didymin treatment remarkably improved neurobehavioral performance and decreased BBB disruption and brain water content. Microglial activation and neutrophil infiltration in the peri-hematoma tissue after ICH were strikingly mitigated by Didymin as well. At the molecular level, administration of Didymin significantly unregulated the expression of Rkip and downregulated the expression of pyroptotic molecules and inflammatory cytokines such as Nlrp3 inflammasome, GSDMD, caspase-1, and mature IL-1β, TNF-α, and MPO after ICH. Besides, Didymin treatment decreased the number of Caspase-1-positive microglia and GSDMD-positive microglia after ICH. Inversely, Locostatin, an Rkip-specific inhibitor, significantly abolished the anti-pyroptosis and anti-neuroinflammation effects of Didymin. Moreover, Rkip binding with Asc could interrupt the activation and assembly of the inflammasome. Mechanistically, inhibition of Caspase-1 by VX-765 attenuated brain injury and suppressed microglial pyroptosis and neuroinflammation by downregulation of GSDMD, mature IL-1β, TNF-α, and MPO based on Locostatin-treated ICH. Taken together, Didymin alleviated microglial pyroptosis and neuroinflammation, at least in part through the Asc/Caspase-1/GSDMD pathway via upregulating Rkip expression after ICH. Therefore, Didymin may be a potential agent to attenuate neuroinflammation via its anti-pyroptosis effect after ICH.

研究证实，神经炎症（Neuroinflammation）对脑出血（Intracerebral Hemorrhage, ICH）后脑损伤具有重要调控作用。既往研究表明，狄迪明（Didymin）在急性肝损伤、高血糖诱导的内皮功能障碍及细胞死亡过程中均表现出明确的抗炎活性。然而，狄迪明在脑出血后小胶质细胞焦亡（microglial pyroptosis）与神经炎症中的具体作用仍不明确。本研究旨在探讨狄迪明对脑出血小鼠模型中小胶质细胞焦亡介导的神经炎症的影响，并揭示其潜在分子机制。本研究结果显示，狄迪明给药可显著改善小鼠神经行为学表现，降低血脑屏障（Blood-Brain Barrier, BBB）破坏程度与脑含水量。同时，狄迪明可显著减轻脑出血后血肿周围组织的小胶质细胞活化与中性粒细胞浸润。在分子水平上，狄迪明给药可显著上调Raf激酶抑制蛋白（Raf kinase inhibitory protein, Rkip）的表达，并下调焦亡相关分子及炎性细胞因子的表达，包括NLRP3炎性小体（Nlrp3 inflammasome）、GSDMD、半胱天冬酶-1（Caspase-1）以及成熟IL-1β、肿瘤坏死因子-α（TNF-α）和髓过氧化物酶（Myeloperoxidase, MPO）。此外，狄迪明处理可减少脑出血后半胱天冬酶-1阳性小胶质细胞与GSDMD阳性小胶质细胞的数量。与之相反，Rkip特异性抑制剂Locostatin可显著抵消狄迪明的抗焦亡与抗炎神经炎症作用。进一步研究发现，Rkip与ASC（凋亡相关斑点样蛋白）结合可阻断炎性小体的激活与组装。机制分析显示，在经Locostatin处理的脑出血模型中，使用VX-765抑制半胱天冬酶-1可通过下调GSDMD、成熟IL-1β、TNF-α及MPO的表达，减轻脑损伤并抑制小胶质细胞焦亡与神经炎症。综上，狄迪明可通过上调Rkip表达，至少部分经ASC/Caspase-1/GSDMD通路减轻小胶质细胞焦亡与神经炎症，进而改善脑出血后脑损伤。因此，狄迪明或可成为通过抗焦亡作用减轻脑出血后神经炎症的潜在治疗候选制剂。