Levistilide A ameliorates ischemic brain injury by suppressing the AIM2 inflammasome related pyroptosis

BackgroundNeuroinflammation is a major pathogenic factor in cerebrovascular diseases (CVDs). The AIM2 inflammasome contributes to the progression of CVDs by mediating inflammatory responses and related pyroptosis.PurposeTo clarify the therapeutic potential of levistilide A from Da Chuanxiong Formula (DCX) against CVDs and to elucidate its mechanism of action.MethodsThe neurocognitive protective effects of DCX and levistilide A were evaluated in rat models of chronic cerebral hypoperfusion (CCH) and middle cerebral artery occlusion/reperfusion (MCAO/R). Functional and structural outcomes were assessed using the Morris water maze, TTC staining, and histological and immunofluorescence analyses. DCX constituents and related compounds in serum and brain samples were characterized by UHPLC-Q-TOF-MS/MS and HPLC-QQQ-MS. Potential binding of levistilide A to AIM2 was assessed by molecular docking and validated by drug affinity responsive target stability (DARTS). In vitro neuronal and microglial injury models were used to examine AIM2-associated pyroptosis signaling, BDNF/TrkB, and PSD-95 by qRT-PCR and western blotting. Selective blockade of AIM2 with 4-sulfonic calixarene confirmed its functional involvement.ResultsDCX exhibited significant protective effects against cerebral ischemia, which were partly attributable to its anti-inflammatory properties. From the main components of DCX, we found that levistilide A exhibited superior affinity toward AIM2 (binding energy: -8.09 kcal/mol). DARTS assays demonstrated that levistilide A protected AIM2 from degradation by protease E. In this way, levistilide A markedly downregulated the expression of AIM2, ASC, cleaved caspase-1, GSDMD-N, mature IL-18, and mature IL-1β in OGD/R-challenged PC12 cells and glutamate-exposed HT22 cells. In the presence of 4-sulfonic calixarene, levistilide A further potentiated the suppression of the AIM2 inflammasome and downstream pyroptosis. Concurrently, it enhanced the expression of BDNF, p-TrkB/TrkB, and PSD-95. In vivo experiments confirmed that levistilide A reduced cerebral infarct volume by inhibiting AIM2-mediated pyroptosis in the hippocampus.ConclusionLevistilide A is an active component of DCX with the strongest affinity for the AIM2 inflammasome. By regulating the AIM2/Caspase-1/GSDMD pyroptotic and BDNF/TrkB pathway, it offers a novel therapeutic strategy for CVDs.