Astragaloside IV attenuates post-ischemic neuroinflammation associated with Treg-related regulation of CD86/CD206-associated brain myeloid phenotypic responses and PI3K/AKT signaling alterations.

BACKGROUND: Ischemic stroke triggers persistent neuroinflammation, microglial activation, and peripheral immune changes that aggravate secondary brain injury. Astragaloside IV (AS-IV), a major bioactive constituent of Astragalus membranaceus, has neuroprotective and immunomodulatory effects, but its influence on Tregs and CD86/CD206-associated microglial phenotypic responses remains unclear.

AIM: This study investigated whether AS-IV attenuates post-ischemic neuroinflammation in association with Treg-related immune responses, PI3K/AKT signaling alterations, and CD86/CD206-associated brain myeloid phenotypic responses.

MATERIALS AND METHODS: A mouse middle cerebral artery occlusion (MCAO) model was established using the intraluminal filament method. Neurological deficits, infarct volume, and histopathological injury were evaluated after AS-IV treatment. Western blotting and ELISA were used to assess inflammatory cytokines and PI3K/AKT-associated signaling in ischemic brain tissue. Transcriptomic analysis identified AS-IV-related biological processes and pathways. Flow cytometry quantified CD4+CD25+FOXP3+ regulatory populations and additionally analyzed CD4+FOXP3+ populations in the spleen and cervical lymph nodes, together with CD86+/CD206+ phenotypic responses within the CD11b+CD45^low/int population in brain tissue. Immunofluorescence evaluated brain CD3+FOXP3+ signals and Iba1/CD86 or Iba1/CD206 double-positive signals. Anti-CD25 intervention was used to evaluate the involvement of Treg-related immune responses in AS-IV-associated effects.

RESULTS: AS-IV reduced infarct volume, showed a trend toward improvement in neurological deficit scores, and alleviated histopathological injury after MCAO. It decreased IL-6 and TNF-α and increased IL-10 and TGF-β in ischemic brain tissue. Transcriptomic analysis identified changes associated with the PI3K/AKT signaling pathway and immune regulatory processes, while western blotting showed increased p-PI3K/PI3K and p-AKT/AKT ratios after AS-IV treatment. AS-IV increased Treg proportions in the spleen and cervical lymph nodes and increased CD3+FOXP3+ signals in brain tissue. It also reduced CD86+-associated and increased CD206+-associated brain myeloid phenotypic responses. Anti-CD25 partially attenuated AS-IV-associated improvements in neurological and brain injury outcomes, as well as its effects on inflammatory cytokines and brain myeloid phenotypic responses.

CONCLUSION: AS-IV attenuated post-ischemic neuroinflammation in association with Treg-related immune responses and PI3K/AKT-associated signaling alterations, accompanied by changes in CD86/CD206-associated brain myeloid phenotypic responses. These findings support AS-IV as a potential neuroimmunomodulatory intervention for ischemic stroke.