BACKGROUND: Acute ischemic stroke (AIS) is a leading global cause of disability and mortality, with limited therapeutic options beyond reperfusion strategies. Evidence suggests that mitophagy and astrocyte polarization play critical roles in neuronal survival and inflammatory regulation after stroke. Xuming tongmai (XMTM) decoction has shown clinical potential in improving AIS outcomes, but the underlying mechanisms remain unclear. This study aimed to investigate the molecular mechanisms of XMTM in the middle cerebral artery occlusion (MCAO) model rats, with a specific focus on its role in regulating astrocyte polarization and mitophagy.
METHODS: In vivo, the MCAO model rats were established and administered 12.90 g/kg and 25.80 g/kg XMTM decoction, with 20 mg/kg aspirin serving as the positive control. In vitro, an oxygen-glucose deprivation/reperfusion (OGD/R) model was induced in astrocytes. Serum containing XMTM (XMTM serum) was prepared from rats administered XMTM decoction. The optimal working concentration of XMTM serum was determined using the CCK-8 assay before cell treatment. OGD/R-induced astrocytes were treated with negative serum, XMTM serum, Mdivi-1 (a mitophagy inhibitor), si-NC, si-PINK1, or their combination. The therapeutic effects of XMTM on MCAO rats were evaluated through pathological analysis, cerebral infarct volume measurement, and neurological deficit scoring. Astrocyte polarization was assessed using A1-type markers (C3, iNOS) and A2-type markers (Arg1, S100A10), while XMTM serum components were characterized by LC-MS/MS. Mitophagy activity was determined by measuring the mitochondrial membrane potential (MMP) and quantifying the expression levels of PINK1, Parkin, LC3II/I, p62, and reactive oxygen species (ROS).
RESULTS: 25.80 g/kg XMTM decoction significantly reduced cerebral infarct volume, ameliorated neurological deficits, and improved pathological outcomes in AIS rats. LC-MS/MS results revealed that the main components of XMTM in the blood were alkaloids, anthraquinones, flavonoids, and phenolic acids. Both in vivo and vitro experiments demonstrated that XMTM upregulated Arg1 and S100A10 expression but downregulated C3 and iNOS, activated the PINK1/Parkin signaling pathway, and induced mitophagy in astrocytes. However, these beneficial effects of XMTM serum were abolished by si-PINK1 and mdivi-1.
CONCLUSION: XMTM decoction exerts its neuroprotective effects in AIS rats by targeting the PINK1/Parkin-mediated mitophagy pathway, which subsequently promotes the polarization of astrocytes toward the neuroprotective A2 phenotype.
