Astragalin alleviates neuroinflammation in LPS-injured HT22 cells and CFA mice by regulating neuronal excitability and autophagy via CXCR4-Beclin1/VPS34 axis.

Abstract

This study demonstrates that astragalin (AST) alleviates neuroinflammation via the CXCR4-Beclin1/VPS34 axis. Bioinformatic and molecular docking analyses indicated its involvement in autophagy pathways and a potential direct CXCR4-Beclin1 interaction. In vitro, HT22 cells were exposed to lipopolysaccharide (LPS)-induced inflammation, then treated with AST. Cell viability was determined with the Cell Counting Kit-8 (CCK-8) assay, while protein levels were analyzed through western blot and immunofluorescence. The involvement of key pathways was verified using the CXCR4 agonist NUCC-390 and the autophagy inhibitor chloroquine (CQ). For in vivo evaluation, an inflammatory pain model was induced in mice by injecting Complete Freund's Adjuvant (CFA) intraplantarly. Subsequently, AST was administered at 60 mg/kg daily for 14 days. L4-L6 spinal cord tissues were collected for analysis. The results indicated that AST significantly enhanced cell viability, reduced TNF-α expression, and restored morphological alterations in LPS-injured HT22 cells. Furthermore, AST inhibited neuronal excitability by downregulating c-Fos and promoting autophagic activity, as evidenced by elevated Beclin1 and LC3 levels along with reduced p62 expression. These effects were mediated through the CXCR4-Beclin1/VPS34 axis, as both NUCC-390 and CQ negated the protective actions of AST. In CFA mice, AST similarly downregulated TNF-α, c-Fos, and CXCR4 while upregulating Beclin1 and VPS34 in the spinal cord, corroborating its role in modulating neuronal excitability and autophagy in vivo. In conclusion, AST mitigates neuroinflammation in both LPS-injured HT22 cells and CFA mice by inhibiting neuronal hyperexcitability and promoting autophagy via the CXCR4-Beclin1/VPS34 axis, suggesting a promising therapeutic potential for inflammatory pain.