Senegenin attenuates LPS-induced neuroinflammation and microglial cell death via Akt phosphorylation and suppression of cGAS-STING-NLRP3 signaling: Network pharmacology and experimental validation.

Senegenin is a major bioactive aglycone derived from Polygala tenuifolia Willd. (Polygalaceae), a traditional Chinese medicinal herb ("Yuan Zhi") widely used for the treatment of cognitive impairment and central nervous system disorders. Although senegenin has been reported to exert neuroactive and anti-inflammatory effects, the intracellular mechanisms underlying its regulation of microglia-driven neuroinflammation remain incompletely understood. This Study aimed to characterize the effects of senegenin on inflammatory responses in lipopolysaccharide (LPS)-stimulated microglia and to elucidate the molecular mechanisms underlying its anti-inflammatory activity, with particular emphasis on Akt and the cGAS-STING pathway. An integrative network pharmacology strategy was employed to predict senegenin-associated molecular targets and signaling pathways, followed by mechanistic validation in LPS-stimulated N9 microglial cells. Oxidative stress, mitochondrial function, DNA damage, inflammasome activation, and inflammatory signaling were assessed using flow cytometry, immunofluorescence, immunoblotting, and biochemical assay. Network pharmacology analysis identified 110 overlapping targets, including CASP3, PIK3CA, and HIF1A, with significant enrichment in the PI3K-Akt and HIF-1 signaling pathways. Experimental validation demonstrated that senegenin markedly attenuated LPS-induced reactive oxygen species production, preserved mitochondrial membrane potential (Δψm), and reduced mitochondrial and nuclear DNA damage. These effects were associated with restoration of Akt phosphorylation and coordinated suppression of the cGAS-STING-TBK1-IRF3 signaling axis, as evidenced by reduced phosphorylation of STING, TBK1, and IRF3, along with diminished IFN-β expression. Senegenin further inhibited NF-κB/NLRP3 inflammasome priming and activation, shifted apoptotic signaling toward cell survival (decreased Bax and increased Bcl-2), and reduced the expression of pro-inflammatory mediators, including TNF-α, IL-6, IL-1β, COX-2, and iNOS. This Study demonstrates that senegenin attenuates LPS-induced microglial inflammatory activation by limiting oxidative stress and DNA damage, thereby suppressing cGAS-STING-dependent inflammatory signaling and associated cell death pathways, potentially through Akt phosphorylation. Although confined to a cellular model, these findings provide mechanistic insight into the ethnopharmacological basis of senegenin and establish a foundation for future in vivo investigations into its neuroprotective relevance in neurological disorders.