Paeoniflorin Alleviates inflammatory injury in mastitis by targeting AKT1-Mediated mitochondrial damage and epithelial cell apoptosis.

Abstract

Peony can clear heat, promote blood circulation, and relieve pain, and peony-containing formulas exert favorable therapeutic effects on mastitis. Paeoniflorin is the primary bioactive component of peony. This study aimed to investigate the therapeutic targets and underlying mechanism of paeoniflorin (Pae) against mastitis, leveraging network pharmacology to guide experimental validation. A mouse mastitis model was established using lipopolysaccharide (LPS). Three concentrations of Pae were administered to evaluate its dose-dependent therapeutic effects, with dexamethasone (DXMS) serving as the positive control drug. Pae treatment significantly ameliorated mastitis in mice, reduced the expression of inflammatory cytokines, and alleviated cellular injury. Network pharmacology analysis revealed that AKT serine/threonine kinase 1 (AKT1) signaling was the most significantly enriched pathway, strongly correlated with both Pae and mastitis. Molecular dynamics simulation and CO-IP experiments confirmed the direct binding between Pae and AKT1. In vitro, we used bovine mammary epithelial cells, in which LPS was applied to induce inflammatory injury, to further verify the protective effects of Pae against cellular damage. The results showed that Pae protected against LPS-induced cellular injury in an AKT1-dependent manner. Mechanistically, Pae suppresses AKT1 phosphorylation and promotes AKT1 protein degradation. Collectively, through an integrated approach of network pharmacology and experimental validation, we identified that the therapeutic mechanism of Pae against mastitis is mediated by direct targeting of AKT1. Pae can bind to AKT1 and subsequently inhibit AKT1-mediated mitochondrial damage and epithelial apoptosis.

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