Salidroside alleviates LPS-induced inflammatory injury in MAC-T cells by promoting mitophagy and improving mitochondrial function via the AMPK/mTOR pathway

Mastitis severely impairs dairy production, and lipopolysaccharide (LPS), a major endotoxin, induces mitochondrial dysfunction and inflammatory injury in bovine mammary epithelial cells (BMECs). Mitophagy, which is regulated by the AMPK/mTOR axis, removes damaged mitochondria to restrict excessive inflammation. Although salidroside (SAL) has anti-inflammatory activity, whether it protects against LPS-triggered inflammatory injury in mammary epithelial cells via this pathway remains unclear. Here, we investigated the protective effects of SAL on LPS-stimulated MAC-T cells. Pretreatment with 200 μM SAL markedly reduced the secretion of proinflammatory cytokines (TNF-α, IL-6, IL-1β, and IL-18) induced by 5 μg/mL LPS. Mechanistically, SAL maintained mitochondrial function by restoring ATP levels, restoring mitochondrial membrane potential, and inhibiting abnormal mPTP opening. Moreover, SAL increased the LC3B-II/I ratio and Beclin-1 expression and promoted p62 degradation. Tandem mRFP-GFP-LC3 assays verified that SAL restored functional autophagic flux; moreover, SAL upregulated PINK1 and Parkin expression, indicating mitophagy activation. The results of inhibition experiments demonstrated that 3-methyladenine or cyclosporin A eliminated the protective effects of SAL. The inhibition of AMPK by Compound C blocked SAL-induced mitophagy and reversed its beneficial effects on mitochondrial homeostasis and inflammation. In summary, SAL protected MAC-T cells against LPS-induced inflammatory injury in an AMPK/mTOR-dependent manner by driving PINK1/Parkin-mediated mitophagy to eliminate damaged mitochondria and maintain mitochondrial homeostasis.