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.
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Funding
the National Key Research and Development Program of China(2023YFD1301000)
the Open Project of the Key Laboratory of Utilization of Livestock and Forage Resources around the Tarim Basin, Ministry of Agriculture and Rural Affairs(BSGJSYS202401)
the Basic Scientific Research Project of Yazhou Bay National Laboratory(2310GL01)
the National Dairy Technology Innovation Center Project (2022-KYGG-3)
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