Sinomenine hydrochloride ameliorates fatty acid–induced bovine mammary epithelial cells' oxidative stress and inflammation via enhancing autophagy activity

ABSTRACT: During the transition period, dairy cows often experience negative energy balance (NEB), leading to excessive mobilization of adipose tissue and elevated circulating nonesterified fatty acids (NEFA). These excess NEFA can be taken up by the mammary gland, triggering redox imbalances and pro-inflammatory cascades that compromise its function. In nonruminants, sinomenine hydrochloride (SINH) has been shown to modulate oxidative stress and inflammation across a range of pathological conditions. However, its effects on NEFA-induced injury in bovine mammary epithelial cells (BMEC) remain largely unexplored. Therefore, this study aimed to determine whether SINH can alleviate NEFA-induced oxidative stress and inflammation in BMEC and to investigate the underlying molecular mechanisms. Primary BMEC isolated from healthy cows were treated with various concentrations of SINH (0, 40, 80, 120, 200, or 400 μM) for 24 h or with 120 μM SINH for different durations (0, 6, 12, 24, 36, or 48 h) to assess dose- and time-dependent effects. For the injury model, cells were co-treated with 1.2 mM NEFA and different concentrations of SINH for 24 h. To investigate the role of autophagy, cells were pretreated with 5 mM 3-methyladenine (3-MA), an autophagy inhibitor, for 1 h before co-treatment with 120 μM SINH and 1.2 mM NEFA for an additional 24 h. The SINH treatment significantly attenuated NEFA-induced oxidative stress, as evidenced by increased activities of antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase, along with upregulated expression of antioxidant proteins such as heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. Concurrently, SINH reduced malondialdehyde content and intracellular reactive oxygen species levels. Inflammatory responses were also mitigated by SINH, as indicated by decreased protein expression of NLR family pyrin domain containing 3 (NLRP3), caspase-1, and phosphorylated nuclear factor kappa B (NF-κB), as well as downregulated mRNA expression of proinflammatory cytokines including IL6, IL1B, IL18, and tumor necrosis factor-α (TNFA). Notably, SINH also restored the mRNA expression of anti-inflammatory cytokine IL10. Importantly, SINH reversed the NEFA-induced inhibition of autophagy in BMEC. However, pharmacological blockade of autophagy with 3-MA abrogated the protective effects of SINH, as reflected by reduced antioxidant enzyme activities and re-elevated oxidative and inflammatory markers. In conclusion, SINH may serve as a promising therapeutic agent to alleviate oxidative stress and inflammation in the mammary gland by restoring autophagic activity during the transition period in dairy cows.