Therapeutic Potential of Thiosemicarbazone Derivative in LPS‐Induced Mastitis: Dual Modulation of Inflammatory Response and Blood–Milk Barrier Integrity

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Mastitis is a prevalent inflammatory disorder in women, this condition exhibits a particularly elevated prevalence among breastfeeding women, which significantly compromises mammary health and lactation function. This research explored the anti-inflammatory properties of YWJ-6 in lipopolysaccharide (LPS)-induced mastitis, its protective role on the blood-milk barrier (BMB), and the underlying mechanisms. An LPS-induced mastitis rat model was established. H&E staining assessed mammary tissue damage, and myeloperoxidase (MPO) activity was measured. RT-qPCR and IHC were used to demonstrate interleukin 1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNFα) expression. These data indicated that inflammatory injury was alleviated. Following drug treatment, MPO expression and the mRNA/protein expression of these inflammatory factors were suppressed. Immunofluorescence staining analyzed the distribution of Zonula Occludens-1 (ZO-1), Occludin, and Claudin-3. WB quantitatively detected ZO-1 and Occludin expression, confirming that YWJ-6 promoted tight junction (TJ) protein expression restoration and distribution integrity, repairing the damaged BMB. Consistently, Evans blue extravasation assay demonstrated that YWJ-6 reduced mammary vascular permeability, providing direct functional evidence for BMB protection. mIHC detected the colocalization of CD68, MPO, and nuclear factor-kappa B (NF-κB) p65 subunit (NF-κB p65), showing that YWJ-6 modulated inflammatory cell infiltration and p65 activation. RT-qPCR, WB, and ELISA assays in HC11 and RAW264.7 cells confirmed that YWJ-6 downregulated inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and IL-1β, IL-6 and TNF-α expression in HC11 cells, and suppressed agent responsible for inflammation secretion in RAW264.7 cells. WB and immunofluorescence showed that YWJ-6 inhibited inhibitor of NF-κB alpha (IκBα) and NF-κB p65 phosphorylation in both HC11 and RAW264.7 cells. In conclusion, YWJ-6 exerts dual protective effects by targeting NF-κB signaling pathway activation, providing experimental evidence for mastitis treatment in future.