Chinese herbal Jin-Ying-Tang attenuates the inflammatory response by inhibiting the activation of TLR4/MyD88/TRAF-6/NIK pathway at the mRNA level in LPS-stimulated mouse mammary epithelial cells

Abstract

Introduction: The effects of Jin-Ying-Tang (JYT) on Toll-like Receptor 4 (TLR4) signalling transduction of lipopolysaccharide (LPS)-stimulated mouse mammary epithelial cells (MECs) in vitro were examined. Material and Methods: The cytotoxicity of JYT (0.06-62.50 mg/mL) on mouse MECs was determined by MTT assay. The MECs were co-cultured with LPS in the presence or absence of JYT (39.10 μg/mL, 391 μg/mL, 3910 μg/mL). The concentrations of interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in the culture supernatants were detected by ELISA. The mRNA expression of TLR4 and downstream TLR4 signalling molecules such as myeloid differentiation factor 88 (MyD88), tumour necrosis factor receptor associated factor 6 (TRAF-6), inhibitor κB (IκB), and nuclear factor κB inducing kinase (NIK) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: The results showed that the IC50 of JYT on MECs was 12.25 mg/mL and JYT could significantly decrease the concentrations of IL-6 and TNF-α in LPS-stimulated MECs (P < 0.05). The mRNA expression of TLR4, MyD88, TRAF-6, IκB, and NIK was also significantly decreased when the LPS-stimulated MECs were cocultured at appropriate concentrations of JYT (P < 0.05, P < 0.01). Conclusion: These observations indicate a potential mechanism through which JYT attenuates the systemic inflammatory response to LPS-stimulated mouse mammary epithelial cells by inhibiting the activation of TLR4/MyD88/ TRAF-6/NIK pathway at the mRNA level.