s Nuezhenide Exerts Anti-Inflammatory Activity through the NF-ΚB Pathway

Background: Nuezhenide (NZD), an iridoid glycoside isolated from Ilex pubescens Hook. & Arn. var. kwangsiensis Hand.-Mazz., used as a traditional Chinese medicine for clearing away heat and toxic materials, displays a variety of biological activities such as anti-tumor, antioxidant, and other life-protecting activities. However, a few studies involving anti-inflammatory activity and the mechanism of NZD have also been reported. In the present study, the anti-inflammatory and antioxidative effects of NZD are illustrated. Objective: This study aims to test the hypothesis that NZD suppresses LPS-induced inflammation by targeting the NF-ΚB pathway in RAW264.7 cells. Methods: LPS-stimulated RAW264.7 cells were employed to detect the effect of NZD on the release of cytokines by ELISA. Protein expression levels of related molecular markers were quantitated by western blot analysis. The levels of ROS, NO, and Ca²⁺ were detected by flow cytometry. The changes in mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were observed and verified by fluorescence microscopy. Using immunofluorescence assay, the translocation of NF-ΚB/p65 from the cytoplasm into the nucleus was determined by confocal microscopy. Results: NZD exhibited anti-inflammatory activity and reduced the release of inflammatory cytokines such as nitrite, TNF-α, and IL-6. NZD suppressed the expression of the phosphorylated proteins like IKKα/β, IΚBα, and p65. Besides, the flow cytometry results indicated that NZD inhibited the levels of ROS, NO, and Ca²⁺ in LPS-stimulated RAW264.7 cells. JC-1 assay data showed that NZD reversed LPS-induced MMP loss. Furthermore, NZD suppressed LPS-induced NF-B/p65 translocation from the cytoplasm into the nucleus. Conclusion: NZD exhibits anti-inflammatory effects through the NF-ΚB pathway on RAW264.7 cells.