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
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