Methanol extracts of Xanthium sibiricum roots inhibit inflammatory responses via the inhibition of nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) in murine macrophages

Xanthium sibiricum has been used as a traditional Chinese medicine for the treatment of appendicitis, bronchitis, arthritis, and other inflammatory ailments. However, its pharmacological activity related to an anti-inflammatory effect remain unknown. This present study aims to investigate the anti-inflammatory effect of methanol extracts of X. sibiricum roots (MXS), and to further determine its underlying mechanism of action in order to assess the medicinal value of X. sibiricum roots. To assess the anti-inflammatory activity of MXS in lipopolysaccharides (LPS)‑stimulated RAW 264.7 macrophages, the production of nitric oxide (NO) was measured using the Griess reagent system. The levels of pro-inflammatory cytokines and mediators were quantified using an Enzyme-linked immunosorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR). Subsequently, immunoblotting analyses were employed to detect inflammatory mediators as well as to elucidate the underlying regulatory mechanisms suppressed by MXS. MXS inhibited LPS-stimulated NO production and inducible nitric oxide synthase (iNOS) expression in RAW 264.7 macrophages within the non-cytotoxic concentration range (50–400μg/ml). In addition, mRNA and protein levels of pro-inflammatory cytokines such as interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were significantly suppressed by MXS at the concentration of 400μg/ml. Furthermore, MXS (200μg/ml) clearly reduced the phosphorylation levels of the inhibitor of kappa Bα (IκBα) and signal transducer and activator of transcription 3 (STAT3), without affecting changes in the phosphorylation levels of mitogen-activated protein kinases (MAPKs). When five major components (betulin, betulinic acid, β-sitosterol, stigmasterol, and scopoletin) of MXS were separately investigated, stigmasterol and β-sitosterol seemed to play major inhibitory roles in the LPS-induced production of inflammatory mediators such as NO, IL-6, and TNF-α. Our results demonstrate that MXS has an anti-inflammatory property in LPS-stimulated RAW 264.7 macrophages, and its anti-inflammatory activity is exerted by the regulation of nuclear factor-κB (NF-κB) and STAT3 signaling pathways. [Display omitted]