The anti-inflammatory activity by suppressing the TRAF6/MAPKs pathway of trishizukaol a from Sarcandra glabra

Sarcandra glabra (Thunb.) Makino (Chloranthaceae) is abundant and shows important clinical effects. Traditionally, S.glabra is used to treat diseases involving inflammation, such as bone fracture and joint swelling. Lindenane-type sesquiterpenoids and dimers are the major anti-inflammatory components in S. glabra. Trishizukaol A (TSA), is an abundant lindenane sesquiterpenoid trimer in S.glabra, but its anti-inflammatory activities and mechanisms are poorly understood. The study was undertaken to unveil the inhibition of inflammation and mechanism of TSA in RAW 264.7 macrophages stimulated by lipopolysaccharide (LPS). Griess reagent and ELISA were utilized to measure nitric oxide (NO) production and inflammatory cytokines, respectively. Signal proteins such as JNK, nuclear factor E2-related factor 2 (Nrf2) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were quantitatively evaluated in western blot experiments. Flow cytometry was used to determine the concentration of reactive oxygen species (ROS). More importantly, Drug Affinity Responsive Target Stability (DARTS) assay and molecular docking were conducted to investigate the potential targets of TSA. TSA effectively reduced the NO production (half-maximal inhibitory concentration (IC50) at 12.53 ± 0.31 μM). In addition, TSA restrained the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), and it could up-regulate the levels of interleukin-10 (IL-10). TSA also decreased ROS levels by enhancing the levels of Nrf2 protein and its related target genes. Meanwhile, TSA regulated the nuclear translocation of nuclear factor-κB (NF-κB) by suppressing the MAPKs signaling pathway. Importantly, TSA may suppress the inflammation through the TRAF6/MAPKs pathway. TSA suppressed the inflammatory mechanism mediated by the TRAF6/MAPKs pathway. Our research first revealed the anti-inflammatory effect of a lindenane sesquiterpenoid trimer, providing a therapeutic drug candidate for inflammatory diseases. Furthermore, the lindenane-type sesquiterpenoid trimers may be among the main anti-inflammatory components in S. glabra.