ABSTRACT
Objective: To study the chemical constituents of 95% ethanol aqueous extract of Chuanxiong Rhizoma and the bioactivities of inhibition on nitric oxide (NO) production in lipopolysaccharides (LPS)-activated murine macrophage RAW264. 7 and mouse microglia BV2 cell lines. Methods: The compounds were separated and purified by silica gel column and high performance liquid chromatographies, and their structures were determined by spectroscopic data analysis. Using LPS-activated RAW264. 7 and BV2 cell line models in vitro, all of the isolated compounds were evaluated for the inhibition against NO production. Results: Three butylphthalide derivatives were obtained and identified as Z-3', 8', 3'a, 7'a-tetrahydro-6, 3', 7, 7'a-diligustilide-8'-one (1), Z, Z'-3.3'a, 7. 7'a-diligustilide (2), and chuanliguspirolide (3), respectively. For the inhibition of NO production in the LPS-activated two cell lines, the half inhibitory concentration (IC50) values of compounds 1-3 and indomethacin as a positive control drug in RAW 264. 7 cell line model were (31.60 ± 2.62), (21.20 ± 0.61), (30.12 ± 2.90), and (54.62 ± 7.53) μmol/L, respectively, while IC50 values of compounds 1-3 and curcumin as a positive control drug in BV2 cell line model were (21.99 ± 4.40), (15.43 ± 1.34), (12.20 ± 3.40), and (10.58 ± 1. 41) μmol/L, respectively. Conclusion: Compound 3 named as chuanliguspirolide is a new one. The results of bioactivity assays indicated that compounds 1-3 are potential anti-inflammatory agents.
ABSTRACT
Introduction: Neurodegeneration resulting from pathogen invasion or tissue damage has been associated with activation of microglia, and exacerbated by the release of neurotoxic mediators such as pro-inflammatory cytokines, chemokines and reactive oxygen species. Activation of microglia stimulated by lipopolysaccharide is mediated in part by GSK-3 signaling molecule. Induced IL-10 expression via GSK-3 inhibition is noteworthy since IL-10 has been remarkably shown to suppress inflammation. Objectives: We aimed to inactivate microglia through inhibition of GSK-3 signaling and to determine its effects on the production of pro- and anti-inflammatory mediators. Methods: LPS-stimulated BV-2 cells were treated with a GSK-3 inhibitor (LiCl, NP12, SB216763 or CHIR99021). Inhibition of GSK-3 was determined by the phosphorylation status of GSK-3β. The effects of GSK-3 inhibition on microglial inflammatory response were investigated by examining various mediators and CD200R marker. Production of nitric oxide (NO), glutamate and pro- and anti-inflammatory cytokines were measured using flow cytometry, Griess assay, glutamate assay and Cytometric Bead Array (CBA) respectively. Results: GSK-3β signaling in LPS-stimulated microglia was blocked by GSK-3 inhibitor through increased phosphorylation at Serine 9 residue. GSK-3 inhibitors had also led to reducing in microglia activity via increased expression of CD200R. Inhibition of GSK-3 also diminished inflammatory mediators such as nitric oxide (NO), glutamate, pro-inflammatory cytokines (TNF-α and IL-6) and chemokine, MCP-1. Reduction of pro-inflammatory mediators by GSK-3 inhibitor was coincided with increased IL-10 production. Conclusions: Suppression of microglia-mediated inflammatory response was facilitated by GSK-3 inhibition with associated increased in IL-10 production.