Inhibition of Neuroinflammation by the Methanolic Extract of Zanthoxylum Zanthoxyloides Root and Skimmianine

Studies have demonstrated the role of neuroinflammation in causing neurotoxicity and neuronal damage in neurodegenerative diseases and cerebral malaria (CM) patients. Zanthoxylum zanthoxyloides (Z. Z) is reputed for its anti-inflammatory activity. This study investigated the effect of Z.Z. and it’s bioactive, skimmianine (SK) on neuroinflammation in BV2 microglia cells stimulated with lipopolysaccharide (LPS) and synthetic hemozoin (sHZ). Cultured mouse microglia (BV2) cells were stimulated with LPS (100 ng/ml) and sHZ (400 μg/ml) following pre-treatment with Z. zanthoxyloides (4, 6 and 8 μg/ml) and skimmianine (10, 20, and 30 μM). Levels of nitric oxide (NO), PGE2, pro IL-1β, IL-1β, IL-6, TNFα and IL-10 were measured in cell culture supernatants. Protein expressions of iNOS, COX-2, NF-КB phospho-p65 subunit, phospho-IҡBα, total IҡBα, phospho-IKK, total IKK, caspase-1 and NLRP3 in extracts were investigated with western blotting. HEK293 cells were transfected with Cignal NF-κB (LUC) (CCS-013L) vector and reporter gene assays were used to investigate the effect of Z.Z and SK on luciferase activity of NF-КB in the nucleus. Isolation of SK was monitored with solid-phase-extraction (SPE), ELISA and HPLC. Structure elucidation of the isolated compound was determined by NMR analysis and mass spectrometry. Z.Z extract and SK reduced the levels of PGE2, IL-1β, IL-6, TNFα IL-1β, pro-IL-1β, caspase-1 protein and NLRP3 protein and increased the levels of IL-10 in BV2 cells stimulated with LPS and sHZ. Further experiments showed that Z. Z and SK decreased the expression of iNOS, COX-2 protein and inhibited NF-B and NLRP3signaling pathway in both LPS and sHZ-stimulated BV2 cells. These results suggest that Z.Z and SK inhibit neuroinflammation through NF-B and NLRP3 signalling pathway in stimulated BV2 cells. This study suggests that bioactive compounds in Z. zanthoxyloides extract are potential phytochemical leads for treating neuroinflammation mediated neurodegenerative diseases and CM.