Abstract
Purpose: To investigate the antioxidant and anti-neuroinflammatory potential of Saussurea lappa Clarke (SLC-EA) extract in LPS-stimulated BV-2 microglial cells.Methods: Cell viability was measured by using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay while antioxidant activity was evaluated by using the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity. Lipopolysaccharide (LPS) was used to stimulate BV-2 microglia. Griess assay was employed to assess nitric oxide (NO) production. iNOS (inducible NO synthase) expression and TNF-α (tumor necrosis factor-alpha) cytokine production were measured by ELISA (enzyme-linked immunosorbent assay) and immuno blot analysis, respectively.Results: Pretreatment of 100 mg/ml of SLC-EA (p < 0.001) was inhibited Nitric Oxide (NO) by 1 ug/ml of LPS-treated murine BV-2 cells. The expression of iNOS and TNF-α were reduced by SLC-EA concentration dependent manner (p < 0.001 at 100 mg/ml). SLC-EA were scavenged 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicals in a dose-dependent manner with an IC50 value of approximately 51.4 μg/ml.Conclusion: The results indicate that SLC-EA extract exhibits strong antioxidant properties and inhibits excessive pro-inflammatory cytokine due probably to the antioxidant phenolic compounds present in SLC-EA extract. Further work in exploring the in-depth mechanisms of SLC-EA extract in regulating inflammatory signaling pathways in treating neuroinflammatory diseases is necessary.
 Keywords: Saussurea lappa, Antioxidant, Neuroinflammation, Microglia, TNF-α, iNOS
Highlights
Stress related stimulating cell signaling and untimely generating of the cell death pathways involved in the beginning of some neurological diseases [1]
The capacity of SLC-ethyl acetate (EA) to scavenge DPPH was measured by electron spin resonance (ESR) spectroscopy
Content of quercetin in the SLC-EA was 6.103 μg/mg. These results showed that the ethyl acetate fraction of Saussurea lappa extract significantly inhibited the increased production of nitric oxide (NO), suppressed the expression of Inducible nitric oxide synthase (iNOS) protein level and attenuated the increased
Summary
Stress related stimulating cell signaling and untimely generating of the cell death pathways involved in the beginning of some neurological diseases [1]. S. lappa extract was found to decrease proinflammatory mediators in LPS-stimulated murine macrophage cells [17]. In the present study the ethyl acetate fraction obtained from S. lappa Clarke (SLC-EA) was investigated for its anti-neuroinflammatory effects in LPS–stimulated. LPS-stimulated murine BV-2 microglial cells was investigated using SLC-EA extract. The anti-oxidant activity of the SLC-EA extract was determined using the stable radical DPPH (2, 2-diphenyl-1-picrylhydrazyl, Sigma-Aldrich, St. Louis, MO, USA). The radical scavenging capacity was measured by using a reaction mixture set up by aliquots of the SLC-EA extract and a DPPH methanolic solution as described previously [19]. At 4 h of post LPS treatment, the cells were harvested and the supernatants were subjected to evaluate tumor necrosis factor --α (TNF-α) levels using murine TNF-α ELISA kit from BD Biosciences (San Jose, CA, USA). Statistical significance (p < 0.05 for all analyses) was assessed by ANOVA using Instat 3.05 (GraphPad, San Diego, CA), followed by Student–Newman–Keuls analysis
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