Turmeric Extract (Curcuma longa) Mediates Anti-Oxidative Effects by Reduction of Nitric Oxide, iNOS Protein-, and mRNA-Synthesis in BV2 Microglial Cells

Abstract

Plant-derived products have been used since the beginnings of human history to treat various pathological conditions. Practical experience as well as a growing body of research suggests the benefits of the use of turmeric (Curcuma longa) and some of its active components in the reduction of oxidative stress, a mechanism leading to neurodegeneration. In this current study, we investigated the effects of a preparation of Curcuma longa, and its constituents curcumin, tetrahydrocurcumin, and curcumenol, in one of the molecular pathways leading to oxidative stress, which is the release of NO, a free radical involved in stress conditions, using the BV2 microglial cell line. The concentration-dependent reduction of NO is linked to reduced amounts of iNOS protein- and mRNA-synthesis and is possibly mediated by the phosphorylation of mitogen-activated protein kinases (MAPK) such as p42/44 or p38 MAPK. Therefore, the use of turmeric extract is a promising therapeutic option for diseases linked to the dysregulation of oxidative stress, with fewer side-effects in comparison to the currently used pharmacotherapeutics.

Keywords: BV2; NO; curcuma; curcumin; iNOS; microglia; oxidative stress; turmeric.

Figure 1

Effects of TE on cell viability of LPS-stimulated BV2 cells (ATP assay). TE was added 30 min before stimulation with 10 ng/mL LPS for 24 h. ATP levels and thus cell viability were measured by production of luminescent light following the firefly luciferase enzymatic reaction of Luciferin to Oxyluciferin using ATP. Values are presented as the mean ± SEM of four independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc test and * p < 0.05 and *** p < 0.001, compared to untreated cells.

Figure 2

Effects of different concentrations of TE and its constituents curcumin, THC, and curcumenol (one single high dose, respectively) (A), and different doses of curcumin (B), on NO-release in LPS-stimulated BV2 cells. The substances were added 30 min before stimulation with 10 ng/mL LPS. NO-release was measured as described below. Values are presented as the mean ± SEM of at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc tests and * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared to LPS.

Figure 3

Effects of TE on iNOS protein levels in LPS-stimulated BV2 cells. TE was added 30 min before stimulation with 10 ng/mL LPS. After 24 h, Western Blot analysis was performed. Values are presented as the mean ± SEM of three independent experiments, and statistical analysis was performed using one-way ANOVA with Bonferroni post hoc test compared to LPS.

Figure 4

Effects of TE on iNOS mRNA expression in LPS-stimulated BV2 cells. TE was added 30 min before stimulation with 10 ng/mL LPS for 4 h. RNA was then isolated, and iNOS mRNA levels were determined using qPCR. Values are presented as the mean ± SEM of at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc tests and * p < 0.05 and **** p < 0.0001, compared to LPS.

Figure 5

Effects of TE on p38 MAPK (A) and Erk 1/2 (B) phosphorylation in LPS-stimulated BV2 cells. TE was added 30 min before stimulation with 10 ng/mL LPS for 30 min. Western Blot analysis was performed for p-p38/MAPK (A) and phospho-Erk 1/2 (B). Values are presented as the mean ± SEM of at least three independent experiments, and protein levels were referenced to β-actin. Statistical analyses were performed using one-way ANOVA with Bonferroni post hoc test and * p < 0.05 and ** p < 0.01, compared to LPS.