Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes

Abstract

The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells.

Keywords: ARE reporter; Ashwagandha; Açaí; NF-κB reporter; Quercetin; Sutherlandia.

Fig. 1. Cytokines and LPS induce NF-κB-dependent luciferase activity in DI TNC1 astrocytes

DI TNC1 astrocytes were stably transfected with a luciferase reporter gene driven by the NF-κB promoter. Cells were treated with (A) TNFα, IL-1β, and IFNγ (10 ng/mL each) or LPS (100 ng/mL) for 6 h,(B) quercetin (0-10 μM), or (C) rutin, cyanidin and cyanidin-3-O-glucoside (0 and 100 μM each) for 1 h followed by stimulation with LPS (100 ng/mL) for 6 h. Luciferase activity was determined, as described in Methods. Results are expressed as the mean ± SEM (n=3) and analyzed by one-way ANOVA (A) or two-way ANOVA (B, C) with Bonferroni post-tests. (A) ***p<0.001 vs. control. (B) *p<0.05, **p<0.01, ***p<0.001 vs. LPS alone.

Fig. 2. Effects of Ashwagandha (A), Sutherlandia (B), and Açaí (C) extracts on LPS-induced NF-κB-dependent luciferase activity in DI TNC1 astrocytes

DI TNC1 astrocytes stably transfected with a luciferase reporter gene driven by the NF-κB promoter were treated with botanical extracts for 1 h followed by stimulation with LPS (100 ng/mL) for 6 h. Luciferase activity was determined, as described in Methods. Results are expressed as the mean ± SEM (n=3). Data were analyzed by one-way ANOVA followed by Bonferroni post-tests. **p<0.01, ***p<0.001 vs. LPS alone.

Fig. 3. Effects of quercetin (A), rutin, cyanidin and cyanidin-3-O-glucoside (B) on LPS-induced ARE-dependent luciferase activity in DI TNC1 astrocytes

DI TNC1 astrocytes stably transfected with a luciferase reporter gene driven by the Nrf2/ARE were treated with quercetin (0-10 μM), rutin, cyanidin or cyanidin-3-O-glucoside (0 and 100 μM) for 1 h followed by LPS (100 ng/mL) or vehicle for 6 h. Luciferase activity was determined, as described in Methods. Results are expressed as the mean ± SEM (n =3) and analyzed by two-way ANOVA with Bonferroni post-tests where “a” denotes significant differences between LPS+polyphenol vs. polyphenol alone (p<0.05 for 0 and 2.5 μM quercetin and p<0.01 for all the other comparisons); “b” denotes significant differences between LPS+polyphenol vs. LPS alone (p<0.05 for rutin; p<0.01 for cyanidin; p<0.001 for quercetin and cyanidin-3-O-glucoside); “c” denotes significant differences as compared to 0 μM polyphenol (p<0.001 for quercetin and p<0.05 for each on Fig. 3B).

Fig. 4. Effects of Ashwagandha (A), Sutherlandia (B), and Açaí (C) extracts on LPS-induced ARE-dependent luciferase activity in DI TNC1 astrocytes

DI TNC1 astrocytes stably transfected with a luciferase reporter gene driven by the Nrf2/ARE were treated with the indicated concentration of botanical extracts for 1 h followed by LPS (100 ng/mL) or vehicle for 6 h. Luciferase activity was determined, as described in Methods. Results are expressed as the mean ± SEM (n=3) and analyzed by two-way ANOVA with Bonferroni post-tests, where “a” denotes significant differences between LPS+botanical extracts vs. extracts alone (p<0.05 for Sutherlandia; p<0.01 for Ashwagandha; p<0.001 for Açaí); “b” denotes significant differences between LPS+botanical extracts vs. LPS alone (p<0.001); “c” denotes significant differences as compared to 0 μg/mL extract (p<0.001 for each comparison except for Açaí at 6.25 μg/mL, where p<0.01).

Fig. 5. Botanicals stimulate Nrf2 and HO-1 expression in untransfected DI TNC1 astrocytes

Untransfected DI TNC1 astrocytes were treated with quercetin (A, B), or Ashwagandha (C, D), or Sutherlandia (E, F) or Açaí (G, H) extracts at the indicated concentrations for 6 h. Nrf2 (A, C, E,F) and HO-1 (B, D, F, H) expression was determined by Western blot analysis, as described in Methods. Representative blots and quantitative analysis (bar graphs) are shown. Results are expressed as the mean ± SEM (n=3-6) and analyzed by one-way ANOVA with Bonferroni post-tests, where *p<0.05, **p<0.01, ***p<0.001 vs. control.

Fig. 5. Botanicals stimulate Nrf2 and HO-1 expression in untransfected DI TNC1 astrocytes

Untransfected DI TNC1 astrocytes were treated with quercetin (A, B), or Ashwagandha (C, D), or Sutherlandia (E, F) or Açaí (G, H) extracts at the indicated concentrations for 6 h. Nrf2 (A, C, E,F) and HO-1 (B, D, F, H) expression was determined by Western blot analysis, as described in Methods. Representative blots and quantitative analysis (bar graphs) are shown. Results are expressed as the mean ± SEM (n=3-6) and analyzed by one-way ANOVA with Bonferroni post-tests, where *p<0.05, **p<0.01, ***p<0.001 vs. control.