Anti-Neuroinflammatory Potential of a Nectandra angustifolia (Laurel Amarillo) Ethanolic Extract

Abstract

Microglia, the resident macrophage-like population in the CNS, plays an important role in the pathogenesis of many neurodegenerative disorders. Nectandra genus is known to produce different metabolites with anti-inflammatory, anti-oxidant and analgesic properties. Although the species Nectandra angustifolia is popularly used for the treatment of different types of inflammatory processes, its biological effects on neuroinflammation have not yet been addressed. In this study, we have investigated the role of a Nectandra angustifolia ethanolic extract (NaE) in lipopolysaccharide (LPS)-induced neuroinflammation in vitro and in vivo. In LPS-activated BV2 microglial cells, NaE significantly reduced the induced proinflammatory mediators TNF-α, IL-1β, IL-6, COX-2 and iNOS, as well as NO accumulation, while it promoted IL-10 secretion and YM-1 expression. Likewise, reduced CD14 expression levels were detected in microglial cells in the NaE+LPS group. NaE also attenuated LPS-induced ROS and lipid peroxidation build-up in BV2 cells. Mechanistically, NaE prevented NF-κB and MAPKs phosphorylation, as well as NLRP3 upregulation when added before LPS stimulation, although it did not affect the level of some proteins related to antioxidant defense such as Keap-1 and HO-1. Additionally, we observed that NaE modulated some activated microglia functions, decreasing cell migration, without affecting their phagocytic capabilities. In LPS-injected mice, NaE pre-treatment markedly suppressed the up-regulated TNF-α, IL-6 and IL-1β mRNA expression induced by LPS in brain. Our findings indicate that NaE is beneficial in preventing the neuroinflammatory response both in vivo and in vitro. NaE may regulate microglia homeostasis, not only restraining activation of LPS towards the M1 phenotype but promoting an M2 phenotype.

Keywords: Nectandra angustifolia extract; ROS; microglia; migration; neuroinflammation; phagocytosis.

Figure 1

Effect of NaE extract on the viability of BV2 microglia. (A) Experimental steps. (B) BV2 cells were treated with the indicated doses of the NaE extract in the absence or presence of 0.1 μg/mL LPS for 24 h. Cell viability was measured using an MTT assay. Values are expressed as the mean ± SD of the three independent experiments. (C) Representative photomicrographs of cells incubated with medium (control) or NaE (10, 25 and 50 µg/mL) for 24 h. Images were taken at 100× and 40× magnification. N = 3.

Figure 2

Effect of NaE extract on LPS-modulated signaling proteins in BV2 microglia. (A) Experimental steps. Cells were pre-treated with the indicated doses of the NaE extract for 1 h at 37 °C, and then were stimulated with or without 0.1 μg/mL of LPS for (B) 30 min or (C) 24 h. (B) Activation of the MAPKs family was verified using specific antibodies of phosphorylated ERK, p38, JNK and P70S6K proteins. (C) Expression levels of NLRP3 and phosphorylated NFkB-p65 were detected using specific antibodies. β-actin was used as the internal control. N = 4. Western blot bar graphs are in Supplementary Material Figure S2.

Figure 3

Anti-inflammatory effect of NaE on BV2 cells. (A) Experimental steps. BV2 cells were incubated for 1 h at 37 °C with the indicated doses of the NaE extract and then stimulated with or without 0.1 μg/mL of LPS for 24 h. (B) Expression of COX-2 and iNOS analyzed by western blot using specific antibodies. (C) NO accumulation in the cell culture supernatants analyzed by a Griess assay. Values are means ± SD (n = 3–5). *** p < 0.001 vs. controls; and +++ p < 0.001 vs. LPS-treated cells. Expression of (D) HO-1 and SOD2 as well as (E) Keap-1 analyzed by western blot using a specific antibody. Western blot bar graphs are in Supplementary Material Figure S3.

Figure 4

NaE extract modulates the M1 phenotype induced by LPS in BV2 cells. (A) Experimental steps. BV2 cells were incubated for 1 h at 37 °C with the indicated doses of the NaE extract, and then stimulated with or without 0.1 μg/mL of LPS for 24 h. (B) Levels of IL-6, TNF-α, IL-1β and IL-10 were determined in the culture supernatants using commercial ELISA kits. Values are means ± SD (n = 3–5). *** p < 0.001 vs. controls; and +++ p < 0.001, ++ p < 0.01 and + p < 0.05 vs. LPS-treated cells. (C) Expression of CD14 on cell surface was evaluated by flow cytometry analysis. The graph represents MFI fold change values of CD14 normalized to untreated control cells. *** p < 0.001 vs. controls; and + p < 0.05 vs. LPS-treated cells. (D) Expression of YM-1 was evaluated by Western blot. (E) Representative photomicrographs showing changes in cell morphology after LPS stimulation in the presence or absence of NaE. Cells were photographed using a Nikon Eclipse TS100 microscope (200× and 400× magnification). N = 3. Western blot bar graphs are in Supplementary Material Figure S4.

Figure 5

NaE extract modulates LPS-induced oxidative stress in BV2 microglia. (A) Experimental steps. Cells were pre-treated with the indicated doses of the NaE extract for 1 h and then stimulated or not with 0.1 μg/mL of LPS for 24 h. (B) After treatments, cells were incubated at 37 °C in the dark for 20 min with a culture medium containing 20 μM DHE to monitor intracellular superoxide anion production using flow cytometry. Representative histograms. (C) Quantified levels of ROS. (D) Lipid peroxidation (MDA + 4-HNE) levels. Values are expressed as the mean ± SD of the three independent experiments. *** p < 0.001 vs. controls; and +++ p < 0.001 and + p < 0.05 vs. LPS-treated cells.

Figure 6

Effect of NaE extract on BV2 microglia functions. (A) Experimental steps. BV2 cells were pre-treated with the indicated doses of the NaE extract for 1 h at 37 °C and then stimulated or not with 0.1 μg/mL of LPS. (B) Migration assay: Data were reported as BV2 cell migration into the scratch area at 3 h and 24 h post-scratch. Values are means ± SD of the three independent experiments. *** p < 0.001 and * p < 0.05 vs. control; and +++ p < 0.001 vs. LPS-treated cells. (C) Representative images showing cell migration in the different conditions at 24 h. Magnification, 40×. (D,E) Phagocytosis assay using YG-microparticles and flow cytometry analysis. (D) Representative histogram. MFI: mean fluorescence intensity. (E) Graph represents fold change in mean fluorescence intensity (MFI) of YG-microparticles phagocytized calculated with respect to unstimulated controls cells. * p < 0.05 vs. control cells. N = 3.

Figure 7

Effect of NaE extract on LPS-induced neuroinflammation in mice. (A) Experimental steps. Mice were treated with vehicle, dexamethasone (2 mg/kg) (Dex), NaE extract (50 mg/kg) or NaE extract (5 mg/kg) 30 min prior to intraperitoneal injection of LPS (3 mg/kg). (B) IL-1β, IL-6 and TNF-α mRNA was determined in brain tissue at 3 h after i.p. LPS injection by RT-qPCR analysis. Values were calculated using the ∆∆Ct method normalized to GAPDH and the control group (only vehicle administration) and expressed as mean ± SD (n = 6). *** p < 0.001 vs. the control group; and +++ p < 0.001 vs. the LPS-treated group.