Anti-neuroinflammatory effects of ethanolic extract of black chokeberry (Aronia melanocapa L.) in lipopolysaccharide-stimulated BV2 cells and ICR mice

Abstract

Background/objectives: One of the mechanisms considered to be prevalent in the development of Alzheimer's disease (AD) is hyper-stimulation of microglia. Black chokeberry (Aronia melanocapa L.) is widely used to treat diabetes and atherosclerosis, and is known to exert anti-oxidant and anti-inflammatory effects; however, its neuroprotective effects have not been elucidated thus far.

Materials/methods: We undertook to assess the anti-inflammatory effect of the ethanolic extract of black chokeberry friut (BCE) in BV2 cells, and evaluate its neuroprotective effect in the lipopolysaccharide (LPS)-induced mouse model of AD.

Results: Following stimulation of BV2 cells by LPS, exposure to BCE significantly reduced the generation of nitric oxide as well as mRNA levels of numerous inflammatory factors such as inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α). In addition, AD was induced in a mouse model by intraperitoneal injection of LPS (250 µg/kg), subsequent to which we investigated the neuroprotective effects of BCE (50 mg/kg) on brain damage. We observed that BCE significantly reduced tissue damage in the hippocampus by downregulating iNOS, COX-2, and TNF-α levels. We further identified the quinic acids in BCE using liquid chromatography-mass spectrometry (LCMS). Furthermore, we confirmed the neuroprotective effect of BCE and quinic acid on amyloid beta-induced cell death in rat hippocampal primary neurons.

Conclusions: Our findings suggest that black chokeberry has protective effects against the development of AD.

Keywords: Phytochemicals; inflammation; microglia; neurons; quinic acid.

Fig. 1. Effects of black chokeberry extract on cell viability and production of nitric oxide (NO) in lipopolysaccharide-stimulated BV2 microglial cells.

(A) The effects of BCE on cell viability were determined using the LIVE/DEAD Cell Viability Assay Kit. Morphological changes in BV2 cells were observed using microscopy (upper panel). The surviving cells were identified by their green fluorescence (bottom panel). (B) The effects of BCE on cell viability were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (n = 4). Cell viability is expressed as 100% in the untreated group. (C) Cells were co-treated with BCE and 500 ng/mL LPS for 48 hours. NO production was measured using the Griess reagent assay (n = 6). NO was quantified based on the sodium nitrate standard curve. Data are expressed as mean ± SD. ^*Significant difference when compared to LPS-only treatment (P < 0.05). BCE, black chokeberry extract; LPS, lipopolysaccharide.

Fig. 2. Effects of black chokeberry extract on expression levels of inflammatory factors and pro-inflammatory cytokines in lipopolysaccharide-stimulated BV2 cells.

(A-D) BV2 cells were seeded in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum for 24 hours, following which they were cultured in the absence or presence of LPS (500 ng/mL) and black chokeberry extract (BCE) (300 µg/mL) for 24 hours. (A-D) The relative levels of mRNA were calculated using 2^−ΔΔCt values and normalized to that of GAPDH. (E) Proteins were separated and immunoblotted for protein expression analysis (COX-2, IL-1β, TNF-α, nuclear factor kappa-light-chain-enhancer of activated B cells, and GAPDH). ^*Significant difference when compared to LPS-only treatment (P < 0.05). LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase 2; IL-1β, interleukin 1 beta; TNF-α, tumor necrosis factor alpha; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 3. Effects of black chokeberry extract on lipopolysaccharide-induced neuroinflammation.

Representative photomicrographs of mouse brain cross-sections taken 7 days after an intraperitoneal injection of LPS. (A) Analysis of hippocampus morphology using hematoxylin and eosin staining. (B) Immunohistochemical staining for inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α) was performed in the cross-sections. Shading represents the intensity of brown color from iNOS, COX-2, IL-1β and TNF-α expression in the analyzed groups. Protein expression in the LPS group was considered to be 100%. Data are expressed as mean ± SD (n = 3). ^*Significant difference when compared to the LPS group (P < 0.05). BCE, black chokeberry extract; LPS, lipopolysaccharide; DG, dentate gyrus; CA1, cornu amonis 1; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase 2; IL-1β, interleukin 1 beta; TNF-α, tumor necrosis factor alpha.

Fig. 4. LC-MS spectrum of black chokeberry extract and the chemical structure of quinic acid. MW: molecular weight.

Fig. 5. Effects of black chokeberry extract and quinic acid on cell viability in Aβ-stimulated primary neuronal cells.

(A) The effects of BCE and quinic acid on cell viability of primary neuronal cells were determined using the LIVE/DEAD Cell Assay Kit. The cell morphologies were compared to the untreated cell group. (B-D) Primary neuronal cells were cultured in medium containing BCE (10 µg/mL, 30 µg/mL, 100 µg/mL, or 300 µg/mL) and Aβ (20 µM) for 48 hours. The primary neuronal cells were also cultured in medium containing quinic acid (0.1 µM, 1 µM, or 10 µM) and Aβ (20 µM) for 48 hours. Cell viability were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell viability is expressed as 100% in the untreated group. Data are expressed as mean ± SD. ^*Significant difference when compared to the Aβ 25-35 group (P < 0.05). BCE, black chokeberry extract; Aβ 25-35, amyloid beta peptide 25-35.