Aronia melanocarpa Extract Fermented by Lactobacillus plantarum EJ2014 Modulates Immune Response in Mice

Abstract

The present study aimed to assess the immunomodulatory effects of fermented Aronia melanocarpa extract (FAME) on RAW 264.7 cells and BALB/c mice. Aronia melanocarpa fruit was fermented with Lactobacillus plantarum EJ2014 by adding yeast extract and monosodium glutamate for 9 days at 30 °C to produce γ-aminobutyric acid (GABA). After fermentation, significant GABA production was noted, along with minerals, polyphenols, and flavonoids (p < 0.05). The polyphenol content was confirmed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. RAW 264.7 cells were stimulated with lipopolysaccharide (LPS, 1 μg/mL) in the presence or absence of FAME, and proinflammatory cytokine contents were measured by qPCR. In the in vivo experiment, female BALB/c mice were administered 125, 250, and 500 mg/kg of FAME for 21 days. FAME treatment increased neutrophil migration and phagocytosis (p < 0.05). It also increased splenocyte proliferation, CD4⁺ and CD8⁺ T-cell expression, and lymphocyte proliferation. Furthermore, it increased IFN-γ, IL-2, and IL-4 cytokine levels in a dose-dependent manner (p < 0.05). However, it decreased TNF-α and IL-6 levels (p < 0.05). These results indicate that FAME fortified with GABA including bioactive compounds exerts anti-inflammatory effects by inhibiting proinflammatory cytokines in RAW 264.7 cells and modulates immune response in mice. Thus, FAME could be a potential therapeutic agent for inflammatory disorders.

Keywords: Aronia melanocarpa; GABA; Lactobacillus plantarum; RAW 264.7 cells; fermentation; immunomodulation; polyphenols; proinflammatory cytokines.

Figure 1

In vitro experimental scheme.

Figure 2

Outline of the in vivo experiment.

Figure 3

Fermentation effects on GABA production pattern in FAME. Thin-layer chromatogram (TLC) for MSG standard (0.25–1%), GABA standard (0.25–1%), and 5-fold dilution of FAME samples collected at different time intervals (0, 1, 3, 5, 7, and 9 days) during fermentation.

Figure 4

Production of NO and expression of different cytokines in LPS-treated RAW 264.7 cells upon treatment with FAME. Effects of FSE on LPS-induced NO production (B). Expression of proinflammatory cytokine mRNA determined by conventional PCR and qRT-PCR: iNOS (A,C), TNF-α (A,D), IL-1β (A,E), IL-6 (A,F), and Cox-2 (A,G). Expression of TLR4 mRNA (A,H) and cell viability (I). The data are presented as the mean ± SEM of three independent experiments. Different letters (a–f) above the bars indicate significant differences (p < 0.05) between groups.

Figure 5

Effects of FAME on neutrophil migration (A) and phagocytic activity (B). Data are expressed as the mean ± SEM (n = 3). Different letters (a–e) above the bars indicate significant differences (p < 0.05) between groups.

Figure 6

Effects of FAME on mouse splenocyte proliferation (A,B). (A) Total splenocyte count of different groups. (B) Ex vivo activation of splenocytes by LPS and Con A after 72 h of incubation. Data are expressed as the mean ± SEM (n = 3). Different letters (a–d) above the bars indicate significant differences (p < 0.05) between groups.

Figure 7

Flow cytometric immunophenotyping analysis of CD4⁺ (PE-conjugated) and CD8⁺ (FITC-conjugated) T-cell expression in splenocytes harvested from control and FAME-treated mice. (A) The percentages of CD4⁺ (region Q1) and CD8⁺ (region Q4) T cells in the spleen of mice. (B) The percentages of CD4⁺ cells and (C) the percentages of CD8⁺ cells. Data are expressed as the mean ± SEM (n = 3). Different letters (a–d) above the bars indicate significant differences (p < 0.05) between groups.

Figure 8

Cytokine profile of plasma obtained from mice treated with FAME (A–E). (A) IFN-γ, (B) IL-2, (C) IL-4, (D) IL-6, and (E) TNF-α. Data are expressed as the mean ± SEM (n = 6). Different letters (a–d) above the bars indicate significant differences (p < 0.05) between groups.