Immune-Enhancement and Anti-Inflammatory Activities of Fatty Acids Extracted from Halocynthia aurantium Tunic in RAW264.7 Cells

Abstract

Halocynthia aurantium, an edible ascidian species, has not been studied scientifically, even though tunicates and ascidians are well-known to contain several unique and biologically active materials. The current study investigated the fatty acid profiles of the H. aurantium tunic and its immune-regulatory effects on RAW264.7 macrophage cells. Results of the fatty acid profile analysis showed a difference in ratios, depending on the fatty acids being analysed, including those of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). In particular, omega-3 fatty acids, such as eicosatrienoic acid n-3 (ETA n-3), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), were much higher than omega-6 fatty acids. Moreover, the H. aurantium tunic fatty acids, significantly and dose-dependently, increased the NO and prostaglandin E2 (PGE₂) production in RAW264.7 cells, for immune-enhancement without cytotoxicity. In addition, these fatty acids regulated the transcription of immune-associated genes, including iNOS, IL-1β, IL-6, COX-2, and TNF-α. These actions were activated and deactivated via Mitogen-activated protein kinase (MAPK)and NF-κB signaling, to regulate the immune responses. Conversely, the H. aurantium tunic fatty acids effectively suppressed the inflammatory cytokine expressions, including iNOS, IL-1β, IL-6, COX-2, and TNF-α, in LPS-stimulated RAW264.7 cells. Productions of COX-2 and PGE₂, which are key biomarkers for inflammation, were also significantly reduced. These results elucidated the immune-enhancement and anti-inflammatory mechanisms of the H. aurantium tunic fatty acids in macrophage cells. Moreover, the H. aurantium tunic might be a potential fatty acid source for immune-modulation.

Keywords: Halocynthia aurantium; MAPK; NF-κB pathway; fatty acids; immunomodulation; tunic.

Figure 1

Fatty acid composition of tunic from Halocynthia aurantium. Data are presented as means ± standard deviation (n = 5). The letters a, b, c, d, e indicate a significant difference (p < 0.05) between the amount of fatty acid (where, a > b > c > d > e).

Figure 2

The cytotoxic effect and NO production of fatty acids from the H. aurantium tunic. (A) The cytotoxic effect of H. aurantium fatty acids on macrophage proliferation in RAW264.7 cells; (B) The cytotoxic effect of H. aurantium fatty acids on macrophage proliferation in LPS-stimulated RAW264.7 cells; (C) The effect of H. aurantium fatty acids on nitric oxide production in RAW264.7 cells; (D) The effect of H. aurantium fatty acids on nitric oxide production in LPS-stimulated RAW264.7 cells. RAW264.7 cells were stimulated with or without 1 μg/mL of LPS for 24 h. Significant differences are p < 0.01, compared with dimethyl sulfoxide (DMSO) or LPS (*).

Figure 3

Quantification of immune genes in relative expression (fold). (A) Relative expression of H. aurantium fatty acids in RAW264.7 cells; (B) Relative expression of H. aurantium fatty acids in LPS-stimulated RAW264.7 cells. Significant differences are p < 0.01 compared with DMSO or LPS (*).

Figure 4

The effect of H. aurantium fatty acids on proteins associated with NF-κB and MAPK pathways. (A) Western blot of proteins from RAW264.7 cells; (B) Relative band intensity of proteins from RAW264.7 cells; (C) Western blots of proteins from LPS-stimulated RAW264.7 cells; (D) Relative band intensity of proteins from LPS-stimulated RAW264.7 cells.

Figure 5

Quantification of PGE₂ production from (A) RAW264.7 cells and (B) LPS-stimulated RAW264.7 cells. Significant differences are p < 0.01 compared with DMSO or LPS (*).