Lipophilic Bioactive Compounds Transported in Triglyceride-Rich Lipoproteins Modulate Microglial Inflammatory Response

Abstract

Microglial cells can contribute to Alzheimer's disease by triggering an inflammatory response that leads to neuronal death. In addition, the presence of amyloid-β in the brain is consistent with alterations in the blood-brain barrier integrity and triglyceride-rich lipoproteins (TRL) permeation. In the present work, we used lab-made TRL as carriers of lipophilic bioactive compounds that are commonly present in dietary oils, namely oleanolic acid (OA), α-tocopherol (AT) and β-sitosterol (BS), to assess their ability to modulate the inflammatory response of microglial BV-2 cells. We show that treatment with lab-made TRL increases the release and gene-expression of IL-1β, IL-6, and TNF-α, as well as NO and iNOS in microglia. On the other hand, TRL revealed bioactive compounds α-tocopherol and β-sitosterol as suitable carriers for oleanolic acid. The inclusion of these biomolecules in TRL reduced the release of proinflammatory cytokines. The inclusion of these biomolecules in TRL reduced the release of proinflammatory cytokines. AT reduced IL-6 release by 72%, OA reduced TNF-α release by approximately 50%, and all three biomolecules together (M) reduced IL-1β release by 35% and TNF-α release by more than 70%. In addition, NO generation was reduced, with the inclusion of OA by 45%, BS by 80% and the presence of M by 88%. Finally, a recovery of the basal glutathione content was observed with the inclusion of OA and M in the TRL. Our results open the way to exploiting the neuro-pharmacological potential of these lipophilic bioactive compounds through their delivery to the brain as part of TRL.

Keywords: bioactive compounds; inflammation; microglia; oxidative stress; triglyceride-rich lipoprotein.

Conflict of interest statement

J.S.P. has received a speaker honorarium from Organización Interprofesional del Aceite de Orujo de Oliva. No other conflicts of interest are disclosed.

Figures

Figure 1

Oil Red O microphotographs of untreated (a) BV-2 microglial cells or treated with TRL (b) for 24 h at 37 °C.

Figure 2

Cytokine release in BV-2 cells stimulated by TRL (positive control) or TRL carrying oleanolic acid (TRL-OA), α-tocopherol (TRL-AT), β-sitosterol (TRL-BS) or a mixture of the three compounds (TRL-M) for 24 h: (a) Interleukin-1 β (IL-1β); (b) Interleukin-6 (IL-6); (c) Tumour necrosis factor-α (TNF-α); Saline (0.9%, pH 7.4) was used as references for calculations. Values are expressed as mean ± SD of three independent experiments. Different signs indicate significant difference (* p < 0.05, *** p < 0.001 vs. saline; # p < 0.05 vs. TRL, ## p < 0.01 vs. TRL, ### p < 0.001 vs. TRL) by one-way ANOVA analysis and Tukey’s post-hoc test.

Figure 3

Cytokine RNA expression of cytokines in BV-2 cells stimulated by TRL (positive control) or TRL carrying oleanolic acid (TRL-OA), α-tocopherol (TRL-AT), β-sitosterol (TRL-BS), or a mixture of the three compounds (TRL-M) for 24 h: (a) Interleukin-1 β (IL-1β); (b) Interleukin-6 (IL-6); (c) Tumor necrosis factor-α (TNF-α); Saline (0.9%, pH 7.4) was used as references for calculations. Values are expressed as mean ± SD of three independent experiments. Different signs indicate significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. saline) by one-way ANOVA analysis and Tukey’s post-hoc test.

Figure 4

(a) Nitrite production and inducible nitric oxide synthase; (b) (iNOS) gene expression in BV-2 cells stimulated by TRL or TRL carrying oleanolic acid (TRL-OA), α-tocopherol (TRL-AT), β-sitosterol (TRL-BS) or a mixture of the three compounds (TRL-M) for 24 h, Saline (0.9%, pH 7.4) was used as references for calculations. Values are expressed as mean ± SD of three independent experiments. Different signs indicate significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. saline; # p < 0.05, ## p < 0.01, vs. TRL) by one-way ANOVA analysis and Tukey’s post-hoc test.

Figure 5

Intracellular concentrations of total glutathione (GSH + GSSG) in BV-2 cells stimulated by TRL or TRL carrying oleanolic acid (TRL-OA), α-tocopherol (TRL-AT), β-sitosterol (TRL-BS) or a mixture of the three compounds (TRL-M) for 24 h, Saline (0.9%, pH 7.4) was used as references for calculations Values are expressed as mean ± SD of three independent experiments. Different signs indicate significant difference (** p < 0.01, vs. saline; ## p < 0.01, vs. TRL) by one-way ANOVA analysis and Tukey’s post-hoc test.