Polyphenols journey through blood-brain barrier towards neuronal protection

Abstract

Age-related complications such as neurodegenerative disorders are increasing and remain cureless. The possibility of altering the progression or the development of these multifactorial diseases through diet is an emerging and attractive approach with increasing experimental support. We examined the potential of known bioavailable phenolic sulfates, arising from colonic metabolism of berries, to influence hallmarks of neurodegenerative processes. In silico predictions and in vitro transport studies across blood-brain barrier (BBB) endothelial cells, at circulating concentrations, provided evidence for differential transport, likely related to chemical structure. Moreover, endothelial metabolism of these phenolic sulfates produced a plethora of novel chemical entities with further potential bioactivies. Pre-conditioning with phenolic sulfates improved cellular responses to oxidative, excitotoxicity and inflammatory injuries and this attenuation of neuroinflammation was achieved via modulation of NF-κB pathway. Our results support the hypothesis that these small molecules, derived from dietary (poly)phenols may cross the BBB, reach brain cells, modulate microglia-mediated inflammation and exert neuroprotective effects, with potential for alleviation of neurodegenerative diseases.

Figure 1

Blood-brain barrier transport of human bioavailable (poly)phenol metabolites. (a) Schematic experimental design used to assess (poly)phenol metabolites transport across the BBB. (b) Endothelial transport of human bioavailable polyphenol metabolites after 2 h of incubation. Endothelial transport was evaluated by LC-Orbitrap MS and is presented as percentage (%) determined by the ratio of the lower compartment concentration and the sum of the upper and lower compartments concentrations. Statistical differences for p < 0.01 are denoted from a-f. (c–e) Immunofluorescence detection of major efflux transporters in HBMEC line: (c) P-gp, in green, (d) MRP1, in red and (e) BCRP, in green. Nuclei stained with DAPI (blue). Scale bar: 40 µm. (f–h) HBMEC intracellular accumulation of specific efflux transporters’ substrates in the presence of the respective inhibitors: (f) 1 µM of verapamil (P-gp inhibitor), (g) 1 µM MK-571 (MRP1 inhibitor) or (h) 1 µM of Ko 143 (BCRP inhibitor). Statistical differences are denoted as ***p < 0.001, **p < 0.01 and *p < 0.05 relatively to control cells. Endothelial transport of (i) Cat-sulf and (j) Pyr-sulf when co-incubated with efflux transporters inhibitors. Statistical differences in the presence of inhibitors are denoted as *p < 0.05 relatively to “No inhibitor”. (k) P-gp substrate accumulation for Cat-sulf and Pyr-sulf compared with verapamil. Intracellular accumulation of P-gp substrate, Rhodamine 123 was evaluated after pre-incubation of cells with the bioavailable (poly)phenol metabolites. Statistical differences are denoted as ***p < 0.001 relatively to control. (l) Endothelial transport of Cat-sulf (solid line) and Pyr-sulf (dashed line) along time. Statistical differences along time are denoted as ***p < 0.001, relatively to 2 h of incubation in Cat-sulf, or ^###p < 0.001, relatively to 2 h of incubation in Pyr-sulf. All values are means ± SD, n=3.

Figure 2

Blood-brain barrier endothelial cells metabolization of Pyr-sulf and Cat-sulf. (a) Putative pathways and enzymes that could be involved in endothelial metabolism into novel phenolic compounds. Proposed metabolization route of the compounds was designed based on canonical enzymatic reactions described in KEGG (Kyoto Encyclopedia of Genes and Genomes). ARS - Arylsulfatase; GST - Glutathione S-transferase; GGT - Gamma glutamyl transferase; AP - Aminopeptidase; Cys-NAT – Cysteine N-acetyl transferase; COMT - Catechol O-methyl transferase; UDPG – UDP-Glucuronosyl transferase. Relative quantification (peak areas) of the novel phenolic metabolites appearing in upper (grey) and lower (black) compartments along time after addition of (b–f) Pyr-sulf or (g–j) Cat-sulf in upper compartment, namely (b) Glutathionyl-pyrogallol, (c) Acetylcysteine-pyrogallol, (d) 2-O-methylcatechol-O-sulfate, (e) Glutathionyl-catechol and (f) Acetylcysteine-2-O-methylcatechol, (g) 2-O-methylcatechol, (h) Catechol-1-O-β-D-glucoronic acid, (i) Glutathionyl-2-O-methylcatechol, and (j) Acetylcysteine-2-O-methylcatechol. Note: the compound Acetylcysteine-2-O-methylcatechol was detected in both samples (panels’ f and j).

Figure 3

Cytoprotective potential of Cat-sulf and Pyr-sulf. (a) HBMEC line submitted to oxidative stress (300 µM H₂O₂); (b) primary mouse cerebellar granule cells exposed to glutamate excitotoxicity (100 µM glutamate); (c) 3D aggregates containing neurons and astrocytes exposed to oxidative injury (300 µM t-BHP). Cells were pre-incubated with 5 µM of each bioavailable polyphenol metabolite for 24 h and then injured with the respective lesion. Cell viability was assessed and is presented as percentage relatively to control. Statistical differences are denoted as ***p < 0.001, **p < 0.01 and *p < 0.05 relatively to control and as ^###p < 0.001, ^##p < 0.01 and ^# p< 0.05 relatively to each lesion (H₂O₂, glutamate or t-BHP). (d-f) Alterations in protein markers of the neuronal (β-III tubulin) and astrocytic (GFAP) population of 3D aggregates towards the t-BHP lesion without and with pre-incubation with idebenone (Ide), a control drug, and with Pyr-sulf. (d) Representative western blot and (e) β-III tubulin and (f) GFAP fold changes in protein levels normalized to GAPDH. Statistical differences are denoted as ***p < 0.001, **p < 0.01 and *p < 0.05 relatively to control and as ^###p < 0.001 relatively to t-BHP. Western blots were analyzed under the same experimental conditions. Data are presented as the means ± SD, n = 3.

Figure 4

Effects on neuroinflammation by Cat-sulf and Pyr-sulf. Pro-inflammatory markers were evaluated, namely (a) TNF-α release, (b) intracellular superoxide production, (c) nitric oxide, and (d) CD40 quantified in N9 microglial cells. Cells were pre-incubated for 6 h with each of the bioavailable (poly)phenol metabolite and then challenged with 300ng/mL of LPS. Statistical differences are denoted as ***p < 0.001, **p < 0.01 and *p < 0.05 relatively to lesion (LPS). (e) Microglial NF-κB p65 translocation into the nucleus after 60 minutes of LPS stimulation. Cells were pre-treated with Cat-sulf or Pyr-sulf for 6 h before LPS-stimulation. NF-κB (red); Nuclei (blue) stained with DAPI. Each capture is representative of at least 3 independent biological replicates. Scale bar: 10 µm. (f–i) Microglial NF-κB p65 phosphorylation ratio and IκBα fold change in protein levels. (f) IkBα protein levels along time after LPS stimulation and (g) after 60 min of LPS stimulation with representative western blots. (h) NF-κB activation profile along time after LPS stimulation looking at NF-κB p65 phosphorylation (ser536) ratio along time after LPS stimulation and (i)after 60 min of LPS stimulation with representative western blots. Cells were pre-treated either with Pyr-sulf or Cat-sulf before LPS stimulation. Control cells (white triangles, solid line), LPS-stimulated cells (black triangles, solid line), cells treated with Cat-sulf prior to LPS stimulation (black circles, dashed line), cells treated with Pyr-sulf prior to LPS stimulation (black squares, dotted line). Statistical differences are denoted as *p < 0.05 and **p < 0.01 relatively to lesion (LPS). Western blots were analyzed under the same experimental conditions. Data are presented as the means ± SD, n = 3.