Blood brain barrier permeability of (-)-epigallocatechin gallate, its proliferation-enhancing activity of human neuroblastoma SH-SY5Y cells, and its preventive effect on age-related cognitive dysfunction in mice

Abstract

Background: The consumption of green tea catechins (GTCs) suppresses age-related cognitive dysfunction in mice. GTCs are composed of several catechins, of which epigallocatechin gallate (EGCG) is the most abundant, followed by epigallocatechin (EGC). Orally ingested EGCG is hydrolyzed by intestinal biota to EGC and gallic acid (GA). To understand the mechanism of action of GTCs on the brain, their permeability of the blood brain barrier (BBB) as well as their effects on cognitive function in mice and on nerve cell proliferation in vitro were examined.

Methods: The BBB permeability of EGCG, EGC and GA was examined using a BBB model kit. SAMP10, a mouse model of brain senescence, was used to test cognitive function in vivo. Human neuroblastoma SH-SY5Y cells were used to test nerve cell proliferation and differentiation.

Results: The in vitro BBB permeability (%, in 30 min) of EGCG, EGC and GA was 2.8±0.1, 3.4±0.3 and 6.5±0.6, respectively. The permeability of EGCG into the BBB indicates that EGCG reached the brain parenchyma even at a very low concentration. The learning ability of SAMP10 mice that ingested EGCG (20 mg/kg) was significantly higher than of mice that ingested EGC or GA. However, combined ingestion of EGC and GA showed a significant improvement comparable to EGCG. SH-SY5Y cell growth was significantly enhanced by 0.05 µM EGCG, but this effect was reduced at higher concentrations. The effect of EGC and GA was lower than that of EGCG at 0.05 µM. Co-administration of EGC and GA increased neurite length more than EGC or GA alone.

Conclusion: Cognitive dysfunction in mice is suppressed after ingesting GTCs when a low concentration of EGCG is incorporated into the brain parenchyma via the BBB. Nerve cell proliferation/differentiation was enhanced by a low concentration of EGCG. Furthermore, the additive effect of EGC and GA suggests that EGCG sustains a preventive effect after the hydrolysis to EGC and GA.

Keywords: (−)-epigallocatechin gallate; 8-oxodG, 8-oxodeoxyguanosine; BBB, blood-brain barrier; Blood-brain barrier permeability; Brain plasticity; C, (+)-catechin; Cognitive dysfunction; EC, (−)-epicatechin; EGC, (−)-epigallocatechin; EGCG, (−)-epigallocatechin gallate; GA, gallic acid; GTC, green tea catechin; Green tea catechin; LC-MS/MS, liquid chromatography tandem-mass spectrometry; LPO, lipid peroxidation; MRM, multiple reaction-monitoring; Nerve cell proliferation; SAMP10, senescence-accelerated mouse prone 10..

Graphical abstract

Fig. 1

Effect of GTCs, EGCG, EGC and GA on learning in aged SAMP10 mice. The learning time of SAMP10 mice was examined using a step-through test system. The time needed to acquire the avoidance response was measured in mice that ingested GTCs, EGCG, EGC and GA (black or gray columns) and in control mice (open column). Each value represents the mean±SEM (n=8–23). Asterisks represent a significant difference (* p<0.05, Bonferroni's t-test).

Fig. 2

Effect of EGCG, EGC and GA on cell growth of human SH-SY5Y neuroblastoma cells. A cell suspension (5×10⁴ cells/well) was plated in a 24-well plate. EGCG, EGC and GA dissolved in 0.01% DMSO were added to the culture medium to make the final concentration of 0.01–1.0 µM, and cultured for 48 h at 37 °C. The number of cells treated with EGCG (a), EGC (b), GA (c), and EGC and GA combined (d) are shown. Each value represents the mean±SEM. Asterisks represent a significant difference (* p<0.05, Bonferroni's t-test).

Fig. 3

Effect of EGCG, EGC and GA on neurite outgrowth of human SH-SY5Y neuroblastoma cells. A cell suspension (2.5×10⁴ cells/well) was plated in a 24-well plate. EGCG, EGC and GA dissolved in 0.01% DMSO were added to the culture medium to make a final concentration of 0.05 µM and cultured for 72 h at 37 °C. Photos of control cells (a), and cells treated with EGC (b), GA (c), EGC+GA (d), or EGCG (e). Neurite length (f) and neurite number (g) of cells treated with catechins. Scale bar is 50 µm. Each value represents the mean±SEM. Asterisks and # represent significant differences with the control (*) and with EGC+GA (#) (p<0.05, Bonferroni's t-test).

Fig. 4

Levels of oxidative damage in the brain. LPO (a), and 8-oxodG (b) in the brain. Each value represents the mean±SEM (n=4). Asterisks represent a significant difference (* p<0.05, Bonferroni's t-test).