Pomegranate Extract Administration Reverses Loss of Motor Coordination and Prevents Oxidative Stress in Cerebellum of Aging Mice

Abstract

The cerebellum is responsible for complex motor functions, like maintaining balance and stance, coordination of voluntary movements, motor learning, and cognitive tasks. During aging, most of these functions deteriorate, which results in falls and accidents. The aim of this work was to elucidate the effect of a standardized pomegranate extract during four months of supplementation in elderly mice to prevent frailty and improve the oxidative state. Male C57Bl/6J eighteen-month-old mice were evaluated for frailty using the "Valencia Score" at pre-supplementation and post-supplementation periods. We analyzed lipid peroxidation in the cerebellum and brain cortex and the glutathione redox status in peripheral blood. In addition, a set of aging-related genes in cerebellum and apoptosis biomarkers was measured via real-time polymerase chain reaction (RT-PCR). Our results showed that pomegranate extract supplementation improved the motor skills of C57Bl/6J aged mice in motor coordination, neuromuscular function, and monthly weight loss, but no changes in grip strength and endurance were found. Furthermore, pomegranate extract reversed the increase in malondialdehyde due to aging in the cerebellum and increased the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio in the blood. Finally, aging and apoptosis biomarkers improved in aged mice supplemented with pomegranate extract in the cerebellum but not in the cerebral cortex.

Keywords: aging; cerebellum; frailty; motor coordination; oxidative stress; pomegranate extract.

Figure 1

Effect of PE administration on glutathione redox status in mice’s blood. The figure shows (A) GSH, (B) GSSG, and (C) GSH/GSSG levels in blood in mice after 18 months, 22 months, and 22 months of supplementation with PE. Results are expressed as mean ± SEM (n = 5–6 per group). * p < 0.05, ** p < 0.01.

Figure 2

Effect of PE supplementation on functional studies. (A) Maximum force on the grip strength test; (B) Running time on treadmill; (C) Running speed on treadmill; (D) Motor coordination on the rotarod test; (E) Sensory motor evaluation on the ladder-climbing test. Results are expressed as mean ± SD (n = 8–9 per group). * p < 0.05, ** p < 0.01 and *** p < 0.005. (ns: not significant).

Figure 3

Effect of PE supplementation on weight loss. Data regarding weight loss are expressed as percentage of weight loss after 4 months of supplementation with PE. Results are expressed as mean ± SEM (n = 8–9 per group). * p < 0.05.

Figure 4

Effect of PE on lipid peroxidation. MDA levels (µmol/mg protein) were measured by HPLC in cerebellum homogenates (A) and in brain cortex homogenates (B) of mice at 10 and 22 months of age. Results are expressed as mean ± SEM (n = 4–7 per group). * p < 0.05. (ns: not significant).

Figure 5

The effect of PE supplementation on the expression of aging biomarkers in cerebellum. (A) C1qa, (B) Ctss, (C) Lzp-s, (D) Gfap, (E) C4b. Results are expressed as mean ± SEM (n = 7–10 per group). * p < 0.05, ** p < 0.01.

Figure 6

The effect of pomegranate extract supplementation on the expression of aging biomarkers in cortex. (A) C1qa, (B) Ctss, (C) Lzp-s, (D) Gfap, (E) C4b. Results are expressed as mean ± SEM (n = 5–6 per group). * p < 0.05, ** p < 0.01.

Figure 7

The effect of PE supplementation on the expression of B2m in cerebellum (A) and brain cortex (B). Results are expressed as mean ± SEM (n = 5–6 per group). * p < 0.05.

Figure 8

Connections between cell processes and six aging biomarkers. The solid line means expression, and the dashed line means regulation. Relations are colored by effect. Red represents a negative effect, and green represents a positive effect.

Figure 9

The effect of PE supplementation on the expression of apoptosis biomarkers in cerebellum and cortex. (A) Casp3, (B) Casp8, and (C) Casp9 in cerebellum and (D) Casp3, (E) Casp8, and (F) Casp9 in cortex. Results are expressed as mean ± SEM (n = 6–8 per group). * p < 0.05, ** p < 0.01. (ns: not significant).