Metformin alleviates neurocognitive impairment in aging via activation of AMPK/BDNF/PI3K pathway

Abstract

Slowing down age-related neurocognitive impairment has been a challenge. We evaluated the therapeutic effects of metformin in D-galactose-induced aging. Additionally, we studied the potential molecular mechanisms that could be responsible for metformin's anti-aging effects. Thirty male rats were equally divided into: 1-control group, which received saline solution, 2-D-galactose (D-gal) group, which received D-galactose (100 mg/kg/day) by gastric lavage for eight weeks, and 3-D-galactose + Metformin (D-gal + Met) treated group, which received D-galactose + metformin (200 mg/kg/day) by gastric lavage for eight weeks. Neurocognitive assessment was done. Measurement of inflammatory, oxidative stress, and BDNF biomarkers was performed. AMPK and PI3K genes expression were assessed. Hippocampal tissues were dissected for histopathological and immunohistochemical studies. D-gal resulted in neurocognitive impairments, elevation of inflammatory biomarkers, altered oxidative stress markers, decreased BDNF, decreased expression of synaptophysin and Bcl2 with increased expression of Caspase-3, and down-regulation of AMPK and PI3K genes. Neurodegenerative changes were present in the hippocampus. Metformin restored significantly D-gal induced neurodegenerative changes. We concluded that metformin could alleviate age-induced neurocognitive deficit via amelioration of neuroinflammation, attenuation of oxidative stress, reduction of apoptosis, as well as promotion of synaptic plasticity. These mechanisms could be mediated via the activation of the AMPK/BDNF/PI3K pathway.

Figure 1

Effect of metformin on working memory and preference for novelty in d-galactose-induced aging rat model in novel object test (*significant when compared to control group, ^#significant when compared to D-gal group). Data are shown as means + SD (n = 10). ANOVA was used to make group comparisons; Significance = P < 0.05).

Figure 2

Effect of metformin on D-galactose-induced aging rat model in Morris water maze (*significant when compared to control group, ^#significant when compared to D-gal group. Data are shown as means + SD (n = 10). ANOVA was used to make group comparisons; Significance = P < 0.05).

Figure 3

Effect of metformin on anxiety-like behavior in D-galactose-induced aging rat model in EPM test (*significant when compared to control group, ^#significant when compared to D-gal group). Data are shown as means + SD (n = 10). ANOVA was used to make group comparisons; Significance = P < 0.05).

Figure 4

Metformin ameliorates the oxidative stress and inflammatory status in D-galactose-induced aging rat model (*significant when compared to control group, ^#significant when compared to D-gal group). Data are shown as means + SD (n = 10). ANOVA was used to make group comparisons; Significance = P < 0.05).

Figure 5

Metformin improves brain BDNF level in D-galactose-induced aging rat model. (*significant when compared to control group, ^#significant when compared to D-gal group). Data are shown as means + SD (n = 10). ANOVA was used to make group comparisons; Significance = P < 0.05).

Figure 6

Gene modifying effect of metformin in D-galactose-induced aging rat model (*significant when compared to control group, ^#significant when compared to D-gal group). Data are shown as means + SD (n = 10). ANOVA was used to make group comparisons; Significance = P < 0.05).

Figure 7

Effect of metformin on hippocampal tissue structure in D-galactose induced-aging rat model. (a) Section of the hippocampal tissue in the control group showed unremarkable pathological changes. (b) Section of the hippocampal tissue in the D-gal group showed many neuronal degeneration (green circles), and apoptotic bodies (blue circles) with massive oedema (blue arrows) and gliosis (green arrows). (c) Section of the hippocampal tissue in the D-gal + Met group showed few neuronal degeneration (green circles) and few apoptotic bodies (blue circles) with mild oedema (blue arrows) and mild focal gliosis (green arrows). (H&E × 200 for A, B and C). (Number of rats = 10/group).

Figure 8

Synaptophysin immunostaining in hippocampal tissues of the studied groups. (a) Section of the hippocampal tissue in the control group showed intense cytoplasmic expression of synaptophysin in neural cells. (b) Section of the hippocampal tissue in the D-gal group showed mild cytoplasmic expression of synaptophysin in neural cells. (c) Section of the hippocampal tissue in the D-gal + Met group showed moderate cytoplasmic expression of synaptophysin in neural cells (Synaptophysin × 200 for A, B and C). (d) Represents the percentage of synaptophysin expression in the above-mentioned groups (*significant when compared to control group, ^#significant when compared to D-gal group). Data are shown as means + SD (n = 10). ANOVA was used to make group comparisons; Significance = P < 0.05).

Figure 9

Caspase-3 immunostaining in hippocampal tissues of the studied groups. (a) Section of the hippocampal tissue in the control group showed mild cytoplasmic expression of Casepase-3 in glial and neural cells. (b) Section of the hippocampal tissue in the D-gal group showed marked cytoplasmic expression of synaptophysin in neural cells. (c) Section of the hippocampal tissue in the D-gal + Met group showed moderate nucleocytoplasmic expression of Casepase-3 in neural cells (Casepase-3 × 200 for A, B and C). (d) Represents the percentage of Casepase-3 expression in the above-mentioned groups (*significant when compared to control group, ^#significant when compared to D-gal group). Data are shown as means + SD (n = 10). ANOVA was used to make group comparisons; Significance = P < 0.05).

Figure 10

Bcl-2 immunostaining in hippocampal tissues of the studied groups. (a) Section of the hippocampal tissue in the control group showed intense cytoplasmic expression of Bcl-2 in neural cells. (b) Section of the hippocampal tissue in the D-gal group showed mild focal cytoplasmic expression of Bcl-2 in neural cells. (c) Section of the hippocampal tissue in the D-gal + Met group showed moderate cytoplasmic expression of Bcl-2 in neural cells (Bcl-2 × 200 for A, B and C). (d) Represents the percentage of Bcl-2 expression in the above-mentioned groups (*significant when compared to control group, ^#significant when compared to D-gal group). Data are shown as means + SD (n = 10). ANOVA was used to make group comparisons; significance = P < 0.05).