Metformin Alleviated Aβ-Induced Apoptosis via the Suppression of JNK MAPK Signaling Pathway in Cultured Hippocampal Neurons

Abstract

Both diabetes and hyperinsulinemia are confirmed risk factors for Alzheimer's disease. Some researchers proposed that antidiabetic drugs may be used as disease-modifying therapies, such as metformin and thiazolidinediones, although more evidence was poorly supported. The aim of the current study is to investigate the role of metformin in Aβ-induced cytotoxicity and explore the underlying mechanisms. First, the experimental results show that metformin salvaged the neurons exposed to Aβ in a concentration-dependent manner with MTT and LDH assay. Further, the phosphorylation levels of JNK, ERK1/2, and p38 MAPK were measured with western blot analysis. It was investigated that Aβ increased phospho-JNK significantly but had no effect on phospho-p38 MAPK and phospho-ERK1/2. Metformin decreased hyperphosphorylated JNK induced by Aβ; however, the protection of metformin against Aβ was blocked when anisomycin, the activator of JNK, was added to the medium, indicating that metformin performed its protection against Aβ in a JNK-dependent way. In addition, it was observed that metformin protected the neurons via the suppression of apoptosis. Taken together, our findings demonstrate that metformin may have a positive effect on Aβ-induced cytotoxicity, which provides a preclinical strategy against AD for elders with diabetes.

Figure 1

Concentration-dependent cytotoxicity of cultured hippocampal neurons induced by exposure under different Aβ concentrations from 20 to 400 μM. Metformin alleviated Aβ-induced cytotoxicity also in a dose-dependent manner. (a) and (b) Quantitative analysis of MTT and LDH release assay showed that Aβ decreased cell viability from 50 to 200 μM (^∗ p < 0.05, ^∗∗ p < 0.01 versus that of control, n = 6 per group). (c) and (d) Quantitative analysis of MTT and LDH release assay represented that metformin pretreatment decreased injuries induced by 200 μM Aβ in a concentration-dependent way. As shown in (c) and (d), 100 mM metformin can increase the neuronal viability distinctively.

Figure 2

Metformin rescued Aβ- (200 μM) induced hyperphosphorylation of MAPK JNK. (a), (b), and (c) Quantitative analysis of western blot assay showed that Aβ increased the phosphorylation level of JNK, which was reserved by metformin (^∗ p < 0.05, n = 5), while there was no significant difference in the phosphorylation level of ERK1/2 or P38 between the experimental groups (p > 0.05, n = 5).

Figure 3

Metformin salvaged Aβ-induced cytotoxicity via MAPK JNK signaling pathway. (a) and (b) Quantitative analysis of MTT and LDH represented that Aβ increased the phosphorylation of JNK, which could be reserved by metformin treatment (^∗ p < 0.05, n = 5). In addition, anisomycin treatment blocked metformin-involved protection, suggesting the depression of JNK had a vital role in the effect of metformin.

Figure 4

Metformin decreased Aβ-induced cytotoxicity by suppression of apoptosis. Representative results of JC-10 staining. Quantitative analysis of fluorescence intensity showed that Aβ increased the phosphorylation level of JNK, which was reserved by metformin (^∗ p < 0.05, n = 5). However, when β-lapachone, the activator of apoptosis, was added into the medium, the metformin-involved protection was blocked, indicating metformin played its role via decreasing apoptosis; scale bars, 50 μm.