Injury of cortical neurons is caused by the advanced glycation end products-mediated pathway

Abstract

Advanced glycation end products lead to cell apoptosis, and cause cell death by increasing endoplasmic reticulum stress. Advanced glycation end products alone may also directly cause damage to tissues and cells, but the precise mechanism remains unknown. This study used primary cultures of rat cerebral cortex neurons, and treated cells with different concentrations of glycation end products (50, 100, 200, 400 mg/L), and with an antibody for the receptor of advanced glycation end products before and after treatment with advanced glycation end products. The results showed that with increasing concentrations of glycation end products, free radical content increased in neurons, and the number of apoptotic cells increased in a dose-dependent manner. Before and after treatment of advanced glycation end products, the addition of the antibody against advanced glycation end-products markedly reduced hydroxyl free radicals, malondialdehyde levels, and inhibited cell apoptosis. This result indicated that the antibody for receptor of advanced glycation end-products in neurons from the rat cerebral cortex can reduce glycation end product-induced oxidative stress damage by suppressing glycation end product receptors. Overall, our study confirms that the advanced glycation end products-advanced glycation end products receptor pathway may be the main signaling pathway leading to neuronal damage.

Keywords: advanced glycation end products; advanced glycation end products receptor; antibody; apoptosis; brain injury; cortical neurons; neural regeneration; neuroregeneration; oxidative stress; oxidative stress injury; pathway.

Figure 1

Morphology of neurons in the rat cerebral cortex following primary culture. (A) Cortical neurons cultured for 2 days: adherent cells appeared plump (black arrow, × 100). (B) Cortical neurons cultured for 7 days: long and dense axons formed networks (black arrow, × 100). (C) Cy3-neuron-specific enolase fluorescent staining: neuronal bodies appeared plump, with the presence of processes, which interacted and formed a network as seen by confocal microscopy (white arrow, × 600).

Figure 2

Effect of receptor of advanced glycation end-products antibody (Ab-RAGE) on apoptosis of neurons in the rat cerebral cortex after intervention with advanced glycation end products (AGEs). Nuclei were blue after Hoechst fluorescence staining. (A) AGEs direct action group: as shown by arrows. Apoptotic nuclei were darkly stained, with the presence of chromatin margination. (B) Ab-RAGE pretreatment group: decreased number of apoptotic cells; arrow exhibits slight changes in neuronal morphology. (A, B) Hoechst fluorescent staining (confocal microscopy, × 600). (C) The number of apoptotic cells with increasing concentration of AGEs. Neuronal apoptosis number in the AGEs direct action group and Ab-RAGE pretreatment group increased gradually. ^a P < 0.05, vs. bovine serum albumin (BSA) control group; ^b P < 0.01, vs. AGEs direct action group. Mean ± SD, n = 3, one-way analysis of variance and two sample t-test.

Figure 3

Inhibition of hydroxyl free radicals after pretreatment and posttreatment with antibody of receptor of advanced glycation end-products (Ab-RAGE). Results show that the ability of inhibiting hydroxyl free radicals was strongest in the Ab-RAGE pretreatment group, followed by Ab-RAGE posttreatment group. The ability was weakest in the advanced glycation end products (AGEs) direct action group.

Figure 4

Changes in the ability to inhibit hydroxyl free radicals after pretreatment and posttreatment with receptor of advanced glycation end-products antibody (Ab-RAGE). With increasing concentrations of advanced glycation end-products (AGEs), the ability to inhibit hydroxyl free radicals gradually reduced in each group. ^a P < 0.05, vs. bovine serum albumin (BSA) control group; ^bP < 0.05, vs. Ab-RAGE pretreatment group; ^c P < 0.05, vs. AGEs direct action group; mean ± SD, n = 3, one-way analysis of variance and two sample t-test.

Figure 5

Malondialdehyde content in cells after pretreatment and posttreatment with antibody of receptor of advanced glycation end-products (Ab-RAGE). Malondialdehyde content was highest in the advanced glycation end products (AGEs) direct action group, followed by Ab-RAGE posttreatment group. Malondialdehyde content was lowest in the Ab-RAGE pretreatment group.

Figure 6

Changes in malondialdehyde content in cells after pretreatment and posttreatment with antibody of receptor of advanced glycation end-products (Ab-RAGE). ^a P < 0.05, vs. bovine serum albumin (BSA) control group; ^bP < 0.05, vs. Ab-RAGE pretreatment group; ^c P < 0.05, vs. AGEs direct action group; mean ± SD, n = 3, one-way analysis of variance and two sample t-test.