Abstract
Insulin is one of the most promising neuroprotectors. A significant gap in understanding the mechanism of its action is the lack of data on whether it is able to prevent autophagic neuronal death. The aim of our work was to evaluate the contribution of autophagy and apoptosis to the death of rat cerebral cortex neurons in culture under oxidative stress and to study the ability of insulin to prevent this death and inhibit autophagy and apoptosis in neurons. The influence of hydrogen peroxide and insulin on the level of two main autophagy markers (LC3B-II and SQSTM1/p62) and apoptosis marker (cleaved сaspase-3) was studied. To assess the viability of neurons, the MTT test was used, and Western blotting was applied to measure the level of marker proteins. It was found that oxidative stress caused the activation of autophagy and apoptosis in neurons. This is manifested in a significant increase of the autophagy marker LC3B-II and apoptosis marker (cleaved сaspase-3) and in a decrease in the SQSTM1/p62 protein level. The content of SQSTM1/p62, which is involved in the formation of autophagosomes, decreases with the activation of autophagy, as this protein is degraded in lysosomes. Hydrogen peroxide causes autophagic and apoptotic death of neurons, as the inhibitors of autophagy (3-methyl adenine) and apoptosis (z-DEVD-FMK) were shown to increase the viability of neurons in conditions of oxidative stress. Insulin, in its turn, prevents the death of neurons and hinders autophagy, causing a decrease of the level of lipidated form LC3B-II and the increase of the SQSTM1/p62 protein level, it hinders apoptosis as well decreasing the level of cleaved caspase-3. The protective effect of insulin is mediated by the activation of specific signaling pathways associated with receptors of insulin and IGF-1, as the inhibitor of these receptors BMS-754807 completely blocks the neuroprotective effect of insulin. Thus, the pronounced activation of autophagy under oxidative stress is one of the causes of neuron death, and the protection of neurons by insulin is associated with the suppression of not only apoptotic, but also autophagic cell death.
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