The pathogenesis of diabetic central neuropathy is not completely clear. The high fatty acids, high blood glucose and abnormal increase of glycation end products caused by hyperglycemia induce oxidative stress. Excess produced or insufficient eliminated ROS will directly cause biological membrane lipid peroxidation, protein and enzyme degeneration, mitochondrial dysfunction, which lead to neuronal cell damage, scabbard film, axon swelling degeneration and even broke off, neurons chromatin dissolved, cytoplasm vacuoles degeneration necrosis, mitochondrial dysfunction, nuclear pyknosis and nerve cells apoptosis (2). Abnormal changes in brain electrophysiology, imaging and pathology caused by diabetic metabolic factors such as glucose and lipid metabolism disorder, insulin resistance, vascular disease and obesity are closely related to the occurrence and development of DE.
Melatonin is a strong antioxidant, with small molecular volume and amphiphilic nature, which is very easy to enter the cells, especially the mitochondria, and participate in mitochondrial energy metabolism to reduce mitochondrial damage caused by oxidative stress (11-13). Relevant studies have shown that melatonin can directly interact with electrons or induce the production of antioxidant enzymes to reduce the damage of oxidative respiratory chain, enhance mitochondrial function and reduce oxidative stress even reduce cell apoptosis (11). In addition, melatonin can help restore the function of islet B cells, improve insulin sensitivity and glucose tolerance, reduce hyperinsulinemia, and reduce insulin resistance. Melatonin may be a potential diabetes treatment drug.
In the hippocampus and cortex of diabetic rats, lipid peroxides increased, and rats showed obvious cognitive impairment. However, these manifestations were significantly reduced or even disappeared after the treatment with melatonin or antioxidants vitamin E (14, 15). Melatonin can also alleviate peripheral neuralgia caused by diabetes and improve diabetic neuropathy (16). In addition, melatonin has neuroprotective effects. Patients suffered from AD showed a reduction in secretion of melatonin, which slowed down the synthesis of the protein of Aβ protein and delay the progression of this disease (17). Activating the receptor of melatonin presented a protective effect on spinal cord injury (18). In addition, melatonin showed satisfactory neuroprotection and antioxidation effects on cultured primary cortical neurons deprived of oxygen and sugar, and animal brain injury induced by cerebral ischemic stroke (19). In this study, we found that melatonin could improve the survival rate of cortical neurons and reduce the apoptosis rate of neurons in fetal rats under high-glucose environment, indicating that melatonin could protect neurons in cortex of fetal rats under high glucose environment.
The cerebral cortex is an important anatomical basis for learning and memory and it is vulnerable to various adverse factors. The thickness and volume of cerebral cortex were significantly reduced in diabetic patients, which is the structural basis for learning and memory impairment in diabetic patients (20). High glucose environment can cause apoptosis of cortex and hippocampal neurons (21-22). Bax and Bcl-2 family are very important apoptotic regulatory genes. Bax is apoptosis promoting gene, and Bcl-2 is apoptosis inhibiting gene. In addition, Bcl-2 protein family can not only regulate cell apoptosis but also affect other cell processes, such as cell cycle, calcium signaling, glucose stability, autophagy and etc, which play an important role in cell survival and death (23). Caspase-3 is the main executor of apoptosis, and activation of caspase-3 can cause downstream apoptosis cascade reaction. In this study, we found that the high glucose environment can lead to apoptosis of cortical neurons, and regulated the expression of Bax, Caspase-3 and Bcl-2, and the hypertonic state of mannitol had no effect on neuronal apoptosis and protein levels, suggesting that it is high glucose rather than hyperosmosis directly leads to apoptosis and the changes of relevant protein expression in neurons, Melatonin can reverse the change of Bax, caspase-3 and bcl-2 proteins in cortical neurons induced by high-glucose environment and reduce neuron apoptosis.
Autophagy is an important degradation pathway for phagocytosis of aging proteins or organelles in the cytoplasm, which is degraded by autophagy lysosome to complete the metabolic needs of the body and the renewal of aging organelles (24).. Different from other cells, neurons don't have the function of re-division, and can't dilute harmful substances in cells by way of division. Therefore, the autophagic lysosome degradation pathway is particularly important in neurons (3). At present, studies have found that the abnormal autophagy function plays an important role in the occurrence and development of nervous system diseases, such as PD and HD. The regulation of autophagy activity can delay the progress of the disease through devouring damaged mitochondria, reducing oxidative stress and inhibiting apoptosis (4, 5). The excessive activation of autophagy can promote apoptosis. In addition, autophagy and apoptosis can exist simultaneously and interact with each other (25). Inhibition of autophagy activity in cerebral ischemia reperfusion rats can improve their neural function (26). At present, there are few studies on autophagy in diabetic encephalopathy, and changes in autophagy activity of cells may be one of the important mechanisms of diabetic encephalopathy (7, 27, 28). Many studies have shown that the neuron apoptosis and cognitive decline in diabetic rats are related to the excessive activation of autophagy, and inhibition of autophagy activity can improve the cognitive function of diabetic mice (7, 27, 28). Diabetic retinopathy and diabetic hearing impairment were associated with elevated levels of autophagy in diabetic nerve cells (6, 29). This study showed that the expression of Beclin-1 and LC3B, two landmark molecules of autophagy, could be increased in high glucose environment, suggesting that high glucose environment could induce autophagy. Melatonin can decrease Beclin-1 and LC3B in high glucose environment, and reduce the apoptosis rate of cortical neurons. It suggests that melatonin can protect cortical neurons by down-regulating autophagy activity. This study is similar to the results of other studies (7, 27, 28). However, some studies showed that the autophagy activity of neurons in diabetic neuropathy and diabetic rats with microvascular disease decreases, and the activation of autophagy can reduce the damage of neurons (30-32). We believe that the differences in these findings are related to the degree of autophagy activation. Moderate autophagy activation has neuroprotective effects, but excessive autophagy activation can induce programmed cell death.
Akt/mTOR signaling pathway is an important mechanism for the negative regulation of autophagy. Bcl-2 inhibits autophagy initiation by binding to Beclin-1 in the structural domain of BH3 protein, and mTOR is required to participate in the binding of Beclin1 to BH3 protein. Activation of mTOR directly inhibits autophagic initiation, and mTOR regulates autophagic related protein phosphorylation and thus blocks autophagy (33-35). Studies found that diabetic cognitive dysfunction is related to mTOR signaling pathway, and activation of PI3K/Akt/mTOR signaling pathway can improve nerve defects in diabetic cerebral ischemia reperfusion rats and hyperglycemia induced neurotoxicity of PC-12 cells (22, 36). In addition, activation of Akt/mTOR signaling pathway can promote the growth of posterior axons and improve the functional recovery after stroke (37). It also found that melatonin can regulate AMPK/mTOR signaling pathway and play a protective role in myocardial ischemia reperfusion (13), reduce the neuron apoptosis induced by focal cerebral ischemia in mice via activating the PI3K/Akt signaling pathway (38), and alleviate nerve defects caused by middle cerebral artery occlusion in adult male rats through activating Akt/mTOR signaling pathway (39). Therefore, we believe that melatonin can regulate Akt/mTOR signaling pathway, which has also been demonstrated in this study. In this study, we found that high glucose environment inhibited the Akt/mTOR signaling pathway of cortical neurons, reduced the expression of Bcl-2, and over-activated the autophagy level of cortical neurons. Melatonin can activate Akt/mTOR pathway, up-regulate the expression of Bcl-2, inhibit the over-activation of autophagy and show neuroprotective effects.
To sum up, melatonin can reduce the apoptosis of cortical neurons under the high-sugar environment by activating Akt/mTOR pathway and following the down-regulation of autophagy, which has a neuroprotective effect.