Protective Effect of Resveratrol Against Corticosterone-induced Neurotoxicity in PC12 Cells

Abstract Objective Resveratrol(RES) is a natural polyphenol which possesses an anti-depressant effect. However, the mechanisms of its anti-depressant effect remain unclear. The aim of the study is to investigate the potential mechanisms in the neuro-protective efficiency in the corticosterone-induced pheochromacytoma 12 (PC12) cells. Methods PC12 cells were treated with 200 μM of corticosterone in the absence or presence of different concentrations of RES for 24 h. Then, cell viability was measured by Cell Counting Kit-8 assay. Apoptosis of PC12 cells was measured by Annexin V-FITC and Propidium iodide (PI) labelling. The expression of apoptosis-related proteins including Bax, Bcl-2, caspase-3 was determined by western blotting. Results The results showed that treatment with 200 μM of corticosterone induced cytotoxicity in PC12 cells. However, different concentrations of RES (2.5μmol/L, 5μmol/L and 10 μmol/L) significantly increased the cell viability, suppressed the apoptosis of PC12 cells, down-regulated Bax and caspase-3 protein expression, and up-regulated Bcl-2 protein expression, compared to the model group (p<0.05). Conclusion Resveratrol has a protective effect on corticosterone-induced neurotoxicity in PC12 cells, which may be related to the apoptosis via inhibition of apoptosis-related proteins and displays the antidepressant-like effect.


Introduction
Depression is one of the most common mental disorders, more than 300 million people worldwide suffering from depression.
Depression leads to a decline in social ability and causes severe burden [1,2]. The pathogenesis of depression has not been fully elucidated. Some acceptable mechanisms include the monoamine neurotransmitter hypothesis, the hypothalamic-pituitaryadrenal (HPA) axis activation hypothesis, nerve-inflammation hypothesis, cytokine hypothesis [3][4][5]. Antidepressant drugs such as serotonin reuptake inhibitors can inhibit the activation of HPA axis [6][7]. However, long-term use antidepressant can cause lots of side effects. The discovery of new antidepressants with high efficiency and low toxicity is one of the important research topics in psychopharmacological field. Resveratrol, known as trans-3,4,5-trihydroxy-stilbene, is a kind of biological polyphenols, mainly derived from peanuts, grapes and mulberry [8][9][10][11].
Researches have been shown that RES could improve depression behavior [12][13][14][15][16]. The mechanisms may associate with the regulation of brain derived neurotrophic fatcor, HPA axis, an increase of 5-HT, reduction of inflammatory factors, neurons protection [17][18][19][20].However, the exact mechanism is still unknown. The PC12 cell line is a popular cell model which is common use in a variety of studies.
It exerts typical neuron-like properties and produces glucocorticoid receptors [21]. It also has been shown that high concentrations of corticosterone can induce cellular damage of PC12 cells [22,23]. Further, the antidepressants have been demonstrated to protect against cytotoxicity induced by corticosterone in PC12 cells [24]. Research also showed that cytoprotective effect is a common action pathway for antidepressants [25].
Thurs, in the present study, PC12 cell line was first applied to investigate the neuroprotective effect of RES and its potential mechanisms.  Statistical analysis SPSS 20.0 statistical analysis software package was used to process the data. Data were expressed as means ± SD, and multiple group comparisons were performed using t-test. p < 0.05 was considered to indicate a statistically significant difference.

RES attenuates cell apoptosis of corticosterone-induced PC12 cells
In terms of exploring the effects of RES on cell apoptosis in corticosterone-induced PC12 cells, Annexin V-FITC, PI and flow cytometry

Discussion
Depression is a mood disorder characterized by persistently feelings of low self-esteem, pessimism,and despair. About 15% of patients with depression commit suicide.
The pathogenesis of depression has not been fully elucidated until now. The currently accepted mechanisms include a mono amine neurotransmitter hypothesis, the HPA axis activation hypothesis, nerve-inflammation hypothesis, cytokine hypothesis, neural plasticity reduction hypothesis, the limbic system loop hypothesis, etc. The activation of HPA axis hypothesis considered that patients with depression activated pathological HPA axis excessively, which was closely associated with suicidal behavior of depression [27].
Antidepressant drugs such as serotonin reuptake inhibitors can inhibit the activation of HPA axis [28][29].
In the study, it is found that RES increased the cell viability, reduced apoptosis, and relieved the neurotoxicity of corticosterone on mitochondria into cytoplasm which activate the caspase-related apoptosis cascade, resulting in mitochondrial-dependent apoptosis [30][31][32][33].
In order to further explore the molecular mechanism of RES in inhibiting corticosteroneinduced PC12 cell apoptosis, Bcl-2, Bax and caspase-3 proteins were detected by western blot in this study. The results showed that the concentrations of resveratrol range from 2.5 to10 μmol/L inhibit apoptosis, increase Bcl-2 protein expression, reduce Bax and Caspase 3 protein expression, which indicated that the regulation of RES on neuron apoptosis is mainly by increasing the ratio of Bcl-2 / Bax and inhibiting the activation of Caspase pathway.
We investigate the potential cytoprotective mechanism of RES. As a small molecule, RES is expected to pass the plasma membrane and accumulate within cells. When PC12 cells were stimulated by corticosterone, a large amount of reactive oxygen species (ROS) was produced. These excessive ROS would damage the mitochondria and cytomembrane, leading to apoptosis. Due to the antioxidant effect, RES suppressed oxidative stress, reduced the content of ROS. Moreover, we also found that RES could inhibit mitochondrial apoptotic pathways by reducing Bax and caspase-3 expression and increasing Bcl-2 expression. The results of our study showed that RES is closely related to mitochondrial pathway, but whether it is associated with other regulation pathways still needs further investigations.
In the study, we first demonstrated that RES exerts neuroprotective effect in the corticosterone-induced PC12 cells and its mechanism may be related to the apoptosisrelated proteins.