Involvement Of PI3K/Akt/FoxO3a And PKA/CREB Signaling Pathways In The Protective Effect Of Fluoxetine Against Corticosterone-induced Cytotoxicity In PC12 Cells

Background:Depression is a chronic mental disorder characterized by feelings of sadness, losing of interests, despair, lacking of initiative and hypomnesia. In recent years, a large number of experimental and clinical observations have supported the notion that the hypothalamic-pituitary-adrenal (HPA) axis in depressed individuals is more active than normal. Hyperactivation of the HPA axis may stimulate the secretion of adrenocorticotrophic hormone from the pituitary and subsequently increased level of cortisol, and hippocampal neurons express a high level of glucocorticoid receptors, thus, dysregulation of the HPA axis can result in overproduction of cortisol that binds and activates glucocorticoid receptors leading to damage of hippcampal neurons. According these, protecting hippcampal neurons from damage becomes an important issue for depression therapeutic.There are large number of studies indicate that many antidepressants may mediate a neuroprotective effect against various insults through promoting neurons proliferation, maturation and survival, reducing apoptotic cell death and improving learning and memory. These substantial findings support the protective and pro-survival effects of antidepressants in neuronal cells, and the point of the potential neuroprotective effects of antidepressants has been a new key toward depression. The phosphatidylinositol-3-kinase/protein kinase B/Forkhead box 03 (PI3K/Akt/FoxO3a) pathway is one of the major pro-survival signaling pathways that mediate the effect of many growth factors and other stimuli, plays an important role in protecting hippcampal neurons through effecting downstream target genes, promoting cell survival and inhibiting cell apoptosis. As for PKA/CREB/BDNF signaling pathway, it has been discovered closely related to depression and neuroprotective. PKA stimulated the phosphorylation of CREB, subsequently activates the downstream signaling molecules such as BDNF. BDNF then binding with its receptor TrkB and activates intracellular signaling cascade, thereby promoting neurogenesis and synaptic plasticity.Objective:PC12 cells, which possess typical neuronal features and express a high level of glucocorticoid receptors, have been widely used in various neurological studies. So in our study, we used PC12 cells treated with corticosterone to simulate the damage of hippcampal neurons due to hyperactivation of HPA axis, and used classical antidepressants fluoxetine to investigate if PI3K/Akt/FoxO3a signaling pathway involved in depression. Besides, explore the relationship between PI3K/Akt/FoxO3a signaling pathway and PKA/CREB/BDNF signaling pathway.Methods:1. Using PC12 cells treated with corticosterone to establish a stable cell injury models, and investigate the protective effect of fluoxetine, MTT and Hoechst 33342 staining detect the cell viability and cell state.Firstly, to study the effect of CORT on the viability of PC12 cells, cells were treated with various concentrations of CORT (from 0 to 1000μM) for 24 h and the cell viability was measured by MTT assay; to explore the antagonistic effects of RU486 on corticosterone, PC12 cells were pretreated with RU486 and then corticosterone was added into the medium;Then, to investigate the protective effects of fluoxetine against CORT, cells pretreated with fluoxetine were treated with 500μM CORT and then cell viability and cell state were respectively determined by MTT and Hoechst 33342 staining.2. To see the potential signaling pathways involved in the protection of fluoxetine, cells were treated with H89 (10μM,40 min), LY294002 (50μM,40 min) and KRX-0401 (45μM,40 min) before the treatment of fluoxetine and CORT, and then cell viability was determined.3. To study the temporal effect of fluoxetine on the phosphorylation of Akt, FoxO3a and CREB, cells were treated with fluoxetine (10μM) for 0-40 min or fluoxetine (1-10μM) for 20 min;To investigate whether PKA, PI3K and Akt were involved in fluoxetine-induced phosphorylation of CREB, Akt and FoxO3a, cells were pretreated for 40 min with H89 (10μM,40 min), LY294002 (50μM,40 min) and KRX-0401 (45μM,40 min) prior to stimulation with fluoxetine (10μM) for 20 min, Western blot detect the phosphorylation of Akt,FoxO3a and CREB.4. To study the temporal effect of fluoxetine on the phosphorylation of Akt, FoxO3a and CREB when CORT-induced cell injury, cells were co-treated with fluoxetine (10μM) and CORT for 20 min;Also, in the case of CORT exist, to investigate whether PKA, PI3K and Akt were involved in fluoxetine-induced phosphorylation of CREB, Akt and FoxO3a, cells were pretreated for 40 min with H89 (10μM,40 min), LY294002 (50μM,40 min) and KRX-0401 (45μM,40 min) prior to stimulation with fluoxetine (10μM) and CORT for 20 min, Western blot detect the phosphorylation of Akt, FoxO3a and CREB.5. To study the effect of fluoxetine on the downstream target gene of FoxO3a and CREB, cells were pretreated for 40 min with H89 (10μM,40 min), LY294002 (50μM,40 min) and KRX-0401 (45μM,40 min) prior to stimulation with fluoxetine (10μM) and CORT for 24 h, RT-PCR detect the mRNA levels of Bim、Bax、Puma and BDNF.Results:1. CORT caused cell death in PC12 cells while fluoxetine reversed the effect of CORT.(1) As shown in MTT, CORT induced cellular toxicity in a concentration-dependent manner in PC12 cells, which was significant at 250μM (P<0.05).To establish stable results, we selected 500μM CORT for subsequent studies.(2) When pre-treated with fluoxetine, the cell viability was enhanced as the concentration increased. The significant survival promoting effect of fluoxetine was observed at 3μM (P<0.05), and reached maximal at 10μM (P<0.05). Consistent with these findings in MTT assay, Hoechst staining observed through microscopy showed that CORT caused nuclear condensation and fragmentation while fluoxetine prevented the cell apoptosis induced by CORT.(3) Glucocorticoid receptor antagonist RU486 antagonized the neurotoxicity of CORT concentration dependently, which was significant at 1μM (P<0.05), indicated that the glucocorticoid receptor was involved in the effect of corticosterone.2. Effects of PI3K/Akt and PKA signal pathways on the protective effect of fluoxetine against CORT.As shown above, fluoxetine produced an apparent neuroprotection against CORT, we then assessed the role of these two pathways in the survival promoting effect of fluoxetine in PC12 cells. Cells were pre-treated with the PKA inhibitor H89, PI3K inhibitor LY294002 and the Akt inhibitor KRX-0401 then stimulated with fluoxetine (10μM) and CORT (500μM) for 24 h. The protective effects of fluoxetine against cell death induced by CORT was attenuated by the pretreatment of LY294002, KRX-0401 and H89 (P<0.05), suggesting the involvement of both PKA pathway and PI3K/Akt pathways in the protective effect of fluoxetine.3. Fluoxetine stimulated the phosphorylation of Akt, FoxO3a and CREB in PC12 cells through PI3K/Akt/FoxO3a signaling pathway and PKA/CREB signaling pathway respectively.(1) Fluoxetine time (P<0.001) and concentration-dependently (P<0.001) stimulated the phosphorylation of Akt at Ser473, FoxO3a at Ser253 and CREB at Ser133 in PC12 cells.(2) As results shown in above, LY294002, KRX-0401 or H89 blocked the protective effect of fluoxetine. We next investigated whether inhibition of PI3K/Akt pathway or inhibition of PKA attenuated the phosphorylation of Akt/FoxO3a and CREB. The results from western blot showed that both LY294002 and KRX-0401 blocked the phosphorylation of Akt and FoxO3a, and H89 attenuated the phosphorylation of CREB induced by fluoxetine (P<0.001).4. Fluoxetine reversed the CORT-induced attenuate phosphorylation of Akt, FoxO3a as well as CREB in PC12 cells.(1) We then found that CORT inhibited the phosphorylation of Akt, FoxO3a as well as CREB in PC12 cells, and western blot results suggested that the inhibitory effect of CORT insult was reversed by fluoxetine in a concentration-dependent manner (P<0.001).(2) As the same as above, when we apply inhibitions, such as LY294002, KRX-0401 and H89, the phosphorylation of Akt, FoxO3a and CREB were blocked (P<0.001).5. Treatment of fluoxetine attenuated the mRNA levels of apoptotic proteins through PI3K/Akt pathway and enhanced the mRNA level of BDNF through PKA/CREB pathway.As results from RT-PCR shows, we found that CORT significantly increased the mRNA levels of Bim, Bax and Puma (P<0.001), and decreased the mRNA level of BDNF (P<0.001), while fluoxetine blocked the effect of CORT by decreased the mRNA level of Bim, Bax and Puma and increased the mRNA level of BDNF. We also found that these reactions of fluoxetine were canceled by LY294002, KRX-0401 and H89 significantly, suggesting the involvement of PI3K in the effect of fluoxetine and the role of PKA in the production of BDNF induced by fluoxetine.Conclusion:1. Studies on the cellular morphology and viability suggest that fluoxetine has a significant neuroprotective effect against CORT.2. Fluoxetine reversed the CORT-induced attenuate phosphorylation of Akt, FoxO3a as well as CREB in PC 12 cells.3. LY294002, KRX-0401 and H89 blocked the effect of fluoxetine, suggesting involvement of PI3K/Akt/FoxO3a signaling pathway and PKA/CREB/BDNF signaling pathway in the effect of fluoxetine.