The Protective Effect Of Testosterone On The Apoptotic Damage Induced By Aβ25-35via The Upregulation Expression Of Seladin-1through Androgen Receptor Mediated Genomic Pathway, Nongenomic PI3-K/Akt/GSK3β/β-catenin Signaling Pathway And MAPK/ERK/CREB Sig

ObjectivesThe previous study indicated that DHCR24/Seladin-1was an important neuroprotective effector. Seladin-1might be a downstream target gene of androgen in the central nervous system. However, the molecular mechanisms that androgen modulate the expression of seladin-1remain incompletely defined. In our experiment, we will examine the ability of androgen to protect C6glial cells from Aβ25-35toxicity using the MTT reduction assay and Hoechst-PI staining. Besides, we will determine whether androgen regulates the expression of DHCR24/Seladin-1gene via AR-mediated genomic pathway, Nongenomic PI3-K/Akt/GSK3(3and MAPK/ERK/CREB signaling pathway in C6Glial Cells.MethodsIn this paper, we employed rat C6astroglioma cells as a glial cell model in our experiment. The mechanisms of androgen on Seladin-1expression are investigated using a glial cell model (C6glial cells) in vitro by Western blot and quantitative real-time PCR.OutcomesPart oneAP25-35obviously lowered the cell activity of the C6glial cells in vitro, while androgen prevented the effect of AP25-35in C6cells. Besides, testosterone significantly blocked the apoptotic activity of C6cells induced by Aβ25-35.The anti-apoptotic effect of androgen was associated with the upregulation expression of Seladin-1in C6cells.Part twoIn this paper, we showed that the expression of Seladin-1was significantly increased by testosterone at all concentrations tested at the protein and mRNA levels in C6cells, the selective AR antagonist flutamide obviously inhibited the effect in a concentration-dependent manner. Part threeIn our experiment, we found that testosterone significantly increased the phosphorylation level of V-akt murine thymoma viral oncogene (Akt), a key effector of the phosphoinositide3-kinase (PI3-K)/Akt signaling pathway, while a specific PI3-K inhibitor LY294002obviously prevented the activation of Akt phosphorylation in C6glial cells. Furthermore, our outcomes also showed that testosterone induced the phosphorylation of Glycogen synthase kinase3beta (GSK-3β), and increased the expression of nuclear β-catenin, a key regulatory enhancer of GSK-3(3in the GSK3p/β-catenin signaling pathway. While PI3-K inhibitor LY294002blocked the phosphorylation of GSK-3p.Meantimes, we also found that a specific PI3-K inhibitor LY294002could obviously lower the expression of nuclear β-catenin induced by testosterone. Taken together, our findings supported that testosterone induced the activation of the PI3-K/Akt/GSK3β/β-catenin signaling pathway in C6cells.In addition, in the upstream signaling pathway of the PI3-K/Akt, the PI3-K inhibitor LY294002also markedly blocked the up-regulation expression of seladin-1gene induced by testosterone at the protein and mRNA levels in C6cells. Besides, in the GSK3p/p-catenin signaling pathway, selective GSK3P inhibitor SB216763could obviously up-regulate the expression of nuclear P-catenin protein and seladin-1gene. The above data demonstrated that androgen regulated the expression of seladin-1gene by the GSK3β/β-catenin signaling pathway via the PI3-K/Akt.Part fourHowever, we also found that testosterone significantly increased the phosphorylation level of ERK1and ERK2. The effect of testosterone on the activation of ERK1and ERK2is short and swift. Besides, testosterone also obviously promoted the phosphortlation activation of cAMP response element binding protein (CREB), a key downstream effector of the MAPK/ERK signaling pathway. While the MEK inhibitor U0126markedly inhibited the effect of testosterone on the activation of the CREB phosphorylation. Our data supported that testosterone lead to the activation of the MAPK/ERK/CREB signaling pathway.Finally, we showed that the expression of Seladin-1was significantly increased by testosterone at the protein and the mRNA levels, while MEK inhibitor U0126 markedly blocked the effect of testosterone in a concentration-dependent manner.ConclusionsIn conclusion, according to our data, we concluded that seladin-1might be a downstream target gene of androgen. The protective effect of testosterone on the apoptotic damage induced by Aβ25-35via the upregulation expression of seladin-1through Androgen Receptor mediated genomic pathway, Nongenomic PI3-K/Akt/GSK3β/β-catenin and MAPK/ERK/CREB signaling pathway in C6Glial Cells.