The Protective Effect Of Dihydrotestosterone（DHT） On C6Cell Apoptosis Induced By Aβ_25-35 Via PI-3K/Akt Signaling Pathway

[Background]Alzheimer’s Disease（AD） is a common and disabled disease with high rising incidence rate, which clinical treatment is not improved much in recent years. AD can cause progressive memory impairment, cognitive decline and behavioral abnormalities. Until recently, there are still not effective prevention and treatment. Several clinical epidemiological data suggest that along with aging, age-related decline of serum testosterone levels shows a positive correlation with the rising incidence of AD. Supplementary androgens can significantly reduce the risk of AD. More and more study suggest that dihydrotestosterone （DHT） plays a wide range of neurobiological role such as suppressing AD pathological damage, repairing damaged neurons and synaptic plasticity in preventing and curing AD. Therefore, as a potential drug androgen has been more and more using for AD’s prevention and therapy caused a widespread concern at home and abroad. Further elucidating the neuroprotective effect of androgen and its molecular mechanism may provide a very important theoretical and clinical basis for us to explore a new medical treatment for ADs.[Objective]Observing the neuroprotective role of dihydrotestosterone （DHT） on C6apoptosis induced by Aβ_25-35 in vitro and finding the anti-apoptosis molecular mechanism of androgens and PI-3K/Akt signaling pathway in it’s potential effect.[Method]Part1To detect the androgen receptor （AR） by indirect immunofluorescence staining; To observe AR expression in C6cells using inverted fluorescence microscope and laser scanning confocal microscope. Part2DHT intervention C6cells in different concentration gradient and time, Western Blotting detects Akt phosphorylation activation on C6cells; Further study the effect of PI-3K inhibitor LY294002on DHT inhibiting the expression of P-Akt in C6cells.Part3Different concentrations of Aβ25-35act on cultured C6cells, utilizing Annexin Ⅴ--FITC/PI double staining flow cytometry method to observe cell apoptotic rate induced by Aβ25-35; addition of DHT, using flow cytometry and Hochest33342nuclear staining method both quantitative and qualitative detect anti-apoptosis activities of DHT on C6cells induced by Aβ25-35; Further special PI-3K inhibitor （LY294002）、DHT and Aβ25-35cointervene C6cells by AnnexinV--FITC/PI double staining flow cytometry to observe the effect of PI-3K/Akt signaling pathway on anti-apoptosis of DHT.Part4Treat C6cells with DHT and Aβ25-35,then WB detect apoptosis-related protein Seladin-1, Bax, Bcl-xl expression on C6cells to further clarify the potential neuroprotective molecular mechanism of DHT in CNS.[Results]Part1There were a widespread、high levels of AR expression on the membrane and cytoplasm of C6cells by indirect immunofluorescence staining under inverted fluorescence microscope and laser scanning confocal microscopy.Part2Western Blotting showed DHT with concentrations between10-3μM-100μM can significantly phosphorylate Akt in C6cells （P<0.05）, and the most obvious is the10-1μM; further processing DHT of10-1μM on C6cells found that Akt phosphorylation starts at30minutes of incubation, and at1hour reaches a maximum （P<0.05）. In addition, we observed that PI-3K inhibitor LY294002at10μM can block the action of DHT on Akt kinase by WB detection（P<0.05）, while Akt kinase phosphorylation is significantly inhibited at50μM. The results suggest that DHT activate PI-3K/Akt signal pathway may by act on the AR receptor.Part3By AnnexinⅤ--FITC/PI double staining flow cytometry method found that after 24hours Aβ_25-35 concentrations between6.75μM to50μM can induce apoptosis in C6cells. The SPSS analysis showed its statistically significant （P<0.05）. That Aβ_25-35 at concentration of25μM exhibit apoptosis activity is statistically significant. DHT can inhibit apoptotic rate of C6cells induced by Aβ_25-35 by flow cytometry （P<0.05）. while C6cells treated by Aβ_25-35 during the early process of apoptosis cell shrinkage and Karyopyknosis were visible by fluorescent microscope using Hochest33342nuclear staining. C6apoptotic rate decreases significantly after pretreating with DHT. Further flow cytometry study shows that PI-3K inhibitor LY294002can inhibit effect of DHT on inhibiting apoptosis induced by Aβ_25-35（P<0.05）. The above results suggest that the neuroprotective effect of DHT on C6cells may generated by activate PI-3K/Akt signaling pathway.Part4WB detection found that Aβ_25-35 significantly downregulate apoptosis protective protein Seladin-1, Bcl-xl expression while upregulate pro-apoptotic protein Bax expression; further study exhibit DHT has opposite results. The above results suggest that Aβ_25-35 induce apoptosis activity by regulating thr expression of apoptosis-related protein Seladin-1, Bcl-xl and Bax protein.While DHT may inhibit the apoptosis induced by Aβ_25-35 through regulate expression of apoptosis-related protein.[Conclusion]According to results above, in the C6cell model, we found that Aβ_25-35 can induce C6cells apoptosis in vitro. DHT has a significant anti-apoptosis activity of C6cells induced by Aβ_25-35 while P13K inhibitor LY294002can significantly block this anti-apoptotic protective effect. Therefore, we hypothesized that DHT exhibit anti-apoptosis action by activating PI3K-Akt signaling pathway; It’s molecular mechanism may be through downregulating expression of apoptosis protective protein Seladin-1, Bcl-xl and upregulating pro-apoptotic protein Bax protein’s expression.