The Effect Of DHT On Apoptosis Induced By U18666A Via PI3- K/Akt Signaling Pathway In C6 Glial Cell Lines

[Background]Neurodegenerative Disease(ND) is always associated with age-related decline of serum testosterone levels,include Alzheimer’s Disease(AD),Anxiety,depression,and cerebral atrophy. More and more study show that dihydrotestosterone (DHT) have a wide range of Neurobiology effect in the prevention and treatment of ND,which is contribute to suppress pathological damage, repair damaged neurons and synaptic plasticity.Thers is little studies of neuroprotective mechanism of androgen recently.The mechanism include: (1) adjust the release of amyloid proteins in the brain;(2)resist oxidative stress; (3)antiapoptosis. (4)promote the outgrowth and differentiation of neuron.Futher 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 ND.[Objective]Our aim is to explore the neuroprotective effect of dihydrotestosterone (DHT) on C6 apoptosis induced by U18666A in vitro. Besides, to explore the molecular mechan-ism of DHT, we further investigate whether DHT modulate the expression of apoptosis-r elated proteins such as Seladin-1, Survivin, Bax, BCL-XL and Caspase-3 mediated by n on-genomic PI3-K/Akt pathway, which may contribute to androgen neuroprotection.[Method]Part1We use the method of AnnexinV-FITC/PI double staining flow cytometry and Hochest33342 staining to detect the proapoptptic effect of U18666A with different concentration in C6 cells. Part 2After the pretreatment of C6 cells with DHT and LY294002, we use the method of An nexinV-FITC/PI double staining flow cytometry and Hochest33342 staining to detect the anti-apoptptic effect of DHT in C6 cells apoptosis induced by U18666A,and the sup -pression of DHT anti-apoptptic effect induced by PI3K inhibitor (LY294002).Part 3We use Western Blot to detect phosphorylation activation of Akt induced by DHT,and t he suspression of DHT phosphorylation activation effect induced by LY294002.Further, we use Western Blot to detect apoptosis related molecules expression include Seladin-1, Bcl-2 family proteins,IAP family proteins and Caspase-3 after treatment with DHT and LY294002. Our aim is to discuss the role of apoptosis related molecules expression via P 13-K/Akt signaling patheway in androgen antiapoptotic effect.[Results]Part 1The result of immunofluorescence demonstrated that amount of C6 cells significantly increased induced by U18666A.The result of AnnexinV-FITC/PI double staining flow cytometry method demonstrated that apoptotic rate of C6 cells significantly increased induced by U18666A with different concentration(0.5μg/ml, 1.0μg/ml,2.5μg/ml) (P<0.05).Part 2After the pretreatment of C6 cells with DHT (10-2μM) one hour prior to treatment with U18666A for 48h,the result of immunofluorescence demonstrated that the amount of C6 cells was significantly decreased.LY294002(50μM) can suppress DHT anti-apoptotic effect.The result of AnnexinV-FITC/PI double staining flow cytometry method showed that th e apoptotic rate of C6 cells was significantly decreased by DHT in contrast to U1866-6A group (P<0.05). LY294002(50μM) can suppress DHT anti-apoptotic effect(P<0.05).Part 3The result of Western Blot showed that DHT can significantly activate the phosphoryl-ation of Akt in contrast to U18666A(P<0.05),which can suspressed by LY294002(P<0.0 5).In addition, the result of Western Blot demonstrated:1) U18666A can significantly downregulate the expression of antiapoptosis Protein Sel-adin-1 (P<0.05), and upregulate the expression of proapoptosis Protein Caspase-3 in con trast to control group(P<0.05).2) DHT can significantly upregulate the expression of antiapoptosis Protein Seladin-1,BCL-XL and Survivin(P<0.05), and downregulate the expression of proapoptosis Protein Bax, Caspase-3(P<0.05) in contrast to U18666A group.3) LY294002 can significantly suspress downregulation of proapoptosis Protein Caspase-3 and Bax induced by DHT(P<0.05),and suspress upregulation of antiapoptosis Protein Seladin-1, BCL-XL and Survivin(P<0.05).[Conclusion]According to results above, in the C6 cell model, we found that:1) U18666A can induce C6 cells apoptosis in vitro.2) DHT has a significant anti-apoptosis activity induced by U18666A in C6 cells w-hile PI3K inhibitor LY294002 can significantly inhibit this anti-apoptotic effect. The result showed that PI3K/Akt signaling pathway was participated in the mech-anism of DHT anti-apoptosis activity induced by U18666A in C6 cells.3) DHT can significantly upregulate the expression of antiapoptosis Protein Seladin-1, BCL-XL and Survivin, downregulate the expression of proapoptosis Protein Cas-pase-3 and Bax. This effect can be blocked by LY294002. Therefore, we conclude that DHT exhibit anti-apoptosis action by the regulation of the downstream ap-optotic related factors(Survivin,Seladin-1, BCL-XL、Bax、Caspase-3)expression through activating PI3K-Akt signaling pathway;...