Studies On Molecular Mechanisms Of Celastrol Intervention In Cadmium-induced Neuronal Apoptosis Via Inhibiting Ca²⁺-CaMKâ…¡-dependent Akt/mTOR Pathway

The present study, using cellular and molecular biological techniques and methods of cell culture, fluorescence staining, RNA interference, MTS assay, Western blotting, etc., systematically investigated protective action of celastrol during progress of cadmium (Cd)-induced neuronal apoptosis, discussed the mechanism of celastrol against Cd-induced neuronal apoptosis based on Cd activation of Akt/mTOR pathway, and further studied the role and molecular mechanisms of celastrol regulation of Ca2+ and CaMKII in blocking Cd-activated Akt/mTOR pathway and protecting from Cd-induced neuronal apoptosis. The results were summarized as follows:1 Celastrol intervenes in apoptotic cell death via inhibiting Cd-induced [Ca2+]i elevation in neuronal cellsPC12 cells and primary neurons were pretreated with/without celastrol (1μM) for 1h, followed by expoure to Cd (10 and 20 μM) for 8 h or 24 h. Fluo-3/AM, a fluorescent probe, was used to evaluate fluorescent intensity of intracellular free Ca2+ ([Ca2+]i). Cell viability and apoptosis was assayed using MTS reagents and DAPI staining, respectively. Expression of cleaved-caspase-3 and cleaved-PARP proteins was determined using Western blotting. The results showed that Cd indcued [Ca+]i elevation, cell viability reduction and apoptosis increase, meanwhile, caused decreases of cleaved-caspase-3 and cleaved-PARP in PC 12 cells and primary neurons. Pretreatment with celastrol reversed Cd-evoked the events. These findings suggest that celastrol intervenes in apoptotic cell death via inhibiting Cd-induced [Ca2+]i elevation in neuronal cells.2 Celastrol intervenes in Cd-induced neuronal apoptosis by preventing Akt-mediated activation mTOR pathwayPC 12 cells and primary neurons, or PC 12 cells infected with Ad-dn-Akt or Ad-GFP, were pretreated with/without Akt inhibitor X (20 μM) for 1 h and then with/without celastrol (1 μM) for 1 h, followed by exposure to Cd (10 μM and/or 20 uM) for 8 h or 24 h. Expression of Akt/mTOR-related proteins and cleaved-caspase-3 protein was determined using Western blotting. Cell apoptosis was assayed using DAPI staining. The results showed that celastrol, Akt inhibitor X, expression of dn-Akt markedly inhibited Cd-induced phosphorylation of Akt, S6K1 and 4E-BP1, as well as increases of cleaved-caspase-3 and apoptosis in the cells, and Akt inhibitor X, or expression of dn-Akt strengthened the inhibitory effects of celastrol on Cd-induced the events. These data give us a hint that celastrol intervenes in Cd-induced neuronal apoptosis by preventing Akt-mediated activation mTOR pathway.3 Celastrol intervenes in Cd-induced neuronal apoptosis via inhibiting Ca2+-dependent Akt/mTOR pathwayPC 12 cells and primary neurons were pretreated with/without BAPTA/AM (20 μM) or EGTA (100μM) for 1 h and then with/without celastrol (1 μM) for 1 h, followed by exposure to Cd (10 μM) 8 h or 24 h. Expression of Akt/mTOR-related proteins and cleaved-caspase-3 protein was determined using Western blotting. Cell apoptosis was assayed using DAPI staining. [Ca2+]i fluorescent intensity was detected using fluorescent probe Fluo-3/AM. The results showed that BAPTA/AM, EGTA or celastrol significantly relieved Cd-induced [Ca2+]i elevation, and BAPTA/AM or EGTA strengthened the inhibitory effect of celastrol on Cd-elevated [Ca2+]i. BAPTA/AM or EGTA also obviously blocked Cd-induced phosphorylation of Akt, S6K1 and 4E-BP1, as well as increases of cleaved-caspase-3 and apoptosis in the cells, and potentiated the inhibitory activity of celastrol. Our data suggested that celastrol intervenes in Cd-induced neuronal apoptosis via inhibiting Ca2+-dependent Akt/mTOR pathway.4 Celastrol intervenes in Cd-induced neuronal apoptosis through suppressing Ca2+-CaMKⅡ-dependent Akt/mTOR pathwayPC 12 cells and primary neurons were pretreated with/without BAPTA/AM (20 μM) or EGTA (100μM) for 1 h and then with/without celastrol (1 μM) for 1 h, followed by exposure to Cd (10 and/or 20 μM) 8 h, or pretreated with/without KN93 (10μM) and/or celastrol (1 μM) for 1 h and then exposed to Cd (10 μM) for 8 h or 24 h. Expression of Akt/mTOR-related proteins and cleaved-caspase-3 protein was determined using Western blotting. Cell apoptosis was assayed using DAPI staining. The results showed that BAPTA/AM, EGTA or KN93 enhanced the inhibitory effect of celastrol Cd-induced CaMKII phosphorylation. Inhibition of CaMKII with KN93 significantly potentiated celastrol prevention of Cd-induced phosphorylation of Akt, S6K1 and 4E-BP1, as well as increases of cleaved-caspase-3 and apoptosis in the cells. These findings support the idea that that celastrol intervenes in Cd-induced neuronal apoptosis through suppressing Ca2+-CaMKII-dependent Akt/mTOR pathway.