Studies On Mechanisms Of Resveratrol’s Prevention Against Cadmium-induced Neuronal Apoptosis Based On Akt/mTOR Pathway

The present study, using cellular and molecular biology techniques and methods including cell culture, trypan blue staining, DAPI and TUNEL staining, RNA interference, Western blot, etc., and employing PC 12 cells and primary murine neurons as experimental objects, systematically studied the effect of resveratrol on cadmium-induced apoptosis in neuronal cells, and deeply explored resveratrol’s prevention from Cd-induced neuronal apoptosis by targeting mTOR signaling and an important role of S6K1 in the process. The detailed results were summarized as follows:1. Resveratrol protects against Cd-induced neuronal apoptosis by inhibiting Akt/mTOR pathwayPC 12 cells and primary murine neurons were pretreated with resveratrol (100μM) for 1 h, followed by exposure to Cd (10 and 20 μM) for 8 h or 24 h. The related proteins of Akt/mTOR pathway were determined by Western blotting, and cell viability and apoptosis were evaluated using trypan blue and DAPI staining,respectively. The results showed that Cd-induced phosphorylation increase of Akt,mTOR, S6K1, S6 and 4E-BP1 in a concentration-dependent manner, which was potently blocked by resveratrol. Consistently, resveratrol markedly attenuated Cd-induced cell viability reduction and apoptosis in neuronal cells. Our data suggest that resveratrol protects against Cd-induced neuronal apoptosis ’by inhibiting Akt/mTOR pathway.2. Resveratrol prevents from Cd-induced neuronal apoptosis via targeting mTOR signalingPC 12 cells and primary murine neurons were pretreated with rapamycin (200 ng/ml) for 48 h and then with/without resveratrol (100 μM) for 1 h, followed by exposure to Cd (10 and 20 μM) for 8 h or 24 h. The related proteins were determined by Western blotting, and cell viability and apoptosis were evaluated using trypan blue and TUNEL staining, respectively. The results showed that rapamycin enhanced resveratrol’s inhibition of Cd-induced phosphorylation of Akt, mTOR, S6K1,4E-BP1 and elevation of cleaved-caspase-3. Consistently, rapamycin is also effective in enhancing resveratrol resistance to cadmium-induced neuronal activity and TUNEL-positive cells.These findings suggest that resveratrol prevents from Cd-induced neuronal apoptosis via targeting mTOR signaling.3. S6K1 plays an important role in resveratrol’s protection against Cd-induced neuronal apoptosisPC 12 cells infected with Ad-S6K1-ca and Ad-LacZ (as control) were pretreated with resveratrol (100 μM) or PP242 (1 μM) for 1 h and then exposed to Cd (10 μM)for 8 h or 24 h. The related proteins were determined by Western blotting,and cell viability and apoptosis were evaluated using trypan blue and TUNEL staining,respectively. The results showed that sustained activation of S6K1 expression did not affect Akt phosphorylation performance, but could enhance basal and cadmium-induced S6K1, S6 phosphorylation and cleaved-caspase-3 expression and resistance to resveratrol or PP242 inhibition of cadmium-induced these events.Consistently, sustained activation of S6K1 expression increases cadmium-induced neuronal cell activity reduction and apoptosis, and is clearly resistant to resveratrol or PP242 inhibitory activity. Our data imply that S6K1 plays an important role in resveratrol’s protection against Cd-induced neuronal apoptosis.