Studies On Molecular Mechanisms Of Resveratrol’s Prevention Of Cadmium-induced NOX2-ROS-mediated Akt-Erk1/2 Against Apoptosis In Neuronal Cells

The present study,using cellular and molecular biology techniques and methods including cell culture,trypan blue exclusion,TUNEL and DAPI staining,ROS fluorescent labeling,immunocytochemical analysis,Western blotting etc.;and employing PC 12 cells and primary murine neurons as experimental objects,synthetically studied the molecular mechanisms of resveratrol’s regulation of cadmium(Cd)-induced NOX2-ROS-mediated Akt-Erk 1/2 pathway against apoptosis in neurons cells.The detailed results were summaried as follows:1.Resveratrol prevented Cd-induced NOX2-dependent ROS against apoptosis in neuronal cellsPC 12 cells and mouse primary neurons were pretreated with/without resveratrol(100 μM)for 1 h and then exposed to Cd(10 and 20 μM)for 12 h or 24 h,or pretreated with/without ROS scavenger NAC(5 mM)and/or resveratrol(100 μM)for 1 h and then exposed to Cd(10 μM)for 12 h or 24 h.Expression of NOX2 in cells was detected by immunocytochemistry,intracellular ROS level was evaluated by ROS fluorescent probe CM-H2DCFDA,cell apoptosis was determined by TUNEL and DAPI staining,Caspase-3/7 activity was assayed,and the expression of related proteins was analyzed by Western blotting.The results showed that resveratrol significantly inhibited Cd-induced elevation of NOX2 and caspase activity in neuronal cells;NAC enhanced the inhibitory effects of resveratrol on Cd-induced the upregulation of NOX2 and its regulatory proteins,the increase of ROS levels,and apoptosis in neuronal cells.The results uncover that resveratrol prevents Cd-induced NOX2-dependent ROS against apoptosis in neuronal cells.2.Resveratrol prevented against Cd-induced neuronal apoptosis through NOX2-ROS-mediated Akt-Erk pathwayPC 12 cells and mouse primary neurons,or PC 12 cells infected with Ad-dn-Akt and Ad-GFP/Ad-LacZ(as control),respectively,were pretreated with/without resveratrol(100 μM)for 1h and then exposed to Cd(10 and 20 μM)for 12 h,or pretreated with/without APO(50 μM)and/or resveratrol(100 μM)for 1 h and then exposed to Cd(10 μM)for 12 h or 24 h,or pretreated with/without U0126(5 μM)and/or resveratrol(100 μM)for 1 h and then exposed to Cd(10 μM)for 12 h or 24 h.Intracellular ROS level was evaluated by ROS fluorescent probe CM-H2DCFDA,cell apoptosis and viability was determined by TUNEL staining and trypan blue exclusion,and the expression of related proteins was analyzed by Western blotting.The results showed that resveratrol significantly inhibited Cd-induced activation of Akt and Erk1/2;Inhibition of Erk1/2 by U0126 or expression of dn-Akt obviously protected from Cd-induced neuronal apoptosis,and potentiated resveratrol’s protective effects;Inhibition of NOX2 by APO strengthened resveratrol’s inhibition of Cd-induced activation of Akt and Erk1/2 against apoptosis in neuronal cells.The data indicate that resveratrol prevents against Cd-induced neuronal apoptosis through NOX2-ROS-mediated Akt-Erk pathway.