Studies On Molecular Mechanisms Of Celastrol’s Prevention Of Cadmium-induced Activation Of JNK Pathway From Apoptosis In Neuronal Cells

The present study, using cellular and molecular biology techniques and methods including cell culture、TUNEL and DAPI staining、Western blotting、ROS fluorescent staining and imaging, etc., and employing PC 12 cells and primary murine neurons as experimental objects, systematically investigated that celastrol prevented against apoptosis via intervening in PP2A-JNK pathway, resisting inactivated PP5-dependent activation of JNK pathway, and targeting inhibition of NOX2-ROS-mediated PP5-JNK pathway in neuronal cells induced by Cd. The detailed results were summarized as follows:1. Celastrol rescues from Cd-induced neuronal apoptosis via intervening in PP2A-JNK pathwayPC 12 cells and primary neurons were chosen and treated with Cd (10 μM) for 4 h following pretreatment with/without celastrol (0-1 μM) for 1 h, or pretreated with/without Okadaic acid (OA, 100 nM) for 1 h and then with/without celastrol (1 pM) for 1 h, followed by exposure to Cd (10 μM) for 4 h or 24 h. In some cases, PC 12 cells infected with Ad-dn-PP2A, Ad-PP2A-wt or Ad-GFP (as control), respectively, were pretreated with/without JNK inhibitor SP600125 (20 μM) for 1 h and then with/without celastrol (1 μM) for 1 h, followed by exposure to Cd (10 μM) for 4 h or 24 h. Cell apoptosis was evaluated by DAPI staining, and expression of the related protein was determined by Western blotting. The results showed that celastrol markedly attenuated Cd-induced inhibition of PP2A activity and activation of JNK pathway. Inhibition of PP2A by OA resisted celastrol’s inhibition of Cd-induced phosphor-PP2A,demethylated-PP2A, phosphor-JNK and apoptosis. SP600125 potentiated celastrol’s prevention of Cd-induced activation of JNK and apoptosis. Ectopic expression of dominant negative PP2A attenuated celastrol’s blockage of Cd-induced activation JNK pathway and apoptosis, whereas overexpression of PP2A enhanced the inhibitory effects of celastrol on Cd-induced the events. The results uncover that celastrol rescues from Cd-induced neuronal apoptosis via intervening in PP2A-JNK pathway.2. Celastrol prevents against neuronal apoptosis by resisting Cd-inactivated PP5-dependent activation of JNK pathwayPC12 cells and primary neurons were chosen andt reated with Cd (10 and 20 μM)for 12 h following pretreatment with/without celastrol (1 μM) for 1 h. In some cases,PC 12 cells infected with Ad-PP5, Ad-dn-c-Jun or Ad-GFP, respectively, were treated with Cd (10 μM) for 12 h following pretreatment with/without celastrol (1 μM), JNK inhibitor SP600125 (20 μM) or caspase inhibitor zVAD-fmk (100 μM) for 1 h. Then,cell viability was analyzed by using MTS, the number of TUNEL-positive cells was quantified by in situ detection of fragmented DNA using TUNEL staining, and expression of the related protein was determined by Western blotting. The results showed that celastrol significantly ameliorated Cd-caused inactivaciton of PP5.Overexpression of PP5 partially potentiates celastrol’s inhibition of Cd-induced JNK activation and apoptosis in neuronal cells. Ectopic expression of dominant negative c-Jun enhanced celastrol’s inhibition of Cd-induced JNK activation and apoptosis in neuronal cells. The findings indicate that celastrol prevents against neuronal apoptosis by resisting Cd-inactivated PP5-dependent activation of JNK pathway.3. Celastrol ameliorates Cd-induced neuronal apoptosis through targeting NOX2-ROS-mediated PP5-JNK pathwayPC 12 cells and primary neurons were pretreated with/without celastrol (1 μM)for 1 h and then exposed to Cd (10 and/or 20μM) for 12 h or 24 h. In some case, PC 12 cells and primary neurons were pretreated with/without NAC (5 mM) or NOX2 inhibitor apocynin (50 μM) for 1 h and then celastrol (1 μM) for 1 h,followed by exposure to Cd (10 μM) for 12 h or 24 h. Then,fluorescence imaging for reactive oxygen species (ROS) intensity was captured by using ROS probe CM-H2DCFDA,the number of TUNEL-positive cells was quantified by in situ detection of fragmented DNA using TUNEL staining, Caspase-3/7 activities were detected using Caspase-3/7 Assay Kit, the related signal alteration was determined by Western blotting. The results showed that celastrol obviously inhibited Cd-induced ROS generation in neuronal cells. The ROS scavenger NAC attenuated Cd-induced ROS and apoptosis in neuronal cells and potentiated the inhibitory effects of celastrol activity. NAC enhanced celastrol blockage of Cd-upregulated NOX2-dependent ROS production.Apocynin inhibited Cd-induced NOX2 upregulation and ROS generation in neuronal cells in a concentration-dependent manner, and potentiated celastrol’s prevention of Cd-inudced the events. Apocynin strengthened celastrol’s prevention of Cd-induced inactivation of PP5 and activation of JNK from neuronal apoptosis by. The results uncover that celastrol ameliorates Cd-induced neuronal apoptosis through targeting NOX2-ROS-mediated PP5-JNK pathway.