Celastrol ameliorates Cd‐induced neuronal apoptosis by targeting NOX2‐derived ROS‐dependent PP5‐JNK signaling pathway

Celastrol, a plant‐derived triterpene, has neuroprotective benefit in the models of neurodegenerative disorders that are characterized by overproduction of reactive oxygen species ( ROS ). Recently, we have reported that cadmium (Cd) activates c‐Jun N‐terminal kinase ( JNK ) pathway leading to neuronal cell death by inducing ROS inactivation of protein phosphatase 5 ( PP 5), and celastrol prevents Cd‐activated JNK pathway against neuronal apoptosis. Therefore, we hypothesized that celastrol could hinder Cd induction of ROS ‐dependent PP 5‐ JNK signaling pathway from apoptosis in neuronal cells. Here, we show that celastrol attenuated Cd‐induced expression of NADPH oxidase 2 ( NOX 2) and its regulatory proteins (p22 phox , p40 phox , p47 phox , p67 phox , and Rac1), as well as the generation of ROS in PC 12 cells and primary neurons. Also, N ‐acetyl‐ l ‐cysteine, a ROS scavenger, potentiated celastrol's inhibition of the events in the cells triggered by Cd, implying neuroprotection by celastrol via blocking Cd‐evoked NOX 2‐derived ROS . Further research revealed that celastrol was involved in the regulation of PP 5 inactivation and JNK /c‐Jun activation induced by Cd, as celastrol prevented Cd from reducing PP 5 expression, and over‐expression of wild‐type PP 5 or dominant negative c‐Jun strengthened celastrol's inhibition of Cd‐induced phosphorylation of JNK and/or c‐Jun, as well as apoptosis in neuronal cells. Of importance, inhibiting NOX 2 with apocynin or silencing NOX 2 by RNA interference enhanced the inhibitory effects of celastrol on Cd‐induced inactivation of PP 5, activation of JNK /c‐Jun, ROS , and apoptosis in the cells. Furthermore, we noticed that expression of wild‐type PP 5 or dominant negative c‐Jun, or pretreatment with JNK inhibitor SP 600125 reinforced celastrol's suppression of Cd‐induced NOX 2 and its regulatory proteins, and consequential ROS in neuronal cells. These findings indicate that celastrol ameliorates Cd‐induced neuronal apoptosis via targeting NOX 2‐derived ROS ‐dependent PP 5‐ JNK signaling pathway. Our data highlight a beneficial role of celastrol in the prevention of Cd‐induced oxidative stress and neurodegenerative diseases.