Studies On Mechanisms Of NAC And Rap Prevention Of Apoptosis By Suppressing Oxidative Stress And Activation Of MTOR Pathway In Cadmium-induced Neurons

The present study studied was divided into two series. First, the primary neurons were chosen as object of study. We examined the mechanisms of N-acetyl-L-cysteine (NAC) and Rapamycin (Rap) prevention of cadmium (Cd)-induced apoptosis by suppressing reactive oxygen species (ROS) generation and activation of mammalian target of rapamycin (mTOR) pathway in in vitro neurons. In the second series, the histology and ultrastructure of cerebral cortex and/or hippocampus in chronic Cd-exposed mice were observed by light microscopy (LM) and transmission electron microscopy (TEM). Consistently, NAC inhibited 4E-BP1 phosphorylation of the cerebral cortex and hippocampus tissue cells in chronic Cd-exposed mice were discussed. The results were as follows:1 NAC and Rap prevents Cd-induced apoptosis by suppressing ROS production and activation of mTOR pathway in neuronsThe primary cortical neurons were isolated and seeded in 96- or 6-well flat-bottomed plates at a density of 1×105 or 2×106 cells/well for culture of 6 days. Then cells were exposed to different concentration of Cd (0-120μM) for 24 h, or were treated with 10 or 20μM Cd for 4 h or 24 h post pretreatment with NAC (5 mM) for 1 h or with Rap (0.2 p.g/ml) for 48 h. Cell viability was evaluated using an MTT assay, intracellular ROS generation was detected using fluorescent probe CM-H2DCFDA, and morphological analysis was taken to assess the protective effect of NAC and Rap on Cd-induced neurons. Simultaneously, Western Blotting was used to investigate protein expression associated with mTOR pathways. We found Cd induced a concentration-dependent increase of apoptosis in neurons, which was associated with ROS production and activation of mTOR pathway in Cd-induced neurons. Pretreatment with NAC, a ROS scavenger or with mTOR inhibitor Rap, we observed that NAC and Rap significantly rescued Cd-induced neuronal apoptosis by inhibiting ROS production and activation of mTOR pathways. The findings reveal that NAC and Rap prevent Cd-induced apoptosis by suppressing ROS production and activation of mTOR pathway in neurons.2 Studies on protective effects of NAC administered on the cerebral tissue and cell injuries in chronic Cd-exposed miceSixty-four healthy adult ICR mice were chosen and randomly divided into 8 sets:control group (treated with 0.9% physiological saline),10,25, or 50 mg/L CdGl2 group, NAC group, NAC+10,25, or 50 mg/LCdCl2 group. CdCl2 was dissolved into water and mice with ad libitum access to food and water. NAC (150 mg/Kg body weight) was dissolved in 0.9% NaCl (15 mg/ml) and administered by intraperitoneal injection every 1 days. The entire experiment lasted 4 weeks. Changes of cerebral cortex and hippocampus in mice were observed by light microscopy (LM) for histology, transmission electron microscopy (TEM) for ultrastructure, and immunohistochemistry for phosphorylation of 4E-BP1. We showed NAC protected the disorganization of the cerebral cortex and hippocampus in chronic Cd-exposed mice at LM and TEM level. The cerebral cortex in control and NAC groups contained a sharply defined three zones. In contrast, Cd-exposed mice's one displayed a hypotrophic cortical plate, a reduced ventricular zone, and a loose band of cells positioned below the subplate in the normally clear intermediate zone. NAC apparently protected the disorganization of the cerebral cortex in the morphological and the vacuolization of neuron were reduced significantly. An abnormal laminar pattern was also evident in the hippocampus. The hippocampus also displayed striking heterotopia in the CA1-CA2 regions. Consistent with these observations, TEM revealed the change of cytoplasmic organelles includings welling and hollowed mitochondria. The results of immunohistochemistry exhibited Cd-induced a marked increase phosphorylation of 4E-BP1, at the same time, there was considerable decreased phospho-4EBPl levels by NAC combined with CdC12 in mouse brain. These data suggests NAC protected the disorganization of the cerebral cortex and hippocampus and suppressed mTOR associated with phosphorylation of 4E-BP1.