| Autophagy is a physiological and evolutionarily conserved phenomenon responsible for the removal of long-lived proteins and damaged organelles by the lysosome. Recent studies have shown that environmental stress can induce autophagy, which helped maintain cell homeostasis. However, massive and persistent autophagy can kill proteins and organelles by a caspase-independent form of cell death. Cadmium, one of the highly toxic heavy metals has nerve toxicity. with symptoms including neuroethology defects learning disabilities and hyperactivity and so on. The vitro studies conducted in animal models have focused on the induction of severe apoptosis and necrosis, and as a result, the role of autophagy remains unclear. The present study surveyed whether autophagy is responsible for the cytotoxicity of cadmium and investigated the mechanisms underlying autophagic pathways by using the PC-12cells and rat cerebral cortical neurons as a neuronal model, which will offer theoretic evidences for future clarifying the mechanism in neurotoxicity of cadmium.1. The influence of cadmium to neuron cells viabilityTo explore PC-12cell culture and the influence of cadmium to neuron cells viability, PC-12cells were cultured by modified method and toxic effects of cadmium on cell viability of PC-12cells and primary neurons was evaluated using MTT assay in different concentrations (0,2.5,5,10,20μmol/L for24h) and times (20μmol/L Cd for0,1,2,4,6,8,12,24h), respectively. The results indicated that PC-12cells was a few scattered lightly attached cells, with the shape of a substantially circular and peripheral halo of light. Cells bagan to cluster after inoculation at24h, agglomerate phenomenon is more obvious at72h. cells logarithm growing phase of PC-12cells was43h, consistent with the ATCC. It was suggested that PC-12cells can be can be used for further study. Within a certain range of concentrations and times, cell viability decreased in a dose-and time-dependent manner after treatment with Cd in PC-12cells and primary neurons. These results indicate that cadmium has significant toxic effects on nerve cells.2. Detection of autophagy induced by cadmiumTo survey whether cadmium can induce autophagy, PC-12cells and primary neurons were exposured to cadmium of different concentrations (0,2.5,5.10,20μmol/L) for4h and20umol/L for various times (0,2,4,6,8,12,24h), LC3, an autophagy-specific protein were examined by western blot. After treatment20μmol/L cadmium for4h, the presence of LC3puncta were observed by immunofluorescence, acidic vesicular organelles (AVOs) were researched by acridine orange (AO) staining, autolysosome were investigated by MDC staining. Cells were treated with20μmol/L cadmium for4h and24h, organelles changes were detected by TEM. The date showed that the levels of LC3-Ⅱ proteins increased in a dose-dependent manner. As cells were treated with20μmol/L cadmium for0h to24h, LC3-Ⅱ proteins reached a peak for4h and then gradually decreased. Specific punctate distribution of endogenous LC3-II was observed, given that more dots appeared in cadmium-treated cells than in control cells (p<0.01). The increase red fluorescent AVOs were found in cadmium-treated cells compare with control. MDC fluorescence accumulation spots were discovered that were not appeared in control, after4h of cadmium exposure, neurons exhibited several autophagosomes that were absent in control cells. However, apoptotic changes such as chromatin condensation and cytoplasmic vacuolization were obvious after treatment with20μmol/L cadmium for24h. In sum, these results indicate that cadmium induces autophagy and apoptosis in PC-12cells and primary neurons.3. The role of autophagy in neurotoxicity induced by cadmiumIn order to investigate the role of autophagy in neurotoxicity induced by cadmium, PC-12cells and primary neurons were pretreated with5μmol/L chloroquine (CQ), an inhibitor of autophagy for0.5h, followed by treatment with cadmium for another4h, or pretreated with rapamycin (RAP), an inducer of autophagy for24h, followed by treatment with cadmium for another24h. Western blot and MTT analysis were performed. In comparison with the cadmium group for4h, the results showed that CQ-mediated autophagy inhibition increased the levels of LC3-Ⅱ proteins (p<0.05) and decreased cell viability induced by cadmium treatment (p<0.05or p<0.01). However, compare with the cadmium group for24h, autophagy activators, such RAP, increase the levels of LC3-Ⅱ proteins (p<0.05or p<0.01) and recoveryed the viability (p <0.05or p<0.01) induced by cadmium treatment. The findings suggest that autophagy plays a positive role in the reduction of cadmium-induced cytotoxicity.4. The relationship between neuronal autophagy and apoptosis induced by cadmiumTo elucidate the relationship between autophagy and apoptosis, PC-12cells and primary neurons were pretreated with5μmol/L CQ for0.5h, followed by treatment with cadmium for another4h, or pretreated with RAP and Z-VAD-fmk for24h, followed by treatment with cadmium for another24h, the apoptosis rate was examined using flow cytometry in PC-12cells and morphological changes were detected in primary neurons. Data indicated that compare with control group, the percentage of apoptosis was increased from4.5%to45.8%for24h (p<0.01). Furthermore, after24h of Cd exposure, the percentage of apoptosis was effectively reduced by Z-VAD-fmk (p<0.01), futher demonstrate that cadmium can induce apoptosis in PC-12cells. Cotreatment CQ and cadmium for4h, the augmentation of of apoptosis ratio resulting from4.5%to33.7%(p<0.01). Furthermore, co-treatment with cadmium and RAP reduced the apoptosis ratio from45.8%to14.7%(p<0.01), indicating that autophagy hinders apoptosis in cadmium-treated neuron cells. In comparison with the control primary neurons group, the cells in cadmium-treated for24h and cotreatment CQ and cadmium for4h showed typical morphological changes of apoptosis with nucleus crimpled and chromatin condensedation, even nucleus disintegratation. However, co-treatment with cadmium and RAP or Z-VAD-fmk, cell injuries were alleviated, indicating that autophagy hinders apoptosis in Cd-treated neurons.5. class Ⅲ PI3K/Beclin-1/Bcl-2signaling in cadmium-induced autophagy of neuronsTo investigate the class Ⅲ PI3K/Beclin-1/Bcl-2signaling pathways involved in the induction of autophagy, PC-12cells and primary neuron cells were treated with20μmol/1Cd for0,2,4,6,8,12,24h, relevant proteins were examined by immunoblot analysis. After treatment cadmium with LY294002, a PI3K inhibitor for6h, indicated proteins and LC3punctate were investigated. As results showed, in cells treated with20μmol/1Cd for0-24h, the protein levels of Beclin-1and class Ⅲ PI3K were firstly upregulated then gradually decreased. However, Bcl-2was downregulated within24h. LY294002significantly decreased the expression of Beclin-1, and class Ⅲ PI3K (p<0.05or p<0.01), and promoted Bcl-2protein expression in cadmium-treated PC-12cells and cerebral cortical neurons. Concomitantly, autophagocytosis was also obviously decreased after treatment with cadmium and LY294002compared to cadmium alone. This finding suggest that class Ⅲ PI3K/Beclin-l/Bcl-2signaling plays a positive regulation in Cd-induced autophagy and may be involved in elongation of autophagosomal membrane.6. ERK signal pathways in cadmium-induced autophagy of neuronsTo research the relationship between ERK signal and autophagy, PC-12cells were treated with20μmol/l Cd for0,0.5,1,2,4h, PC-12cells and primary neuron cells were treated with20μmol/L Cd4h following pre-incubation with U0126, a ERK inhibitor for0.5h, ERK, LC3and AVOs were examined. The results showed that, in PC-12cells treated with20μmol/l cadmium for0-4h, P-ERK1/2were rapid increased at0.5h, and maintained until4h. U0126significantly decreased the levels of P-ERK1/2, LC3-Ⅱ and AVOs, confirming that ERK is a upstream signaling molecule of autophagy, its activation can favor autophagy induction.7. The impact of oxidative stress on neuronal autophagy induced by cadmiumTo study the impact of oxidative stress on autophagy induced by cadmium, autolysosome and LC3-Ⅱ were observed by fluorescent staining and western blot, the level of ROS,△ψ and DNA fragmentation were analyzed by flow cytometry, LDH leakage was examined by LDH kit after PC-12cells were treated cadmium with/without CQ and/or NAC. The results showed that CQ treatment induces intense autolysosome accumulation. Compared to the cadmium for4h, NAC significantly inbibited LC3-Ⅱ expression. Compared to the normal group, the24h of cadmium exposure and the4h of CQ-cadmium treatment had a marked generation of ROS (p<0.01), declination of△ψ(p<0.01), increase of DNA fragmentation (p<0.01) and LDH leakage (p<0.01). However, compare with CQ-cadmium treatment, NAC can obviously release ROS (p <0.05) and increase DNA fragmentation (p<0.05). These results indicate that ROS and△ψplay an essential role in the regulation of QD-induced autophagy. At the early stage of cadmium expourre, ROS can make for induction of cytoprotective autophagy. |
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