Role Of Fas/FasL Signaling Pathway In Cadmium Induced Neural Apoptosis And Protect Effect Of NAC

Cadmium (Cd) is a common accumulating heavy metal toxicant in environment, causing multi-system and-organ damage. Brain is one of significant target organ for Cd toxicity, it cause blood brain barrier dysfunction, lead Cd enter brain tissue induce neuronal apoptosis. Death receptor pathway is one of the classic cell apoptosis pathway and Fas/FasL signaling pathway is the most significant one. Many studies have confirmed that activation of Fas/FasL signaling pathway could sensitize the mitochondrial apoptotic pathway. Our previous investigation have confirmed that Cd-induce apoptosis through mitochondrial apoptotic pathway in never cells. While the role of Fas/FasL signaling pathway in Cd-induced neuronal apoptosis and effects on mitochondrial apoptotic pathway is still unclear. In current experiment, the model of primary neurons and PC-12 cells cultured in vitro were used to explore the mechanism of apoptosis induced by Cd via Fas/FasL signaling pathway and mechanism of mediating mitochondrial apoptotic pathway, and the protection of NAC in the above process.1. The role of Fas/FasL signaling pathway in Cd-induced neuronal apoptosisTo explore the role of Fas/FasL signaling pathway in Cd-induced neuronal apoptosis, primary neurons and PC-12 cell line were treated with CdAc2 (0,5,10,20 μmol/L) for different times (0,6,12 and 24 h). The western blot was used to measure the expression of Fas/FasL signaling pathway related protein, Fas, FasL, FADD and cleaved caspase-8. The dates showed that Cd increased the protein level of Fas, FasL, FADD and cleaved caspase-8 significantly (p<0.05 or p<0.01).To further study the role of Fas/FasL signaling pathway in Cd-induced neuronal apoptosis, primary neurons and PC-12 cells were treated with 10 μmol/L Cd and 100 μmol/L Z-IETD-FMK (caspase-8 specific inhibitor) or Anti-FasL (FasL neutralizing antibody) for 24 h. Flow cytometry (FCM), Annexin V staining and hoechst 33258 staining were used to detect apoptotic rate. The results showed that Z-IETD-FMK effectively inhibit cell shrinkage and fall off, axons, dendrites and neural networks disappearance of neurons induced by Cd (p<0.01). Anti-FasL significantly decreased the apoptotic rate induced by Cd (p<0.01). It demonstrated that Fas/FasL signaling pathway plays an important role in Cd-induced neuronal apoptosis.2. The role of Fas/FasL signaling pathway in Cd-induced mitochondrial apoptotic pathway of neuronalThe role of Fas/FasL signaling pathway in Cd-induced mitochondrial apoptotic pathway of neuronal, the primary neurons and PC-12 cells were treated with 10 μmol/L CdAc2 and 40 μmol/L Z-IETD-FMK or 10 mg/L Anti-FasL for 24 h. The release of cyt c, expression of tBID, cleaved caspase-9, cleaved caspase-3, cleaved PARP, Bax and Bcl-2 were detected by western blot assay. Mitochondrial membrane potential(ΔΨm) were detected by FCM and fluorescence probe. The results showed that Z-IETD-FMK effectively prevented ΔΨm disruption and prevented release of cyt c, expression of tBID, cleaved caspase-9, cleaved caspase-3, cleaved PARP and decrease of Bcl-2/Bax ratio induced by Cd (p<0.05 or p<0.01). Anti-FasL prevented the expression of cleaved caspase-9, cleaved caspase-3 and cleaved PARP (p<0.01). It demonstrated that the activation of Fas/FasL signaling pathway mediates the mitochondrial apoptotic pathway in Cd-induced neuronal cell apoptosis.3. The protective effect of NAC on Cd-induced Fas/FasL signaling pathway of neuronalTo explore protective effect of NAC on Cd-induced Fas/FasL signaling pathway, PC-12 cells were treated with 10 μmol/L CdAc2 and/or 100 μmol/L NAC for 24 h. FCM and hoechst 33258 staining were used to detect the apoptotic rate. The western blot was used to measure the expression of Fas/FasL signaling pathway related protein, Fas, FasL, FADD and cleaved caspase-8. The results showed that NAC effectively inhibit increase of apoptotic rate and expression of Fas, FasL, FADD and cleaved caspase-8 induced by Cd (p<0.01). It demonstrated that NAC can protect Cd-induced PC-12 apoptosis through prevent activation of Fas/FasL signaling pathway.