The Regulation Of Fas/FasL In MAPK Pathway And Mitochondrial Apoptotic Pathway Of PC12 Cells Induced By Cadmium

Cadmium(Cd)is a common environmental and industrial heavy metal pollutant,and acute or chronic accumulation of Cd can cause damage to a variety of tissues and organs.The brain is one of the important target organs of Cd toxicity.Existing studies have confirmed that Cd can increase the blood-brain barrin permeability and lead to neuronal apoptosis.Fas/FasL signaling pathway is one of the most important death receptor pathways and plays an important role in process of apoptosis.Studies have confirmed that cell apoptosis via MAPK pathway and mitochondrial pathway is mediated by Fas/FasL signaling pathway.Our previous investigations have confirmed that Cd can induce neuronal apoptosis through Fas/FasL signaling pathway,MAPK pathway and mitochondrial pathway,but the mechanism of the relationship between Fas/FasL signaling pathway and MAPK pathway,mitochondrial pathway in Cd-induced neuronal apoptosis is still unclear.In this study,rat pheochromocytoma cell line(PC 12 cells)was used as neuronal cell model to explore the role of Fas/FasL in Cd-induced apoptosis of PC 12 cells and the regulation mechanism of Fas/FasL on MAPK pathway and mitochondrial pathway.1.The establishment of Ientivirus-mediated Fas gene silencing PC12 cell lineIn order to obtain the Fas gene silencing PC 12 cell line,PC 12 cells were infected with three Fas shRNA lentivirus with different interference targets for 12 hours,followed by cultured in the complete culture medium containing puromycin(working concentration of 4 mg/L)for 48 hours.Under fluorescence microscopy the infection efficiency of lentivirus was determined by counting the number of cells expressing green fluorescent protein in 100 cells.The transcription of Fas gene was detected by qRT-PCR,and the expression of Fas protein was detected by Western-blot.The results showed that shRNA NC lentivirus and Fas shRNA lentivirus 3 could efficiently infect PC 12 cells with an infection multiplicity of 10,and the infection efficiency could reach 80%.shRNA NC lentivirus did not affect Fas gene transcription and protein expression in PC 12 cells,Fas shRNA lentivirus could effectively inhibit Fas gene transcription and protein expression in PC 12 cells.They could be used as negative control group(NC group)and Fas gene silencing group(Fas shRNA group)for subsequent experiments.2.The role of Fas/FasL in Cd-induced apoptosis of PC12 cellsTo study the role of Fas/FasL in Cd-induced PC 12 cells apoptosis,NC group cells and Fas shRNA group cells were treated with 10 ?mol/L CdAc2,or PC 12 cells were treated with 10?mol/L CdAc2 in the presence or absence of 10 mg/L Anti-FasL for 12 hours.The expression of Fas/FasL signaling pathway-related proteins,Fas,FasL,FADD,Cleaved caspase-8,Daxx and ASK1 were detected by Western-blot.The changes of nuclear morphology were detected by Hoechst 33258 staining under a laser scanning confocal microscope and the apoptotic rate was detected by flow cytometry.The results showed that Fas shRNA significantly inhibited the increased expression of Fas/FasL signaling pathway-related proteins induced by Cd in PC 12 cells(P<0.01).Fas shRNA and Anti-FasL inhibited Cd-induced typical morphological indicative of apoptosis with nucleus crimpled and chromatin condensation,even nucleus disintegratation,and significantly inhibited the increase of apoptotic rate induced by Cd in PC 12 cells(P<0.01).It indicates that Fas/FasL plays an important role in Cd-induced PC 12 cells apoptosis.3.The regulation of Fas/FasL in Cd-induced MAPK apoptotic pathway of PC12 cellsTo study the regulation of Fas/FasL in Cd-induced MAPK apoptotic pathway of PC 12 cells,NC group cells and Fas shRNA group cells were treated with 10 ?mol/L CdAc2,or PC 12 cells were treated with 10 ?mol/L CdAc2 in the presence or absence of 40?mol/L Z-IETD-FMK(caspase-8 specific inhibitor)for 12 hours.The expression and their phosphorylation levels of MAPK pathway-related proteins,ERK1/2,JNK1/2 were detected by Western-blot.The results showed that Fas shRNA and Z-IETD-FMK significantly inhibited the increased phosphorylation level of ERK1/2 and JNK1/2 induced by Cd in PC12 cells(P<0.01).It indicates that PC12 cells apoptosis induced by Cd via MAPK pathway is mediated by Fas/FasL.4.The regulation of Fas/FasL in Cd-induced mitochondrial apoptotic pathway of PC12 cellsTo study the regulation of Fas/FasL in Cd-induced mitochondrial apoptotic pathway of PC 12 cells,NC group cells and Fas shRNA group cells were treated with 10 ?mol/L CdAc2,or PC 12 cells were treated with 10 ?mol/L CdAc2 in the presence or absence of 10 mg/L Anti-FasL for 12 hours.The expression of Bax,Bcl-2,AIF,Endo G,the release of Cyt C and the activation of tBID,caspase-9,caspase-3,PARP were detected by Western-blot.Mitochondrial membrane potential(??m)were detected by flow cytometry.Nuclear translocation of AIF was detected by immunofluorescence.The results showed that Fas shRNA significantly inhibited the increase of tBID/BID and Bax/Bcl-2 ratios,the release of Cyt C,the activation of caspase-9,caspase-3,PARP,the expression of AIF,Endo G(P<0.05 or P<0.01)and nuclear translocation of AIF induced by Cd in PC12 cells.Anti-FasL significantly inhibited the increase of tBID/BID and Bax/Bcl-2 ratios,the disruption of ??m(P<0.01).It indicates that PC 12 cells apoptosis induced by Cd via mitochondrial pathway is mediated by Fas/FasL.Conclusions:Fas/FasL plays an important role in the apoptosis of PC 12 cells induced by Cd.Fas shRNA and Anti-FasL can significantly inhibit the apoptosis of PC 12 cells induced by Cd.It indicates that PC 12 cells apoptosis induced by Cd via mitochondrial pathway and MAPK pathway is mediated by Fas/FasL.