Studies On Molecular Mechanisms Of Metformin Amelioration Of Cadmium-induced Accumulation Of Autophagosomes-dependent Apoptosis In Neuronal Cells

The present study,using cellular and molecular biology techniques and methods including cell culture,RNA interference,GFP-LC3/m Cherry-GFP-LC3 fluorescent labeling,MDC staining,TUNEL and/or DAPI staining,cell viability assay,Western blotting,etc.,and employing PC12 cells and primary murine neurons as experimental objects,by establishing cellular models of cadmium(Cd)exposure,systematically investigated molecular mechanisms of metformin’s amelioration of Cd-induced accumulation of autophagosomes-dependent apoptosis in neuronal cells.The detailed results were summarized as follows: 1 Study on metformin’s intervention in Cd-induced accumulation of autophagosomes against apoptosis in neuronal cellsPC12 cells and primary murine neurons,as experimental subjects,were pretreated with different concentrations of metformin(0-1.5 m M)for 24 h and then exposed to Cd(10 μM)for 4 h,12 h,or 24 h.Expressions of Atg5,LC3,p62,cleaved-caspase-3 and cleaved-PARP proteins were assayed by using Western blotting,fluorescence-labeled autophagosomes were counted by infection with Ad-GFP-LC3,fluorescent intensity of autophagosomes was monitored by MDC staining,and cell viability and apoptosis were evaluated using MTS experiment and TUNEL/DAPI staining.The results showed that metformin resisted Cd-induced viability reduction and apoptosis,inhibited Cd-evoked increases in expressions of cleaved-caspase-3,cleaved-PARP,LC3-II,p62 and Atg5,and attenuated Cd-elicited accumulation of autophagosomes in neuronal cells.These results uncover that metformin intervenes in Cd-induced accumulation of autophagosomes against apoptosis in neuronal cells.2 Study on mechanisms of metformin’s amelioration of Cd-induced accumulation of autophagosomes-dependent apoptosis by regulating Atg5 and autophagic flux in neuronal cellsPC12 cells and primary murine neurons,or PC12 cells infected with lentiviral sh RNA Atg5 or sh RNA GFP,as experimental subjects,were pretreated with metformin(1 m M)for 24 h and/or chloroquine(CQ)(2.5 μM)for 1 h,followed by exposure to Cd(10 μM)for 4 h,12 h,or 24 h.Expressions of Atg5,LC3,p62 and cleaved-caspase-3 proteins were assayed by using Western blotting,fluorescence-labeled autophagosomes were counted by infection with Ad-GFP-LC3,and cell apoptosis were evaluated using TUNEL staining.The results showed that down-regulation of Atg5 significantly enhanced metformin’s inhibition of the basal and Cd-induced expressions of LC3-II and cleaved-caspase-3,reinforced metformin’s amelioration of Cd-induced autophagosome accumulation and apoptosis in neuronal cells.Impairment of autophagic flux by CQ potentiated Cd-induced expressions of LC3-II,p62 and cleaved-caspase-3 in neuronal cells,and resisted inhibitory effects of metformin on the events.These results suggest that metformin ameliorates Cd-induced accumulation of autophagosomes-dependent apoptosis by regulating Atg5 and autophagic flux in neuronal cells.3 Study on mechanisms of metformin’s amelioration of Cd-induced accumulation of autophagosomes-dependent apoptosis by activating AMPK blockage of impaired autophagic flux in neuronal cellsPC12 cells and primary murine neurons,or PC12 cells infected with Ad-AMPK-ca or Ad-Lac Z,as experimental subjects,were pretreated with metformin(0-1.5 m M or 1 m M)for 24 h and/or AICAR(2 m M)for 1 h,followed by exposure to Cd(10 μM)for 4 h,12 h,or 24 h.Expressions of AMPK,p-AMPK,ACC,p-ACC,Atg5,LC3,p62 and cleaved-caspase-3 proteins were assayed by using Western blotting,fluorescence-labeled autophagosomes were counted by infection with Ad-GFP-LC3,manifestation of autophagic flux was detected by infection with Ad-m Cherry-GFP-LC3 for double fluorescence labeling,and cell apoptosis were evaluated using TUNEL staining.The results showed that metformin activated AMPK,and resisted Cd inactivation of AMPK in neuronal cells.Activation of AMPK with AICAR or expression of constitutively active AMPK(AMPK-ca)potentiated metformin’s inhibition of Cd-induced expressions of Atg5,LC3-II and cleaved-caspase-3 in neuronal cells,enhanced metformin’s amelioration of Cd-induced autophagosome accumulation and apoptosis in the cells.Expression of AMPK-ca strengthened metformin’s resistance of impaired autophagic flux in neuronal cells in response to Cd.These results suggest that metformin ameliorates Cd-induced accumulation of autophagosomes-dependent apoptosis by activating AMPK blockage of impaired autophagic flux in neuronal cells.