Studies On Molecular Mechanisms Of Magnesium Isoglycyrrhizinate's/Metformin's Resistance Of Cadmium-induced Cell Apoptosis Based On PP2A/PP5/AMPK-JNK Signaling Pathway

The present study,using cellular and molecular biology techniques and methods including cell culture,RNA interference,Western blotting,protein fluorescent labeling,HE staining,DAPI and TUNEL staining,trypan blue exclusion,cell viability assay,etc.,and employing ICR mice as well as L02 cells,AML-12 cells,PC12 cells,SH-SY5Y cells and mouse primary neurons as experimental objects,by establishing cadmium(Cd)exposure model in vivo and in vitro,synthetically studied the roles of magnesium isoglycyrrhizinate(Mg IG)and metformin in resisting Cd-induced apoptosis in hepatocytes and neuronal cells,and deeply dissected the molecular mechanisms of Mg IG's and metformin's prevention against apoptosis by controlling ROS-mediated PP2A-JNK and PP5/AMPK-JNK signaling pathways in the cells,respectively,The detailed results were summarized as follows:1 Mg IG prevents Cd-induced activation of JNK pathway from hepatocyte apoptosisL02 and AML-12 cells,or L02 or AML-12 cells infected with Ad-GFP,Ad-dn-c-Jun,respectively,were pretreated with/without Mg IG(10-10⁴?g/ml or100-800?g/ml or 400?g/ml)for 4 h,or pretreated with/without SP600125(20?M)for 1 h and then Mg IG(400?g/ml)for 4 h,followed by exposure to Cd(50?M)for 6h or 24 h.The cell morphology was monitored,the number of live cells was counted by trypan blue exclusion,cell apoptosis was assessed by DAPI and TUNEL staining,and expressions of related proteins were detected by Western blotting.The results showed that Mg IG obviously prevented Cd-elicited cell morphological shrinkage,cell viability reduction,cleaved-PARP elevation,and apoptosis increase in L02 and AML-12 cells.Mg IG prevented Cd-activated JNK pathway against hepatocyte apoptosis.This was supported by the findings that JNK inhibitor SP600125 and expression of dn-c-Jun potentiated Mg IG's resistance to Cd-induced cell viability reduction and apoptosis in hepatocytes.It is suggested that Mg IG prevents Cd-induced activation of JNK pathway against apoptosis in hepatocytes.2 Mg IG inhibits Cd induction of ROS-dependent PP2A-JNK signaling pathway from apoptosis in hepatocytesL02 and AML-12 cells,or L02 or AML-12 cells infected with Ad-GFP and Ad-PP2A-wt,respectively,were pretreated with/without Mg IG(10-10⁴?g/ml or100-800?g/ml)for 4 h,or pretreated with/without NAC(5 m M)or okadaic acid(100n M)for 1 h and then Mg IG(400?g/ml)for 4 h,followed by exposure to Cd(50?M)for 6 h,12 h or 24 h.The level of ROS in cells was detected by CM-H₂DCFDA probe,the number of living cells was counted by trypan blue exclusion,cell apoptosis was evaluated by DAPI staining,expressions of related proteins were detected by Western blotting.The results showed that Mg IG reversed Cd and/or okadaic acid-induced PP2A inactivation,JNK activation and apoptosis in hepatocytes;Overexpression of PP2A enhanced Mg IG's inhibition of Cd-induced JNK activation-dependent hepatocyte apoptosis;Mg IG inhibited Cd-induced ROS production in hepatocytes;NAC potentiated Mg IG's prevention of Cd-induced ROS-dependent PP2A-JNK pathway against apoptosis in hepatocytes.The data imply that Mg IG inhibits Cd induction of ROS-dependent PP2A-JNK signaling pathway from apoptosis in hepatocytes.3 Metformin amelioration of neuronal apoptosis in hippocampus of brain is associated with changes of ROS and PP5/AMPK-JNK signaling pathway in mouse model of Cd exposureThirty-six adult healthy ICR male mice were randomly divided into six groups,including normal control group(0.9%physiological saline),metformin treatment group(200 mg/kg body weight/day),two Cd treatment groups(0.5 and 1 mg/kg body weight/day),two metformin(200 mg/kg body weight/day)/Cd(0.5 and 1 mg/kg body weight/day)treatment groups.The experimental drugs were administered by intragastrical administration for 6 weeks.HE and Nissl staining were used to observe the pathological changes of brain tissues,the apoptosis of hippocampal neurons was evaluated by TUNEL staining,the change of ROS levels was assayed by biochemical analysis in brain tissues,the expressions of AMPK,JNK,cleaved-caspase-3 and other related proteins were detected by Western blotting in brain tissues.The results showed that metformin had protective effects on the damages of brain tissues in Cd-exposed mice;Metformin amelioration of neuronal apoptosis in hippocampus of brain is involved in regulating changes of PP5/AMPK-JNK signaling pathway in mouse model of Cd exposure;Metformin inhibited ROS levels in the brain tissues of Cd-exposed mice.The data reveal that metformin amelioration of neuronal apoptosis in hippocampus of brain is associated with changes of ROS and PP5/AMPK-JNK signaling pathway in mouse model of Cd exposure.4 Metformin resists Cd-induced apoptosis in neuronal cellsPC12 cells,SH-SY5Y cells and primary neurons were pretreated with/without metformin(1 m M)for 24 h,and then exposed to Cd(10?M and 20?M)for 4 h or 24h.Cell activity was determined by using MTS assay,cell apoptosis was evaluated by DAPI and TUNEL staining as well as mitochondrial membrane potential,caspase-3/7activity was assayed,and expressions of cleaved-caspase-3 and cleaved PARP were detected by Western blotting.The results showed that metformin significantly suppressed Cd-induced decreases of cell viability and mitochondrial membrane potential in PC12 cells,SH-SY5Y cells and primary neurons,as well as blocked Cd-elicited elevations of cleaved-caspase-3/cleaved-PARP expression and caspase-3/7activity,and increase of apoptosis in the cells.The findings suggest that metformin resists Cd-induced apoptosis in neuronal cells.5 Metformin prevents against Cd-induced neuronal apoptosis by regulating PP5-JNK signaling pathwayPC12 cells,SH-SY5Y cells and primary neurons,or PC12 cells infected with Ad-GFP,Ad-dn-c-Jun and Ad-PP5,respectively,were pretreated with/without metformin(1 m M)for 24 h,or pretreated with/without metformin(1 m M)for 24 h and then SP600125(20?M)for 1 h,followed by exposure to Cd(10 and/or 20?M)for 4 h or 24 h.Cell viability was determined by MTS assay,cell apoptosis was evaluated using DAPI staining,expression of phospho-JNK(Thr183/Tyr185)was analyzed by immunofluorescence assay,and expression of related proteins were detected by Western blotting.The results showed that metformin inhibited Cd-induced activation of JNK pathway in neuronal cells;Inhibition of JNK with SP600125 or ectopic expression of dominant negative c-Jun(dn-c-Jun)strengthened metformin's prevention against Cd-induced neuronal apoptosis;Metformin reversed Cd-induced PP5 inactivation;Overexpression of PP5 potentiated metformin's inhibition of Cd-induced activation of JNK pathway-dependent neuronal apoptosis.The data suggest that metformin prevents against Cd-induced neuronal apoptosis by regulating PP5-JNK signaling pathway.6 Metformin blocks Cd-induced activation of JNK pathway-dependent neuronal apoptosis by regulating AMPKPC12 cells,SH-SY5Y cells and/or primary neurons,or PC12 cells infected with Ad-dn-AMPK?,Ad-AMPK?-ca or Ad-GFP,respectively,were pretreated with/without metformin(1 m M)for 24 h,or pretreated with/without metformin(1m M)for 24 h and then AICAR(2 m M)for 1 h,followed by exposure to Cd(10and/or 20?M)for 4 h or 24 h.Cell viability was determined by MTS assay,cell apoptosis was evaluated using DAPI staining,and expression of related proteins were detected by Western blotting.The results showed that metformin resisted Cd-induced inactivation of AMPK in neuronal cells;Activation of AMPK by AICAR enhanced metformin's inhibition of Cd-activated JNK pathway-dependent neuronal apoptosis;Ectopic expression of dn-AMPK?resisted metformin's inhibition of Cd-activated JNK pathway-dependent neuronal apoptosis,whereas expression of AMPK?-ca potentiated metformin's inhibition of Cd-activated JNK pathway-dependent neuronal apoptosis.These results demonstrate that metformin blocks Cd-induced activation of JNK pathway-dependent neuronal apoptosis by regulating AMPK.7 Metformin impedes Cd-induced neuronal apoptosis by regulating mitochondrial ROS-mediated PP5/AMPK-JNK signaling pathwayPC12 cells,SH-SY5Y cells and/or primary neurons were pretreated with/without metformin(1 m M)for 24 h,or pretreated with/without metformin(1 m M)for 24 h and then NAC(5 m M)/Mito-TEMPO(10?M)for 1 h,followed by exposure to Cd(10 and/or 20?M)for 4 h or 24 h.The level of ROS was detected by CM-H₂DCFDA probe,cell apoptosis was evaluated by DAPI staining,and expressions of related proteins were detected by Western blotting.The results showed that metformin inhibited Cd induction of excessive ROS in neuronal cells;NAC potentiated metformin's inhibition of Cd-induced ROS mediation of PP5/AMPK-JNK signaling pathway against neuronal apoptosis;Mito-TEMPO enhanced metformin's prevention of Cd-induced ROS-dependent PP5/AMPK-JNK signaling pathway from neuronal apoptosis.The findings suggest that metformin impedes Cd-induced neuronal apoptosis by regulating mitochondrial ROS-mediated PP5/AMPK-JNK signaling pathway.