Research On MAPK Mediated Oxidative Stress And Apoptosis Of Oviduct Magnum Epithelial Cells In Layers Exposed To Vanadium In Vitro

Vanadium(V)induced oxidative stress and apoptosis in oviduct magnum of laying hens,then caused egg quality decline,and had a potential harm to human health.Three experiments were conducted to investigate the possible signal pathway for V-induced oxidative stress and apoptosis of oviduct magnum epithelial cells(OMECs)in layers.It provided some theoretical foundation for revealing the mechanism of V-induced egg quality decline as well as for V detoxification in practice.Experiment 1:Establishment of an oxidative stress model induced by vanadium in oviduct magnum epithelial cells of layersThis study was conducted to establish a V+5-induced oxidative stress model by using primary OMECs of laying hens.OMECs were isolated from Lohman layers(2-3 weeks before onset of laying),cultured and characterized subsequently.The cultured OMECs were then treated with 0,25,50,100,250,and 1000 ?mol/L V for 1 h,4 h,12 h,24 h and 48 h to measure cell viability to detemine the optimistic time.Then the OMECs were then treated with 0,25,50,100,250,and 1000 ?mol/L V for 12 h(determined by measured cell viability)to measure apoptosis ratio as well as cellular ROS,SOD,MDA,CAT and LDH release.The results showed that:1)cell island formed after 24 h and showed classical flagstone morphology.Cultured cells were positive to anti-CK18,anti-OVA,anti-ESR1 and anti-PGR antibody,while were negative to anti-vimentin antibody.2)Cell viability rate decreased after 50 and 100 ?mol/L V treated for 12 h(P<0.05),which were lower(P<0.05)than that of 25 ?mol/L and higher(P<0.05)than that of 250 and 1000 ?mol/L V.3)The apoptosis rate increased(P<0.05)as V levels increased,with the 1000 ?mol/L V treatment has the highest apoptosis rate.4)The cellular ROS,FITC mean values in 100 and 250 ?mol/L V groups were significantly higher than in 0 ?mol/L V treatment(P<0.05),meanwhile FITC mean value in 250 ?mol/L V was significantly higher than in 100?mol/L V treatment(P<0.05).5)Compared with 0 ?mol/L V,cells treated with 50?mol/L V had lower(P<0.05)SOD activity,while 100,250 and 1000 ?mol/L V groups significantly increased MDA content(P<0.05).The activity of CAT in 50,100,250 and 1000 ?mol/L V treatments were significantly lower than in 0 ?mol/L V treatment(P<0.05).The amount of released LDH increased as V levels increased,with the 1000 ?mol/L V treatment had the highest value(P<0.05),and 100 ?mol/L V group had higher value than in 0 ?mol/L(P<0.05).Experiment 2:The possible molecular mechanism of MAPK mediated oxidative stress on OMECs exposed to vanadium inducedThe cultured OMECs were divided into 8 treatments including 0 ?mol/L V(control),100 ?mol/L V(V100),V100+P38MAPK inhibitor(SB203580),SB203580,V100+ERK1/2 inhibitor(U0126),U0126,V100+JNK inhibitor(SP600125)and SP600125.The OMECs were treated with V100 for 12 h after their pretreatement with the MAPKs inhibitors.Cell viability,ROS generation,antioxidant enzymes activities,related genes mRNA and proteins expression were assessed.Results:1)V100 reduced cell viability(P<0.05),while pretreated inhibitors SB203580 and U0126 ameliorated the adverse effects of vanadium on cell viability(P<0.05).2)Pretreatment with these MAPK inhibitors effectively suppressed V-induced apoptosis and ROS generation(P<0.05).3)Inhibitor U0126 tend to increase the superoxide dismutase activity after treatment with V100(P=0.06);SB203580(P<0.05)and SP600125(P=0.08)increased catalase activity;and both SB203580 and U0126 increased glutathione peroxidase activity(P<0.05).The concentrations of malondialdehyde and lactose dehydrogenase activity were lower after pretreatment with the MAPK inhibitors than after treatment with V100 alone(P<0.05).4)V100 down-regulated P38,ERK1/2,JNK,NrfZ,sMaf,GCLC,NQO1,and HO-1 mRNA expression(P<0.05),whereas pretreatment with SP600125 upregulated P38,ERK1/2,JNK,Nrf2,GCLC,and HO-1 mRNA expression(P<0.05),with SB203580 up-regulated JNK,NQOl,and HO-1 mRNA expression(P<0.05),and with U0126 upregulated ERK1/2,GCLC,and HO-1 mRNA expression(P<0.05).5)V100 had little effect on P38,ERK1/2,JNK,Nrt2,sMaf and HO-1 proteins,but enhanced the phosphorylation of the P38,ERK1/2,JNK,and Nrt2 proteins,whereas SP600125 blocked the phosphorylation of these proteins and SB203580 blocked the phosphorylation of P38 and Nrf2.Experiment 3:The possible mechanism of MAPK mediated apoptosis onOMECs exposed to vanadiumThe cultured OMECs were divided into 8 treatments including 0 ?mol/L V(control),100 ?mol/L V(V100),V100+P38MAPK inhibitor(SB203580),SB203580,V100+ERK1/2 inhibitor(U0126),U0126,V100+JNK inhibitor(SP600125)and SP600125.The OMECs were treated with V100 for 12 h after their pretreatment with the MAPKs inhibitors.Apoptosis,LDH activity in supernatant,mitochondrial membrane potential and RT-PCR for related genes' mRNA were measured.The results showed:1)V100 increased apoptosis of OMECs(P<0.05),three MAPK inhibitors suppressed V100-induced apoptosis to some extent(/P<0.05).2)LDH activity increase by the supplementation of V100(P<0.05),P38MAPK,ERK1/2 or JNK inhibitor all decreased the LDH activity(P<0.05)treated by V.3)V100 enhanced mitochondrial membrane potential depolarization(P<0.05),SB203580 and U0126 can alleviated V100-induced mitochondrial membrane potential decrease(P<0.05).4)V100 downregulated B-cell lymphoma-2(Bcl-2)and poly ADP-ribose polymerase 1(PARP1)mRNA expression(P<0.05),meanwhile upregulated Bcl-2 associated x(Bax),cytochrome C(Cyt C)and cysteine aspartase 3(Caspase-3)mRNA expression(P<0.05).All supplemented inhibitors alleviated the up-regulation of V100 for Bax and Caspase-3 mRNA expression level and down-regulation of V100 for Bcl-2 mRNA expression(P<0.05)o SB203580 and U0126 upregulated mRNA level of Cyt C treated by V100(P<0.05),except SP600125.And SB203580 had a similar upregulation for PARP1 mRNA level(P<0.05).All MAPKs inhibitors prevented from V-induced upregulation of PPARy mRNA level.In conclusion,100 ?mol/L V treating OMECs for 12h was used as an ideal V-induced oxidative stress model in present study.The possible reason for V-induced oxidative stress in OMECs was because V induced exceccive ROS generation,which resulted in activating the phosphorylation of P38 and JNK,then phosphor-P38 and phosphor-JNK mediated the Nrf2-ARE pathway to decrease the antioxidant enzymes production.On the other hand,due to phosphorylation of P38MAPK and ERK1/2 triggered by V in OMECs,the proportion of Bax/Bcl-2 was enhanced,which regulated mitochondrial membrane potential decrease,then released Cyt C into cytosol,recruited and activated Caspase-3,then cleaved PARP1,led to apoptosis thereafter.Therefore,MAPK pathway plays a critical role in V-induced oxidative stress and apoptosis in OMECs cells.