To Investigate The Effects Of Sodium Para-aminosalicylic Acid On Apoptosis Of PC12 Cells Induced By Lead Exposure Based On MAPK Pathway

OBJECTIVE To explore the effects of lead acetate(Pb)and sodium para-aminosalicylate acid(PAS-Na)on Pb-exposure injury,apoptosis related genes and proteins expression of PC12 cells in vitro culture.METHODS PC 12 cells were cultured to its logarithmic growth phase,then were randomly exposed to(1)0、1、10、100、1000 μmol·L-1Pb(Ac)2 for 24 h.(2)0、5、50、100、500 μmol·L-1 PAS-Na for 24h;morpHology of PC 12 cells was observed,and the effect of Pb(Ac)2 or PAS-Na on the survival of PC12 cells was determined by MTT analysis.PC12 cells were randomly divided into the normal control,Pb model,Pb + PAS-Na 20,100,500 μmol·L-1 intervention groups.Cells in normal control group and normal +PAS-Na 500 μmol·L-1 group were cultured with normal medium for 24 h.The former with the normal medium,and the latter with 500 μmol·L-1PAS-Na medium cultured for another 24 h.Lead acetate model group,Pb + PAS-Na 20,100,500 μmol·L-1 intervention groups were incubated with medium containing lead acetate(the final concentration of 10 μmol·L-1)for 24 h,and fed the culture solution,then added to corresponding concentration of PAS-Na for another 24 h.Subsequently,MTT assay was used to observe the viability of PC 12 cells,intracellular glutathione(GSH)content was estimated by assay kit.Apoptosis of PC 12 was detected by Hoechst33342 fluorescence staining and AnnexinV/PI double staining flow cytometry.Real time quantitative PCR(RT-PCR)was used to determin the mRNA expression of P53,Caspase-3,Caspase-9 and MAPK14.Western blotting(WB)was performed to detect protein expression of P53,Bax,Bcl-2,Capsase-3,Caspase-9 and T-/P-JNK,T-/P-ERK,T-/P-P38proteins.RESULTS(1)Pb induced the morphological damage and the dose-response descended cell viability of PC 12 cells.(2)A high concentration(500μmol·L-1)PAS-Na may reduce the survival rate of PC12 cells,and the other dose PAS-Na groups was not different from the control group.(3)Compared with the normal control group,the survival rate of cells and the content of intracellular GSH decreased in Pb model group(P<0.05).Typical morphologic characteristic was found by Hochest33342 staining,and apoptosis rate increased(P<0.05).The mRNA expression of P53,Caspase-3,Caspase-9 and MAPK14 were increased significantly(P<0.05).Western blotting showed that the level of P53,Bax,activated Capsase-3,activated Caspase-9 and p-JNK protein expression were increased,while p-ERK and Bcl-2 decreased in Pb model group(P<0.05).In normal + PAS-Na 5OOμmol·L-1 group,the above indexes did not change significantly.Compared with the Pb model group,survival rate of PC 12 cells and intracellular GSH content were increased in Pb +PAS-Na 100 and 500μmol·L-1 intervention group(P<0.05),while the apoptosis rate were decreased(P<0.05).PAS-Na intervention or treatment was down regulated gene changes induced by lead in different degree,which in lead acetate + PAS-Na 100 and 500 mol L-1 group,the mRNA expression of P53,Caspase-3,Caspase-9 and MAPK14 decreased obviously(P<0.05).PAS-Na intervention may up-regulate expression of p-ERK and Bcl-2,down-regulation of P53,Bax,activated Capsase-3,p-JNK protein,but it has little effect on the expression of activated Caspase-9 and P38 protein.Conclusion(1)Pb exposure reduced the survival rate and apoptosis rate of PC12 cells.PAS-Na had a protective effect on the survival rate and apoptosis of Pb-exposed PC 12 cells.(2)Pb exposure could decrease the content of GSH in PC 12 cells,which were inhibited by PAS-Na.(3)Pb exposure caused increasing mRNA expression of P53,Caspase-3,Caspase-9 and MAPK14,which were effectively inhibited by PAS-Na.(4)By activated JNK phosphorylation and decreased ERK activity,Pb may through the JNK and ERK MAPK pathway involved in apoptosis;lead can also induce the apoptosis by the up-regulation of P53,Caspase-9 and Caspase-3 express,but decreasing the ratio of Bax/Bcl-2.PAS-Na intervention have antagonistic effect on MAPK pathway and apoptosis related protein changes.