The Role Of MAPK Signaling Pathway In Necroptosis Induced By Aluminum In PC12 Cells

Objective:To explore the effect of MAPK signaling pathway in necroptosis induced by Al(mal)3 in PC12 cells by adding inhibitor of apoptosis(z-VAD-fmk),necroptosis(Nec-1),P38,JNK and ERK.Methods:1.Cells culture and treatment:(1)The PC12 cells were cultured in a 37 °C,5% CO2 incubator.(2)Appropriate PC12 cells in the logarithmic growth phase were exposed with Al(mal)3(200 μmol/L)for 24 h,and divided into blank control group,solvent control group(DMSO group),apoptosis inhibitor(z-VAD-fmk)group,necroptosis inhibitor(Nec-1)group,Al(mal)3 group,Al(mal)3+z-VAD-fmk group,and Al(mal)3+ Nec-1 group.The inhibitors of z-VAD-fmk and Nec-1 were incubated for 1 h before treatment with aluminum,in concentrations of 20 μmol/L and 60 μmol/L,respectively.(3)Appropriate amount of PC12 cells in the logarithmic growth phase were treated with Al(mal)3(200μmol/L)for 24 h.The corresponding inhibitors of P38,JNK and ERK(SB203580 /SP600125 / U0126)were added before the treatment with Al(mal)3 for 1h,the concentrations of inhibitors were 625 nmol/L,10 μmol/L and 15 μmol/L,respectively.2.Cell viability of each treatment group was determined by CCK-8.The apoptosis rate and necrosis rate were measured by flow cytometry.The morphological changes of the cells were detected by AO-EB method.The genes of P38,JNK and ERK were detected by RT-PCR.The expression of P38,JNK,ERK,p-P38,p-JNK and p-ERK protein weredetected by Western-blot.Results:1.(1)AO-EB staining results: The nuclear chromatin was green and showed normal structure in the blank control group and the DMSO group.Compared with the blank control group,the nucleus of the Al(mal)3 group was orange-red and showed normal and solidified or fragmented structure increased,that is,apoptotic cells and necrotic cells increased.Compared with the Al(mal)3 group,the apoptotic cells of the Al(mal)3+zVAD-fmk group decreased,the cells with the nuclear chromatin stained with orange color and chromatin shrinking were reduced,the necrotic cells of the Al(mal)3+ Nec-1 group reduced,the cells with orange staining and normal structure reduced.(2)CCK-8 cell viability test: Compared with the blank control group,the cell viability of Al(mal)3group,Al(mal)3+ z VAD-fmk group and Al(mal)3+ Nec-1 group decreased by 36.91%,13.8% and 18.94%,and the difference was statistically significant(P<0.05).Compared with Al(mal)3 group,the cell viability of Al(mal)3 + zVAD-fmk group and Al(mal)3 +Nec-1 group increased by 23.11% and 17.97%,respectively,and the difference was statistically significant(P<0.05).(3)Flow cytometry: Compared with the blank control group,the necrosis rate of z VAD-fmk group,Nec-1 group,Al(mal)3 group,Al(mal)3 +zVAD-fmk group and Al(mal)3 + Nec-1 group decreased,and the difference was statistically significant(P<0.05).The apoptotic rate and necrosis rate of Al(mal)3 +zVAD-fmk group and Al(mal)3 + Nec-1 group were significantly higher than those of Al(mal)3,and the difference was statistically significant(P<0.05).(4)RT-PCR: The relative expression of P38,JNK and ERK genes in the different treatment groups slightly changed,but the difference was not statistically significant(P>0.05).(5)Western-blot assay Results: The protein expression of P38,JNK,ERK and p-JNK in PC12 cells treated with Al(mal)3 was not statistically significant(P>0.05),and the differences of p-P38 and p-ERK were statistically significant(P<0.05).Compared with the normal control group,the expression of p-P38 in Al(mal)3 group,Al(mal)3 + zVAD-fmk group and Al(mal)3 +Nec-1 group increased,the difference was statistically significant(P<0.05).Compared with Al(mal)3 group,the expression of p-P38 in Al(mal)3 + z VAD-fmk group and Al(mal)3 +Nec-1 group decreased,the difference was statistically significant(P <0.05).Compared with the normal control group,the expression of p-ERK in Al(mal)3 group,Al(mal)3 +zVAD-fmk group and Al(mal)3 + Nec-1 group decreased(P<0.05).The expression levels of p-ERK in Al(mal)3 + z VAD-fmk group and Al(mal)3 + Nec-1 group were significantly higher than those in group Al(mal)3 Statistically significant(P<0.05).2.(1)AO-EB results showed that the nuclear chromatin of the Al(mal)3 + SB203580 inhibitor group was lower than that of the Al(mal)3 group.The nuclear chromatin of the Al(mal)3 + SP600125 inhibitor group was no significant changes in color;Al(mal)3+U0126 inhibitor group of nuclear staining of orange cells increased.(2)CCK-8 cell viability test: Compared with the blank control group,the viability of Al(mal)3,Al(mal)3 +SB203580 inhibitor,group Al(mal)3 + SP600125 inhibitor andAl(mal)3+U0126 inhibitor group decreased(P<0.05).The cell viability of Al(mal)3+SB203580 inhibitor group was significantly higher than that of Al(mal)3 group(P<0.05),and the difference was statistically significant(P<0.05).The cell viability of the inhibitor group was higher than that of the control group(P <0.05),but the difference was not significant(P>0.05).(3)Flow cytometry: Compared with the blank control group,the apoptotic rate of Al(mal)3group,Al(mal)3 + SB203580 inhibitor group,Al(mal)3 + SP600125 inhibitor group and Al(mal)3 + U0126 inhibitor group were increased,and the difference was statistically significant(P<0.05).Compared with Al(mal)3,the apoptotic rate and necrosis rate of Al(mal)3 + SB203580 inhibitor group were decreased,and the difference was statistically significant(P<0.05).The apoptotic rate and necrosis of Al(mal)3 + SP600125 inhibitor group was changed,but the difference was not statistically significant(P>0.05).The apoptotic rate and necrosis rate of Al(mal)3 + U0126 inhibitor group increased(P<0.05).The difference was statistically significant(P<0.05).(4)Western-blot assay Results: There was no significant difference in the relative expression of P38 protein between the groups(P>0.05).The difference of p-P38 expression was statistically significant(P<0.05).Compared with the blank control group,the phosphorylation of P38 in SB203580 inhibitorgroup,Al(mal)3 group and Al(mal)3+ SB203580 inhibitor group were changed,and the difference was statistically significant(P<0.05)The phosphorylation of P38 in SB203580 inhibitor group and Al(mal)3+ SB203580 inhibitor group decreased,and the difference was statistically significant(P<0.05).The expression of JNK was not significantly different(P>0.05).The control group,DMSO solvent control group,SP600125 inhibitor group,Al(mal)3 group and Al(mal)3 +SP600125 were no phosphorylation.There was no significant difference in the relative expression of ERK protein between the groups after adding ERK inhibitor U0126(P>0.05).The difference of p-ERK expression was statistically significant(P<0.05).Compared with the blank control group,the phosphorylation of ERK in U0126 inhibitor group,Al(mal)3 group and Al(mal)3 + U0126 inhibitor group were decreased,and the difference was statistically significant(P<0.05),and the phosphorylation level of ERK group was significantly lower than that of the control group(P <0.05).Conclusions:(1)Maltol aluminum can induce apoptosis and necroptosis in PC12 cells.(2)Activation of P38 MAPK signaling pathway and inactivation of ERK signaling pathway can induce apoptosis and necroptosis of PC12 cells.JNK signaling pathway has no effect on apoptosis and necroptosis of PC12 cells.