| Objective: 1.To explore the effect of maltol aluminum on TNFR1,RIP1 and RIP3 in PC12 cells.2.To study the effects of TNFR1,RIP1 and RIP3 on necroptosis of PC12 cells induced by maltol aluminum,which were transfected with TNFR1,RIP1 and RIP3 siRNA,respectively.Methods: 1.PC12 cells were cultured in vitro.The cells were treated with Z-VAD-FMK and Nec-1,and the Al(mal)3 was exposed for 24 h.The experimental group were divided into the blank control,DMSO solvent control,200 μM Al(mal)3,Z-VAD-FMK(20 μM),Z-VAD-FMK+Al(mal)3,Nec-1(60 μM)and Nec-1+Al(mal)3.AO-EB were used to observe the cell morphology.The cell viability was determined with CCK-8,and the apoptotic rate and necrotic rate was detected by flow cytometry.TNFR1,RIP1 and RIP3 gene were detected by q RT-PCR.Western blot analysis was performed to confirm protein expression of TNFR1,RIP1 and RIP3.2.Transfection of TNFR1 siRNA: The PC12 cells were treated with Z-VAD-FMK and Nec-1,and the Al(mal)3 was exposed for 24 h.The experimental group were divided into the blank control,negative control,200 μM Al(mal)3,TNFR1 siRNA,TNFR1 siRNA+Al(mal)3,TNFR1 siRNA+Z-VAD-FMK,TNFR1 siRNA +Z-VAD-FMK+Al(mal)3,TNFR1 siRNA+Nec-1 and TNFR1 siRNA+Nec-1+Al(mal)3.The cell viability was determined with CCK-8,and the apoptotic rate and necrotic rate was detected by flow cytometry.TNFR1,RIP1 and RIP3 gene were detected by q RT-PCR.Western blot analysis was performed to confirm protein expression of TNFR1,RIP1 and RIP3.3.Transfection of RIP1 siRNA: the methods,grouping and detection indicators were similar with 2.4.Transfection of RIP3 siRNA: the methods,grouping and detection indicators were similar with 2.ResμLts: 1.Effect of maltol aluminum on PC12 cells:(1)Apoptosis and necrosis were observed by AO-EB staining: cells in groups that treated without Al(mal)3 were in good condition and chromatin was basically bright green.Apoptotic cells and necrotic cells in Al(mal)3 group were significantly increased;Z-VAD-FMK+Al(mal)3 had decreased number of apoptotic cells and increased number of necrotic cells;Nec-1+Al(mal)3 group’s necrotic rate were decreased,but apoptotic rate were increased.(2)The differences of cell viability among groups were statistically significant(P<0.001).Compared with the control group,the cell viability of Al(mal)3-exposed groups was significantly decreased(P<0.05).The cell viability of groups,which treated with inhibitors,was significantly higher than that of the control group(P<0.05).(3)The differences of apoptotic rate and necrotic rate among the seven groups was statistically significant(P<0.001).Compared with the control group,the apoptotic rate of each Al(mal)3-exposed group was increased(P<0.05),and Al(mal)3 and Z-VAD-FMK+Al(mal)3 have increased the rate of necrosis(P<0.05).The apoptotic rate of Z-VAD-FMK+Al(mal)3 group was decreased(P<0.05),and the same condition occurred in Nec-1+Al(mal)3 group for necrotic rate(P<0.05),compared with Al(mal)3 group.(4)The expression of TNFR1 among groups was significantly different(P <0.001).Compared with the control group,the expression of TNFR1 mRNA and protein increased significantly(P<0.05),and the expression of TNFR1 protein in Nec-1 group decreased slightly(P<0.05).When inhibitor joint with Al(mal)3 the expression of TNFR1 was decreased,compared with Al(mal)3(P<0.05).The expression of TNFR1 in inhibitor and Al(mal)3 combined group was significantly higher than that in single inhibitor-treated group(P<0.05).(5)The differences of RIP1 mRNA and protein expression among groups was statistically significant(P<0.001).Compared with the control group,the expression of RIP1 protein in Nec-1 treated groups was significantly decreased regardless aluminum exposed or not(P<0.05),while other Al(mal)3-exposed groups appeared opposite resμLts.Compared with Al(mal)3 group,the expression of RIP1 mRNA and protein in Z-VAD-FMK+Al(mal)3 group increased(P<0.05),but decreased in Nec-1+Al(mal)3 group(P<0.05).Compared with the inhibitor-treated groups,the expression of RIP1 mRNA and protein increased in corresponding group that treated with both inhibitor and Al(mal)3 after transfection of TNFR1 siRNA(P<0.05).(6)The differences of RIP3 mRNA and protein expression among groups was statistically significant(P<0.001).Compared with the control group,the expression of RIP3 mRNA and protein in Nec-1 group decreased(P<0.05),and the expression of RIP3 protein in each Al(mal)3-exposed group increased(P<0.05).Compared with Al(mal)3 group,the expression of RIP3 mRNA and protein in Z-VAD-FMK+Al(mal)3 group increased(P<0.05),but slightly decreased in Nec-1+Al(mal)3 group(P<0.05).Compared with the inhibitor-treated group,the expression of RIP3 mRNA and protein increased in corresponding group that treated with both inhibitor and Al(mal)3(P<0.05).2.Effect of transfection of TNFR1 siRNA on Al(mal)3-exposed PC12 cells:(1)The differences of cell viability among groups were statistically significant(P<0.001).Compared with the control,the cell viability of Al(mal)3-exposed groups was significantly decreased(P<0.05),The cell viability of that treated with inhibitors,was significantly higher than that of the control group(P<0.05).Compared with TNFR1 siRNA group,the cell viability of other groups that incubated with TNFR1 siRNA and Al(mal)3 were significantly decreased(P<0.05).(2)The differences of apoptotic rate and necrotic rate among the groups was statistically significant(P<0.001).Compared with the control,the apoptotic rate of each group was increased except for TNFR1 siRNA+Z-VAD-FMK treatment group(P<0.05),and the necrotic rate decreased after treated with Al(mal)3 except for Nec-1+Al(mal)3 group(P<0.05).Compared with Al(mal)3 group,transfection of TNFR1 siRNA and Al(mal)3 increased the apoptotic rate and decreased the necrotic rate of PC12 cells(P<0.05).Compared with TNFR1 siRNA group,the rate of necrosis decreased after treatment with inhibitors(P<0.05).Compared with TNFR1 siRNA+Al(mal)3 group,treated with both Nec-1 and Al(mal)3 decreased necrotic rate and increased the rate apoptosis;while there was a completely opposite resμLt when PC12 cells exposed in Z-VAD-FMK and Al(mal)3 after transfection of TNFR1 siRNA.(3)The expression of TNFR1 among groups was significantly different(P<0.001).Compared with the control group,TNFR1 mRNA and protein expression levels were significantly decreased after transfection of TNFR1 siRNA(P<0.05).TNFR1 gene silencing efficiency was over 70%.Compared with TNFR1 siRNA group,the expression of TNFR1 mRNA and protein in each aluminum exposed group increased after transfection.(4)The differences of RIP1 mRNA and protein expression among groups was statistically significant(P<0.001).The expression of RIP1 mRNA and protein in groups that without aluminum treated and the group treated with Nec-1and Al(mal)3 were significantly decreased compared with the control group.Compared with the TNFR1 siRNA group,the expression of RIP1 mRNA and protein in groups that without aluminum treated were decreased,but the groups those exposed in Al(mal)3 were increased after transfection of TNFR1 siRNA(P<0.05).Compared with TNFR1 siRNA+Al(mal)3 group,the expression of RIP1 mRNA and protein in Nec-1+Al(mal)3 group was decreased after transfection of TNFR1 siRNA(P<0.05).(5)The differences of RIP3 mRNA and protein expression among groups was statistically significant(P<0.001).Compared with the control group,the expression of RIP3 mRNA and protein in each group decreased after transfection of TNFR1 siRNA.Compared with the TNFR1 siRNA group,the expression level of RIP3 mRNA in each aluminum exposed group increased significantly after transfection of TNFR1 siRNA(P <0.05),TNFR1 siRNA+Al(mal)3 group has increased on expression of RIP3 protein(P <0.05).3.Effect of transfection of RIP1 siRNA on Al(mal)3-exposed PC12 cells:(1)The differences of cell viability among groups were statistically significant(P<0.001).Compared with the control,the cell viability of Al(mal)3-exposed groups was significantly decreased(P<0.05).Compared with Al(mal)3 group,the viability of cells exposed to RIP1 siRNA and Al(mal)3 showed an increased tendency,especially those treated with inhibitors.(2)The differences of apoptotic rate and necrotic rate among the groups wasstatistically significant(P<0.001).Compared with the control,the apoptotic rate of each Al(mal)3-exposed group was increased.When RIP1 was transfected,the necrotic rate of Nec-1 treated groups were lower than that of the control group,while there was a completely opposite result when PC12 cells were exposed in Al(mal)3 and Z-VAD aftertransfection of RIP1 siRNA.Compared with Al(mal)3 group,both apoptotic rate and necrotic rate of each Al(mal)3-exposed group were decreased after transfection of RIP1 siRNA(P<0.05).Compared with RIP1 siRNA group,the apoptotic rate of all Al(mal)3-exposed groups increased(P<0.05);and the rate of necrosis increased after treatment with RIP1 siRNA and Al(mal)3but not Nec-1.(P<0.05).Compared with RIP1 siRNA+Al(mal)3 group,both apoptotic rate and necrotic rate decreased after treated with RIP1 siRNA,inhibitors and Al(mal)3.(3)The expression of TNFR1 among groups was significantly different(P<0.001).The expression of TNFR1 mRNA in aluminum absence groups was not significantly change.The groups treated with Al(mal)3 were significantly increased compared with the control group.After transfection of RIP1 siRNA,the expression of TNFR1 protein decreased,especially in the groups treated with Nec-1(P<0.05).(4)The differences of RIP1 mRNA and protein expression among groups was statistically significant(P<0.001).RIP1 mRNA and protein expression levels were significantly decreased after transfection of RIP1 siRNA(P<0.05).RIP1 gene silencing efficiency was over 85%.(5)The differences of RIP3 mRNA and protein expression among groups was statistically significant(P<0.001).Compared with the control group,RIP3 mRNA and protein expression levels were significantly decreased after transfection of RIP1 siRNA(P<0.05).Compared with the RIP1 siRNA group,the expression of RIP3 mRNA and protein decreased in Nec-1 treated group,but increased in each Al(mal)3-exposed groups after transfection of RIP1 siRNA(P<0.05).4.Effect of transfection of RIP3 siRNA on Al(mal)3-exposed PC12 cells:(1)The differences of cell viability among groups were statistically significant(P<0.001).Compared with the control,the cell viability of Al(mal)3-exposed groups was significantly decreased(P<0.05).Compared with Al(mal)3 group,the cell viability decreased after transfection of RIP3 siRNA.Compared with the inhibitor-treated groups,the cell viability increased in corresponding group that treated with both inhibitor and Al(mal)3 aftertransfection of RIP3 siRNA(P<0.05).(2)The differences of apoptotic rate and necrotic rate among the groups was statistically significant(P<0.001).Compared with the control,the apoptotic rate and necrotic rate of each Al(mal)3-exposed group was increased(P<0.05).Compared with Al(mal)3 group,the rate of apoptosis increasing and the rate of necrosis decreasing occurred in all Al(mal)3-exposed groups(P<0.05).Compared with RIP3 siRNA group,the apoptotic rate of Z-VAD-FMK group decreased(P<0.05),and necrotic rate of Nec-1 group decreased,both apoptotic rate and necrotic rate increased after transfection of RIP3 siRNA.Compared with RIP3 siRNA+Al(mal)3 group,the rate of necrosis decreased after treated with RIP3 siRNA,inhibitors and Al(mal)3,but the rate of apoptosis was not significantly changed.(3)The expression of TNFR1 among groups was significantly different(P <0.001).The expression of TNFR1 mRNA in aluminum absence groups was not significantly changed.After transfection of RIP3 siRNA,the groups treated with Al(mal)3 were significantly increased,compared with the control group(P<0.05).Compared with Al(mal)3 group,the expression of TNFR1 protein decreased after transfection of RIP3 siRNA.(4)The differences of RIP1 mRNA and protein expression among groups was statistically significant(P<0.001).When RIP3 was transfected,expression of RIP1 protein in Nec-1+Al(mal)3 group and the groups which aluminum absence was lower than that of the control group,while there was a completely opposite resμLt when PC12 cells were exposed in Al(mal)3(P<0.05).Compared with Al(mal)3 group,the expression of RIP1 mRNA and protein decreased in groups with inhibitors and Al(mal)3 after transfection of RIP3 siRNA.(5)The differences of RIP3 mRNA and protein expression among groups was statistically significant(P<0.05).RIP3 mRNA and protein expression levels were significantly decreased after transfection of RIP3 siRNA(P<0.05).RIP3 gene silencing efficiency was over 99%.Compared with RIP3 siRNA group,the expression of RIP3 mRNA in each aluminum exposed group increased after transfection of RIP3 siRNA.The expression of RIP3 protein in each group was not statistically significant after transfection of RIP3 siRNA(P>0.05).Conclusions: 1.Maltol aluminumcan induce apoptosis and necroptosis in PC12 cells.2.In PC12 cells,maltol aluminum may induce necroptosis via the TNFR1-RIP1 / RIP3 signaling pathway. |
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