Study On Effect Of RIP3-PGAM5-DRP1 Signaling Pathway On Necroptosis Of PC12 Cells Induced By Aluminum Maltol

Objective:To explore the effects of aluminum maltol on RIP3,MLKL,PGAM5,DRP1,p-DRP1（Ser637）protein in PC12 cells.To study the role of RIP3 and PGAM5 proteins in the necroptosis of PC12 cells caused by aluminum maltol by using RIP3 inhibitors and using siRNA to interfere with the PGAM5 gene.Methods:The RIP3 inhibitor,GSK’872,was used to inhibit the activity of RIP3 protein in PC12cells,and siRNA interfered with the expression of PGAM5 gene in PC12 cells.Set groups separately.Dose exposure group:0μM Al（mal）₃,100μM Al（mal）₃ exposed group,200μM Al（mal）₃ exposed group,400μM Al（mal）₃ exposed group.Inhibitor intervention group:normal control group,DMSO solvent control group,GSK’872 group,400μM Al（mal）₃exposed group,GSK’872+400μM Al（mal）₃ exposed group.Si RNA intervention group:NControl control group,PGAM5 siRNA group,400μM Al（mal）₃ exposed group,PGAM5siRNA+400μM Al（mal）₃ exposed group.The CCK-8 method was used to detect cell viability.The AnnexinⅤ-FITC/PI double staining method was used to detect the necrosis rate of cells.Lactate dehydrogenase（LDH）assay kit was used to detect the activity of lactate dehydrogenase.Use the reactive oxygen species（ROS）test box to detect the viability of ROS.The q RT-PCR method was used to detect the expression level of PGAM5 gene to screen siRNA.Confocal laser was used to observe the changes of cell mitochondrial membrane potential and the data were analyzed with Image J.Western blot was used to detect the expression of RIP3,MLKL,PGAM5,p-DRP1（Ser637）,and DRP1 protein.Results:1.Effects of aluminum maltol on PC12 cells:（1）400μM Al（mal）₃ can decrease the viability of PC12 cells,and the difference is statistically significant（P<0.05）.（2）400μM Al（mal）₃ increased the necrosis rate of PC12 cells（P<0.05）.（3）400μM Al（mal）₃ enhanced the LDH activity of PC12 cells（P<0.05）.（4）The mitochondrial membrane potential of PC12cells was decreased by 200μM and 400μM Al（mal）₃,and the mitochondrial membrane potential of the 400μM Al（mal）₃group was lower than that of the 200μM Al（mal）₃ group（P<0.05）.（5）200μM and 400μM Al（mal）₃ staining increased the ROS content of PC12 cells（P<0.05）.（6）Aluminum maltol will increase the relative expression of DRP1,RIP3,MLKL,and PGAM5,and decrease the relative expression of p-DRP1（Ser637）（P<0.05）.2.After treatmented with RIP3 inhibitor GSK’872,effects of aluminum maltol on PC12cells:（1）The GSK’872 inhibitor reduces the increase in the necrosis rate of PC12 cells caused by aluminum maltol.（2）The GSK’872 inhibitor reduced the LDH activity caused by aluminum maltol（P<0.05）.（3）The GSK’872 inhibitor can increase the mitochondrial membrane potential caused by aluminum maltol from（0.71±0.03）to（0.89±0.03）（P<0.05）.（4）The GSK’872 inhibitor can reduce ROS caused by aluminum maltol（P<0.05）.（5）After the intervention of GSK’872 inhibitor,the relative expression of DRP1,RIP3,MLKL,and PGAM5 protein did not increase after PC12 cells were stained with aluminum maltol（P>0.05）.The expression level of p-DRP1（Ser637）protein was higher than that of the aluminum maltol exposure group（P<0.05）.3.After siRNA interfered with PGAM5,effects of aluminum maltol on PC12 cells:（1）The necrosis rate of PC12 cells treated with PGAM5 gene treated by siRNA interference was lower than that of untreated PC12 cells treated with aluminum maltol（P<0.05）.（2）Compared with the NControl control group,interference with PGAM5 gene can reduce the activity of lactate dehydrogenase in PC12 cells stained with aluminum maltol（P<0.05）.（3）After interfering with the expression of PGAM5 gene,the mitochondrial membrane potential of PC12 cells stained with aluminum maltol increased（P<0.05）.（4）After interfering with the expression of PGAM5 gene,there was no significant difference in the ROS content of PC12 cells stained with aluminum maltol compared with the non-intervention group（P>0.05）;compared with the PC12 cell group stained with aluminum maltol alone,The content of ROS in the aluminum maltol group that interfered with the PGAM5 gene was lower than that of the aluminum maltol group（P<0.05）.（5）After interfering with the expression of PGAM5 gene,the relative expression levels of RIP3 and MLKL protein in PC12 cells stained with aluminum maltol were not significantly different from those in the aluminum maltol exposed group（P>0.05）;after the intervention,The relative expression levels of PGAM5 protein and DRP1 protein in the aluminum maltol exposure group were lower than those in the aluminum maltol exposure group（P<0.05）;after the intervention treatment,the p-The relative expression of DRP1（Ser637）protein was higher than that of the aluminum maltol exposed group in the non-intervention treatment group（P<0.05）.Conclusion:1.Aluminum maltol can cause necroptosis in PC12 cells2.Aluminum maltol may cause necroptosis in PC12 cells through the RIP3-PGAM5-DRP1 signaling pathway.