Effects Of HSP90 Regulation Of RIP1/RIP3/MLKL Pathway On Necroptosis Induced By Aluminum Maltol

Objective:To investigate whether heat shock protein 90（HSP90）affects necroptosis induced by maltol aluminum[Al（mal）₃]by regulating interaction protein 1（RIP1）/interaction protein 3（RIP3）/mixed kinase domain like protein（MLKL）pathway.Methods:32 C57BL/6 mice were randomly divided into a control（saline）group and a low dose of 20μmol/kg Al（mal）₃groups,medium dose 40μmol/kg Al（mal）₃groups,high dose group of 80μmol/kg Al（mal）₃were administered intraperitoneally.Morris water maze experiment was used to test the spatial learning and memory abilities of mice;The pathological morphological changes of mouse brain tissue were observed by Nissl staining;The protein expression levels of HSP90,RIP1,RIP3,and MLKL in the hippocampus were measured by Western blotting.Using different concentrations of maltol aluminum(0μmol/L、100μmol/L、200μmol/L、400μmol/L Al（mal）₃and PC12cells were treated with 1200μmol/L Maltol,and then small interfering RNA（si RNA）was used to interfere with the HSP90 m RNA of PC12 cells.The transfection group was divided into blank control group,negative control group（NC group）,transfection reagent group（R4000 group）,and 50 nmol/L si RNA group+400μmol/L Al（mal）₃,50 nmol/L si RNA group,400μmol/L Al（mal）₃groups.CCK-8 was used to detect the effects of aluminum exposure at different times and doses on the viability of PC12 cells;Flow cytometry was used to test the apoptosis and necrosis of PC12 cells after exposure and intervention;RT-PCR was used to detect the expression of HSP90 m RNA to determine the transfection time and concentration;The protein expression levels of HSP90,RIP1,RIP3,MLKL and p-MLKL in the cells were measured by Western blotting.Results:1.1 Compared with normal saline control group mice,the weight of mice in the middle and high dose groups decreased slightly,but the difference was not statistically significant（P>0.05）.In the water maze experiment,the number of times the mice in each group crossed the platform was tested by H,and the result showed that H=9.499,P=0.023.The difference between the groups was statistically significant（P<0.05）.With the increase of Al（mal）₃concentration,the memory ability of mice decreased.The search strategy graph shows that the search strategy of the control group mice presents a"random trend linear"pattern as the number of training increases,but the search strategy of the mice exposed to aluminum presents a"random edge"pattern.1.2 In the Nissl staining experiment,it was observed that with the increase in the concentration of maltol aluminum exposure,the number of hippocampal neurons in mice gradually decreased,the arrangement was disordered,and Nissl bodies decreased.In the high dose aluminum exposed group,the number of cells in the hippocampus of mice was significantly reduced,the cell arrangement was significantly disordered,and the intercellular stratification was unclear.1.3 Western blotting results showed that the expression levels of RIP1,RIP3,MLKL,and HSP90 proteins in the hippocampus of mice were significantly higher in the high-dose aluminum exposed group than in the control group（P<0.05）.The expression levels of RIP3,MLKL,and HSP90 proteins in the hippocampus of mice exposed to medium and high doses of aluminum were significantly higher than those in the control group（P<0.05）.However,there was no statistically significant difference in the expression levels of various proteins in the hippocampus of mice exposed to low doses of aluminum compared to the control group（P>0.05）.1.4 The results of cell viability testing showed that the viability of PC12 cells gradually decreased with the increase of the dose of Al（mal）₃exposure at different times.After 12 hours of exposure,there was no significant difference in cell viability among different dose groups（P>0.05）.After 24 hours of exposure,The cell viability of the group of 400μmol/L Al（mal）₃has decreased to 70%-80%.1.5 When exposed to maltol aluminum at different times and doses:under the light microscope,it was found that in the same exposure time group,In the 0μmol/L Al（mal）₃group,the cell morphology was good and the number of cells was large,the cell body was small,the axons were relatively thin and long,and the cells were tightly connected.With the increase of the dose of Al（mal）₃,when the dose of Al（mal）₃gradually increased,the number of cells gradually decreased,the length of axons began to shorten,and the connection between cells decreased,at 400μmol/L Al（mal）₃group the changes of cell morphology were particularly significant;At different exposure times,there was no significant difference in the changes between cells in each group after 12 hours of exposure（P>0.05）.Compared to other exposure times,at 48 hours of exposure,it was found that there were fewer adherent cells and significantly more floating cells.And The morphological changes of cells in 400μmol/L Al（mal）₃group were still the most significant.1.6 Annexin VITC/PI apoptosis detection reagent was used to detect the effect of maltol aluminum exposure on cell necrosis in PC12 cells at 12,24,36,and 48 hours.The results showed that there was no significant difference in cell necrosis rates among the dose groups at 12 hours of maltol aluminum exposure（P>0.05）.Compared with the control group,The necrosis rate of PC12 cells in the groups of 200μmol/L、400μmol/L Al（mal）₃were significantly different（P<0.05）.After 36 hours of exposure,there was no statistically significant difference in the necrosis rate of PC12 cells among the groups（P>0.05）.Exposure for 48 hours,The necrosis rate of the group of 100μmol/L PC12 cells increased.Compared with the control group,the necrosis rate of PC12 cells exposed to 200μmol/L、400μmol/L Al（mal）₃had no significant difference（P>0.05）.But The necrosis rate of PC12 cells in 1200μmol/L Maltol group was significantly increased（P<0.05）.1.7 Using Western blot assay to detect the expression levels of HSP90,RIP3,MLKL,and p-MLKL proteins in PC12 cells exposed to different doses of maltol aluminum at different times,it was found that there was no statistically significant difference in the protein levels in each dose group after 12 hours of exposure（P>0.05）;After 24 hours of exposure,compared to the control group,The expression levels of proteins HSP90,RIP3,MLKL,and p-MLKL in the 400μmol/L Al（mal）₃group was increased and significantly different（P<0.05）;After 36 hours of exposure,compared to the control group,There was a statistically significant difference in the expression level of protein RIP3 among the group of 400μmol/L Al（mal）₃（P<0.05）,There was a statistically significant difference in the increased expression level of protein p-MLKL among the groups of 100μmol/L、200μmol/LAl（mal）₃（P<0.05）;Exposure for 48 hours,There was a statistically significant difference in the increased expression level of protein HSP90 among the group of 400μmol/L Al（mal）₃（P<0.05）,The expression levels of proteins RIP3,MLKL,and p-MLKL were increased in the group of 100μmol/L Al（mal）₃was significantly different（P<0.05）.1.8 After si RNA intervention in PC12 cells for 36 hours and continued exposure for24 hours,flow cytometry was used to detect the necrosis rate of the cells after intervention.The results showed that there was no statistically significant difference among the blank control group,NC group,and R4000 group（P>0.05）.Compared with NC group,there was no significant difference in the necrosis rate of cells in the si RNA+Al（mal）₃groups（P>0.05）,while the necrosis rate of cells in the si RNA intervention group decreased significantly,with a statistically significant difference（P<0.05）;The cell necrosis rate in Al（mal）₃groups was significantly higher than that in other groups,and the difference was statistically significant（P<0.05）.1.9 After si RNA intervention in PC12 cells for 36 hours,PC12 cells were continuously exposed for 24 hours,and protein immunoblotting was performed after sample collection.The results showed that there was no significant difference in the expression levels of HSP90,RIP1,RIP3,MLKL,and p-MLKL between NC group and R4000 group compared with the blank control group（P>0.05）.Compared with NC group,the expression of HSP90,RIP1,RIP3,MLKL,and p-MLKL proteins in si RNA group decreased significantly（P<0.05）;The expression of HSP90,RIP1,RIP3,MLKL,and p-MLKL proteins in Al（mal）₃groups increased significantly（P<0.05）;There was no significant difference in the expression levels of HSP90,RIP1,RIP3,MLKL,and p-MLKL proteins among the three groups of si RNA+Al（mal）（P>0.05）.Compared with the Al（mal）₃groups,there was a statistically significant difference in the expression level of proteins in the other groups（P<0.05）.Conclusion:1.Al（mal）₃can cause necroptosis of neural cells in C57BL/6 experimental mice and damage their spatial memory abilities.HSP90 is involved in this process.2.The key protein RIP1/RIP3/MLKL for Al（mal）₃-induced necroptosis is regulated by HSP90,and HP90 can further affect Al（mal）₃-induced necroptosis.