The Molecular Mechanism Of MiRNA-195-5p Regulating ERK Involvement In Abnormal Phosphorylation Of Tau Protein By Aluminum Maltol In PC12 Cells

Objective:In this study,adrenal pheochromocytoma cells（PC12）were cultured and exposed to Al（mal）₃ to interfere with ERK andm RNA-195-5p,respectively,to explore the role of miRNA-195-5p regulating ERK in process of Al（mal）₃-induced tau hyperphosphorylation.Methods:PC12 cells were selected and Al（mal）₃ was used for exposure.The experimental groups were divided into control group,100μmol/L Al（mal）₃ exposure group,200μmol/L Al（mal）₃exposure group,400μmol/L Al（mal）₃ exposure group,exposure for 24 hours;ERK activation inhibitor U0126 for intervention,the experimental group is divided into control group,200μmol/L Al（mal）₃ exposure group,DMSO solvent control group,50μmol/L U0126intervention group,50μmol/L U0126 intervention group+200μmol/L Al（mal）₃ exposure group,first pretreated with U0126 for 1h,and then Al（mal）₃ exposure for 24h;miRNA-195-5p plasmid for intervention,experimental groups are divided into control group,200μmol/L Al（mal）₃ exposure group,blank plasmid group,miRNA-195-5p overexpression group,miRNA-195-5p overexpression group Group+200μmol/L Al（mal）₃ exposure group,Al（mal）₃ was exposed for 24h after miRNA-195-5p intervention for 36h;After exposure and intervention,the cell morphology was observed by light microscope.The cell viability was detected by CCK8.The distribution of aluminum in the cell and the expression of tau protein were observed by immunofluorescence.The ERK,P-ERK,tau5,PHF and NFT were detected by Western blotting.The ERK m RNA and miRNA-195-5p was detected by RT-PCR.Results:1.Results of different doses of aluminum maltol group1.1 Distribution of aluminum in cells:The fluorescence signal shows that aluminum is mainly distributed in the cytoplasm.As the concentration of Al（mal）₃ increases,the fluorescence signal gradually increases.1.2 Cell morphology:As the concentration of Al（mal）₃ increases,the number of cells decreases,axons decrease,break,and cell bodies become rounded.1.3 Cell viability:As the concentration of Al（mal）₃ increases,cell viability decreases.Compared with the control group,the cell viability of the 200μmol/L Al（mal）₃ exposure group and the 400μmol/L Al（mal）₃ exposure group decreased 7.95%,12.31%,and 17.12%,respectively（P<0.05）.1.4 The expression of miRNA-195-5p:As the concentration of Al（mal）₃ increases,the expression of miRNA-195-5p gradually decreases.Compared with the control group,the expression of miRNA-195-5p in 100μmol/L Al（mal）₃ exposure group,200μmol/L Al（mal）₃exposure and 400μmol/L Al（mal）₃ exposure group decreased 14.70%,28.00%,and 40.90%,respectively（P<0.05）.1.5 Expression of ERK m RNA,total ERK and P-ERK protein:With the increase of Al（mal）₃concentration,the expression of ERK m RNA and total ERK protein expression were not change,the expression of P-ERK protein gradually increased.Compared with the control group,the expression of P-ERK protein in the 200μmol/L Al（mal）₃ and 400μmol/L Al（mal）₃exposure groups increased 1.74 times and 1.99 times respectively（P<0.05）.1.6 Expression of tau5,PHF,and NFT protein:With the increase of Al（mal）₃ concentration,the expression of tau5,PHF,and NFT protein showed an increasing trend.Compared with the control group,the expression of tau5 protein in 200μmol/L Al（mal）₃ exposure group and400μmol/L Al（mal）₃ exposure group increased 1.22 times and 1.46 times,respectively（P<0.05）;Compared with the control group,the expression of PHF protein in 100μmol/L Al（mal）₃ exposure group,200μmol/L Al（mal）₃ exposure group and 400μmol/L Al（mal）₃exposure group increased 1.18 times,1.53 times and 2.50 times,respectively（P<0.05）;Compared with the control group,the expression of NFT protein in 200μmol/L Al（mal）₃exposure group and 400μmol/L Al（mal）₃ exposure group increased 1.39 times and 2.42 times,respectively（P<0.05）.1.7 Immunofluorescence:The results show that tau5 is mainly located in the cytoplasm,PHF is mainly located in the nucleus,and NFT is distributed in both the nucleus and the cytoplasm.With the increase of Al（mal）₃ concentration,the fluorescence signal of tau5,PHF and NFT gradually increased.After Al（mal）₃ treatment,both tau5 and NFT showed large fluorescent plaques in the cytoplasm;while PHF showed protein transfer from the nucleus to the cytoplasm,and a weak fluorescence signal appeared in the cell cytoplasm..2.Results of U0126 intervention experiment2.1 Cell morphology:Compared with the control group,the number of cells in the Al（mal）₃exposure group gradually decreased,the axons were reduced,broken,and the cell bodies became round.After 50μmol/L U0126 treatment,the number of cells increased,the cell bodies became larger,and the cell connections increased.2.2 ERK m RNA,total ERK and P-ERK protein expression:ERK m RNA and total ERK protein expression in each group did not change.Compared with the control group,the expression of P-ERK protein in 200μmol/L Al（mal）₃ increased 1.74 times（P<0.05）and in the U0126 intervention group decreased 31.92%（P<0.05）.Compared with the 200μmol/L Al（mal）₃ exposure group,the expression of P-ERK in the U0126+200μmol/L Al（mal）₃ group was reduced 57.50%（P<0.05）.2.3 Expression of tau5,PHF,and NFT protein:Compared with the control group,the expression of tau5,PHF,and NFT protein in the 200μmol/L Al（mal）₃ exposure group increased by 1.22 times,1.53 times,and 1.39 times,respectively（P<0.05）.The expression of tau5,PHF,and NFT protein in the 50μmol/L U0126 treatment group were reduced by17.90%,16.50%,and 56.54%（P<0.05）,respectively.Factorial analysis showed that P-ERK and Al（mal）₃ had an interaction effect on the expression of tau5,PHF,and NFT proteins.P-ERK participated in Al（mal）₃ exposure to cause abnormal expression of tau5,PHF,and NFT proteins.2.4 Immunofluorescence:The results showed that tau5 was mainly located in the cytoplasm,PHF was mainly located in the nucleus,and NFT was distributed both in the nucleus and cytoplasm.Compared with the 200μmol/L Al（mal）₃ group,the fluorescence signal and the fluorescent plaques of tau5,PHF,and NFT in the U0126+200μmol/L Al（mal）₃ group decreased.After U0126 treatment,tau5 mainly showed that the overall fluorescence density was reduced and the protein distribution was uniform;PHF mainly showed that the fluorescence signal in the cytoplasm disappeared;NFT mainly showed that the overall fluorescence signal was weakened,and no large fluorescent plaques were seen in the cytoplasm.3.Results of miRNA-195-5p intervention experiment3.1 Cell morphology:Compared with the control group,Al（mal）₃ poisoning gradually reduced the number of cells,reduced and broken axons,and rounded the cell body.After the overexpression of miRNA-195-5p,the number of cells increased,the axon mutation was longer,and the cell More connections.3.2 The expression of miRNA-195-5p:Compared with the control group,the expression of miRNA-195-5p in the miRNA-195-5p overexpression group increased by 1428.63 times（P<0.05）.Compared with 200μmol/L Al（mal）₃ group,miRNA-195-5p expression of miRNA-195-5p overexpression+200μmol/L Al（mal）3 group increased 1826.31 times（P<0.05）.3.3 ERK m RNA,total ERK and P-ERK protein expression:ERK m RNA and total ERK protein expression did not change in each group;compared with the control group,the expression of P-ERK protein in 200μmol/L Al（mal）₃ increased 1.74 times（P<0.05）,and the P-ERK protein expression in the miRNA-195-5p overexpression group decreased 16.69%（P<0.05）.Factorial analysis showed that miRNA-195-5p and Al（mal）3 had an interaction effect on the expression of P-ERK protein expression.Mi RNA-195-5p participates in the abnormal expression of P-ERK protein caused by Al（mal）₃.3.4 The expression of tau5,PHF,and NFT protein:Compared with the control group,the expression of tau5,PHF,and NFT protein in the 200μmol/L Al（mal）₃ exposure group increased 1.22 times,1.53 times,and 1.39 times,respectively（P<0.05）)and the expression of tau5,PHF,and NFT protein in the miRNA-195-5p overexpression group decreased13.90%,21.31%,18.27%,respectively（P<0.05）.Factorial analysis showed that miRNA-195-5p and Al（mal）₃ had an interaction effect on the expression of tau5,PHF,and NFT.Mi RNA-195-5p participates in Al（mal）₃ exposure to cause abnormal expression of tau5,PHF,and NFT proteins.3.5 Immunofluorescence:The results showed that tau5 was mainly located in the cytoplasm,PHF was mainly located in the nucleus,and NFT was distributed both in the nucleus and cytoplasm.Compared with the 200μmol/L Al（mal）₃ group,the fluorescence signals of tau5,PHF,and NFT in the miRNA-195-5p mimics+200μmol/L Al（mal）₃ group were weakened,and the fluorescent plaques were reduced.After the intervention of miRNA-195-5p mimics,tau5 mainly showed the decrease of overall fluorescence density;PHF mainly showed the disappearance of fluorescence signal in the cytoplasm;NFT mainly showed the disappearance of large fluorescent plaques in the cytoplasm.Conclusion:1.Aluminum maltolate can enter cells and is mainly distributed in the cytoplasm;tau5 protein is mainly distributed in the cytoplasm;double helix filaments（PHF）are mainly distributed in the nucleus;neurofibrillary tangles（NFT）are both in the nucleus and cytoplasm.In addition,tau5 and NFT showed increased protein expression in the cytoplasm after maltol aluminum exposure,and the expression of PHF not only increased but also shifted to the cytoplasm.2.The phosphorylation of tau protein by aluminum first occurs at the phosphorylation of Ser396,and the phosphorylation of Ser202 and Thr205 appears with the increase of aluminum concentration.3.Aluminum maltolate can reduce the expression of miRNA-195-5p,thereby activating phosphorylated ERK and causing abnormal phosphorylation of tau protein.Therefore,the intervention of miRNA-195-5p or inhibition of ERK activation can effectively improve the cytotoxicity caused by aluminum maltol,which provides a scientific basis for the prevention and treatment of cognitive impairment.