Research Of The Mechanism Of JAK2/STAT3 Signaling Pathway And Klotho Gene In PC12 Cells Apoptosis Induced By Cadmium

Objectives:At the cellular level,exploring the mechanismof action of the JAK2/STAT3signaling pathway,the cholinergic system,and the Klotho gene in the process of cadmium-induced PC12 cells apoptosis,to provide clues for studying the mechanismof cadmium-induced cognitive impairment.Methods:In this research,PC12 cells were used as the research material,and the MTT cell viability test was used to determine the PC12 cadmiumexposure dose and the inhibitor concentration.The experiments were divided into（1）0μM,10μM,and20μM Cd Cl₂ groups,（2）0h,12h,and 24h Cd Cl₂ groups,（3）blank control group,solvent control group,20μM Cd Cl₂ group and 20μM Cd Cl₂+10μM AG490（JAK2/STAT3 pathway inhibitor）group,（4）blank control group,20μM Cd Cl₂group and 20μM Cd Cl₂+10μM Mc N-A 343（M1 muscarinic acetylcholine receptor agonist）group.After collecting the treated cells,Annexin V-FITC/PI double-stained the cells of each treatment group were analyzed for the rate of apoptosis by flow cytometry.Reverse transcription real-time fluorescence quantitative polymerase chain reaction（RT-qPCR）was used to detect the relative mRNA expression of JAK2,STAT3 and Klotho genes in each group of cells.Western blot（WB）was used to detect the expression levels of JAK2,p-JAK2,STAT3,p-STAT3 and Klotho protein.Enzyme-linked immunosorbent assay（ELISA）detected the activity of acetylcholine（ACh）,choline acetyltransferase（ACh E）and choline acetyltransferase（Ch AT）in the cholinergic system.One-way ANOVA was used for comparison among groups,and LSD method was used for comparison between groups with SPSS 22.0.Results:1.（1）The results of different doses（0μM,10μM and 20μM）and different time（0h,12h and 24h）cadmiumtreatment PC12 cells showed that the cadmium-induced apoptosis rate of PC12 cells increased with increasing dose and time（p<0.05）.（2）RT-qPCR results showed that the relative expression levels of JAK2and STAT3 gene mRNA decreased with the increase of cadmiumexposure dose and time,and the mean between each group was statistically significant（p<0.05）;The mRNA relative expression of the Klotho gene was 1.00±0.10,1.45±0.08and 0.81±0.04 in the 0μM,10μM and 20μM groups,and 1.00±0.07,1.39±0.05 and 0.75±0.05 in the 0h,12h and 24h groups,respectively.It was a trend of decreasing after increasing,and the differences between groups were statistically significant（p<0.05）.（3）WB results showed that with the increase of cadmiumexposure dose and time,the expression of JAK2 and STAT3 protein in PC12 cells was decreased（p<0.01）,but the expression levels of phosphorylated protein in both were increased（p<0.01）;Klotho protein of PC12 cells expression was decreased with increasing dose and time of cadmiumexposure（p<0.01）.（4）With the increase of cadmiumexposure dose and time,the relative expression of apoptosis-related protein Cleared caspase 3 was increased significantly（p<0.01）.（5）With the increase of cadmiumexposure dose and time,the concentrations of acetylcholine（ACh）（F=241.672,p<0.01）and acetylcholinesterase（ACh E）（F=102.998,p<0.01）in the cell culture mediumwere decreased.And the concentration of choline acetyltransferase（Ch AT）（F=325.519,p<0.01）in the cell lysate was decreased.2.（1）The JAK2/STAT3 signaling pathway inhibitor AG490 was given to PC12 cells under cadmiumexposure.Among the blank control group,solvent control group,20μM Cd Cl₂ group and 20μM Cd Cl₂+10μM AG490 group,the solvent control group compared with the blank control group,there was no statistically significant difference in cell survival rate and apoptosis rate（p>0.05）;and compared with the Cd Cl₂ treatment group,the survival rate of Cd Cl₂ and AG490 treatment group decreased from72.99%to 66.84%（p<0.05）,and the apoptosis rate increased from21.50%to 29.18%（p<0.05）.（2）There is no difference in the mRNA relative expression of the three target genes between the solvent control group and the blank control group（p>0.05）,and compared with the Cd Cl₂ treatment group,the decrease in the relative expression of JAK2 mRNA with the addition of AG490 was statistically different（p<0.05）,and there was no statistical difference in the relative expression of STAT3 and Klotho gene mRNA between the two groups（p>0.05）.（3）Compared with the blank control group,there was no significant change in the expression level of each target protein in the solvent control group（p>0.05）;compared with the 20μM Cd Cl₂ group,the expression levels of JAK2 and STAT3 protein were slightly reduced after the addition of AG490,and the expression level of phosphorylated protein of themwere significantly reduced（p<0.05）.（4）In the blank control group and the solvent control group,there was no significant difference in the expression level of apoptosis-related protein Cleaved caspase 3;and compared with the 20μM Cd Cl₂group,the expression level of Cleared caspase 3 increased significantly after the addition of AG490（p<0.05）.（5）Compared with the 20μM Cd Cl₂ group,the activity of the cholinergic systemcontinued to be decrease after the addition of AG490,which showed that the concentrations of ACh and ACh E in cell culture mediumwere decreased（p<0.05）,and the concentration of Ch AT in lysate was decreased（p<0.05）.3.（1）PC12 cells were treated with M1 muscarinic acetylcholine receptor（M1 mACh R）agonist Mc N-A 343 under cadmiumexposure.The control group,20μM Cd Cl₂ group and 20μM Cd Cl₂+10μM Mc N-A 343 group were detected by flow cytometer.Compared with the 20μM Cd Cl₂ group,the positive rate of apoptosis in the treatment group supplemented with Mc N-A 343 decreased from8.86%to 3.32%（p<0.05）,and the cell survival rate increased from89.88%to96.19%（p<0.05）.（2）Compared with the Cd Cl₂ treatment group,the relative expression levels of STAT3 and Klotho gene mRNA in PC12 cells were decreased（p<0.05）in the group with Mc N-A 343 agonist,and the relative expression levels of JAK2 gene mRNA did not change significantly between the two groups.（3）Compared with the 20μM Cd Cl₂ treatment group,the expression levels of JAK2and STAT3 protein in PC12 cells added with agonist Mc N-A 343 were not significantly different（p<0.05）,and the expression levels of the two phosphorylated proteins were also the same;the expression level of Klotho protein was decreased slightly,but the difference was not statistically significant（p>0.05）.（4）Compared with the 20μM Cd Cl₂ treatment group,the expression level of Cleaved caspase 3 in PC12 cells with the addition of agonist Mc N-A 343was significantly reduced（p<0.05）.（5）Compared with the Cd Cl₂ treatment group,the activity of the cholinergic systemof PC12 cells with the agonist Mc N-A 343was increased,manifested by the increase of the concentration of ACh and ACh E in the cell culture medium（p<0.05）,and the increase of the concentration of Ch AT in the lysate（p<0.05）.Conclusions:1.Cadmiumcan mediate the decrease of cholinergic systemactivity by activating PC12 JAK2/STAT3 signaling pathway,leading to the apoptosis of PC12 cells.2.High dose of cadmiumcan cause the expression of Klotho protein in PC12cells to decrease,which may lead to the impairment of related function.