Regulation Of MGluR1 On The Expression Of PKC And NMDAR In Aluminum-exposed PC12 Cells

Objective:To investigate the effects of metabotropic glutamate receptor 1（mGluR1）on the expression of protein kinase C（PKC）and N-methyl-D-aspartate receptor（NMDAR）of rat adrenal medullary pheochromocytoma differentiated cell line（PC12）exposed to maltolate aluminum[Al（mal）₃].Methods:1.PC12 cells in logarithmic growth phase were divided into blank control group（normal PC12 cells）,agonist group（100μmol/L mGluR1 agonist）,inhibitor group（100μmol/L mGluR1 inhibitor）,maltol aluminum exposure group（200μmol/L maltol aluminum）,aluminum exposure plus agonist group（100μmol/L mGluR1 agonist and200μmol/L maltol aluminum）,aluminum exposure plus inhibitor group(100μmol/L mGluR1 inhibitor and 200μmol/L maltol aluminum,the exposure time is 24h.The mRNA expression levels of PKC and NMDAR subunits in PC12 cells were determined by real-time fluorescent quantitative PCR.The protein expression levels of PKC and NMDAR subunits in PC12 cells were determined by Western Blot.The PKC enzyme activity of PC12 cells of each group were determined by ELISA.2.（1）PC12 cells in logarithmic growth phase were divided into blank control group,negative transfection group and 3 positive transfection groups.The blank control group did not being given any treatment.The negative transfection group was transfected the empty plasmid without the target siRNA.The positive transfection group was transfected with plasmid containing 001-siRNA,002-siRNA,and 003-siRNA.After incubation for24 hours,the expression level of mGluR1 mRNA in PC12 cells was detected by RT-PCR,and the best interference sequence was screened.（2）The PC12 cells in the logarithmic growth phase were divided into blank control group,negative transfection group and 4 positive transfection groups.The blank control group did not being given any treatment.The negative transfection group was transfected the empty plasmid without the target siRNA.The positive transfection group was transfected with the plasmid containing 003-siRNA and incubated for 12 h,24 h,36 h and 48 h,respectively.The expression of mGluR1 mRNA in PC12 cells was detected by RT-PCR.The expression of mGluR1 protein in PC12 cells was detected by Western Blot,and the optimal interference time was screened.（3）PC12 cells in logarithmic growth phase were divided into blank control group（normal PC12 cells）,solvent control group（transfection reagent control）,negative control group（blank plasmid control）,positive control group（100 nmol/L mGluR1-siRNA）,Maltol aluminum exposure group（200μmol/L maltol aluminum）and aluminum exposure plus mGluR1-siRNA group（100nmol/L mGluR1-siRNA and200μmol/L maltol aluminum）,the exposure time was 24h.The mRNA expression levels of PKC and NMDAR subunits in PC12 cells of each group were determined by real-time fluorescent quantitative PCR.The protein expression levels of PKC and NMDAR subunits in PC12 cells were determined by Western Blot.The PKC enzyme activity of PC12 cells of each group were determined by ELISA.Results:1.（1）Effects of mGluR1 agonism and antagonism on the expression of PKC in PC12cells exposed to aluminum.The expression of PKC mRNA in the maltol aluminum exposure group,the aluminum exposure plus agonist group and the aluminum exposure plus inhibitor group decreased by 39%,45%and 38%,respectively,compared with the blank control group（P<0.05）.The expression of PKC protein in the maltol aluminum exposure group,the aluminum exposure plus agonist group and the aluminum exposure plus inhibitor group decreased by 39%,48%and 51%,respectively,compared with the blank control group（P<0.05）.The PKC activity of the maltol aluminum exposure group and the aluminum exposure plus inhibitor group was lower than that of the blank control group,and the difference was statistically significant（P<0.05）.Compared with the maltol aluminum exposure group,the PKC activity of PC12 cells in the aluminum exposure plus agonist group was up-regulated by 116%（P<0.05）.（2）Effects of mGluR1 agonism and antagonism on the expression of NMDAR in PC12 cells exposed to aluminum.Compared with the blank control group,the expression of NMDAR1 mRNA in the maltol aluminum exposure group and the aluminum exposure plus agonist group decreased by 21%and 32%,respectively（P<0.05）.The expression of NMDAR1 mRNA was up-regulated in the aluminum exposure plus inhibitor group compared with the maltol aluminum exposure group（P<0.05）.The expression of NMDAR2A mRNA in the maltol aluminum exposure group,the aluminum exposure plus agonist group and the aluminum exposure plus inhibitor group decreased by 38%,31%and 35%,respectively,compared with the blank control group（P<0.05）.For NMDAR2B,the expression of NMDAR2B mRNA in the maltol aluminum exposure group,the aluminum exposure plus agonist group and the aluminum exposure plus inhibitor group decreased by 26%,32%and 60%,respectively,compared with the blank control group（P<0.05）.Compared with the maltol aluminum exposure group,the mRNA expression of NMDAR2B was down-regulated in the aluminum exposure plus inhibitor group（P<0.05）.Compared with the blank control group,the protein expression of NMDAR1 in the maltol aluminum exposure group and the aluminum exposure plus agonist group all decreased by 31%（P<0.05）.The protein expression of NMDAR1 was up-regulated by36%in the aluminum exposure plus inhibitor group compared with the maltol aluminum exposure group（P<0.05）.The protein expression of NMDAR2A in the maltol aluminum exposure group,the aluminum exposure plus agonist group and the aluminum exposure plus inhibitor group decreased by 41%,39%and 36%,respectively,compared with the blank control group（P<0.05）.For NMDAR2B,the protein expression of NMDAR2B in the maltol aluminum exposure group and the aluminum exposure plus inhibitor group decreased by 46%and 56%,respectively,compared with the blank control group（P<0.05）.Compared with the maltol aluminum exposure group,the protein expression of NMDAR2B was up-regulated in the aluminum exposure plus agonist group（P<0.05）.2.（1）Screening of interference sequences and interference time:compared with the blank control group,the sequence silencing efficiency of the three groups was 59%,39%,73%,and the 003-siRNA silencing effect was the best.The mRNA and protein expression of mGluR1 was decreased after transfection of mGluR1-siRNA into PC12cells for 12 h,24 h,36 h and 48 h.The gene silencing efficiency was 29%,72%,63%and 50%,respectively.And gene silencing was most effective 24 h to 36 h after transfection.The protein expression of mGluR1 was reduced by 22%,51%,66%,and58%,respectively.And protein expression of mGluR1 was lowest 36 hours after transfection.（2）Effects of mGluR1 silence on the expression of PKC in PC12 cells exposed to aluminum.The mRNA expression of PKC in the maltol aluminum exposure group and the aluminum exposure plus mGluR1-siRNA group decreased by 30%and 32%,respectively,compared with the blank control group（P<0.05）.The protein expression of PKC in the maltol aluminum exposure group and the aluminum exposure plus mGluR1-siRNA group decreased by 28%and 29%,respectively,compared with the blank control group（P<0.05）.The PKC activity of the positive control group,maltol aluminum exposure group and the aluminum exposure plus mGluR1-siRNA group was lower than that of the blank control,and the difference was statistically significant（P<0.05）.The PKC activity of PC12 cells in the positive control group was higher than the maltol aluminum exposure group while the aluminum exposure plus mGluR1-siRNA was down-regulated by 29%（P<0.05）.（3）Effects of mGluR1 silence on the expression of NMDAR in PC12 cells exposed to aluminum.Compared with the blank control group,the mRNA expression of NMDAR1in the maltol aluminum exposure group decreased by 32%（P<0.05）.The mRNA expression of NMDAR1 was up-regulated in the aluminum exposure plus mGluR1-siRNA group compared with the maltol aluminum exposure group（P<0.05）.The mRNA expression of NMDAR2A in the maltol aluminum exposure group and the aluminum exposure plus mGluR1-siRNA group decreased by 28%and 33%,respectively,compared with the blank control group（P<0.05）.For NMDAR2B,the mRNA expression of NMDAR2B in positive control group,the maltol aluminum exposure group and the aluminum exposure plus mGluR1-siRNA group decreased by13%,33%and 47%,respectively,compared with the blank control group（P<0.05）.The mRNA expression of NMDAR2B of PC12 cells in the positive control group was higher than that in the maltol aluminum exposure group while the aluminum exposure plus mGluR1-siRNA was down-regulated by 21%（P<0.05）.Compared with the blank control group,the positive control group was higher than that in the blank control and the maltol aluminum exposure group decreased by 26%（P<0.05）.The protein expression of NMDAR1 was up-regulated in the aluminum exposure plus mGluR1-siRNA group compared with the maltol aluminum exposure group（P<0.05）.The protein expression of NMDAR2A in the maltol aluminum exposure group and the aluminum exposure plus mGluR1-siRNA group decreased by 30%and34%,respectively,compared with the blank control group（P<0.05）.For NMDAR2B,the protein expression of NMDAR2B in positive control group,the maltol aluminum exposure group and the aluminum exposure plus mGluR1-siRNA group decreased by22%,31%and 47%,respectively,compared with the blank control group（P<0.05）.The protein expression of NMDAR2B of PC12 cells in the aluminum exposure plus mGluR1-siRNA group was down-regulated by 23%from that in the maltol aluminum exposure group（P<0.05）.Conclusions:1.Maltol aluminum inhibits mRNA and protein expression of PKC and NMDAR subunits in PC12 cells.2.Maltol aluminum can regulate the expression of NMDAR and PKC enzyme activity by mGluR1.3.The regulation of NMDAR by mGluR1 mainly acts on NMDAR1 and NMDAR2B.In conclusion,aluminum may further regulate the expression of NMDAR2A and NMDAR2B in NMDAR through mGluR1 regulation of PKC enzyme activity,thereby affecting learning and memory.