Effects Of GSTO1 On Expression Of Related Factors In Hippocampus Cell Line (HT22) Induced By Fluoride

?Objective?The study was aimed to detected the change of m RNA and protein expression level about GSTO1 and the related factors of learning and memory after CREB,BDNF,NCAM and SCF in hippocampus neuron cell(HT22)dye in different concentration and different time of fluoride,establish the time exposed to fluoride and concentration;The study construct the HT22 cell model inhibited by si RNA-GSTO1 to detect the expression of related genes and proteins in learning and memory,and to explore the regulatory mechanism of GSTO1 in fluorine on HT22.?Methods?1.MTT assay for HT22 cell proliferation rate:The well-growing HT22 was randomly divided into7 groups and treated 24 hours later as follows:The control group(C)was cultured in a normal DMEM culture medium,and the fluoride group(F)was supplemented with 10-6,10-5,10-4,10-3,10-2 and 10-1 mol/L fluorinated solutions.Sodium,after 24 h,36h and 48 h of fluorine staining,the cell proliferation rate was detected by MTT assay.2.According to MTT results,the concentration of fluoride was selected,and the effect of fluoride on GSTO1,CREB,BDNF,NCAM,and SCF was tested.The experimental group was set up with control group(C),low fluoride group(10-5 mol/L),medium fluoride group(10-4mol/L),and high fluoride(10-3mol/L).The expression of GSTO1,CREB,BDNF,NCAM and SCF in each group was detected by q RT-PCR and Western Blot.3.Select the GSTO1 expression of the highest fluorine concentration(10-4mol/L)to establish the si RNA-GSTO1 model to detect the expression of silencing GSTO1 on learning and memory-related factors,experimental set up blank control group(C),negative control group(NC-si RNA),Na F group(10-4 mol/L),Silence group(si RNA-GSTO1)and Fluoro-plus silencing group(Na F+si RAN-GSTO1),the expression of GSTO1,CREB,BDNF and NCAM genes and proteins were detected by q RT-PCR and Western Blot.?Results?1.MTT assay results showed that 10-3,10-4 and 10-5mol/L sodium fluoride had significant effects on the proliferation of HT22 cells at 24 h,36h and 48 h,10-2-10-1 mol/L.Sodium fluoride has a significant inhibitory effect on the proliferation of HT22 cells.2.Experimental results in the fluorine model:(1)q RT-PCR results: Compared with the C group,the GSTO1 gene expression in the low-fluoridation group and the middle-fluoride group was increased at 24 h,36h,and 48 h after fluorine was treated with HT22,and the middle-fluoride group was significantly increased,while Significantly lower in the high fluoride group.The overall expression of CREB,BDNF,NCAM,and SCF decreased during the three test periods.There was no significant difference in m RNA expression of SCF and NCAM at 48 hours after the test.(2)Western Blot:GSTO1,CREB and BDNF protein expression was consistent with q RT-PCR gene expression during the three test periods.3.si-GSTO1 model experimental results:(1)Selection of optimal transfection concentration of si RNA: FAM-si RNAs with different concentrations(20,30,50,and 100 n M)were added to adherent cell plates,and the intensity of green fluorescence in each well was observed under fluorescence microscope.Weakly,it was found that the fluorescence was the strongest at the concentration of 50 n M,so 50 n M was selected as the optimal transfection concentration of the si RNA.(2)Screening of effective interference sequences:Three kinds of si-GSTO1 were transfected into HT22 cells with a concentration of 50 n M to detect the expression of GSTO1 gene and protein.The results showed that the expression of GSTO1 gene and protein in si RNA2(si-GSTO1-2)group and si RNA3(si-GSTO1-3)group was significantly lower than that in the blank control group,and the difference in si-RNA3 group was extremely significant.(3)q RT-PCR results:Compared with the blank control group,the expression of GSTO1,CREB,NCAM,and BDNF genes in the Na F group decreased.Consistent with the findings in the second part,the GSTO1,CREB,and BDNF in the silencing group were significantly decreased;Compared with the fluorosis group,the expression of GSTO1,CREB and BDNF in the fluoroplus silencing group was significantly reduced.Western Blot results: Expression changes of GSTO1 and CREB were consistent with q RT-PCR results.? Conclusion ? Fluorine may eventually affect the decline of learning and memory ability of the body by regulating the decrease of the expression of HT22 learning and memory related factors CREB BDNF NCAM and SCF,the decreased expression of learning and memory related factors under certain dose of fluorine may increase under the regulation of GSTO1.