Alginate Oligosaccharides Regulate MiR-34a To Inhibit D-gal-induced Apoptosis Of HT22 Cells

Objective:To investigate the protective effects of Alginate oligosaccharides(AOS)on D-galactose-induced(D-gal)HT22 hippocampal neuron cell apoptosis through micro RNA-34a(miR-34a)and its possible mechanism.Methods:This experiment used HT22 mice hippocampal neuron cell line.The cells were divided into six groups:control group,Model group(D-gal),AOS group,D-gal+AOS group,D-gal+AOS+micro RNA-Negative Control(miR-NC)group and D-gal+AOS+miR-34a mimic group.The D-gal+AOS+miR-NC group and D-gal+AOS+miR-34a mimic group were transfected miR-NC(50n M)and miR-34a mimic(50n M)with Lipofectamine~TMM 3000 respectively,and the medium was changed after 4-6h.In the control group,the cells were given synchronous replacement of medium during the experiment,the cells in D-gal group,D-gal+AOS group,D-gal+AOS+miR-NC group and D-gal+AOS+miR-34a mimic group were exposed to D-gal(100 mmol/L)for 24 h to induce the apoptosis model of HT22 hippocampal neuron cell.After that,the AOS group,D-gal+AOS group,D-gal+AOS+miR-NC group and D-gal+AOS+miR-34a mimic group were treated with AOS(100?g/m L)to intervene the hippocampal neuron cells for 48 hours.After intervention,Annexin V-FITC/PI Apoptosis Detection kit was used to analyze the apoptosis of hippocampal neuron cell;RT-PCR was used to detect the expression of miR-34a;the expressions of Sirt1,p53,Bcl-2,Bcl-xl and Bax proteins were detected by Western blot.The mRNA expressions of cyt-c,caspase-9 and caspase-3 were detected by RT-PCR.Results:1.Apoptotic results showed that in comparison to the control group,the percentage of hippocampal neuron apoptosis in the D-gal group was significantly increased(p<0.01).In comparison to the D-gal group,the proportion of hippocampal neuron apoptosis in the D-gal+AOS group was significantly declined(p<0.01).In comparison to the D-gal+AOS+miR-NC group,the apoptosis rate of hippocampal neurons in the D-gal+AOS+miR-34a mimic group was significantly increased(p<0.01).2.The expression of miR-34a showed that in comparison to the control group,the mRNA expression of miR-34a in the D-gal group was significantly increased(p<0.01).In comparison to the D-gal group,the mRNA expression of miR-34a in the D-gal+AOS group was significantly reduced(p<0.01).In comparison to the D-gal+AOS+miR-NC group,the m RNA expression of miR-34a in the D-gal+AOS+miR-34a mimic group was significantly increased(p<0.01).3.Western blot showed that in comparison to the control group,the protein expression of Sirt1 in the D-gal group was significantly reduced,and the protein expression of p53 in the D-gal group was significantly increased(p<0.01).In comparison to the D-gal group,the protein expression of Sirt1 in the D-gal+AOS group was significantly increased,the protein expression of p53 in the D-gal+AOS group was significantly reduced(p<0.01).In comparison to the D-gal+AOS+miR-NC group,the protein expression of Sirt1 in the D-gal+AOS+miR-34a mimic group was significantly reduced,the expression of p53 protein in the D-gal+AOS+miR-34a mimic group was significantly increased(p<0.05).4.Western blot showed that in comparison to the control group,the protein expression of Bcl-2/Bax and Bcl-xl in the D-gal group were significantly reduced(p<0.01).In comparison to the D-gal group,the protein expression of Bcl-2/Bax and Bcl-xl in the D-gal+AOS group were significantly increased(p<0.01).In comparison to the D-gal+AOS+miR-NC group,the protein expression of Bcl-2/Bax and Bcl-xl in the D-gal+AOS+miR-34a mimic group were significantly reduced(p<0.05).5.RT-PCR results showed that in comparison to the control group,the mRNA expression of cyt-c in the D-gal group was significantly increased(p<0.01).In comparison to the D-gal group,the m RNA expression of cyt-c in the D-gal+AOS group were significantly reduced(p<0.01).In comparison to the D-gal+AOS+miR-NC group,the mRNA expression of cyt-c in the D-gal+AOS+miR-34a mimic group was significantly increased(p<0.01).6.RT-PCR results showed that in comparison to the control group,the mRNA expression of caspase-9 in the D-gal group was significantly increased(p<0.01).In comparison to the D-gal group,the mRNA expression of caspase-9 in the D-gal+AOS group was significantly reduced(p<0.01).In comparison to the D-gal+AOS+miR-NC group,the m RNA expression of caspase-9 in the D-gal+AOS+miR-34a mimic group was significantly increased(p<0.01).7.RT-PCR results showed that in comparison to the control group,the mRNA expression of caspase-3 in the D-gal group was significantly increased(p<0.01).In comparison to the D-gal group,the mRNA expression of caspase-3 in the D-gal+AOS group was significantly reduced(p<0.01).In comparison to the D-gal+AOS+miR-NC group,the m RNA expression of caspase-3 in the D-gal+AOS+miR-34a mimic group was significantly increased(p<0.01).Conclusions:AOS inhibits D-gal-induced apoptosis of HT22 hippocampal neuron cells via mi R-34a,and its mechanism may be related to the miR-34a/Sirt1/p53 signaling pathway.