Protective Role and Mechanism of Salidroside Against High Glucose-induced Apoptosis via NF-κB Pathway in HT22 Hippocampal Neurons
Objective To investigate the effect of salidroside(SAL)on the apoptosis of neurons in the hippocampal area of HT22 under high glucose conditions and to explore the mechanism of NF-κB signaling pathway involved in it.Methods Mouse HT22 hippocampal area neurons were placed in DME/F12 medium containing 10％fetal bovine serum(FBS)and 1％penicillin-streptomycin double antibody,and placed in an incubator at 37℃and 5％CO2 for in vitro culture.HT22 cells were treated with different concentrations of glucose(25,50,100 mM)and SAL(37.5,75,150,300 μM)for 24,48,and 72 h.The proliferative activity of HT22 cells was detected by CCK-8 reagent to screen for the appropriate concentrations of glucose and SAL;the apoptotic rate of HT22 cells was detected by flow cytometry;and the apoptotic rate of HT22 cells was detected by Hoechst 33342 staining.Hoechst 33342 staining was used to detect HT22 cell apoptosis;reactive oxygen species kit was used to detect changes in reactive oxygen species(ROS)content of cells in each group;mitochondrial membrane potential kit(JC-1)was used to detect changes in mitochondrial membrane potential;Western Blot was employed to analyze the expression of phosphorylated NF-κB p65(p-p65),cleaved Caspase-3,Bax and Bcl-2 protein within the cells of each group.Results The high glucose concentration was determined as 50 mM and the SAL concentration was 300 μM through the CCK-8 assay for subsequent experiments.Compared with the control group,after HT22 hippocampal neuron cells were induced by high glucose for 48 h,the cell proliferation viability decreased significantly(P＜0.01),the cell apoptosis rate increased significantly(P＜0.01),the intracellular ROS content increased markedly(P＜0.01),the mitochondrial membrane potential decreased conspicuously(P＜0.01),the expression of p-p65,cleaved Caspase-3,and Bax proteins increased(P＜0.01),and the expression of Bcl-2 protein decreased significantly(P＜0.01).Compared with the high glucose group,the high glucose+SAL group could reverse the above results significantly.Conclusion SAL can inhibit apoptosis of HT22 hippocampal neurons cells under high glucose conditions.The mechanism might be related to inhibiting oxidative stress,suppressing the expression p-p65,restoring the mitochondrial membrane potential,up-regulating the expression of Bcl-2 protein,and down-regulating the expression of Bax and cleaved Caspase-3 proteins.
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