Abstract: BACKGROUND: Previous studies have confirmed that the “metabolic memory” effect induced by a sustained high-glucose environment can significantly exacerbate damage in mouse hippocampal neuronal cell lines HT-22 by regulating histone acetylase activity.
OBJECTIVE: To investigate the effects of glycemic variability and sustained high glucose on apoptosis and the expression of histone deacetylase 4 and silent information regulator 1 in mouse hippocampal neuronal cell lines HT-22.
METHODS: The 6th generation of HT-22 cells were adherent and divided into three groups for culture: a control group was cultured with 25 mmol/L glucose for 3 or 5 days; a high glucose group was cultured with 55 mmol/L glucose for 3 or 5 days; a glycemic variability group was cultured with 25 mmol/L glucose for 12 hours and then switched to 55 mmol/L glucose for 12 hours, with this fluctuation lasting for 3 or 5 days. After 3 days of culture, cell morphology was observed under an optical microscope. After 5 days of culture, cell apoptosis was detected by flow cytometry. After 3, 4, and 5 days of culture, cell viability was detected by cell counting kit-8 assay. After 3 and 5 days of culture, reactive oxygen species levels in cells were detected using a DCFH-DA fluorescent probe, histone deacetylase content in the cell supernatant was detected by ELISA, protein expression of Bax, Bcl-2, Caspase-3, Cleaved Caspase-3, silent information regulator 1, and histone deacetylase 4 in cells was detected by western blot, and mRNA expression of Bax, Bcl-2, Caspase-3, silent information regulator 1, and histone deacetylase 4 in cells was detected by RT-qPCR.
RESULTS AND CONCLUSION: (1) Under an optical microscope, cells in the control group showed good growth, forming a dense network with interconnected synapses; cell growth was inhibited in the high glucose and glycemic variability groups, with reduced synaptic connections between cells. The apoptosis rate in the high glucose group was higher than that in the control and glycemic variability groups (P < 0.05), and the apoptosis rate in the glycemic variability group was higher than that in the control group (P < 0.05). (2) On days 3, 4, and 5 of culture, cell viability in the high glucose group was lower than that in the control and glycemic variability groups (P < 0.05), and cell viability in the glycemic variability group was lower than that in the control group (P < 0.05). (3) After 3 and 5 days of culture, reactive oxygen species levels in the high glucose group were higher than those in the control and glycemic variability groups (P < 0.05), reactive oxygen species levels in the glycemic variability group were higher than those in the control group (P < 0.05), and histone deacetylase content in the cell supernatant of the high glucose and glycemic variability groups was higher than that in the control group (P < 0.05). (4) After 3 and 5 days of culture, the protein and mRNA expression of histone deacetylase 4, Bax, and Caspase-3 in the high glucose and glycemic variability groups were higher than those in the control group (P < 0.05), while the protein and mRNA expression of silent information regulator 1 and Bcl-2 were lower than those in the control group (P < 0.05), and Cleaved Caspase-3 protein expression was higher than that in the control group (P < 0.05). Overall, these findings indicate that glycemic variability may induce HT-22 cell apoptosis by upregulating histone deacetylase 4 expression and downregulating silent information regulator 1 expression.

Key words: glycemic variability, establishment of a neuronal model for glycemic variability, high glucose, apoptosis, histone deacetylase, mouse hippocampal neuronal cells, silent information regulator