Effects and mechanisms of glycemic variability on apoptosis in mouse hippocampal neuronal HT-22 cells

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. OBJECTIVE: To investigate the effects of glycemic variability and sustained high glucose on apoptosis and the expression of histone deacetylase 4 (HDAC4) and silent information regulator 1 (SIRT1) in mouse hippocampal neuronal HT-22 cells. METHODS: Passage 6 HT-22 cells were cultured in three groups after adherence: control group (25 mmol/L glucose for 3 or 5 days), high glucose group (55 mmol/L glucose for 3 or 5 days), and glycemic variability group (alternating 25 mmol/L and 55 mmol/L glucose every 12 hours for 3 or 5 days). After 3 days of culture, cell morphology was observed under an optical microscope. After 5 days, apoptosis was detected by flow cytometry. Cell viability was measured by CCK-8 assay at 3, 4, and 5 days. Reactive oxygen species (ROS) levels were detected using 2,7-dichlorofluorescein diacetate fluorescent probe at 3 and 5 days. Histone deacetylase (HDAC) content in the supernatant was measured by ELISA. Protein expression of Bax, Bcl-2, Caspase-3, Cleaved Caspase-3, SIRT1, and HDAC4 was detected by western blot, and mRNA expression of Bax, Bcl-2, Caspase-3, SIRT1, and HDAC4 was detected by RT-qPCR. RESULTS AND CONCLUSION: (1) Under the optical microscope, control cells grew well, forming a dense network with interconnected synapses; high glucose and glycemic variability groups showed inhibited growth and reduced synaptic connections. Apoptosis rate was higher in the high glucose group than in the control and glycemic variability groups (P < 0.05), and higher in the glycemic variability group than in the control group (P < 0.05). (2) At 3, 4, and 5 days, cell viability was lower in the high glucose group than in the control and glycemic variability groups (P < 0.05), and lower in the glycemic variability group than in the control group (P < 0.05). (3) At 3 and 5 days, ROS levels were higher in the high glucose group than in the control and glycemic variability groups (P < 0.05), and higher in the glycemic variability group than in the control group (P < 0.05). HDAC content in the supernatant was higher in the high glucose and glycemic variability groups than in the control group (P < 0.05). (4) At 3 and 5 days, protein and mRNA expression of HDAC4, Bax, and Caspase-3 were higher in the high glucose and glycemic variability groups than in the control group (P < 0.05), while SIRT1 and Bcl-2 expression were lower (P < 0.05). Cleaved Caspase-3 protein expression was higher in the high glucose and glycemic variability groups than in the control group (P < 0.05). (5) These results indicate that glycemic variability may induce apoptosis in HT-22 cells by upregulating HDAC4 expression and downregulating SIRT1 expression.

1. Introduction

In recent years, with the changes in modern lifestyle and dietary structure, the incidence of diabetic encephalopathy has been increasing year by year. According to the global diabetes map released by the International Diabetes Federation in 2023, the number of diabetic patients worldwide has reached 537 million [1]. Among the many complications of diabetes, diabetic encephalopathy has attracted much attention due to its progressive damage to the central nervous system. Epidemiological predictions show that by 2025, the number of diabetic encephalopathy patients worldwide will exceed 300 million, with more than 3 million deaths each year [2]. Since DEJONG first proposed the concept of diabetic encephalopathy in 1965, the exploration of its pathogenesis has never ceased [3-5]. Current research mostly focuses on brain regions closely related to cognitive function. The hippocampus plays a key role in memory formation and emotional regulation, making it a key target in diabetic encephalopathy research. As the information integration center of the limbic system, the CA1 pyramidal neurons and dentate gyrus granule cells of the hippocampus are particularly sensitive to high-glucose microenvironments. Under hyperglycemic conditions, neuronal cells exhibit a series of problems such as inflammatory responses, oxidative stress, and energy metabolism disorders [6-7]. Existing related research mainly focuses on the effects of hyperglycemia.