高糖诱导HT-22小鼠海马神经元代谢记忆细胞模型的构建及影响

doi:10.11958/20231115

天津医药 ›› 2024, Vol. 52 ›› Issue (1): 44-49.doi: 10.11958/20231115

高糖诱导HT-22小鼠海马神经元代谢记忆细胞模型的构建及影响

段云峰¹^,²(), 许永劼¹^,³, 杨婷婷¹^,², 黄昶煜东¹, 朱丽英²^,³, 李兴⁴, 潘卫¹^,²^,^△()

1.贵州医科大学附属医院产前诊断中心（邮编550004）
2.贵州医科大学医学检验学院
3.贵州医科大学附属医院临床检验中心
4.贵州中医药大学基础医学院

收稿日期:2023-09-11 出版日期:2024-01-15 发布日期:2024-01-18
通讯作者: ^△E-mail：313831139@qq.com
作者简介:段云峰（1997），男，硕士在读，主要从事糖尿病慢性并发症机制研究。E-mail：473989716@qq.com
基金资助:
国家自然科学基金地区科学基金项目(81960151);国家自然科学基金地区科学基金项目(81960822);国家自然科学基金地区科学基金项目(82260165);国家自然科学基金地区科学基金项目(82300920)

Construction and effect of a high glucose induced hippocampal neuron metabolic memory cell model in HT-22 mice

DUAN Yunfeng¹^,²(), XU Yongjie¹^,³, YANG Tingting¹^,², HUANG Changyudong¹, ZHU Liying²^,³, LI Xing⁴, PAN Wei¹^,²^,^△()

1. Prenatal Testing Center, Affiliated Hospital of Guizhou Medical University, Guiyang 550004, China
2. School of Laboratory Medicine, Guizhou Medical University
3. Clinical Laboratory Center of Affiliated Hospital of Guizhou Medical University
4. School of Basis Medicine, Guizhou University of Traditional Chinese Medicine

Received:2023-09-11 Published:2024-01-15 Online:2024-01-18
Contact: ^△E-mail：313831139@qq.com

段云峰, 许永劼, 杨婷婷, 黄昶煜东, 朱丽英, 李兴, 潘卫. 高糖诱导HT-22小鼠海马神经元代谢记忆细胞模型的构建及影响[J]. 天津医药, 2024, 52(1): 44-49.

DUAN Yunfeng, XU Yongjie, YANG Tingting, HUANG Changyudong, ZHU Liying, LI Xing, PAN Wei. Construction and effect of a high glucose induced hippocampal neuron metabolic memory cell model in HT-22 mice[J]. Tianjin Medical Journal, 2024, 52(1): 44-49.

摘要/Abstract

摘要：

目的体外构建高糖诱导HT-22小鼠海马神经元“代谢记忆”细胞模型，探究“代谢记忆”对HT-22细胞凋亡和组蛋白乙酰化的影响。方法以高糖培养基（葡萄糖浓度为55 mmol/L）和常规糖培养基（葡萄糖浓度为25 mmol/L）培养HT-22细胞，分为常糖组（NG 4、6、8组，25 mmol/L葡萄糖分别培养4、6、8 d），高糖组（HG 4、6、8组，高糖培养4、6、8 d），代谢记忆组（HG2NG2、HG2NG4、HG2NG6、HG4NG2、HG4NG4组，即高糖2 d转25 mmol/L葡萄糖培养2、4或6 d，高糖4 d转25 mmol/L葡萄糖培养2 d或4 d）。CCK-8法检测细胞活力变化，检测细胞培养上清液中乳酸脱氢酶（LDH）释放量，筛选出建立“代谢记忆”模型的最佳作用时间。后续将细胞分为NG4组、NG8组、HG4组、HG4NG4组和HG8组，光学显微镜观察各组细胞形态；流式细胞术检测各组细胞凋亡率；酶联免疫吸附试验（ELISA）检测去乙酰化酶（HDAC）和组蛋白乙酰转移酶（HAT）活性；Western blot检测组蛋白去乙酰化酶4（HDAC4）、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）和胱天蛋白酶3（Caspase-3）蛋白表达情况。结果 HG4NG4组为理想的高糖“代谢记忆”细胞模型；NG4、NG8组细胞交织成致密网状，生长状态良好，细胞呈梭形，突触结构明显。而HG4、8组细胞胞体变圆，突触结构消失，生长受抑，HG4NG4组细胞数量增多但形态受损；流式细胞术结果显示，与NG8组相比，HG8、HG4NG4组凋亡率增加（P<0.05）；ELISA结果表明，与NG8组相比，HG8组及HG4NG4组HAT和HDAC的水平均增高（P<0.05），与HG8组相比，HG4NG4组HAT和HDAC差异无统计学意义；Western blot结果显示，与NG8组相比，HG8组、HG4NG4组HDAC4、Bax和Caspase-3蛋白表达增加，Bcl-2蛋白表达水平降低（P<0.05），与HG8组相比，HG4NG4组蛋白表达差异无统计学意义。结论 HT-22小鼠海马神经元细胞经高糖55 mmol/L培养4 d后，再以25 mmol/L葡萄糖培养4 d是理想的高糖“代谢记忆”细胞模型。其机制可能与高糖模型中HDAC、HAT活性及HDAC4表达升高有关。

关键词: 糖尿病, 脑疾病, 细胞凋亡, 组蛋白乙酰转移酶, 组蛋白脱乙酰基酶类, HT-22, 代谢记忆

Abstract:

Objective To construct an in vitro "metabolic memory" cell model of HT-22 mouse hippocampal neurons induced by high glucose, and to investigate the effect of "metabolic memory" on apoptosis and histone acetylation in HT-22 cells. Methods HT-22 cells were cultured in high glucose medium (glucose concentration was 55 mmol/L) and conventional glucose medium (glucose concentration was 25 mmol/L), and cells were divided into the control group (NG 4, 6 and 8 groups, 25 mmol/L glucose was cultured for 4, 6 and 8 days, respectively), the high glucose group (HG 4, 6 and 8 groups, respectively) and the metabolic memory group (HG2NG2, HG2NG4, HG2NG6, HG4NG2 and HG4NG4 groups, high glucose culture for 2 days to 25 mmol/L glucose culture for 2, 4 or 6 days, high glucose culture for 4 days to 25 mmol/L glucose culture for 2 or 4 days). Cell viability was detected by CCK-8 method. The release of lactate dehydrogenase (LDH) in cell culture supernatant was detected, and the optimal time to establish a "metabolic memory" model was selected. Subsequently, cells were divided into the NG4 group, the NG8 group, the HG4 group, the HG4NG4 group and the HG8 group, and the cell morphology of each group was observed by optical microscope. The apoptosis rate was detected by flow cytometry. The activities of deacetylase (HDAC) and histone acetyltransferase (HAT) were detected by enzyme-linked immunosorbent assay (ELISA). Western blot assay was used to detect expression levels of histone deacetylase 4 (HDAC4), B lymphocyte tumor 2 (Bcl-2), Bcl-2 related X protein (Bax) and Caspase-3 protein. Results The HG4NG4 group was the ideal cell model with high glucose metabolic memory. Cells of the NG4 group and the NG8 group were interwoven into a dense network, growing well, with spindle shaped cells and distinct synaptic structures. However, in the HG4 group and the HG8 group, the cell body became round, synaptic structure disappeared and growth was inhibited. In the HG4NG4 group, the number of cells increased but their morphology was damaged. Results of flow cytometry showed that compared with the NG8 group, the apoptosis rates were significantly increased in the HG8 group and the HG4NG4 group (P<0.05). ELISA results showed that compared with the NG8 group, the expression levels of HDAC4, Bax, and Caspase-3 proteins increased in the HG8 group and the HG4NG4 group, while the expression level of Bcl-2 protein significantly decreased (P<0.05). Compared with the HG8 group, there were no significant differences in protein expression levels of HAT and HDAC in the HG4NG4 group. Western blot reslts showed that compared with the NG8 group, the levels of HDAC4, Bax and Caspase-3 protein increased in the HG8 group and the HG4NG4 group (P<0.05). Compared with the HG8 group, there were no significant differences in protein expression levels in the HG4NG4 group. Conclusion HT-22 mouse hippocampal neurons cultured with 55mmol/L high glucose for 4 days, and then cultured with 25 mmol/L glucose for 4 days are the ideal "metabolic memory" cell model. The mechanism may be related to the increased activity of HDAC, HAT and HDAC4 expression in the hyperglycemic model.

Key words: diabetes mellitus, brain diseases, apoptosis, histone acetyltransferases, histone deacetylases, HT-22, metabolic memory

中图分类号:

R587.2

图/表 6

图1 各组细胞存活率的比较 a与相同培养时间的NG组比较，b与相同培养时间的HG组比较，P<0.05。

Fig.1 Comparison of cell survival rates between three groups

图2 光学显微镜下各组细胞形态变化（×200）

Fig.2 Morphological changes of cells in each group under optical microscope (×200)

图3 各组细胞流式图

Fig.3 Flow cytometry of each group

表1 各组间HT-22细胞HAT及HDAC活性比较

Tab.1 Comparison of HAT and HDAC activities of HT-22 cells between five groups （n=3，OD450，$\bar{x}±s$）

组别	HAT	HDAC
NG4组	0.61±0.03	0.61±0.02
NG8组	0.62±0.02	0.61±0.03
HG4组	0.80±0.01^a	0.82±0.01^a
HG8组	0.87±0.06^b	1.05±0.05^b
HG4NG4组	0.87±0.02^b	1.00±0.07^b
F	72.467^＊＊	123.289^＊＊

表2 各组细胞HDAC4及凋亡相关蛋白表达情况

Tab.2 Expression of HDAC4 and apoptosis related proteins in each group of cells （n=3，$\bar{x}±s$）

组别	HDAC4	Bax	Bcl-2	Caspase-3
NG4组	0.37±0.01	0.29±0.01	0.78±0.02	0.28±0.01
NG8组	0.36±0.01	0.28±0.01	0.76±0.02	0.26±0.01
HG4组	0.54±0.06^a	0.53±0.02^a	0.66±0.03^a	0.63±0.01^a
HG8组	0.77±0.03^b	0.60±0.01^b	0.47±0.01^b	0.74±0.01^b
HG4NG4组	0.77±0.02^b	0.61±0.02^b	0.49±0.02^b	0.75±0.03^b
F	113.820^＊＊	427.796^＊＊	127.964^＊＊	464.712^＊＊

图4 HDAC4、Bax、Bcl-2和Caspase-3蛋白表达情况 A：NG4组；B：HG4组；C：NG8组；D：HG8组；E：HG4NG4组。

Fig.4 HDAC4, Bax, Bcl-2 and Caspase-3 protein expression

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高糖诱导HT-22小鼠海马神经元代谢记忆细胞模型的构建及影响

Construction and effect of a high glucose induced hippocampal neuron metabolic memory cell model in HT-22 mice

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