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

doi:10.11958/20231115

Abstract

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

CLC Number:

R587.2

Figures/Tables 6

References 29

[1]	LACHIN J M, NATHAN D M. Understanding metabolic memory:The prolonged influence of glycemia during the Diabetes Control and Complications Trial (DCCT)on future risks of complications during the Study of the Epidemiology of Diabetes Interventions and Complications(EDIC)[J]. Diabetes Care, 2021, 44(10):2216-2224. doi:10.2337/dc20-3097.
[2]	MILER R G, ORCHARD T J. Understanding metabolic memory:A tale of two studies[J]. Diabetes, 2020, 69(3):291-299. doi:10.2337/db19-0514.
[3]	TULIGENGA R H. Intensive glycaemic control and cognitive decline in patients with type 2 diabetes: a meta-analysis[J]. Endocr Connect, 2015, 4(2):R16-24. doi:10.1530/EC-15-0004.
[4]	AREOSA SASTRE A, VERNOOIJ R W, GONZÁLEZ-COLAÇO HARMAND M, et al. Effect of the treatment of Type 2 diabetes mellitus on the development of cognitive impairment and dementia[J]. Cochrane Database Syst Rev, 2017, 6(6):CD003804. doi:10.1002/14651858.CD003804.pub2.
[5]	CUKIERMAN-YAFFE T, MCCLURE L A, RISOLI T, et al. The relationship between glucose control and cognitive function in people with diabetes after a lacunar stroke[J]. J Clin Endocrinol Metab, 2021, 106(4):e1521-e1528. doi:10.1210/clinem/dgab022.
[6]	KATO M, NATARAJAN R. Epigenetics and epigenomics in diabetic kidney disease and metabolic memory[J]. Nat Rev Nephrol, 2019, 15(6):327-345. doi:10.1038/s41581-019-0135-6.
[7]	ZHANG L, CHEN Z W, YANG S F, et al. MicroRNA-219 decreases hippocampal long-term potentiation inhibition and hippocampal neuronal cell apoptosis in type 2 diabetes mellitus mice by suppressing the NMDAR signaling pathway[J]. CNS Neurosci Ther, 2019, 25(1):69-77. doi:10.1111/cns.12981.
[8]	CHANG P, TIAN Y, WILLIAMS A M, et al. Inhibition of histone deacetylase 6 protects hippocampal cells against mitochondria-mediated apoptosis in a model of severe oxygen-glucose deprivation[J]. Curr Mol Med, 2019, 19(9):673-682. doi:10.2174/1566524019666190724102755.
[9]	XU Y, LI H, CHEN G, et al. Radix polygoni multiflori protects against hippocampal neuronal apoptosis in diabetic encephalopathy by inhibiting the HDAC4/JNK pathway[J]. Biomed Pharmacother, 2022, 153:113427. doi:10.1016/j.biopha.2022.113427.
[10]	许雯, 许永劼, 刘歆蕾, 等. TSA对不同糖浓度下小鼠海马神经元HT-22细胞凋亡的影响[J]. 天津医药, 2021, 49(4):349-353.
	XU W, XU Y J, LIU X L, et al. Effects of TSA on the apoptosis of HT-22 cells in mouse hippocampal neurons under different concentrations of glucose[J]. Tianjin Med J, 2021, 49(4):349-353. doi:10.11958/20202807.
[11]	ESIN R G, KHAIRULLIN I K, ESIN O R, et al. Diabetic encephalopathy:current insights and potential therapeutic strategies[J]. Zh Nevrol Psikhiatr ImSS Korsakova, 2021, 121(7):49-54. doi:10.17116/jnevro202112107149.
[12]	VILLENEUVE L M, REDDYM A, LANTINGL L, et al. Epigenetic histone H3 lysine 9 methylation in metabolic memory and inflammatory phenotype of vascular smooth muscle cells in diabetes[J]. Proc Natl Acad Sci U S A, 2008, 105(26):9047-9052. doi:10.1073/pnas.0803623105.
[13]	WANG Z, ZHAO H, GUAN W, et al. Metabolic memory in mitochondrial oxidative damage triggers diabetic retinopathy[J]. BMC Ophthalmol, 2018, 18(1):258. doi:10.1186/s12886-018-0921-0.
[14]	WANG H, DENG J, CHEN L, et al. Acute glucose fluctuation induces inflammation and neurons apoptosis in hippocampal tissues of diabetic rats[J]. J Cell Biochem, 2021, 122(9):1239-1247. doi:10.1002/jcb.29523.
[15]	ZHANG J H, ZHANGJ F, SONG J, et al. Effects of berberine on diabetes and cognitive impairment in an animal model:The mechanisms of action[J]. Am J Chin Med, 2021, 49(6):1399-1415. doi:10.1142/S0192415X21500658.
[16]	许永劼, 许雯, 陈钢, 等. 2种高糖诱导海马神经元模型应用及优势比较[J]. 中国比较医学杂志, 2021, 31(8):1-8.
	XU Y J, XU W, CHEN G, et al. Application and comparison of the advantages of two high-glucose-induced hippocampal neuron models[J]. Chinese Journal of Comparative Medicine, 2021, 31(8):1-8. doi:10.3969/j.issn.1671-7856.2021.08.001.
[17]	YAO Y, SONG Q, HU C, et al. Endothelial cell metabolic memory causes cardiovascular dysfunction in diabetes[J]. Cardiovasc Res, 2022, 118(1):196-211. doi:10.1093/cvr/cvab013.
[18]	任伟伟, 李守宏, 熊洁, 等. 人牙周膜细胞与高糖损伤的代谢记忆效应[J]. 中国组织工程研究, 2017, 21(4):532-537.
	REN W W, LI S H, XIONG J, et al. High glucose induces a metabolic memory in human periodontal ligament cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(4):532-537. doi:10.3969/j.issn.2095-4344.2017.04.007.
[19]	SHEN Y, WEI W, ZHOU D X, et al. Histone acetylation enzymes coordinate metabolism and gene expression[J]. Trends Plant Sci, 2015, 20(10):614-621. doi:10.1016/j.tplants.2015.07.005.
[20]	陈钢, 许永劼, 黄昶煜东, 等. 二苯乙烯苷、大黄素改善高糖诱导下小鼠海马神经元凋亡[J]. 天津医药, 2022, 50(6):561-565.
	CHEN G, XU Y J, HUANG C Y D, et al. Tetrahydroxystilbene glucoside and emodin improves the hippocampal neuronal apoptosis in mice induced by high glucose[J]. Tianjin Med J, 2022, 50(6):561-565.doi:10.11958/20212403.
[21]	ZHENG Z, CHEN H, LI J, et al. Sirtuin 1-mediated cellular metabolic memory of high glucose via the LKB1/AMPK/ROS pathway and therapeutic effects of metformin[J]. Diabetes, 2012, 61(1):217-228. doi:10.2337/db11-0416.
[22]	ZHONG Q, KOWLURU R A. Role of histone acetylation in the development of diabetic retinopathy and the metabolic memory phenomenon[J]. J Cell Biochem, 2010, 110(6):1306-1313. doi:10.1002/jcb.22644.
[23]	JEBASINGH F, THOMAS N. Barker hypothesis and hypertension[J]. Front Public Health, 2021, 9:767545. doi:10.3389/fpubh.2021.767545.
[24]	GAWLIN S K, GAWLIN S D, FILIP M, et al. Relationship of maternal high-fat diet during pregnancy and lactation to offspring health[J]. Nutr Rev, 2021, 79(6):709-725. doi:10.1152/ajpcell.00201.2022.
[25]	ZHAO S, LI J, WANG N, et al. Fenofibrate suppresses cellular metabolic memory of high glucose in diabetic retinopathy via a sirtuin 1-dependent signalling pathway[J]. Mol Med Rep, 2015, 12(4):6112-1168. doi:10.3892/mmr.2015.4164.
[26]	CERIELLO A, IHNNAT M A, THORPE J E. Clinical review 2:The "metabolic memory":is more than just tight glucose control necessary to prevent diabetic complications[J]. J Clin Endocrinol Metab, 2009, 94(2):410-415. doi:10.1210/jc.2008-1824.
[27]	KINNEL B, SINGH S K, OPREA G, et al. Targeted therapy and mechanisms of drug resistance in breast cancer[J]. Cancers(Basel), 2023, 15(4):1320. doi:10.3390/cancers15041320.
[28]	CHATTERJEE S, CASSEL R, SCHNEIDER A, et al. Reinstating plasticity and memory in a tauopathy mouse model with an acetyltransferase activator[J]. EMBO Mol Med, 2018, 10(11):e8587. doi:10.15252/emmm.201708587.
[29]	GÜNAYDIN C, ÇELIK Z B, BILGE S S, et al. SAHA attenuates rotenone-induced toxicity in primary microglia and HT-22 cells[J]. Toxicol Ind Health, 2021, 37(1):23-33. doi:10.1177/0748233720979278.

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^＊＊

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^＊＊

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^＊＊

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^＊＊