Yang Huaitao, Chen Zhifang, Gao Wenhong
Department of Neurosurgery, Jingzhou Hospital Affiliated to Yangtze University, No. 26, Chuyuan Ave, Jingzhou District, Jingzhou, Hubei 434020, P.R. China.
Department of Obstetrics and Gynecology, Jingzhou Hospital Affiliated to Yangtze University, Jingzhou, Hubei 434020, P.R. China.
Transl Neurosci. 2023 Jul 27;14(1):20220276. doi: 10.1515/tnsci-2022-0276. eCollection 2023 Jan 1.
This research was designed to ascertain the function of euchromatic histone lysine methyltransferase 2 (EHMT2) in ischemic stroke-induced neuronal damage and inflammatory response and its regulatory mechanism.
Mouse microglia (BV-2 cells) were induced by oxygen glucose deprivation/reoxygenation (OGD/R) to establish a cellular model, and then co-cultured with HT22 hippocampal neurons. After that, HT22 cell viability and apoptosis were evaluated, followed by the measurement of apoptosis-related factors (B-cell lymphoma-2, Bcl-2 associated X, and cleaved-Caspase 3). Meanwhile, the expression of inducible nitric oxide synthase (M1 microglia polarization marker) and arginase 1 (M2 microglia polarization marker) in BV-2 cells was detected, as well as the levels of inflammatory factors (tumor necrosis factor-α, interleukin [IL]-6, IL-10, IL-1β, and IL-4). Additionally, the expression of EHMT2 and heme oxygenase 1 (HMOX1) in BV-2 cells was assessed by quantitative reverse transcription polymerase chain reaction and western blot, and the binding between EHMT2 and HMOX1 was predicted and verified.
OGD/R treatment led to decreased cell viability and increased cell apoptosis in HT22 cells, and aggravated inflammatory response in BV-2 cells. In OGD/R-induced BV-2 cells, EHMT2 and HMOX1 were increasingly expressed, and knockdown of EHMT2 or HMOX1 in BV-2 cells could inhibit neuronal damage and inflammatory response. Moreover, EHMT2 promoted HMOX1 transcription level by histone methylation.
Collected evidence showed that down-regulation of EHMT2 relieved neuronal damage and inflammatory response by inhibiting HMOX1 expression.
本研究旨在确定常染色质组蛋白赖氨酸甲基转移酶2（EHMT2）在缺血性脑卒中诱导的神经元损伤和炎症反应中的作用及其调控机制。
采用氧糖剥夺/复氧（OGD/R）诱导小鼠小胶质细胞（BV-2细胞）建立细胞模型，然后与HT22海马神经元共培养。之后，评估HT22细胞活力和凋亡情况，随后检测凋亡相关因子（B细胞淋巴瘤-2、Bcl-2相关X蛋白和裂解的半胱天冬酶3）。同时，检测BV-2细胞中诱导型一氧化氮合酶（M1小胶质细胞极化标志物）和精氨酸酶1（M2小胶质细胞极化标志物）的表达，以及炎症因子（肿瘤坏死因子-α、白细胞介素[IL]-6、IL-10、IL-1β和IL-4）的水平。此外，通过定量逆转录聚合酶链反应和蛋白质印迹法评估BV-2细胞中EHMT2和血红素加氧酶1（HMOX1）的表达，并对EHMT2与HMOX1之间的结合进行预测和验证。
OGD/R处理导致HT22细胞活力降低、细胞凋亡增加，并加重BV-2细胞的炎症反应。在OGD/R诱导的BV-2细胞中，EHMT2和HMOX1表达上调，敲低BV-2细胞中的EHMT2或HMOX1可抑制神经元损伤和炎症反应。此外，EHMT2通过组蛋白甲基化促进HMOX1转录水平。
收集的证据表明，EHMT2的下调通过抑制HMOX1表达减轻神经元损伤和炎症反应。
