| 脑络欣通靶向CaMKKβ调控OGD/R后神经元线粒体机制研究 |
| 投稿时间：2025-02-22 修订日期：2025-04-13 点此下载全文 |
| 引用本文：张涵智,刘明明,江慧慧,洪璐,赵雨彤,何玲,陈卫东.脑络欣通靶向CaMKKβ调控OGD/R后神经元线粒体机制研究[J].医学研究杂志,2025,54(9):71-78 |
| DOI：
10.11969/j.issn.1673-548X.2025.09.013 |
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| 基金项目:国家自然科学基金青年科学基金资助项目(82204671)；安徽省高校优秀青年教师培育重点项目(YQZD2024020) |
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| 中文摘要:目的 探讨脑络欣通(Naoluoxintong, NLXT)对氧糖剥夺/再灌注(oxygen-glucose deprivation /reoxygenation, OGD/R)模型下小鼠海马神经元HT22细胞的保护作用和对线粒体功能的影响,以及钙调蛋白依赖性蛋白激酶激酶β(calcium/calmodulin- dependent protein kinase kinase β, CaMKKβ)在其中的作用。方法 培养HT22细胞,建立OGD/R模型,通过CCK-8法筛选不同浓度NLXT含药血清(0~20％)干预HT22细胞的最佳浓度后将细胞分为正常 (Control) 组、模型 (OGD/R) 组、NLXT (OGD/R+10％NLXT) 组、阳性药丁基苯酞(dl-3-n-butylphthalide, NBP)(OGD/R+ NBP 50μmol/L) 组,利用CCK-8法检测各组细胞存活率,倒置荧光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,试剂盒检测各组丙二醛(malondialdehyde, MDA)、超氧化物歧化酶(superoxide dismutase, SOD)、谷胱甘肽(glutathione, GSH)活性或含量。随后引入CaMKKβ抑制剂(STO-609,5μmol/L),将细胞分为Control组、OGD/R组、OGD/R+10％NLXT组、OGD/R+NBP组、OGD/R+STO-609组、OGD/R+STO-609+NLXT组,利用JC-1荧光探针观察各组细胞线粒体膜电位,试剂盒检测终产物Na+-K+-ATP、Ca+-Mg+-ATP酶活性,Western blot法检测各组细胞CaMKKβ蛋白表达变化；实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction, RT-qPCR)法检测各组细胞线粒体DNA拷贝数和CaMKKβ mRNA表达变化。结果 NLXT含药血清浓度为10％时效果最明显,与OGD/R组比较,经过NLXT干预后细胞存活率明显上升(P<0.05),细胞形态特征明显恢复；与Control组比较,OGD/R组细胞凋亡率明显升高(P<0.05),MDA含量显著增加,而SOD、GSH活性显著下降,线粒体膜电位、ATP活性、线粒体DNA拷贝数下降,同时下调了CaMKKβ蛋白及基因的表达(P<0.05)；与OGD/R组比较,NLXT和NBP给药后能够拮抗这些效应并上调了CaMKKβ蛋白和mRNA表达(P<0.05)；此外,加入CaMKKβ抑制剂STO-609后,明显削弱了NLXT对线粒体功能和神经元的保护作用(P<0.05)。结论 NLXT通过激活CaMKKβ增强OGD/R后HT22细胞线粒体功能,调高能量代谢发挥神经保护作用。 |
| 中文关键词:脑络欣通 缺血性脑卒中 线粒体 CaMKKβ 氧糖剥夺/再灌注 |
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| Study on the Mechanisms of NLXT Targeting CaMKKβ to Regulate Neuronal Mitochondrial after OGD/R |
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| Abstract:Objective To investigate the protective effect of Naoluoxintong (NLXT) on mouse hippocampal neuronal HT22 cells in the oxygen-glucose deprivation/reoxygenation (OGD/R) model, focusing on its impact on mitochondrial function and the role of the calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) in it. MethodsHT22 cell were cultured to establish OGD/R model, and the cell were divided into normal group (Control), model group (OGD/R), NLXT group (OGD/R+10% NLXT), positive drug dl-3-n-butylphthalide (NBP) group (OGD/R+ NBP 50μmol/L) after the screening of the optimal concentrations of different concentrations of NLXT-containing serum (0-20%) in interfering with the HT22 cells by the CCK-8 assay. Then, using CCK-8 assay to detect the cell survival rate of each group, inverted fluorescence microscope to observe the morphology of each group, flow cytometry to detect the apoptosis rate of each group, and the kit to detect the activity or content of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) in each group. Nest, the CaMKKβ inhibitor (STO-609,5μmol/L) was introduced, HT22 cells were divided into Control group, OGD/R group, OGD/R+10% NLXT group, OGD/R+NBP group, OGD/R+STO-609group, OGD/R+STO-609+NLXT group. The mitochondrial membrane potential in each group was observed by using JC-1 fluorescent probe, and the end products Na+-K+-ATP, Ca+-Mg+-ATPase activity were detected by the kit. The expression changes of CaMKKβ protein in cells of each group were detected by Western blot, the changes of mitochondrial DNA copy number and CaMKKβ mRNA expression in cells of each group were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Results The optimal concentration of NLXT-containing serum was 10%, which resulted in the most significant effects. Compared with the OGD/R group, NLXT intervention significantly increased cell viability (P<0.05) and restored cellular morphological characteristics. Compared with the Control group, the OGD/R group showed significantly increased apoptosis (P<0.05), elevated MDA levels, and decreased activities of SOD and GSH, alongside a reduction in mitochondrial membrane potential, ATP activity, and mitochondrial DNA copy number. Furthermore, CaMKKβ protein and mRNA expression were downregulated (P<0.05). Compared with the OGD/R group, the NLXT and NBP groups antagonized these effects and upregulated CaMKKβ protein and mRNA expression (P<0.05). Additionally, the application of the CaMKKβ inhibitor STO-609significantly attenuated the protective effects of NLXT on mitochondrial function and neurons (P<0.05). Conclusion NLXT exerts neuroprotective effects by activating the CaMKKβ to enhance mitochondrial function after OGD/R in HT22 cells. |
| keywords:Naoluoxintong Ischemic stroke Mitochondrial CaMKKβ Oxygen-glucose deprivation/reoxygenation |
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