Study on the Mechanisms of NLXT Targeting CaMKKβ to Regulate Neuronal Mitochondrial after OGD/R
10.11969/j.issn.1673-548X.2025.09.013
- VernacularTitle:脑络欣通靶向CaMKKβ调控OGD/R后神经元线粒体机制研究
- Author:
Hanzhi ZHANG
1
;
Ming-ming LIU
;
Huihui JIANG
Author Information
1. 230012 合肥,安徽中医药大学药学院
- Publication Type:Journal Article
- Keywords:
Naoluoxintong;
Ischemic stroke;
Mitochondrial;
CaMKKβ;
Oxygen-glucose deprivation/reoxygenation
- From:
Journal of Medical Research
2025;54(9):71-78
- CountryChina
- Language:Chinese
-
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 cal-cium/calmodulin-dependent protein kinase kinase β(CaMKKβ)in it.Methods HT22 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 con-centrations 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 di-vided 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.Further-more,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-609 significantly 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.
