Ginsenoside Rg1 controls CKLF1-mediated apoptosis to reduce hypoxic/reoxygenation injury in HT22 cells

Jinping Liang¹Xuan Zhu¹Yan Yang¹Yuchen Zhu¹Shasha Liu²Yang Sun¹Boyu Kuang¹Junpeng Long¹Qian Yan¹Yuting Lin¹Qidi Ai^1*Yantao Yang^1*

• ¹Hunan University of Chinese Medicine, Changsha, China
• ²Changsha Hospital for Maternal and Child Health Care, Changsha, Hunan Province, China

Background: Stroke is a prevalent and debilitating neurodegenerative condition. Ginsenoside Rg1 has demonstrated neuroprotective properties in the context of stroke.The upregulation of chemokine-like factor 1 (CKLF1) observed in ischemic stroke positions CKLF1 as a promising therapeutic target. However, limited research has explored whether Rg1 can mitigate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced apoptosis in HT22 cells through the modulation of CKLF1.Methods: In this study, Na2S2O4 was used to treat HT22 cells to establish the OGD/R model. The effects of different concentrations of Rg1 on cell viability were firstly determined by CCK-8 assay to determine its safe administration range. Subsequently, the level of oxidative stress was assessed by detecting LDH release and antioxidant indexes (CAT, SOD, MDA). Western blotting was used to analyse the expression of CKLF1 and apoptosis-related proteins, and TUNEL staining was used to quantify the apoptosis rate. To explore the cell-cell interactions, a Transwell co-culture system of HT22 and BV-2 cells was established.In this study, the optimal parameters for the OGD/R model were determined: 25 mmol/L Na2S2O4 treatment for 2.5 h followed by 2.5 h of reoxygenation, and a cell inoculation density of 1 × 10⁵ cells/mL for 1 day of culture. Based on the safety assessment, 5, 25, and 50 μmol/L Rg1 were selected for intervention.Rg1 significantly decreased LDH release (P≤0.05) and MDA content (P≤0.05) and alleviated oxidative stress.Western blotting showed that Rg1 dose-dependently down-regulated the expression of CKLF1 (P≤0.05) and inhibited Caspase-3 and other apoptotic protein activation. In the HT22/BV-2 co-culture system, Rg1 inhibited microglia activation, as shown by reduced NO and IL-1β secretion (P≤0.05).: Rg1 attenuates OGD/R injury, reduces oxidative stress and apoptosis in HT22 cells by inhibiting CKLF1 expression and alleviates the inflammatory response in activated BV-2 cells, showing therapeutic potential.