560 Saikosaponin A induces HEKa cell apoptosis via ROS generation

Abstract

Background: Psoriasis is a common, chronic and noninfectious skin disease with the complex pathogenesis which is reported as excessive proliferation and aberrant apoptosis of keratinocytes. Saikosaponin A (SSA) is a kind of triterpenoid saponin extracted from Chinese traditional medicine Radix bupleuri, possessing various biological functions such as anti-inflammatory, immune regulation and anti-tumor. But the effect of SSA on keratinocytes is unclear. Objective: To explore the effect of SSA on keratinocytes and to analyze the corresponding mechanism. Methods: 1. The cell viability was detected by MTT assay and the apoptosis was examined using the annexin V-FITC/PI flow cytometry double staining method. Apoptosis-related protein were determined by Western blot. 2. After stimulation with SSA, DCFH-DA and JC-1 were used to detect intracellular reactive oxygen species (ROS) levels and mitochondrial membrane potential. MitoSOX Red and MitoTracker Green were used to observe generation and distribution of ROS in mitochondria by confocal laser scanning microscope; ROS-related protein was detected by Western blot. 3. Antioxidants N-acetyl-cysteine (NAC, 10mM) was added to HEKa cells 1h before SSA(20μM) treatment to identify whether SSA induced the apoptosis of HEKa cells through ROS production. Results: 1. Compared with the control group, SSA significantly inhibited the viability and induced the apoptosis of HEKa cells with changes in BCL-2 family protein levels. 2. SSA promoted ROS production of mitochondria and decreased Δψ, which caused changes in ROS-related protein levels and oxidative stress damage to cells. 3. SSA-treated HEKa cells produced more ROS, but this effect was inhibited by the antioxidants NAC. Furthermore, NAC treatment attenuated HEKa cell apoptosis and proliferation suppression of SSA. Conclusion: SSA can inhibit proliferation and induce apoptosis of HEKa cells through ROS generation, making SSA a potential promising candidate drug for future research.