A Preliminary Study On The Saikosaponin D Induced Hela Cells Autophagy And Its Mechanisms

Autophagy is a lysosome-based degradation process whose primary function isto degrade long-lived proteins and recycle cellular components. There are varioustypes of autophagy, including macroautophagy and microautophagy, as well aschaperone-mediated autophagy (CMA), and they differ in their mechanisms andfunctions. Autophagy has been shown to degrade cargo in selective and non-selectivemanners. Both macroautophagy and microautophagy have the capacity to engulf largestructures through both selective and non-selective mechanisms, whereas CMAdegrades only soluble proteins, albeitin a selective manner. In literature, the term“autophagy” usually indicates macroautophagy. Macroautophagy is characterized bythe formation of double membrane vesicles termed autophagosomes. Autophagy canbe induced by various stimuli, including nutrient starvation, hypoxia and variouschemical reagents such as rapamycin. In yeast, the major physiological function ofautophagy is to adjust the metabolic machinery in response to varying nutrient status,thereby ensuring the cell survival in an ever-changing environment. In mammaliancells, however, the roles of autophagy are more complex. Autophagy has beenimplicated in many different cellular processes as varied as cell survival, death,pathogen clearance, and antigen presentation. Various diseases have been linked todysfunctioning autophagy. There are strong genetic and animal data supporting thedirect connections between autophagy and cancers, neurodegenerative diseases, andchronic inflammation. Albeit the exact mechanisms of autophagy in each of thediseases remain to be determined, modulating autophagy has become a target fortherapeutic approaches for various diseases.Saikosaponin D (SSD) is one of the major triterpenoid saponins derived fromBupleurum falcatum L.(Umbelliferae), a medicinal plant that has been used for treating various inflammatory and infectious diseases by Chinese doctors for over onethousand of years. Other saikosaponins have been isolated and identified from thisplant, including saikosaponins A, B, C, D, M, N, P and T. SSD is believed to be themost active among them, SSD was reported to have immunemodulatory,anti-inflammatory, hepatoprotective, antiviral, and anticancer activities.Because both autophagy and SSD have close relationship with a variety ofdiseases, whether SSD can induce autophagy becomes a very interesting and valuablequestion. This paper uses HeLa cells as a model cell to study whether SSD can induceautophagy, and make a preliminary study of the mechanism.First, HeLa cell vitality was assessed by CCK-8assay.Five well-reeognizedmarkers of autophagy including the MDC and AO fluoreseent intensity, the GFP-LC3puncta formation, the protein levels of LC3-Ⅱ and p62were analyzed. The resultsshow that SSD inhibits the proliferation of HeLa cells in time-and dose-dependentmanner. We find that SSD can induce autophagic vacuoles and GFP-LC3punctaformation. And it also can improve the expression of LC3-Ⅱ and promote thedegradation of p62. Second, the intraeellular ROS generation was estimated byfluorescence plate reader using the fiuoreseent probe DCFH-DA. Protein levels ofLC3-Ⅱ, JNK and p-JNK were determined by western blot. We also studied theinteraction among ROS, JNK and autophagy by using the inhibitors NAC andSP600125. The results show that SSD promotes accumulation of intracellular ROS,resulting in sustained activation of JNK, which in turn leads to autophagy of HeLacells.In conclusion, our results show that SSD induces autophagy in HeLa cells via aROS-dependent JNK activation pathway. And that will lay a foundation for the furtherdevelopment and utilization of SSD.