Toxic Effects Of Betulinic Acid On Neuronal Cells And Possible Mechanism

Research background:Betulinic acid(BA)is a naturally occurring pentacyclic triterpene that exhibits a variety of biological activities including anti-viral,anti-inflammatory,and anti-malarial effects;it has also been found to induce apoptosis in many types of cancer.This anticancer activity has been linked to its ability to directly trigger mitochondrial membrane permeabilization,a central event in the apoptotic process that seals the cell's fate.In contrast to the potent cytotoxicity of BA against a variety of cancer types,normal tissue cells remained relatively resistant to BA.Thus,BA hold great promise as a novel drug for human cancer.However,little is known about the effect of BA on normal neuronal cells.In this study,the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC 12 cells as a model.Methods:In the first part,the primary cortical neurons were obtained from the cortices of embryonic day 18 Sprague-Dawley rats and PC 12 cells were cultured in differentiation medium.We used Cell Counting Kit-8 to measure cell viability and assessed the apoptosis of differentiated PC 12 cells by Flow cytometry using Annexin V-FITC/PI detection kit.In the second part,in order to explore the specific underlying mechanisms we examined the mitochondrial membrane potential(MMP)by flow cytometry using the JC-1 assay kit.After treatment with betulinic acid for different times,the level of cytochrome c and caspase-3 were evaluated by Western blotting.In addition,we detected the effects of BA on intracellular ROS production and the role of ROS in the apoptosis of PC 12 cells induced by BA.Results:Part 1:BA reduced cell viability of primary cortical neurons and PC 12 cells as well as induced PC 12 cell apoptosis.The estimated IC50 values of both primary cortical neurons and PC 12 cells were approximately 50 ?M.Part 2:BA treatment reduced the mitochondrial membrane potential,induced cytochrome c release and caspase-3 activation.BA induced a significant accumulation of ROS in PC 12 cells and ROS may trigger PC 12 cell apoptosis through the mitochondrial apoptotic pathway,which can be significantly blocked by antioxidants.Conclusions:Taken together,our observations suggest that BA exerted cytotoxicity on both primary cortical cells and differentiated PC12 cells.The generation of ROS occurs in the early stage of PC 12 cell apoptosis induced by BA and triggers the mitochondrial apoptotic pathway.