The Effect Mechanisms Of Phytosterols And Phytosterol Oxides In Saccharomyces Cerevisiae

Phytosterols are structurally similar to cholesterol but are found exclusively in plants. More than 100 phytosterols exist naturally in the plant kingdom and many are found in edible foodstuffs. The most common phytosterols in the human diet are β-sitosterol, campesterol, and stigmasterol, etc.. Due to their knowing cholesterol-lowering and anticancer properties, phytosterols have been incorporated in diet to lower the cardiovascular disease risk and prevent cancer development. However, the exact mechanism by which dietary phytosterols offer protection from heart disease and cancer is not fully understood. Due to the lack of commercially available most of individual phytosterols, most researches focused on phytosterols mixture or β-sitosterol, one of the few commerical individual phytosterols. Thus, it is highly needed to carry out the research on other important phytosterols, including campesterol or stigmasterol etc., to gain more comprehensive insights.Sterols, including cholesterol and phytosterols, are unsaturated lipids and prone to oxidation, giving rise to their corresponding cholesterol oxidation products(COPs) and phytosterol oxidation products(POPs). Previous studies have demonstrated that COPs had cytotoxicity, apoptotic and pro-inflammatory effects. COPs are also associated with the initiation and progression of major chronic diseases including atherosclerosis, neurodegenerative processes, diabetes, kidney failure, and ethanol intoxication. Compared with the relatively comprehensive studies of COPs, research on biological effects of POPs are limit, some results are contradictory. It is therefore important to gain better knowledge of the toxicity of POPs and delineate their molecular pathways underlying.Nowadays, the budding yeast Saccharomyces cerevisiae(S. cerevisiae) has been widely used as an in vivo model for elucidating the mechanism of human diseases. In fact, due to the simple genetic manipulation, overexpression and deletion screens, yeast constitutes an excellent model organism for the identification of cell death pathways relevant to human diseases. Furthermore, it becomes clear that the yeast genome encodes orthologs of crucial mammalian apoptosis regulators, including the sole yeast metacaspase(Yca1p), endonuclease G(Nuc1p), the apoptosis-inducing factor(Aif1p), or BH3-only proteins(Ybh3p).In the present study, one kind of important phytosterol oxides(7-keto oxides) were chemically synthetized from a phytosterol mixture(Generol 95R) firstly. Individual phytosterols and phytosterol oxides were obtained by column chromatography and semi-preparative HPLC system, including campesterol, β-sitosterol, 7-ketocampesterol and7-ketositosterol. Their profiles were also characterized. This method may provide a novel strategy for the preparation of individual phytosterol oxides.Since oxidative stress plays a major role in the pathogenesis of human diseases, in this work the probable protective effect of campesterol during oxidative stress conditions was investigated in a yeast model. Here we show that pre-treatment of yeast cells with campesterol resulted in reduced reactive oxygen species(ROS) production, elevated antioxidant enzyme activity as well as decreased level of lipid peroxidation. We compared the effects of campesterol on both wild-type yeast and Δyno1 mutant subjected to oxidative stress. The protective effect of campesterol is NADPH oxidase-dependent, because the Δyno1 mutant abrogated campesterol-conferred protection.The potential toxic effects of 7-ketocholesterol and 7-ketophytosterols were assessed in the budding yeast. Precise measurements of actual cell death in yeast were taken through plating assays or apoptotic markers. The data presented in this study indicated that both 7-ketocholesterol and 7-ketophytosterols can induce apoptosis in yeast cells. Treatment with 7-ketocholesterol or 7-ketophytosterols resulted in an increase in chromatin condensation, the generation of ROS, and externalization of phosphatidylserine. Our studies identified metacaspase function as a major role in 7-ketosterols-mediated apoptosis of yeast cells since deletion of YCA1 abrogated the toxic function of the these compounds. Moreover, 7-ketocholesterol was found to be more cytotoxic than 7-ketophytosterols. In addition, an electrochemical method based on square wave anodic stripping voltammetry(SWASV) was developed to detect the apoptosis of yeast cells. The results were well consistent with that obtained from traditional flow cytometry. This electrochemical technique may have great potentials for detecting cell apoptosis.