Laboratory of Development and Innovation, Butantan Institute, Brazil; Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, Brazil.
Department of Chemistry, Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of São Paulo, Brazil.
Arch Biochem Biophys. 2020 Dec 15;696:108654. doi: 10.1016/j.abb.2020.108654. Epub 2020 Oct 29.
β-Sitosterol (βSito) is the most abundant phytosterol found in vegetable oils, grains such as wheat, beans, and corn, and in many phytosterol-enriched foods. It is prone to oxidation by reactive oxygen species, such as ozone, leading to the formation of oxyphytosterols. A better understanding regarding the biological effects and mechanism of action of oxyphytosterols is required since the beneficial and adverse side effects of these compounds on human health remain highly controversial. In this work, we investigated the biological effects of β-Secosterol (βSec), a new oxyphytosterol generated by the reaction of βSito with ozone. Treatment of HepG2 cells with βSito or βSec (0.1-100 μM) for 24, 48, and 72 h induced a dose-dependent reduction of cell viability in the MTT assay, with βSec showing higher efficacy than βSito. However, βSec presented a lower potency than βSito, showing IC = 37.32 μM, higher than βSito (IC = 0.23 μM) at 48 h. Cell cycle analyses by flow cytometry showed a slight decrease of G0/G1 phase with βSito 0.5 μM, but a significant cell cycle arrest at the G0/G1 phase in the treatment for 48 h with βSec 20 μM (62.69 ± 2.15%, p < 0.05) and βSec 40 μM (66.96 ± 5.39%, p < 0.0001) when compared to control (56.97 ± 2.60%). No suggestion of apoptosis was indicated by flow cytometry data. Also, βSec (20 and 40 μM) reduced the mitotic index. In the laser scanning confocal microscopy analysis no alterations in cell morphology were observed with βSito (0.5 μM). Nevertheless, round-shaped cells, abnormal nuclear morphology with shrinkage, and formation of microtubules clusters were observed in the treatment with βSec, indicating a disruption in the microtubules network organization. N-acetyl-l-cysteine was not able to inhibit any of these cellular effects, indicating a lack of involvement of oxidative stress in the mechanism of action of βSec. Although not further investigated in this study, it was discussed the hypothesis that covalent adduct formation with lysine residues of proteins, could play an important role in the biological effects elicited by βSec. Elucidation of the primary cellular processes induced by βSec provides the essential knowledge to be aware of its potential adverse side effects or therapeutic use of this oxyphytosterol.
β-谷甾醇（βSito）是植物油、小麦、豆类和玉米等谷物以及许多富含植物固醇的食物中含量最丰富的植物固醇。它容易被臭氧等活性氧物质氧化，导致形成氧杂植物固醇。由于这些化合物对人类健康的有益和不利影响仍然存在很大争议，因此需要更好地了解氧杂植物固醇的生物学效应和作用机制。在这项工作中，我们研究了β-豆甾醇（βSec）的生物学效应，βSec 是βSito 与臭氧反应生成的一种新的氧杂植物固醇。用 MTT 法检测 HepG2 细胞在 24、48 和 72 小时内用 βSito 或 βSec（0.1-100μM）处理时，细胞活力呈剂量依赖性降低，βSec 的效果高于 βSito。然而，βSec 的效力低于 βSito，在 48 小时时，IC=37.32μM，高于 βSito（IC=0.23μM）。用流式细胞术进行细胞周期分析显示，βSito 0.5μM 时 G0/G1 期略有下降，但βSec 20μM（62.69±2.15%，p<0.05）和 40μM（66.96±5.39%，p<0.0001）处理 48 小时时 G0/G1 期细胞周期明显停滞，与对照组（56.97±2.60%）相比。流式细胞术数据未提示凋亡。此外，βSec（20 和 40μM）降低了有丝分裂指数。在激光扫描共聚焦显微镜分析中，用 βSito（0.5μM）处理未观察到细胞形态发生变化。然而，在用 βSec 处理时观察到圆形细胞、核形态收缩、微管簇形成，表明微管网络组织紊乱。N-乙酰-L-半胱氨酸不能抑制任何这些细胞效应，表明βSec 作用机制中不存在氧化应激的参与。尽管本研究没有进一步探讨，但讨论了βSec 与蛋白质赖氨酸残基形成共价加合物的假说，这可能在βSec 引起的细胞生物学效应中发挥重要作用。阐明βSec 诱导的主要细胞过程为了解其潜在的不利影响或治疗用途提供了必要的知识。
