In the present study, replicative lifespan and chronological lifespan assays of yeast were used to double-screen antiaging compounds from Gentiana rigescens Franch, a Chinese herb medicine. Inokosterone from G. rigescens Franch extended not only the replicative lifespan of K6001 yeast but also the chronological lifespan of YOM36 yeast. Furthermore, it can enhance the survival ability of mammalian cells. In order to understand the mechanism of action of this compound, this study focused on antioxidative stress and autophagy when performing the analysis. The increased cell survival rate under oxidative stress conditions, antioxidant enzyme activity and gene expression were observed in the inokosterone-treated groups. Meanwhile, the reactive oxygen species (ROS) and lipid peroxidation of yeast were obviously decreased. Additionally, the macroautophagy and mitophagy in YOM38-GFP-ATG8 yeast were increased upon inokosterone treatment, respectively. At the same time, the cleavage-free GFP from GFP-ATG8 in the cytoplasm and the ubiquitin of the mitochondria at the protein level were markedly enhanced after incubation with inokosterone. Furthermore, we investigated the effect of inokosterone on antioxidative stress and autophagy in mammalian cells, and the relationship between ROS and autophagy. The ROS, malondialdehyde (MDA) were significantly decreased, and the autophagosomes in mammalian cells were obviously increased after inokosterone treatment. The autophagosomes in ∆sod1 yeast with a K6001 background had no obvious changes, and the ROS and MDA of ∆sod1 yeast were increased compared with K6001 yeast. The increase of autophagosomes and the reduction of ROS and MDA in ∆sod1 yeast were observed after treatment with inokosterone. Meanwhile, the reduction of the ROS level and the increase of the SOD1 gene expression of K6001 yeast lacking autophagy were observed after treatment with inokosterone. In order to indicate whether the genes related to antioxidant enzymes and autophagy were involved in the antiaging effect of inokosterone, mutants of K6001 yeast were constructed to conduct a lifespan assay. The replicative lifespans of ∆sod1, ∆sod2, ∆uth1, ∆skn7, ∆gpx, ∆cat, ∆atg2, and ∆atg32 of K6001 yeast were not affected by inokosterone. These results suggest that inokosterone exerted an antiaging activity via antioxidative stress and increased autophagy activation; autophagy affected the ROS levels of yeast via the regulation of SOD1 gene expression.

中文翻译：

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Gentiana rigescens Franch 中的 Inokosterone 通过抗氧化应激和线粒体自噬诱导延长酵母和哺乳动物细胞的寿命

在本研究中，酵母的复制寿命和时间寿命测定用于双重筛选来自中草药龙胆草的抗衰老化合物。来自 G. rigescens Franch 的 Inokosterone 不仅延长了 K6001 酵母的复制寿命，还延长了 YOM36 酵母的时间寿命。此外，它可以增强哺乳动物细胞的存活能力。为了了解该化合物的作用机制，本研究在进行分析时重点关注抗氧化应激和自噬。在肌醇酮处理组中观察到氧化应激条件下细胞存活率、抗氧化酶活性和基因表达的增加。同时，酵母的活性氧（ROS）和脂质过氧化明显降低。此外，YOM38-GFP-ATG8酵母中的巨自噬和线粒体自噬在肌醇酮处理后分别增加。同时，细胞质中来自 GFP-ATG8 的无切割 GFP 和蛋白质水平的线粒体泛素在与 inokosterone 孵育后显着增强。此外，我们研究了inokosterone对哺乳动物细胞抗氧化应激和自噬的影响，以及ROS与自噬之间的关系。经吲哚酮处理后，哺乳动物细胞内活性氧、丙二醛（MDA）明显降低，自噬体明显增多。K6001背景的Δsod1酵母中的自噬体没有明显变化，Δsod1酵母的ROS和MDA较K6001酵母有所增加。在用inokosterone处理后观察到Δsod1酵母中自噬体的增加和ROS和MDA的减少。同时，用inokosterone处理后，观察到缺乏自噬的K6001酵母的ROS水平降低，SOD1基因表达增加。为了表明抗氧化酶和自噬相关基因是否参与了肌醇酮的抗衰老作用，构建了K6001酵母突变体进行寿命测定。K6001酵母的Δsod1、Δsod2、Δuth1、Δskn7、Δgpx、Δcat、Δatg2和Δatg32的复制寿命不受肌醇酮的影响。这些结果表明，inokosterone 通过抗氧化应激和增加自噬激活发挥抗衰老活性；自噬通过调节 SOD1 基因表达影响酵母的 ROS 水平。