背景:随着社会老龄化的发展,衰老相关疾病,如老年痴呆症,心血管疾病,糖尿病,正在急剧增加。从天然产物中寻找可以预防人类衰老和这些疾病发生的小分子,我们使用酵母的寿命测定法作为生物测定系统来筛选抗衰老物质。异槲皮苷(IQ),一种抗衰老物质,从罗布麻中分离出来,一种通常在新疆消费的凉茶,中国。
目的:在本研究中,我们利用分子生物学技术阐明了智商的作用机制。
方法:使用K6001酵母的复制寿命和YOM36酵母的时间顺序寿命来筛选和确认IQ的抗衰老作用。此外,活性氧(ROS)和丙二醛(MDA)测定,酵母在压力下的存活测定,实时聚合酶链反应(RT-PCR)和蛋白质印迹分析,突变体的复制寿命测定,如Δsod1、Δsod2、Δgpx、Δcat,K6001的Δskn7,Δuth1,Δatg32,Δatg2和Δrim15,自噬通量分析,并在给予线粒体自噬抑制剂和激活剂后对K6001酵母进行寿命测定。
结果:IQ延长了K6001酵母的复制寿命和YOM36酵母的时间顺序寿命。此外,活性氮物种(RNS)在生长阶段没有变化,但在IQ处理后的固定阶段显着降低。在IQ处理后,酵母在氧化和热应力条件下的存活率有所提高,增加的超氧化物歧化酶(Sod)活性减轻了热应力。此外,IQ降低了酵母的ROS和MDA,同时增加了抗氧化酶的活性。然而,它不能延长Δsod1、Δsod2、Δgpx的复制寿命,Δcat,Δskn7和K6001的Δuth1。智商显著增加自噬和线粒体自噬诱导,细胞质中存在游离绿色荧光蛋白(GFP),以及在蛋白质水平上YOM38酵母线粒体中的泛素化。IQ没有延长K6001的Δatg2和Δatg32的复制寿命。此外,IQ处理导致蛋白质水平的Sch9减少,Rim15和Msn2的核易位增加。
结论:这些结果表明,Sch9/Rim15/Msn信号通路,以及抗氧化应激,抗热应力,和自噬,参与了酵母智商的抗衰老作用。
BACKGROUND: With the development of an aging sociality, aging-related diseases, such as Alzheimer\'s disease, cardiovascular disease, and diabetes, are dramatically increasing. To find small molecules from natural products that can prevent the aging of human beings and the occurrence of these diseases, we used the lifespan assay of yeast as a bioassay system to screen an
antiaging substance. Isoquercitrin (IQ), an
antiaging substance, was isolated from Apocynum venetum L., an herbal tea commonly consumed in Xinjiang,
China.
OBJECTIVE: In the present study, we utilized molecular-biology technology to clarify the mechanism of action of IQ.
METHODS: The replicative lifespans of K6001 yeasts and the chronological lifespans of YOM36 yeasts were used to screen and confirm the
antiaging effect of IQ. Furthermore, the reactive oxygen species (ROS) and malondialdehyde (MDA) assay, the survival assay of yeast under stresses, real-time polymerase chain reaction (RT-PCR) and Western blotting analyses, the replicative-lifespan assay of mutants, such as Δsod1, Δsod2, Δgpx, Δcat, Δskn7, Δuth1, Δatg32, Δatg2, and Δrim15 of K6001, autophagy flux analysis, and a lifespan assay of K6001 yeast after giving a mitophagy inhibitor and activator were performed.
RESULTS: IQ extended the replicative lifespans of the K6001 yeasts and the chronological lifespans of the YOM36 yeasts. Furthermore, the reactive nitrogen species (RNS) showed no change during the growth phase but significantly decreased in the stationary phase after treatment with IQ. The survival rates of the yeasts under oxidative- and thermal-stress conditions improved upon IQ treatment, and thermal stress was alleviated by the increasing superoxide dismutase (Sod) activity. Additionally, IQ decreased the ROS and MDA of the yeast while increasing the activity of antioxidant enzymes. However, it could not prolong the replicative lifespans of Δsod1, Δsod2, Δgpx, Δcat, Δskn7, and Δuth1 of K6001. IQ significantly increased autophagy and mitophagy induction, the presence of free green fluorescent protein (GFP) in the cytoplasm, and ubiquitination in the mitochondria of the YOM38 yeasts at the protein level. IQ did not prolong the replicative lifespans of Δatg2 and Δatg32 of K6001. Moreover, IQ treatment led to a decrease in Sch9 at the protein level and an increase in the nuclear translocation of Rim15 and Msn2.
CONCLUSIONS: These results indicated that the Sch9/Rim15/Msn signaling pathway, as well as antioxidative stress, anti-thermal stress, and autophagy, were involved in the
antiaging effects of IQ in the yeasts.