Isoquercitrin from Apocynum venetum L. Exerts Antiaging Effects on Yeasts via Stress Resistance Improvement and Mitophagy Induction through the Sch9/Rim15/Msn Signaling Pathway

Abstract

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.

Aim of the study: 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.

Keywords: Sch9/Rim15/Msn; anti-thermal stress; antiaging; antioxidative stress; isoquercitrin; mitophagy.

Figure 1

The chemical structure and antiaging effects of isoquercitrin (IQ) on yeasts. (a) The chemical structure of IQ. (b) Effect of IQ on the replicative lifespan of K6001 yeasts. Resveratrol (RES) at 10 μM was used as the positive control. (c) Effect of IQ on the chronological lifespan of YOM36 yeasts. Rapamycin at 1 μM was used as the positive control. *, **, and *** represent significant differences compared to the control group at p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 2

Effect of IQ on reactive nitrogen species (RNS) levels during chronological aging. (a) The growth curve of YOM36 yeasts during chronological aging. (b–e) The changes in the RNS levels after IQ treatment at 1, 2, 3.5, and 5 days. *** indicates significant differences from the control group at p < 0.001.

Figure 3

Effect of IQ on the survival of yeasts under oxidative stress, and evaluation of ROS, MDA levels and antioxidant enzyme activity in yeasts. (a) The colony formation of BY4741 yeasts upon IQ treatment under H₂O₂ stimulation at 11 mM. (b) The survival rate of BY4741 after IQ treatment under oxidative stress induced by 5.5 mM H₂O₂. (c,d) Effects of IQ on reactive oxygen species (ROS) and malondialdehyde (MDA) in yeasts under physiological status. (e–h) Changes in antioxidant enzyme activity in BY4741 yeast after incubation with IQ for 24 h. *, **, and *** indicate significant differences from the control group at p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 4

IQ enhances the Sod activity to synergistically counteract thermal stress in yeasts. (a) Photograph of YOM36 yeasts with or without thermal stimuli after culturing at 28 °C for 24 h. (b) The growth of YOM36 yeasts with IQ treatment after heating at 60 °C for 30 min and culturing at 28 °C for 48 h. (c) The survival rate of YOM36 yeasts under different thermal-stress conditions. (d) The quantitative anti-thermal-stress experimental results after IQ treatment followed by heating at 60 °C for 25 min. * p < 0.05 and ** p < 0.01 represent significant differences compared to the control group. (e,f) The ROS and MDA in YOM36 yeasts after treatment with IQ under room-temperature and thermal-stress conditions. * p < 0.05 and ** p < 0.01 represent significant differences compared with the control group under room-temperature conditions. ### p < 0.001 represents significant difference between the negative control group. (g,h) The T-Sod and CuZn-Sod activities in YOM36 yeasts after treatment with IQ under room-temperature and thermal-stress conditions. * p < 0.05, ** p < 0.01, and *** p < 0.001 represent significant differences compared with the control group under room-temperature conditions. $ p < 0.05, $$ p < 0.01, and $$$ p < 0.001 represent significant differences compared with the control group under thermal-stress conditions. # p < 0.05 and ### p < 0.001 represent significant differences between the negative control group. (i) The growth of K6001 yeasts and its Δsod1 and Δsod2 mutants after IQ treatment with or without heating at 55 °C for 30 min.

Figure 5

Effect of IQ on the replicative lifespans of Δsod1, Δsod2, Δcat, Δgpx, Δskn7, and Δuth1 yeasts with K6001 background. (a–f) The replicative lifespans of Δsod1, Δsod2, Δcat, Δgpx, Δskn7, and Δuth1 yeasts. # p < 0.05, ** p < 0.01, and *** p < 0.001 represent significant differences compared with the negative control group of K6001.

Figure 6

Effect of IQ on autophagy induction in YOM38 yeasts. (a) The fluorescent images of autophagy and mitophagy induced by IQ with or without wortmannin inhibition. (b) The digitized results of YOM38 yeasts containing free green fluorescent protein (GFP) in (a). (c) The statistical results of YOM38 yeasts with the colocalization of free GFP (green) and MitoTracker Red CMXRos (red) in (a). (d) Western blot analysis of GFP-Atg8 and free GFP in YOM38 yeast after treatment with IQ at 1, 10, and 30 μM with or without wortmannin for 22 h. (e) The digital results of free GFP in (d). (f) The changes in ubiquitin in the mitochondria after treatment with IQ with or without wortmannin. (g,h) The replicative lifespans of Δatg2 and Δatg32 of K6001 yeasts. (i) The replicative lifespans of K6001 yeasts after treatment of mitophagy activator GSK3-IN-3 or inhibitor Mdivi-1. The average lifespans of K6001 and mutants are displayed in Supplementary Table S3. * p < 0.05, ** p < 0.01, and *** p < 0.001 represent significant differences compared with the control group without wortmannin. $ p < 0.05 represents significant difference compared with the control group with wortmannin. # p < 0.05, ## p < 0.01, and ### p < 0.001 indicate significant differences between the groups with or without wortmannin inhibition.

Figure 7

Effect of IQ on the expressions of Sch9 and nuclear translocation of GFP-Rim15 and GFP-Msn2. (a) The changes in Sch9 after treatment with IQ for 2 h. (b) The digital results of (a). (c) The replicative lifespans of Δrim15 of K6001 after IQ treatment. (d) The fluorescence images of the colocalization of GFP-Rim15 and nuclear staining with Hoechst 33,342. (e) The statistical results of (d). (f) The fluorescence signals of the colocalization of GFP-Msn2 and nuclear staining with Hoechst 33,342. (g) The statistical results of (f). *, **, and *** indicate significant differences from the control group at p < 0.05, p < 0.01, and p < 0.001, respectively. # indicates significant differences from the control group in Δrim15 of K6001 at p < 0.05.

Figure 8

Rim15 is essential for IQ to exert autophagy-induction and thermal-stress-resistance effects. (a) The fluorescence images showing the autophagosomes in K6001 and ∆rim15 of K6001 stained with CYTO-ID green dye. (b) The digital results of (a). (c) The growth of K6001 and ∆rim15 of K6001 yeasts after IQ treatment with heating at 55 °C for 30 min and culturing at 28 °C for 48 h. * indicates significant differences from the control group of K6001 yeasts at p < 0.05. # and ## represent significant differences between the same treatment group of K6001 and ∆rim15 of K6001 yeasts at p < 0.05 and p < 0.01. $ and $$ indicate significant differences from the control group of ∆rim15 of K6001 yeasts at p < 0.05 and p < 0.01, respectively.

Figure 9

The proposed mechanism of action for IQ. Improvements in stress resistance and mitophagy induction through the Sch9/Rim15/Msn signaling pathway are involved in the antiaging effect of IQ on yeasts.