Gentiopicroside, a Secoiridoid Glycoside from Gentiana rigescens Franch, Extends the Lifespan of Yeast via Inducing Mitophagy and Antioxidative Stress

Abstract

Gentiopicroside (GPS), an antiaging secoiridoid glycoside, was isolated from Gentiana rigescens Franch, a traditional Chinese medicine. It prolonged the replicative and chronological lifespans of yeast. Autophagy, especially mitophagy, and antioxidative stress were examined to clarify the mechanism of action of this compound. The free green fluorescent protein (GFP) signal from the cleavage of GFP-Atg8 and the colocation signal of MitoTracker Red CMXRos and GFP were increased upon the treatment of GPS. The free GFP in the cytoplasm and free GFP and ubiquitin of mitochondria were significantly increased at the protein levels in the GPS-treated group. GPS increased the expression of an essential autophagy gene, ATG32 gene, but failed to extend the replicative and chronological lifespans of ATG32 yeast mutants. GPS increased the survival rate of yeast under oxidative stress condition; enhanced the activities of catalase, superoxide dismutase, and glutathione peroxidase; and decreased the levels of reactive oxygen species and malondialdehyde. The replicative lifespans of Δsod1, Δsod2, Δuth1, and Δskn7 were not affected by GPS. These results indicated that autophagy, especially mitophagy, and antioxidative stress are involved in the antiaging effect of GPS.

Figure 1

Chemical structure and effect of GPS on the lifespan of yeast. (a) Chemical structure of GPS. (b) The changes in the replicative lifespan of K6001 yeast after treatment of GPS. RES at 10 μM is used as the positive control. (c) Effect of GPS on the chronological lifespan of YOM36 yeast. ^∗,^∗∗, and ^∗∗∗ represent significant difference compared with the control group at p < 0.05, p < 0.01, and p < 0.001, respectively. The chronological lifespan assay is performed in the SC medium, and the survival rate less than 5% of each group is defaulted to 100% death.

Figure 2

Effect of GPS on autophagy in yeast. (a) Fluorescent images of YOM38 yeast containing plasmid pR316-GFP-Atg8 after treatment of RES or GPS observed with a two-photon confocal fluorescent microscope. (b) The percentage of YOM38 cells containing plasmid pR316-GFP-Atg8 with autophagosome (green). Three pictures containing more than 60 cells in each group are used for statistical analysis. (c) Western blot analysis of GFP-Atg8 and free GFP in yeast after treatment with RES or GPS for 22 h in the SD medium. ^∗ and ^∗∗∗ indicate significant differences from the corresponding control at p < 0.05 and p < 0.001, respectively. Each experiment is repeated twice.

Figure 3

Effect of GPS on mitophagy in yeast. (a) Fluorescent images of YOM38 yeast containing plasmid pR316-GFP-Atg8 after treatment of GPS through MitoTracker Red CMXRos staining. (b) The percentage of YOM38 cells containing plasmid pR316-GFP-Atg8 with the colocation of autophagosome (green) and MitoTracker Red CMXRos (red). (c, d) The changes in ATG8-GFP, free GFP, and ubiquitin at the protein level in the mitochondria after treatment of GPS. Three pictures containing more than 60 cells in each group are used for statistical analysis.

Figure 4

Effect of GPS on ATG32 gene expression and lifespan of Δatg32 mutant yeast with K6001 and YOM36 backgrounds. (a) Effect of GPS on ATG32 gene expression after 12 h treatment. (b) Effect of GPS on the replicative lifespan of atg32 mutants with K6001 background. (c) Effect of GPS on the chronological lifespan of atg32 mutants with YOM36 background. ^∗, ^∗∗, and ^∗∗∗ indicate significant differences from the corresponding control at p < 0.05, p < 0.01, and p < 0.001, respectively. Each experiment is repeated twice.

Figure 5

Effect of GPS on the survival rate of yeast under oxidative stress condition and antioxidative enzyme activity in yeast. (a) Photograph of yeast growth after treatment of GPS under oxidative stress condition induced by H₂O₂ at 10.5 mM. (b) The survival rate changes in yeast under oxidative conditions at 5 mM H₂O₂. (c–f) The changes in CAT, T-SOD, SOD1, and GPx enzyme activities in yeast after treatment of GPS for 48 h. (g, h) Effect of GPS on ROS and MDA levels. Each experiment is repeated thrice. ∗, ∗∗, and ∗∗∗ indicate significant differences from the corresponding control (p < 0.05, p < 0.01, and p < 0.001), respectively.

Figure 6

Effect of GPS on the replicative lifespans of Δ sod1 (a), Δsod2 (b), Δ uth1 (c), and Δskn7 (d) yeast with K6001 background. The procedure for the replicative lifespan assay is the same as that for the K6001 lifespan assay. Each experiment is conducted thrice. ^∗∗ indicates significant difference between the control group of K6001 and GPS-treated group at p < 0.01.