Involvement of the Sch9/Rim15/Msn2 signaling pathway in the anti-aging activity of dendrobine from Dendrobium nobile Lindl. via modification of oxidative stress and autophagy

Abstract

Background: Aging is an important pathogenic factor of age-related diseases and has brought huge health threat and economic burden to the society. Dendrobium nobile Lindl., a valuable herb in China, promotes longevity according to the record of ancient Chinese materia medica. This study aimed to discover the material basis of D. nobile as an anti-aging herb and elucidate its action mechanism.

Methods: K6001 yeast replicative lifespan assay was used to guide the isolation of D. nobile. The chronological lifespan assay of YOM36 yeast was further conducted to confirm the anti-aging activity of dendrobine. The mechanism in which dendrobine exerts anti-aging effect was determined by conducting anti-oxidative stress assay, quantitative real-time PCR, Western blot, measurements of anti-oxidant enzymes activities, determination of nuclear translocation of Rim15 and Msn2, and replicative lifespan assays of Δsod1, Δsod2, Δcat, Δgpx, Δatg2, Δatg32, and Δrim15 yeasts.

Results: Under the guidance of K6001 yeast replicative lifespan system, dendrobine with anti-aging effect was isolated from D. nobile. The replicative and chronological lifespans of yeast were extended upon dendrobine treatment. In the study of action mechanism, dendrobine improved the survival rate of yeast under oxidative stress, decreased the levels of reactive oxygen species and malondialdehyde, and enhanced the enzyme activities and gene expression of superoxide dismutase and catalase, but it failed to elongate the replicative lifespans of Δsod1, Δsod2, Δcat, and Δgpx yeast mutants. Meanwhile, dendrobine enhanced autophagy occurrence in yeast but had no effect on the replicative lifespans of Δatg2 and Δatg32 yeast mutants. Moreover, the inhibition of Sch9 phosphorylation and the promotion of nuclear translocation of Rim15 and Msn2 were observed after treatment with denrobine. However, the effect of dendrobine disappeared from the Δrim15 yeast mutant after lifespan extension, oxidative stress reduction, and autophagy enhancement.

Conclusions: Dendrobine exerts anti-aging activity in yeast via the modification of oxidative stress and autophagy through the Sch9/Rim15/Msn2 signaling pathway. Our work provides a scientific basis for the exploitation of D. nobile as an anti-aging herb.

Keywords: Anti-aging; Autophagy; Dendrobine; Dendrobium nobile Lindl.; Oxidative stress; Sch9/Rim15/Msn2 signaling pathway.

Fig. 1

The chemical structure and anti-aging activity of dendrobine. A The chemical structure of dendrobine. B Replicative lifespan-prolonging effect of dendrobine on K6001 yeast. C Effect of dendrobine on chronological lifespan in YOM36 yeast. D The photograph of colonies of PC12 cells after treating with different concentrations of dendrobine. E The digital result of D. The experiment was repeated three times and the data from each experiment are displayed as mean ± SEM. The repeats number of each group for replicative lifespan assay was 40, and the repeats number of each group for chorological lifespan assay was five. ^*, ^** and ^*** represent significant difference compared with negative control (p < 0.05, p < 0.01, p < 0.001)

Fig. 2

Increased anti-oxidant capability of yeast upon dendrobine treatment. A Growth status of BY4741 yeast under oxidative stress induced by 9.5 mM H₂O₂; B The survival rates of BY4741 yeast under oxidative stress induced by 5.5 mM H₂O₂; C, D The levels of ROS (C) and MDA (D) in BY4741 yeast upon dendrobine treatment at 24 and 48 h; E–H The effect of dendrobine on anti-oxidant enzyme activities in BY4741 yeast at 24 and 48 h. The experiment was repeated three times and data from each experiment are displayed as mean ± SEM. The repeats number of each group was five. ^*, ^**, ^*** represent significant difference compared with negative control (p < 0.05, p < 0.01, p < 0.001)

Fig. 3

The effect of dendrobine on antioxidant enzyme genes expression. A–D The mRNA abundance of SOD1 (A), SOD2 (B), CAT (C) and GPx (D) at 24 and 48 h. The experiment was repeated three times and data from each experiment are displayed as mean ± SEM. ^*, ^**, ^*** represent significant difference compared with negative control (p < 0.05, p < 0.01, p < 0.001)

Fig. 4

The effects of dendrobine on lifespans of yeast mutants with deletion of antioxidant enzyme genes. A–D Dendrobine failed to prolong the replicative lifespans of Δsod1 (A), Δsod2 (B), Δcat (C) and Δgpx (D) yeast mutants. The experiment was repeated three times and data from each experiment are displayed as mean ± SEM. ^*, ^** represent significant difference compared with negative control (p < 0.05, p < 0.01)

Fig. 5

Enhanced autophagy by dendrobine treatment. A, B Failure of dendrobine to prolong the lifespans of Δatg2 and Δatg32 yeast mutants with K6001 background. C Fluorescent images of yeasts treated with 300 µM RES or 0, 0.1, 1 and 10 µM dendrobine. Punctate green fluorescence is free GFP representing the occurrence of autophagy. D Statistical result of C; ten pictures of each group were selected randomly, and calculated the percentage of cells with free GFP in each group; ^*** represents significant difference compared with negative control (p < 0.001). E The western blot results of GFP-Atg8 and free GFP in yeast after treatment with 200 nM wortmannin (Wor), 300 µM RES and different doses of dendrobine for 22 h. F The digital result of E, ^*, ^** represent significant difference compared with negative control (p < 0.05, p < 0.01). G The western blot results of GFP-Atg8 and free GFP in yeast after treatment with 300 µM RES, 200 nM wortmannin, 200 nM wortmannin plus 1 µM dendrobine, or 1 µM dendrobine at indicated time. H The digital result of G; ^*** represents significant difference compared with dendrobine-treated group at 0 h (p < 0.001). ^##, ^### represent significant difference compared with control group at 22 h (p < 0.01, p < 0.001). The experiment was repeated three times and data from each experiment are displayed as mean ± SEM.

Fig. 6

Sch9/Rim15/Msn2 signaling pathway mediates the anti-aging effect of dendrobine. A The western blot result of phosphorylation level of sfGFP-Sch9-5HA after treatment with RA or dendrobine for 40 min. B The digital result of A. C, E Nuclear translocation of Rim15-GFP (C) and Msn2-GFP (E) upon dendrobine treatment. D, F Statistical results of C and E. Nine images of each group in C and five images of each group in E were selected randomly, calculated the percentage of cells with the overlap of green and blue fluorescence indicating the nuclear translocation of Rim15-GFP or Msn2-GFP. G Failure of dendrobine to extend the replicative lifespan of Δrim15 yeast mutant with K6001 background. The experiment was repeated three times and data from each experiment are displayed as mean ± SEM. ^*, ^**, ^*** represent significant difference compared with negative control (p < 0.05, p < 0.01, p < 0.001)

Fig. 7

The effects of dendrobine on oxidative stress and autophagy are mediated by Rim15. A, B Dendrobine failed to decrease the levels of ROS (A) and MDA (B) in Δrim15 yeast mutant with K6001 background. C Fluorescence images of autophagy in K6001 yeast and Δrim15 yeast mutant with K6001 background after RES or dendrobine treatment. D Quantification result of C. The experiment was repeated three times and data from each experiment are displayed as mean ± SEM. Ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test were utilized to evaluate statistically significant differences among groups. Two-tailed, unpaired, Student’s t-tests were used for comparison between two groups. ^*, ^** represent significant difference compared with negative control (p < 0.05, p < 0.01). ^#, ^##, ^### represent significant difference between K6001 yeast and Δrim15 yeast mutant with K6001 background upon corresponding treatment (p < 0.05, p < 0.01, p < 0.001)

Fig. 8

The proposed action mechanism of dendrobine. Dendrobine produced anti-aging effect via inhibiting activity of Sch9 to increase Rim15 activity and Rim15 nuclear translocation to reduce oxidative stress and enhance autophagy via regulation of transcription factor, such as Msn2