Ginsenoside Prolongs the Lifespan of C. elegans via Lipid Metabolism and Activating the Stress Response Signaling Pathway

Abstract

Panax ginseng is a valuable traditional Chinese medicine in Northeast China. Ginsenoside, the active component of ginseng, has not been investigated much for its effects on aging and its underlying mechanism(s) of action. Here, we investigated the effects of total ginsenoside (TG), a mixture of the primary active ginsenosides from Panax ginseng, on the lifespan of Caenorhabditis elegans (C. elegans). We found that TG extended the lifespan of C. elegans and reduced lipofuscin accumulation. Moreover, TG increased the survival of C. elegans in response to heat and oxidative stress via the reduction of ROS. Next, we used RNA-seq to fully define the antiaging mechanism(s) of TG. The KEGG pathway analysis showed that TG can prolong the lifespan and is involved in the longevity regulating pathway. qPCR showed that TG upregulated the expression of nrh-80, daf-12, daf-16, hsf-1 and their downstream genes. TG also reduced the fat accumulation and promoted lipid metabolism. Moreover, TG failed to extend the lifespan of daf-16 and hsf-1 mutants, highlighting their role in the antiaging effects of TG in C. elegans. The four main constitution of TG were then confirmed by HPLC and included ginsenoside Re, Rg₁, Rg₂ and Rd. Of the ginsenosides, only ginsenoside Rd prolonged the lifespan of C. elegans to levels comparable to TG. These findings provided mechanistic insight into the antiaging effects of ginsenoside in C. elegans.

Keywords: Caenorhabditis elegans; Panax ginseng; lifespan; lipid metabolism; stress-resistant.

Figure 1

Effects of TG on the lifespan of C. elegans and E coil. OP50. (A) Survival of C. elegans treated with 0.1% DMSO (control); 500 μM EGCG (positive control) and 1, 10 and 100 μg/mL TG. TG produced its optimal effects at 10 μg/mL, extending the lifespan of C. elegans by up to 9.11 ± 2.04%, n = 3 (~80 individuals per group), Kaplan–Meier survival analysis with the log-rank test. (B) OP50 in LB medium containing 1, 10 and 100 μg/mL TG. TG did not inhibit the growth of OP50 during any bacterial growth phase. Data were analyzed by a Student’s t-test using GraphPad 8. Values are presented as the mean ± SEM.

Figure 2

Effects of TG on the breeding population, body size, body bending and pharyngeal pumping in C. elegans. (A) Breeding of C. elegans treated with or without TG. The number of eggs were monitored on day 1 of adulthood until day 5. (B) Worms treated with TG showed no significant changes in body size. (C) Worms treated with TG showed no significant changes in body bending. (D) Worms treated with TG showed no significant changes in pharyngeal pumping.

Figure 3

Effects of TG on the lipofuscin accumulation in C. elegans. (A–D) Worms were treated with TG for 8 days at 20 °C. Lipofuscin accumulation was measured using blue (Ex/Em 340/430 nm) and red autofluorescence (Ex/Em 546/600 nm). TG significantly reduced the lipofuscin accumulation in C. elegans. The data were analyzed using a Student’s t-test. The values were shown as the mean ± SEM, ** p < 0.01.

Figure 4

Effects of TG pretreatment on the stress resistance and ROS accumulation in C. elegans. (A) TG significantly increased the survival of worms under heat stress. (B) The worms were treated with or without TG and exposed to 50 mM paraquat. (C) Relative levels of ROS in C. elegans following the treatment with TG. TG decreased the ROS levels in the worms, evidenced by the quantification of the fluorescence intensity. The data were analyzed using a Student’s t-test. The values were shown as the mean ± SEM, * p < 0.05.

Figure 5

Modulated pathways in TG treated in C. elegans. (A) A total of 26 genes were upregulated and 10 genes were downregulated following the TG treatment (FDR < 0.05 and fold change > 1.5). (B) The KEGG analysis of differentially expressed genes in TG-treated C. elegans.

Figure 6

GO analysis of the genes significantly regulated in TG-treated C. elegans.

Figure 7

The KEGG analysis of differentially regulated genes.

Figure 8

TG reduces the fat accumulation and increases the expression of nhr-80, daf-12, daf-16 and hsf-1 in C. elegans. (A) TG enhances the expression of nhr-80, fat-6, daf-12, fard-1, daf-16, sod-3, hsf-1 and hsp-16.2. (B,C) The fat content in C. elegans treated with or without TG. The worms were stained with oil red O. TG significantly reduced the fat content in C. elegans. (D) The mean lifespan of daf-16 (mgDf50) treated with or without TG. TG could not extend the lifespan of daf-16 (mgDf50). (E,F) DAF-16::GFP expression in TG-treated worms. (G) The mean lifespan of hsf-1 (sy441) with or without TG. TG failed to extend the lifespan of hsf-1 (sy441). (H,I). The quantification of HSP-16.2::GFP fluorescence in the presence or absence of TG. TG significantly increases the expression of HSP-16.2 in C. elegans. The data were analyzed using a Student’s t-test. The values were shown as the mean ± SEM, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 9

HPLC chromatograms of TG recorded at 203 nm.

Figure 10

Effects of ginsenoside Rd, Rg₁, Re and Rg₂ on the lifespan of C. elegans. (A) The survival of C. elegans treated with 0.1% DMSO (control) and 1 μg/mL Rd, Rg₁, Re and Rg₂. Worms exposed to ginsenoside Rd survived longer than untreated worms (p < 0.05). (B) Ginsenoside Rg₁, Re and Rg₂ (1 μg/mL) did not extend the lifespan of C. elegans. (C,D) Fluorescence images of DAF-16::GFP expression in ginsenoside Rd-treated and untreated worms. (E) mRNA levels of nhr-80, daf-12 and daf-16 and the downstream genes fat-6, fard-1 and sod-3 in C. elegans treated with ginsenoside Rd. The data were analyzed using a Student’s t-test. The values were shown as the mean ± SEM, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 11

Schematic representation of the mechanism of the TG-extended lifespan.