Ganoderma lucidum stimulates autophagy-dependent longevity pathways in Caenorhabditis elegans and human cells

Abstract

The medicinal fungus Ganoderma lucidum is used as a dietary supplement and health tonic, but whether it affects longevity remains unclear. We show here that a water extract of G. lucidum mycelium extends lifespan of the nematode Caenorhabditis elegans. The G. lucidum extract reduces the level of fibrillarin (FIB-1), a nucleolar protein that correlates inversely with longevity in various organisms. Furthermore, G. lucidum treatment increases expression of the autophagosomal protein marker LGG-1, and lifespan extension is abrogated in mutant C. elegans strains that lack atg-18, daf-16, or sir-2.1, indicating that autophagy and stress resistance pathways are required to extend lifespan. In cultured human cells, G. lucidum increases concentrations of the LGG-1 ortholog LC3 and reduces levels of phosphorylated mTOR, a known inhibitor of autophagy. Notably, low molecular weight compounds (<10 kDa) isolated from the G. lucidum water extract prolong lifespan of C. elegans and the same compounds induce autophagy in human cells. These results suggest that G. lucidum can increase longevity by inducing autophagy and stress resistance.

Keywords: caloric restriction mimetics; dietary supplements; lingzhi; mTOR; medicinal mushrooms.

Figure 1

G. lucidum treatment reduces FIB-1 levels and extends lifespan in C. elegans. (A) Effects of a water extract of G. lucidum (GL) on fibrillarin-1 (FIB-1) levels as monitored by fluorescence microscopy. Synchronized L4 larvae of transgenic C. elegans strain SJL1 expressing FIB-1::GFP (green fluorescent protein) under the FIB-1 gene’s native promoter were cultured for 3 days on agar plates spread with control water, the water extract of GL, or rapamycin (400 μM, Rapa). Data are expressed as arbitrary units (a. u.). (B) FIB-1 levels assessed by Western blots in wild-type N2 C. elegans treated with GL. (C) Quantification of FIB-1 protein levels shown in (B). FIB-1 expression was measured by densitometry and normalized against actin. (D) Lifespan assay of GL-treated nematodes. C. elegans was cultured on agar plates spread with water, GL or rapamycin as above. Survival was assessed for 30 days using an optical microscope based on motility. Representative lifespan curves are shown. See also Supplementary Table 1. (E) Pharyngeal pumping of nematodes following culture with water, GL or rapamycin for 3 days. Pharyngeal contractions were recorded for 1 min under optical microscopy. (F) Size of 3-day old worms. Size was monitored by delineating the worms’ region of interest (ROI) under optical microscopy. Data represent means ± standard deviation. *p<0.05; **p<0.01; ***p<0.001.

Figure 2

G. lucidum extends nematode lifespan by inducing autophagy. (A–D) Effects of G. lucidum (GL) in wild-type and mutant C. elegans. Synchronized L4 larvae of (A) wild-type (WT) N2 C. elegans or mutant strains lacking (B) atg-18, (C) daf-16, or (D) sir-2.1 were maintained on agar plates spread with GL (2 mg/plate) or rapamycin (Rapa, 400 μM), and survival was monitored based on motility (see also Supplementary Table 1). (E) GL induces autophagy in C. elegans. Transgenic DA2123 C. elegans expressing GFP::LGG-1 were treated as above for 3 days, prior to observation under fluorescence microscopy. Approximately 50 cells were examined per treatment. (F) Quantification of fluorescent GFP::LGG-1 puncta following GL treatment based on the experiments shown in (E). (G) GL treatment increases GFP::LGG-1 levels in DA2123 worms as revealed by Western blotting. Membranes were incubated with both anti-GFP and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies, prior to signal detection. (H) Quantification of Western blot signals shown in (G) after normalization against GAPDH. *p<0.05; **p<0.01; ***p<0.001.

Figure 3

G. lucidum inhibits the mTOR pathway in human cells. (A) Effects of G. lucidum (GL) on the mTOR pathway in human Huh7 hepatoma cells. Cells were treated with GL for 4 hrs, prior to Western blot analysis. Expression was normalized against GAPDH. (B) Protein intensity evaluated by densitometry. (C, D) Effects of GL on the mTOR pathway in human IMR-90 lung fibroblasts. Cells were processed as above for (C) Western blotting and (D) densitometry analysis. Statistical analysis was done with one-way analysis of variance (ANOVA). *p<0.05; **p<0.01; ***p<0.001.

Figure 4

G. lucidum induces autophagy in human cells. (A) Effects of G. lucidum (GL) on LC3B-I and LC3B-II in human cells. Huh7 cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) were treated with GL (1%) or DMEM for 12 hrs, prior to treatment with DMEM containing 3-methyladenine (3-MA, 2 mM) for 12 hrs. Protein levels were monitored by Western blot and normalized against actin. (B) GL induces the formation of autophagosomes and autolysosomes in human cells. Huh7 cells expressing monomeric red fluorescent protein (RFP)-LC3 and green fluorescent protein (GFP)-LC3 were treated with GL (1%) for 24 hrs, prior to fluorescence microscopy analysis. In differential interference contrast (DIC) images, cells are delineated in red for clarity. (C) Quantification of fluorescent puncta based on fluorescence microscopy. (D) Representative transmission electron microscopy (TEM) images of GL-treated cells. Huh7 cells were treated with GL (1%) for 24 hrs prior to fixation and thin-sectioning as described in Materials and Methods. Images on the right correspond to the insets delineated by white rectangles in the images on the left. An autolysosome is delineated by a white dashed line for the GL panel. (E) Quantification of autophagosomes and autolysosomes based on TEM. ***p<0.001.

Figure 5

G. lucidum-derived subfraction 10K-2 attenuates FIB-1 expression, extends lifespan, and induces autophagy in C. elegans. (A) Effects of G. lucidum (GL) sub-fractions on FIB-1::GFP intensity. Synchronized L4 larvae of C. elegans SJL1 were treated with water, GL sub-fractions, or rapamycin (Rapa; 400 μM) for 3 days. FIB-1::GFP was monitored by fluorescence microscopy. (B) Lifespan assay of C. elegans treated with GL sub-fractions. Synchronized C. elegans SJL1 larvae were treated with water, GL subfractions 10K-1 (1 mg/plate) or 10K-2 (2 mg/plate), or rapamycin (400 μM). Representative survival curves are shown (see also Supplementary Table 1). (C) GL sub-fraction 10K-2 induces autophagy in C. elegans. DA2123 nematodes were treated with the sub-fractions for 3 days, and GFP::LGG-1 levels were quantified by fluorescence microscopy. Numbers of GFP puncta were counted in 50 seam cells. Data represent means ± standard deviation. Statistical analysis was done using Student’s t test. *p<0.05; ***p<0.001.

Figure 6

G. lucidum-derived sub-fraction 10K-2 represses the mTOR pathway in human cells. (A) Inhibition of the mTOR pathway by G. lucidum (GL) sub-fraction 10K-2. Huh7 liver cells were treated with control medium (i.e., Dulbecco’s modified Eagle’s medium, DMEM), 10K-1 or 10K-2 for 4 hrs, prior to Western blot analysis. (B) Protein intensity was evaluated by densitometry and normalized against actin. (C) Sub-fraction 10K-2 inhibits the mTOR pathway in IMR-90 cells. Cells cultured in Eagle’s miminum essential medium (EMEM) were processed as above for Western blot analysis. (D) Densitometry analysis of the results shown in (C). *p<0.05; **p<0.01; ***p<0.001.

Figure 7

G. lucidum-derived sub-fraction 10K-2 induces autophagy in human cells. (A) G. lucidum (GL) sub-fractions 10K-1 and 10K-2 induce the formation of autophagosomes and autolysosomes in human cells. Huh7 cells expressing mRFP-GFP-LC3 were treated with DMSO or GL sub-fractions 10K-1 and 10K-2 (1 mg/ml) for 24 hrs, prior to fluorescence microscopy observations. (B) Quantification of fluorescent puncta based on fluorescence microscopy analysis. (C) TEM observations of Huh7 cells treated with GL sub-fractions. Cells were treated as above prior to fixation and preparation for TEM analysis. Images on the right correspond to the white rectangles in the images on the left. Autolysosomes are delineated by a white dashed line in the 10K-1 and 10K-2 panels. (D) Quantification of autophagosomes and autolysosomes based on TEM analysis. *p<0.05; **p<0.01; ***p<0.001.