Midlife intervention of Dendrobium officinale extract modulates gut microbiota to activate InR-Nrf2 axis, promoting intestinal health and longevity

Figure 1 Survival of nematodes, flies, and MRC-5 cells after supplementation with medium containing Dendrobium officinale extract. A: Survival curve of nematodes (n = 90 nematodes); B: Mean lifespan of nematodes (n = 90 nematodes); C: Survival curve for female flies (n = 150 flies); D: Mean lifespan for female flies (n = 150 flies); E: Survival curve for male flies (n = 150 flies); F: Mean lifespan for male flies (n = 150 flies); G: Effect on viability of MRC-5 cells induced by Dendrobium officinale extract (DOE) by cell counting kit-8 (CCK-8) assay (n = 3); H: Effect on viability of MRC-5 cells induced by hydrogen peroxide by CCK-8 assay (n = 3); I: CCK-8 assays of damaged MRC-5 cells treated with DOE (n = 3); J: Aging-related galactosidase staining (SA-β-gal), senescent cells are stained dark blue-green, and have increased cell volume and irregular shapes, scale bar = 200 μm. The data are expressed as the mean ± SD. Data were analyzed by log-rank test (survival) or one-way analysis of variance with post-hoc test (multiple comparisons). ^aP < 0.05 vs control group in nematodes. ^bP < 0.001 vs control group in nematodes. ^cP < 0.001 vs control group in female flies. ^dP < 0.01 vs control group in female flies. ^eP < 0.01 vs control group in male flies. ^fP < 0.01 vs control group in male flies. ^gP < 0.05 vs control group in MRC-5 cells. ^hP < 0.01 vs control group in MRC-5 cells. ⁱP < 0.001 vs control group in MRC-5 cells. ^jP < 0.0001 vs control group in MRC-5 cells. ^kP < 0.05 vs model group in MRC-5 cells. C. elegans: Caenorhabditis elegans; DOE: Dendrobium officinale extract; H₂O₂: Hydrogen peroxide; con: Control.

Figure 2 Midlife is the best intervention window for Dendrobium officinale extract to extend lifespan. A: Dendrobium officinale extract (DOE) intervention duration screening strategy for nematodes; B: DOE intervention duration screening strategy for flies; C: Survival curve of DOE treatment for 4 days in nematodes (n = 90 nematodes); D: Survival curve of DOE treatment for 20 days in female flies (n = 150 flies); E: Survival curve of DOE treatment for 20 days in male flies (n = 150 flies); F: Comparison of the average lifespan extension rate of nematodes after 2 days, 4 days, 8 days, and lifelong treatment with DOE (n = 3); G: Comparison of the lifespan extension rate of female flies after 10 days, 20 days, 30 days, 40 days, and lifelong treatment with DOE (n = 3); H: Comparison of the lifespan extension rate of male flies after 10 days, 20 days, 30 days, 40 days, and lifelong treatment with DOE (n = 3); I: Staged DOE intervention strategies in nematodes; J: Staged DOE intervention strategies in flies; K: Survival curve of DOE midlife intervention for 4 days in nematodes (n = 90 nematodes); L: Survival curve of DOE midlife intervention for 20 days in female flies (n = 150 flies); M: Survival curve of DOE midlife intervention for 20 days in male flies (n = 150 flies); N: Comparison of the mean lifespan after intervention with DOE in the early, midlife, late, and lifelong stages in nematodes (n = 3); O: Comparison of the mean lifespan after intervention with DOE in the early, midlife, late, and lifelong stages in female flies (n = 3); P: Comparison of the mean lifespan after intervention with DOE in the early, midlife, late, and lifelong stages in male flies (n = 3). The data are expressed as the mean ± SD. Data were analyzed by log-rank test (survival) or one-way analysis of variance with post-hoc test (multiple comparisons). ^aP < 0.05 vs control group in nematodes. ^bP < 0.01 vs control group in flies. ^cP < 0.0001 vs 2-day group in nematodes. ^dP < 0.0001 vs 10-day group in flies. ^eP < 0.01 vs control group in nematodes with midlife intervention. ^fP < 0.001 vs control group in flies with midlife intervention. ^gP < 0.001 vs midlife group in nematodes. ^hP < 0.0001 vs midlife group in nematodes. ⁱP < 0.01 vs midlife group in flies. ^jP < 0.001 vs midlife group in flies. ^kP < 0.001 vs midlife group in flies. DOE: Dendrobium officinale extract.

Figure 3 Dendrobium officinale extract midlife intervention extends healthy lifespan. A: Motility in nematodes after staged Dendrobium officinale extract (DOE) intervention (n = 90 nematodes); B: Pumping frequency in nematodes after staged DOE intervention (n = 90 nematodes); C and D: Intestinal atrophy in nematodes after staged DOE intervention (n = 90 nematodes); E and F: Intestinal leakage in nematodes after staged DOE intervention (n = 90 nematodes); G and H: Microbial colonization capacity in nematodes after staged DOE intervention (n = 90 nematodes); I: Food intake in flies after staged DOE intervention (n = 3); J: Reproductivity in flies after staged DOE intervention (n = 3); K: Climbing number in flies after staged DOE intervention (n = 3); L and M: Intestinal integrity in flies after staged DOE intervention (n = 3). The data are expressed as the mean ± SD. Data were analyzed by one-way analysis of variance with post-hoc test (multiple comparisons). ^aP < 0.05 vs control group. ^bP < 0.01 vs control group. ^cP < 0.001 vs control group. ^dP < 0.0001 vs control group.

Figure 4 Inhibition of intestinal stem cells proliferation and maintenance of intestinal microbiota homeostasis by Dendrobium officinale extract. A: Representative images obtained at 7 days, 22 days, and 42 days from Dendrobium officinale extract (DOE)-intervened esg-gal4 > UAS-GFP/cyo flies showing midgut enterocytes [4’,6-diamidino-2-phenylindole (DAPI)], intestinal stem cells (ISCs)/entero-blasts [green fluorescent protein (GFP)], and ISCs [Delta (DI)]. Scale bar = 10 μm; B: Quantification of esg-GFP⁺ or DI⁺ cells as percentages of total DAPI-stained cells in female flies (n = 3); C: Quantification of the mitotic index (phospho-histone H3; phospho-histone H3-positive cells per midgut) in female flies (n = 3); D: Bacterial abundance at the phylum level in female flies; E: Relative abundance of the dominant strain in female flies (phylum level); F: Bacterial abundance at the phylum level in male flies; G: Relative abundance of the dominant strain in male flies (phylum level); H: Bacterial abundance at the genus level in female flies; I: Relative abundance of the dominant strain in female flies (genus level); J: Bacterial abundance at the genus level in male flies; K: Relative abundance of the dominant strain in male flies (genus level); L: Growth status of Acetobacter sp. in the gut of flies at 10-day, 20-day, 30-day, 40-day, and 40-day + DOE; M: Quantification of Acetobacter sp. in female flies (n = 3); N: Growth status of Lactobacillus sp. in the gut of flies at 10-day, 20-day, 30-day, 40-day, and 40-day + DOE; O: Quantification of Lactobacillus sp. in male flies (n = 3). The data are expressed as the mean ± SD. Data were analyzed by Student’s t-test (two groups) or one-way analysis of variance with post-hoc test (multiple comparisons). ^aP < 0.05 vs 7-day group. ^bP < 0.01 vs 7-day group. ^cP < 0.0001 vs 7-day group. ^dP < 0.0001 vs 42-day group. ^eP < 0.05 vs control group. ^fP < 0.0001 vs 10-day group. ^gP < 0.01 vs 20-day group. ^hP < 0.05 vs 40-day group. ⁱP < 0.001 vs 40-day group. DOE: Dendrobium officinale extract; DI: Delta; GFP: Green fluorescent protein; DAPI: 4’,6-diamidino-2-phenylindole; PH3: Phospho-histone H3; F: Female; M: Male.

Figure 5 Dendrobium officinale extract activates the skn-1/Nrf2 and Keap1-Nrf2-antioxidant response element signaling pathways to resist oxidative stress. A: Enzyme activity of catalase (CAT) in nematodes (n = 3); B: Enzyme activity of superoxide dismutase (SOD) in nematodes (n = 3); C: Enzyme activity of malondialdehyde (MDA) in nematodes (n = 3); D: Reactive oxygen species (ROS) level in nematodes (n = 3); E: Activity of CAT in flies (n = 3); F: Activity of SOD in flies (n = 3); G: Activity of MDA in flies (n = 3); H: ROS level in flies (n = 3); I: Representative image and quantitative analysis of fluorescence intensity of CL2166 (n = 3); J: Representative graph of LD1 fluorescence intensity and analysis of cell total correlates fluorescence (n = 3); K: Expression of antioxidant genes in nematodes (n = 3); L: Expression of antioxidant genes in female flies (n = 3); M: Expression of antioxidant genes in male flies (n = 3). The data are expressed as the mean ± SD. Data were analyzed by Student’s t-test (two groups) or one/two-way analysis of variance with post-hoc test (multiple comparisons). ^aP < 0.05 vs control group. ^bP < 0.01 vs control group. ^cP < 0.001 vs control group. ^dP < 0.0001 vs control group. DOE: Dendrobium officinale extract; CAT: Catalase; SOD: Superoxide dismutase; MDA: Malondialdehyde; ROS: Reactive oxygen species; mRNA: Messenger RNA; CuZn: Copper zinc; Mn: Manganese; MTH: Methuselah; GclC: Glutamate-cysteine ligase catalytic subunit.

Figure 6 Dendrobium officinale extract antagonizes oxidative stress and prolongs healthy lifespan dependent on Nrf2. A and B: Survival curves of EU1 nematodes (n = 90 nematodes); C: Intestinal atrophy of EU1 with or without Dendrobium officinale extract (DOE) (n = 3); D: Intestinal integrity of EU1 with or without DOE (n = 3); E: Reactive oxygen species (ROS) level of EU1 with or without DOE (n = 3); F: Antioxidant gene expression of EU1 with or without DOE (n = 3); G: Image showing the construction process of Da-gal4 > UAS-CncC RNAi; H: Survival curves for CncC mutant flies (n = 150 flies); I: Intestinal integrity in CncC mutant flies with or without DOE (n = 3); J: ROS level in CncC mutant flies with or without DOE (n = 3); K: Antioxidant gene expression in CncC mutant flies with or without DOE (n = 3). The data are expressed as the mean ± SD. Data were analyzed by log-rank test (survival), Student’s t-test (two groups) or two-way analysis of variance with post-hoc test (multiple comparisons). DOE: Dendrobium officinale extract; CAT: Catalase; SOD: Superoxide dismutase; ROS: Reactive oxygen species; CuZn: Copper zinc; Mn: Manganese; MTH: Methuselah; GclC: Glutamate-cysteine ligase catalytic subunit.

Figure 7 Midlife Intervention with Acetobacter sp. and Lactobacillus sp. mitigates oxidative stress, fosters intestinal homeostasis, and extends healthy lifespan. A: Schematic diagram of advantageous bacterial species replanting; B: Survival curve of early, midlife, late and lifelong intervention with Acetobacter sp. in female flies (n = 150 flies); C: Survival curve of early, midlife, late and lifelong intervention with Lactobacillus sp. in male flies (n = 150 flies); D: Survival curves for wild type (WT) or sterile female flies with or without dominant strain treatment (n = 150 flies); E: Survival curves for WT or sterile male flies with or without dominant strain treatment (n = 150 flies); F: Climbing number in WT or sterile flies with or without dominant strain treatment (n = 3); G and H: Intestinal leakage in WT or sterile flies with or without dominant strain treatment (n = 3); I: Activity of catalase in WT or sterile flies with or without dominant strain treatment (n = 3); J: Activity of superoxide dismutase in WT or sterile flies with or without dominant strain treatment (n = 3); K: Activity of malondialdehyde in WT or sterile flies with or without dominant strain treatment (n = 3); L: Reactive oxygen species level in WT or sterile flies with or without dominant strain treatment (n = 3). The data are expressed as the mean ± SD. Data were analyzed by log-rank test (survival) or one-way analysis of variance with post-hoc test (multiple comparisons). ^aP < 0.05 vs control group. ^bP < 0.01 vs control group. ^cP < 0.05 vs wild type group. ^dP < 0.01 vs wild type group. ^eP < 0.001 vs wild type group. ^fP < 0.05 vs sterile group. ^gP < 0.01 vs sterile group. ^hP < 0.001 vs sterile group. CAT: Catalase; SOD: Superoxide dismutase; MDA: Malondialdehyde; ROS: Reactive oxygen species; WT: Wild type.

Figure 8 Dendrobium officinale extract mediates the regulation of the “InR-Nrf2” signaling axis by gut microbiota through gender dimorphism, in order to combat oxidative stress and extend healthy lifespan. A: Schematic diagram illustrating the potential mechanism of Dendrobium officinale extract (DOE) differential regulation of dominant bacterial species in both sexes; B: Relative expression levels of InR in flies with or without DOE (n = 3); C: Relative expression levels of Dilp2 in flies with or without DOE (n = 3); D: Relative expression levels of Dilp3 in flies with or without DOE (n = 3); E: Relative expression levels of InR in flies after intervention with Acetobacter sp. or Lactobacillus sp. (n = 3); F: Relative expression levels of Dilp2 in flies after intervention with Acetobacter sp. or Lactobacillus sp. (n = 3); G: Relative expression levels of Dilp3 in flies after intervention with Acetobacter sp. or Lactobacillus sp. (n = 3); H: Survival curves for Da-gal4 > UAS-InR RNAi with or without DOE treatment (n = 150 files); I: Intestinal integrity in Da-gal4 > UAS-InR RNAi with or without DOE (n = 3); J: Reactive oxygen species level in Da-gal4 > UAS-InR RNAi with or without DOE (n = 3); K: Relative expression of Keap1-Nrf2-antioxidant response element pathway-related genes in Da-gal4 > UAS-InR RNAi with or without DOE (n = 3); L: Relative expression levels of InR, Dilp2, Dilp3 in Da-gal4 > UAS-CncC RNAi with or without DOE (n = 3). Data were analyzed by log-rank test (survival) or one/two-way analysis of variance with post-hoc test (multiple comparisons). ^aP < 0.05 vs control group. ^bP < 0.01 vs control group. ^cP < 0.001 vs control group. ^dP < 0.0001 vs control group. DOE: Dendrobium officinale extract; mRNA: Messenger RNA; CAT: Catalase; SOD: Superoxide dismutase; ROS: Reactive oxygen species; CuZn: Copper zinc; Mn: Manganese; MTH: Methuselah; GclC: Glutamate-cysteine ligase catalytic subunit.

Figure 9 Molecular mechanism of Dendrobium officinale extract delaying aging related degenerative phenotypes. DOE: Dendrobium officinale extract; CAT: Catalase; SOD: Superoxide dismutase; MDA: Malondialdehyde; ROS: Reactive oxygen species; ARE: Antioxidant response element.