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

Abstract

BACKGROUND

Dendrobium officinale (D. officinale) is a traditional Chinese herb that has been studied extensively for its medicinal properties, including gastrointestinal protection, anti-aging effects, and antioxidant properties. However, the molecular mechanisms by which D. officinale delays aging have not been fully elucidated.

AIM

To investigate the effects of D. officinale extract (DOE) on extending healthy lifespan through the maintenance of intestinal homeostasis, and to explore its intervention window, molecular mechanism, and key components.

METHODS

Based on evaluation systems using human MRC-5 cells, Caenorhabditis elegans, and Drosophila melanogaster, the optimal spatiotemporal intervention window for DOE was determined. Its effects on aging-related markers and stress resistance were assessed. (16S) rDNA sequencing, bioinformatics, immunofluorescence staining, reintroduction of dominant strains, and in vivo gene knockdown strategies were applied to identify and validate potential targets.

RESULTS

We determined that midlife is the optimal intervention window for DOE to extend healthy lifespan. Intervention at this stage delayed the age-related decline in health indicators, including motor and intestinal functions as well as oxidative stress. Mechanistically, DOE remodeled the gut microbiota in a sex-specific microbe host pairing, favoring Acetobacter pomorum in females and Lactobacillus plantarum in males, which differentially regulated glucose and lipid metabolism in both sexes. This remodeling modulated the insulin/insulin-like growth factor-1 signaling pathway, thereby activating the Keap1-Nrf2-antioxidant response element antioxidant signaling pathway. The enhanced antioxidant defense ultimately contributed to the prolonged healthy lifespan. In addition, D. officinale polysaccharide was identified as a potential core component of DOE’s pharmacological activity.

CONCLUSION

DOE regulated the microbiota-InR-Nrf2 axis to counteract oxidative stress, thereby maintaining intestinal function and extending healthy lifespan. These findings provide a molecular basis for the discovery of new antioxidants and anti-aging agents.

Key Words: Aging; Antioxidants; Caenorhabditis elegans; Drosophila melanogaster; Gut microbiota

Core Tip: Dendrobium officinale extract intervention during middle age is the optimal window for cross-species conservation of its benefits, delaying degeneration and extending lifespan. It maintains intestinal stem cell homeostasis and remodels gut microbiota, promoting gender-specific microbe-host pairings (Acetobacter pomorum in females, Lactobacillus plantarum in males) to modulate metabolic differences. This reshaped microbiota activates the InR-Nrf2 axis, antagonizing oxidative stress, ensuring intestinal homeostasis, and prolonging health span.

INTRODUCTION

Population aging poses a global challenge, driving the search for interventions to promote healthy aging and mitigate age-related diseases[1,2]. Among the various theories of aging, the free radical theory has informed widespread exploration of antioxidants[3,4]. Pharmacological activation of the Keap1-Nrf2-antioxidant response element (ARE) pathway has been demonstrated to ameliorate aging and age-related diseases by modulating oxidative stress, inflammation, and cellular senescence[5]. Given the potential of antioxidant-based strategies, traditional Chinese medicine has attracted growing interest due to its rich repository of anti-free radical compounds and favorable safety profile. However, few studies have clarified their anti-aging effects, and evidence regarding their material basis and molecular mechanisms is scarce.

Dendrobium officinale (D. officinale) is a prized medicinal herb historically associated with longevity. The Shen Nong herbal scripture records that D. officinale can be used to “thicken the stomach and intestines, lighten the body, and prolong the lifespan”. Modern research has identified its bioactive components, such as D. officinale polysaccharide (DOP)[6-10], while Cai et al[9] have demonstrated pharmacological activities such as regulating intestinal function and anti-aging. However, previous studies have often relied on single models or drug-induced aging, limiting confirmation of conserved effects and mechanistic understanding. Therefore, there is an urgent need to clarify the material basis, cross-species conservation, optimal time window for intervention, and mechanism of action of D. officinale at its potential targets.

This exploratory study established a multi-model evaluation system comprising MRC-5 cells, Caenorhabditis elegans (C. elegans), and Drosophila melanogaster (D. melanogaster) to define the optimal spatiotemporal window for D. officinale extract (DOE) intervention against natural aging. Integrated 16S rDNA sequencing with gene knockdown and bacterial reassociation techniques was used to elucidate the role of DOE in maintaining intestinal stem cells (ISCs) and gut microbiota homeostasis, as well as determining its underlying mechanism. Focusing on the “microbiota-InR-Nrf2” signaling axis, we propose a mechanistic model whereby DOE delays intestinal and systemic aging by inducing sexually dimorphic remodeling of the gut microbiota, which in turn modulates the metabolic-oxidative stress circuit and reinforces the endogenous antioxidant defense system. Furthermore, we identify the major DOP as the key fraction responsible for healthy lifespan extension, with its operative pathways showing high correlation with DOE targets. Our work not only provides empirical evidence of the therapeutic potential of DOE but also establishes a conceptual framework for the development of precision interventions targeting intestinal aging in both sexes.

MATERIALS AND METHODS

Plant materials and reagents

D. officinale collected from the Yandang Mountain cultivation base in August was powdered by mechanical grinding. The extraction was performed using distilled water (solid-liquid ratio 1:10) through two sequential extractions at 95-105 °C for 1 hour per cycle. The combined extracts were treated with 0.5% (w/w) activated carbon powder (≥ 200 mesh) for 30 minutes, followed by centrifugation for solid-liquid separation. The clarified supernatant was lyophilized (Songwon, Qingdao, Shandong Province, China) at -50 °C to obtain the final extract. We characterized the key components of DOE using high-performance liquid chromatography and liquid chromatography tandem mass spectrometry, and performed content detection, the details of which are shown in the Supplementary material.

Dichloro-dihydro-fluorescein diacetate (cat. No. D6883), hydrogen peroxide (H₂O₂) (cat. No. 1.08600), Lissamine rhodamine B (cat. No. 86186), and Blue Dye 1 (cat. No. 80717) were sourced from Merck KGaA Co., Ltd. (Darmstadt, Germany). A cell counting kit (CCK)-8 (cat. No. AR1199) was purchased from Boster Bioengineering Co., Ltd. (Shanghai, China). Delta (DI) (cat. No. C594.9B) was purchased from Developmental Studies Hybridoma Bank Co., Ltd. (Beijing, China), and phospho-histone H3 Ser¹⁰ (PH3) (cat. No. H0412) from Millipore Sigma Co., Ltd. (Shanghai, China). SA-β-gal staining kit (cat. No. C0602), 4’,6-diamidino-2-phenylindole (DAPI) (cat. No. P0131), and bicinchoninic acid (BCA) working fluid (cat. No. P0011) were purchased from Beyotime Co., Ltd. (Shanghai, China). Catalase (CAT) (D. melanogaster: Cat. No. YX-0330120F; C. elegans: Cat. No. YX-0330120C), superoxide dismutase (SOD) (D. melanogaster: Cat. No. YX-191504F; C. elegans: Cat. No. YX-191504C), malondialdehyde (MDA) (D. melanogaster: Cat. No. YX-130401F; C. elegans: Cat. No. YX-130401C), and reactive oxygen species (ROS) (D. melanogaster: Cat. No. YX-181519F; C. elegans: Cat. No. YX-181519C) kits were obtained from Shanghai Preferred Biotechnology Co., Ltd. (Shanghai, China). Selective medium containing Acetobacter sp. and Lactobacillus sp. was sourced from Qingdao Hope Biotechnology Co., Ltd. (Qingdao, Shandong Province, China). TRIzol (cat. No. 15596018CN) was purchased from Invitrogen Co., Ltd. (Carlsbad, CA, United States). A Prime Script RT kit (cat. No. RR047Q) and SYBR Green polymerase chain reaction (PCR) Master Mix (cat. No. CN830S) were sourced from Takara Biotechnology Co., Ltd. (Dalian, Liaoning Province, China). A chromatography column was purchased from Agilent Co., Ltd. (Santa Clara, CA, United States), an SP8 confocal microscope was purchased from Leica Co., Ltd. (Wetzlar, Germany), and a stereoscopic microscope was purchased from Chongqing Optec Instrument Co., Ltd. (Chongqing, China). Infinite^® 200 Pro microplate reader was purchased from Tecan Group Co., Ltd. (Shanghai, China). TGrinder H24 tissue grinding homogenizer was purchased from Tiangen Biochemical Technology Co., Ltd. (Beijing, China).

Animal stocks and husbandry

The wild-type Canton-S, UAS CncC/Nrf2 RNAi, UAS-GFP/cyo, Da-gal4, esg-gal4, UAS-InR^JF01482 (InR RNAi) stocks used in the experiments were provided by Dr. Yang YF (Institute of Life Sciences, Fuzhou University, Fuzhou, Fujian Province, China). Flies in the control group were maintained on sugar-yeast-agar (SYA) medium [1% agar, 3% yeast, 1.9% sucrose, 3.8% dextrose, 9.1% corn meal, 1% acid mix, and 1.5% methylparaben (all concentrations in w/v)] under controlled environmental conditions (24-25 °C, 60% relative humidity, 12-hour light/dark cycle). Flies in the intervention group were exposed to DOE at various concentrations (2 mg/mL, 2.5 mg/mL, 5 mg/mL, and 10 mg/mL) in basic medium.

C. elegans (wild-type, N2), Escherichia coli (E. coli) (OP50), and E. coli green fluorescent protein (GFP) (OP50 GFP) were sourced from Dr. Shao ZY at the Institute of Brain Science, Fudan University, Shanghai, China. A mutant EU1 [skn-1(zu67)] transgenic CL2166 strain dvIs19 [(pAF15)gst-4p:: GFP:: NLS] III and LD1 ldIs7 [skn-1p:: Skn-1b/c:: GFP + rol-6 (su1006)] were obtained from the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN, United States). Both strains serve as a measurable indicator of elevated oxidative stress and longevity[11,12].

All nematodes were grown on the plates with OP50 as the main food source and maintained under controlled environmental conditions (20 °C and 60% relative humidity). The main components of the nematode growth medium (NGM) were 0.3% sodium chloride (w/v), 0.25% peptone (w/v), 1.7% agar (w/v), 0.1% calcium chloride (1 mol/L, v/v), 1.5% magnesium sulfate (1 mol/L, v/v), 2.5% phosphate-buffered saline (1 mol/L, v/v), and 1% cholesterol (5 mg/mL, v/v). Nematodes in the intervention group were treated with various concentrations of DOE (10 μg/mL, 25 μg/mL, and 50 μg/mL) in OP50.

Cell subculture method

MRC-5 cells were obtained from the American Type Culture Collection (Manassas, VA, United States) and grown in Minimum Essential Medium (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) under controlled environmental conditions (37 °C and 5% carbon dioxide). Pharmacological interventions were started from the PDL7 generation.

Cell viability measurement using the CCK-8 assay

PDL7 cells (2.0 × 10⁵ cells/mL) were used for the cell viability experiment. DOE solution (1 mg/mL in phosphate-buffered saline) was dissolved to a final concentration of 100 μg/mL, 200 μg/mL, 300 μg/mL, 400 μg/mL, 500 μg/mL, 600 μg/mL, or 700 μg/mL (in complete medium). The conditioned medium was aspirated, and 100 μL fresh CCK-8 solution was carefully added to each well. The plates were then incubated at 37 °C for 0.5-4 hours in the dark, and absorption was measured at 450 nm.

β-Galactosidase staining assay

PDL7 cells (2.0 × 10⁵ cells/mL) were used in the β-galactosidase staining experiment. MRC-5 cells were exposed to H₂O₂ and DOE (200 μg/mL). We followed the manufacturer’s instructions for the senescence-associated-β-galactosidase staining kit for cell fixation and staining. The samples were imaged under a microscope to count the proportion of positive (blue-stained) cells.

Ethics statement

This study utilized established invertebrate model organisms (D. melanogaster, C. elegans) and human cell lines. Research involving D. melanogaster and C. elegans does not require approval from an institutional ethics committee. All human cell lines used in this study are widely used and well-documented in the scientific community. The research was conducted in accordance with all relevant institutional biosafety guidelines and regulations.

Lifespan analysis

Ninety nematodes from the same batch were placed in NGM petri dishes, and the medium was replaced with fresh medium daily until all nematodes died. Flies with 2 days of eclosion (150 in each group) were collected in the medium, and the medium was replaced with fresh medium every day. Death was recorded until all the flies had died. According to the death of the flies and nematodes, a survival curve was created using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, United States).

Analyses of motility, pumping frequency, intestinal leakage, and intestinal atrophy in C. elegans

Sixty nematodes (D4 stage) were maintained in NGM with or without DOE until 15 days of age, and the following indicators were observed under a stereomicroscope. The movement ability of the nematodes was evaluated by recording the number of sinusoidal movements within 60 seconds. The pumping frequency was evaluated by recording the number of pharyngeal pumps in the pharyngeal region of the nematode within 60 seconds. For intestinal leakage, the nematodes were removed from the NGM plates and suspended for 3 hours in liquid cultures of standard OP50 bacteria (grown overnight) mixed with blue food dye [2.5% (w/v) Blue Dye 1]. The nematodes were then transferred to NGM plates seeded with OP50 bacteria and analyzed for the presence or absence of blue food dye in the body cavity. To investigate intestinal atrophy, the nematodes were anesthetized using levamisole hydrochloride and photographed, and intestinal atrophy was quantified by measuring the width of the hindgut, lumen, and body according to the following equation: Intestinal atrophy (%) = (W₁ - W₀)/W × 100%, where W₁ is the width of the hindgut, W₀ is the width of the lumen, and W is the width of the body.

Analyses of intestinal microbial colonization and gst-4 fluorescence intensity

Sixty nematodes (D4 stage) were transferred to NGM petri dishes for the gst-4 fluorescence intensity assay[13] or to NGM containing OP50 GFP for the intestinal microbial colonization assay[14] until 15 days of age. The nematodes were placed on agarose pads and anesthetized using levamisole hydrochloride. Fluorescence photographs were taken using a confocal microscope and analyzed to measure fluorescence intensity.

Analysis of skn-1 nuclear translocation in C. elegans

LD1 nematodes were cultured for 15 days on NGM plates supplemented with or without DOE. The nematodes were placed on agarose pads and anesthetized using levamisole hydrochloride. Fluorescence photographs were taken using a confocal microscope and analyzed to measure fluorescence intensity.

Analyses of weight, food intake, and D. melanogaster fecundity

Thirty flies (aged 22 days) were kept in SYA medium with or without DOE until 42 days of age. The flies were weighed in a centrifuge tube, and the weight of the flies was obtained by subtracting the mass of the centrifuge tube. The flies were starved for 5 hours and then transferred to medium containing 0.2% lysine rhodamine B for 2 hours. The degree of redness around the abdomen (indicating the amount of food consumed) was observed under a microscope, and a subjective rating scale (0-5 points) was used for scoring (0, colorless abdomen; 5, dark red abdomen) to identify food intake.

To measure fecundity, male and female flies that emerged for 3 hours were collected (after 48 hours, if there were no larvae in the medium of female flies, they were identified as virgin flies). Mating the male flies to be tested with newborn virgin flies, and mating the female flies to be tested with newborn male flies. The eggs were collected every 12 hours, and the number of eggs was recorded. The reproductive ability of male flies was quantified by the egg-laying rate of their mating virgin flies, while the reproductive ability of female flies was quantified by their own egg-laying rate, as described previously[15,16].

Analysis of D. melanogaster climbing ability

Thirty flies (aged 22 days) were kept in SYA medium with or without DOE until 42 days of age. The flies were anesthetized, placed in vertical columns (length 25 cm, diameter 1.6 cm), and equilibrated at 25 °C for 10 minutes to acclimatize. The flies were shaken to the bases of the columns to unify their starting positions. We recorded the distribution of flies in each region (nine regions, total height 18 cm) within 5 seconds and evaluated their climbing ability by multiplying the score of each region by the number of flies in that region[17].

Analysis of the “Smurf” ratio of D. melanogaster

Thirty flies (aged 22 days) were kept in SYA medium with or without DOE until 42 days of age. The flies were starved for 2 hours and kept overnight in medium supplemented with 2.5% (w/v) Blue Dye 1, and the “Smurf” ratio (dye-colored flies observed outside the digestive tract) was recorded[18].

Immunofluorescence

The flies were washed with 70% ethanol to obtain the midguts. Intestinal samples were fixed in 4% paraformaldehyde at 25 °C for 30 minutes. The midgut was washed three times for 10 minutes each using 0.3% Triton X-100 in phosphate-buffered saline and blocked for 30 minutes with 5% goat serum in phosphate-buffered saline with Tween-20. The samples were stained with DI or anti-PH3 at 4 °C overnight. On the second day, the samples were incubated with secondary antibody for 2 hours at 25 °C. Finally, the samples were fixed with an anti-fluorescence quencher for DAPI and observed under a confocal microscope.

Analyses of the gut microbiota and BugBase prediction of the D. melanogaster phenotype

Thirty flies (aged 22 days) were kept in SYA medium with or without DOE until 42 days of age. The flies were washed in 70% ethanol, after which the midguts were obtained. Total genomic DNA was extracted from the gut samples using the E.Z.N.A.^® Stool DNA Kit (Omega Bio-tek, Guangzhou, Guangdong Province, China) according to the manufacturer’s instructions. The hypervariable V3-V4 regions of the bacterial 16S rDNA were amplified by PCR using the universal primers 338F (5’-ACTCCTACGGGAGGCAGCA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The products were purified using AMPure XT beads (Beckman Coulter Genomics Co. Ltd., Danvers, MA, United States) and quantified using Qubit (Invitrogen). To perform 16S rDNA sequencing, the amplicon pools were prepared for sequencing, and the size and quantity of the amplicon library were assessed using a 2100 Bioanalyzer (Agilent) and the Illumina Library Quantification Kit (Kapa Biosciences, Wilmington, MA, United States). The libraries were sequenced on the Illumina NovaSeq 6000 platform. BugBase phenotype predictions were first normalized by the predicted 16S copy number of operational taxonomic units, after which the microbial phenotypes were predicted using the pre-calculated files provided[19].

Separation and identification of dominant intestinal bacteria in D. melanogaster

The flies were washed using 70% ethanol, after which the midguts were obtained. The midguts were abraded in sterile phosphate-buffered saline and centrifuged for 60 seconds at 600 g. The supernatant was spread on selective medium containing Acetobacter sp. and Lactobacillus sp. Single colonies were obtained after incubation at 37 °C overnight, and the number of colonies was recorded. Genomic DNA was extracted and amplified by PCR, after which the bacterial colony was identified by 16S rDNA sequencing. The species were compared using the NCBI database, and the results showed a match of 99.79% with Acetobacter pomorum and 99.52% with Lactobacillus plantarum.

Bacteria reimplantation and construction of sterile flies

The bacterial isolates were cloned in deMan-Rogosa-Sharpe medium and cultured at 29 °C to an OD600 equal to 1. To inoculate the flies, 40 μL bacterial solution was applied to the surface of standard food and dried at 29 °C. The control samples also contained 40 μL deMan-Rogosa-Sharpe medium. The sterile flies (aged 3 days) were kept in SYA medium inoculated with the bacteria[20] until 42 days of age.

To prepare sterile flies, new eggs were collected and washed according to the following procedure: The eggs were treated with 33% welsh at a rate of 3 times/minute, 50% 84 disinfectant at a rate of 1 time/minute, 70% alcohol at a rate of 2 times/2 minutes, and sterile water at a rate of 2 times/2 minutes in a sterile environment. The treated eggs were hatched and cultured into sterile flies on sterilized medium containing 100 μg/mL ampicillin, 50 μg/mL tetracycline, and 100 μg/mL streptomycin.

In vitro colony-forming unit assay

Based on the intervention concentration of natural medicine extracts in probiotics in vitro, we treated Acetobacter pomorum and Lactobacillus plantarum strains with or without DOE (0.8 mg/mL, 1 mg/mL, 1.2 mg/mL) and observed them after 48 hours of incubation. The bacterial solution was coated onto a selected culture medium and incubated for 48 hours at 30 °C, after which the colonies were counted.

Oxidative stress

Sixty nematodes (D4 stage) were kept in NGM with or without DOE until 15 days of age. The nematodes were washed using M9 buffer and exposed to oxidative stress induced by 30% H₂O₂ (0.1%). The number of surviving nematodes was counted every 30 minutes until all nematodes had died. Sixty flies (aged 22 days) were kept in SYA medium with or without DOE until 42 days of age. The flies were moved to SYA medium with the addition of 5% (w/v) H₂O₂ (for the H₂O₂ test) or 6 mg/mL sodium dodecyl sulfate (SDS) (for the SDS test). The number of dead flies was recorded at 12 hours for the SDS test and at 2 hours for the H₂O₂ test. Survival curves were created using GraphPad Prism 9 software.

Heat shock stress

Sixty nematodes (D4 stage) were kept in NGM with or without DOE until 15 days of age, and 60 flies (aged 22 days) were kept in SYA medium with or without DOE until 42 days of age. The flies and nematodes were transferred to a constant temperature incubator (37 °C) for experimentation. The number of deaths was counted every hour until all flies and nematodes had died, and survival curves were created using GraphPad Prism 9 software.

Antioxidant and ROS assay

Tissues (30 flies or 60 nematodes) were ground with saline at a ratio of 1 mg/900 μL in a tissue grinding homogenizer and centrifuged at 600 g for 10 minutes at 4 °C. SOD, CAT, MDA, and ROS were measured according to the instructions of the enzyme-linked immunosorbent assay kit. The Infinite 200 Pro microplate reader was used to measure the absorbance at 450 nm, and the content proportions were presented in units of pg/mL protein.

Protein measurements

Protein was extracted from the flies and nematodes by grinding. The bovine serum albumin (BSA) standard protein curve was prepared based on a range of different protein concentrations (0 mg/mL, 0.025 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, and 0.5 mg/mL) from the BSA stock concentration (0.5 mg/mL). Furthermore, 2.0 μL protein samples were mixed with 18.0 μL phosphate-buffered saline in a 96-well plate, and 200 μL BCA working was added. After incubation for 20 minutes at 37 °C, the optical density of the protein samples was measured to determine absorbance at a wavelength of 562 nm using the Infinite 200 Pro microplate reader, and the protein concentrations were calculated.

Reverse transcription-quantitative PCR

The flies and nematodes were abraded, and total RNA was obtained by extraction using TRIzol reagent. Reverse transcription was performed using the Prime Script RT kit. Real-time PCR was performed using SYBR Green PCR Master Mix. The data were analyzed according to the 2^-∆∆Ct method, as described previously[21]. The primer sequence is shown in Supplementary Table 1.

Statistical analysis

There were at least three biological replicates per experiment unless otherwise stated. GraphPad Prism 9 was used to analyze the data. Fluorescence intensity was analyzed using Image J software (National Institutes of Health, Bethesda, MD, United States). Survival (lifespan) data were compared using the log-rank (Mantel-Cox) test. For other data, comparisons between two groups were performed using the Student’s t-test, while comparisons among multiple groups were performed using one-way or two-way analysis of variance. The data are expressed as the mean ± SD. P < 0.05 indicated statistical significance.

RESULTS

DOE promoted longevity in multiple species

We first investigated whether DOE exerted an anti-aging effect in C. elegans (N2) and D. melanogaster (Canton-s), both of which are multicellular organisms. By referring to the intake concentrations of D. officinale in previous studies[22,23] and our toxicity screening, we finalized our lifespan experiments using various concentrations of DOE (10 μg/mL, 25 μg/mL, or 50 μg/mL for nematodes; 2.5 mg/mL, 5 mg/mL, or 10 mg/mL for female flies; 2 mg/mL, 2.5 mg/mL, or 5 mg/mL for male flies). The lifespans of all organisms are shown in Supplementary Tables 2 and 3. The mean lifespan was significantly prolonged when the DOE concentration was 25 μg/mL (nematodes show in Figure 1A and B), 5 mg/mL (female flies show in Figure 1C and D), or 2.5 mg/mL (male flies show in Figure 1E and F), but not when other concentrations were used. Therefore, the above-mentioned concentrations were considered optimal and were used in subsequent experiments.

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.

Next, we tested the effect of DOE on human (MRC-5) cells to confirm its species conservation. We found that DOE had no inhibitory effect on cell activity in the range of 100-700 μg/mL, but its inhibitory effect significantly increased at 200 μg/mL (increased by 23%, Figure 1G). H₂O₂ is widely used as an oxidant and to induce senescence during the construction of cellular models of senescence and damage[24]. Analysis of cell proliferation determined the half-maximal inhibitory concentration; the inhibition rate at 700 μmol/L H₂O₂ on cell viability was 58.51% ± 1.61%, and thus we chose 700 μmol/L as the final modeling concentration (Figure 1H). Compared with the model group, 200 μg/mL DOE significantly increased H₂O₂-induced cell viability (Figure 1I). Aging-related galactosidase staining showed that the proportion of stained cells decreased after DOE intervention (Figure 1J). These data suggest that DOE extends the lifespan across species and has the potential for application as a treatment for human aging.

DOE had a key window for promoting longevity in C. elegans and D. melanogaster

Lifelong continuity of functional foods is difficult to achieve. Therefore, based on the C. elegans (N2)-D. melanogaster(Canton-s) dual model system, we screened the optimal duration and key intervention windows for DOE. The duration of DOE was evaluated (2 days, 4 days, 8 days, and lifelong for nematodes; 10 days, 20 days, 30 days, 40 days, and lifelong for flies) based on previous research[25,26] (Figure 2A and B; Supplementary Tables 4 and 5). The results showed that a 4-day intervention in nematodes and a 20-day intervention in flies significantly increased the mean lifespan to the greatest extent possible (by 16% in nematodes, 12% in female flies, 13% in male flies), with no statistically significant difference compared with lifelong intervention (Figure 2C-H and Supplementary Figure 1), and therefore these were selected as the optimal durations. Next, we administered DOE treatment in the early, midlife, and late stages with the optimal intervention duration (Figure 2I and J; Supplementary Tables 6 and 7). In nematodes, the lifespan was significantly prolonged by intervention in the early (12%; Supplementary Figure 1), midlife (23%; Figure 2K), and lifelong (16%; Figure 1C) stages. In the fly experiments, where early (12% in females, 13% in females; Supplementary Figure 1), midlife (15% in females, 17% in males; Figure 2L-P), and lifelong (11% in females, 12% in females; Figure 1D and E) intervention significantly prolonged the lifespan, whereas late intervention (Supplementary Figure 1) did not. Comparing the lifespan extension rates of intervention in different windows, we found that the average lifespan extension rate of midlife intervention was significantly higher than that of other interventions (Figure 2N-P).

To further explore the specificity of midlife intervention, we transferred the 2-day (nematodes) or 10-day (flies) intervention period, which was ineffective in early intervention, to midlife. The results showed that intervening for 2 days/10 days in midlife extended the lifespan by 4% in nematodes, 8% in female flies, and 9% in male flies, which was significantly better than early intervention for the same period, but it was still lower than the effect of 4-day/20-day intervention in midlife (Supplementary Figure 1; Supplementary Tables 6 and 7). According to our experimental results, a phased midlife intervention emerges as a superior strategy for DOE-mediated lifespan extension.

Midlife DOE intervention extended the healthy lifespan

Contemporary research indicates that extended survival is only meaningful when based on a healthy lifespan. We compared the effects of DOE administered at different windows (early, midlife, late, and lifelong) on nematode health and fly physiology. Midlife DOE intervention had the most significant effect on health indicators (Figure 3), whereas late intervention did not improve these indicators. As nematodes age, the frequency of body bends, the speed of swallowing, and intestinal function decline markedly. We found that midlife DOE intervention increased bending frequency by 75% (Figure 3A) and pharyngeal pumping rate by 66% (Figure 3B) compared with the control group. Moreover, intestinal function was significantly rescued, as shown by reduced intestinal atrophy (Figure 3C and D), and leakage (reduced by 31%, Figure 3E and F), as well as decreased bacterial colonization (reduced by 42%, Figure 3G and H). Numerous studies have demonstrated that dietary restriction and reproductivity sacrifice can extend the lifespan. However, in our experiments, DOE had no negative effects on body weight (Supplementary Figure 2A), food intake (Figure 3I), or reproductivity (Figure 3J), and it even enhanced the reproductivity of aging male flies. Aging in flies is accompanied by decreased exercise capacity and impaired organ function[27]. Midlife DOE intervention delayed the declines in climbing ability (increased by 31%, Figure 3K) and intestinal shielding (Figure 3L and M), and the “Smurf” rate decreased from 47% to 18% in female flies and from 18% to 6% in male flies.

The ability to withstand stress resistance is a marker of organismal health that declines with age. We examined the effects of DOE on various stresses in flies and nematodes, including heat stress and oxidative stress. In nematodes and flies (show in Supplementary Figure 3), DOE significantly prolonged the average lifespan under H₂O₂ (Supplementary Figure 3A and D, Supplementary Tables 8 and 9) and SDS (Supplementary Figure 3C, Supplementary Tables 8 and 9) stimulation. Meanwhile, DOE increased the survival rate of female flies under heat shock stress, but it did not affect males (Supplementary Figure 3B and E, Supplementary Tables 8 and 9). These results indicate that middle-aged DOE intervention can effectively prolong healthy lifespan and improve age-related degenerative phenotypes.

DOE restored ISCs proliferation homeostasis in aging D. melanogaster

D. officinale has excellent protective effects in the gastrointestinal tract[28,29], and our experiments confirmed its ability to improve intestinal function in Canton-s (Figure 3I-M) and N2 (Figure 3C-H). Therefore, we explored the effects of DOE on ISCs homeostasis. Aging in flies has a deleterious impact on ISCs in the midgut, affecting stem cell homeostasis. We used GFP driven by esg-gal4 to mark ISCs and enter oblasts simultaneously, and we performed immunofluorescence staining using DI (a specific marker of ISCs), while observing the number of histone-3 (PH3; a commonly used mitotic marker)-positive cells in the midgut. As shown in Figure 4A-C and Supplementary Figure 2B and C, the number of esg-GFP⁺ cells, DI⁺ cells, and PH3⁺ cells increased significantly with aging. However, intervention with DOE significantly inhibited the accumulation of these cells. These results indicated that DOE restored ISCs proliferation homeostasis in aging D. melanogaster.

DOE altered the diversity of the gut microbiota in aging D. melanogaster

Previous DOE studies showed intestinal protection in nematodes and flies. Based on these results, we further explored the effect of DOE on gut microbiota using second-generation microbial sequencing. Rarefaction (Supplementary Figure 4A and B) and Shannon curves (Supplementary Figure 4C and D) showed that the sequencing depth of the samples was sufficient to analyze the gut microbiota. Principal coordinate analysis (Supplementary Figure 4E and F) showed obvious clustering, and the number of operational taxonomic units (Supplementary Figure 4G and H) showed that DOE attenuated the effects of age-related changes in the gut microbiota.

After intervention with DOE, the relative abundance of Firmicutes in female flies and Bacteroidetes in male flies increased significantly at the phylum level (Figure 4D-G). At the genus level, Acetobacter sp. and Lactobacillus sp. are considered to have antioxidant and metabolic-regulating effects[30,31], and they were the dominant microorganisms mediated by DOE (Figure 4H-K). Selective cultivation revealed an age-dependent decline in Acetobacter sp. in females, while DOE significantly increased the bacterial count (P < 0.001; Figure 4L and M). In males, Lactobacillus sp. showed a specific increase at 20 days (P < 0.0001), followed by a daily decrease. DOE restored the bacterial count at 40 days to 89% and 78%, respectively, of the 20-day level (Figure 4N and O).

To further explore the potential pathways of DOE-mediated microbial regulation, we conducted BugBase analysis. As shown in Supplementary Table 10, DOE significantly lowered potential pathogenicity and restored a healthier oxidative stress profile by adjusting the components of the gut microbiota. These findings suggest that DOE enhanced anti-aging capacity by improving the composition and structure of the gut microbiota, a process that is likely to be associated with resistance to oxidative stress.

DOE altered the lifespan via the Keap1-Nrf2-ARE and skn-1/Nrf2 signaling pathways

Based on the predicted results of gut microbiota function, we detected the antagonistic oxidation effect of DOE by measuring oxidative stress-related biomarkers (Figure 5). DOE treatment increased the activity of CAT and SOD by 32% and 25%, respectively, while reducing the levels of MDA and ROS (Figure 5A-H). Next, we explored the potential mechanisms through which DOE promotes resistance to oxidative stress. Keap1-Nrf2-ARE signaling represents a key antioxidant pathway. Under oxidative stress, Keap1 undergoes conformational changes, releasing Nrf2, which translocates to the nucleus and binds ARE to activate downstream gene expression. In C. elegans, we quantified the expression of gst-4, a downstream target gene of skn-1 (a homolog of Nrf2 in nematodes), and assessed the nuclear translocation ratio of skn-1. DOE significantly increased the fluorescence intensity of CL2166 (gst-4:: GFP transgenic nematode) by 51% and enhanced the skn-1 nuclear localization of LD1 (skn-1: GFP transgenic nematode) by 42% (Figure 5I and J). We measured the expression of genes related to the skn-1/Nrf2 pathway and found that the messenger RNA (mRNA) expression was significantly elevated in response to DOE treatment (Figure 5K). To confirm the dependence of DOE on skn-1, we used EU1 [skn-1(zu67)]-mutant nematodes. In EUI-mutant nematodes (Figure 6A), lifespan, intestinal function, and oxidative stress resistance were not rescued (Figure 6B-F, Supplementary Figure 2D and E, Supplementary Table 11).

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.

We continued to evaluate the expression of genes associated with the Keap1-Nrf2-ARE pathway in flies. After DOE intervention, the mRNA expression of the copper/zinc-SOD, manganese-SOD, CAT, glutamate-cysteine ligase catalytic subunit, CncC, Keap1, and dSir2 genes was significantly higher, and the expression of Methuselah was significantly lower (Figure 5L and M). Next, we constructed CncC (a homolog of Nrf2 in D. melanogaster)-mutant flies using UAS-CncC RNAi and Da-gal4 to explore the dependence of DOE on the Keap1-Nrf2-ARE pathway (Figure 6G). Similar to nematodes, the beneficial effects of DOE also disappeared when Nrf2 was interfered (Figure 6H-K and Supplementary Table 11). These results suggest that the DOE-mediated activity was dependent on the skn-1/Nrf2 and Keap1-Nrf2-ARE signaling pathways.

Keap1-Nrf2-ARE activation by DOE and its effect on oxidative stress are mediated by the gut microbiota

DOE maintains gut microbiota homeostasis at the organizational level and reduces oxidative stress at the molecular level. However, the potential interactions between oxidative stress and gut microbiota homeostasis remain unclear. Therefore, we studied the cascade relationship between oxidative stress and the maintenance of gut microbiota homeostasis through the use of dominant strain rewiring technology and the construction of sterile flies.

To elucidate the regulatory effect of gut microbiota, two dominant strains, Acetobacter sp. and Lactobacillus sp., were isolated from the gut microbiota through in vitro culture. They were identified as Acetobacter pomorum and Lactobacillus plantarum, respectively, by 16S rDNA gene sequencing (Figure 7A). Consistent with the optimal intervention window of DOE, middle-aged intervention with predominant strains achieves maximum benefits in terms of lifespan (Figure 7B and C; Supplementary Table 12). The average lifespan was extended by 3.75%, 4.93% in wild-type flies and 13.24%, 13.54% in sterile flies (Figure 7D and E; Supplementary Table 13). In addition, the dominant strains increased climbing ability by 32% (Figure 7F), significantly impacted intestinal damage by 26% (Figure 7G and H), and regulated antioxidant-related indicators (Figure 7I-L). These findings demonstrate that the DOE-regulated dominant strains improve overall health and enhance antioxidant function.

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.

Next, we validated whether DOE activation of Nrf2 targets to counteract oxidative stress is mediated by gut microbiota. Interestingly, after losing the mediation of the gut microbiota, the lifespan-extending effect of DOE (Supplementary Figure 5A and Supplementary Table 14), as well as its effects on climbing ability (Supplementary Figure 5B) and intestinal leakage rate (Supplementary Figure 5C and D), disappeared. In counteracting oxidative stress, ROS levels (Supplementary Figure 4E), antioxidant enzyme activity (Supplementary Figure 5E-H), and relative expression of Keap1-Nrf2-ARE-related genes (Supplementary Figure 5I) were not regulated by DOE. This discovery indicates that DOE regulates the Keap1-Nrf2-ARE signaling pathway to exert antioxidant and anti-aging effects, a process that is mediated by the gut microbiota.

DOE mediated InR-Nrf2 axis regulation by the gut microbiota in a sexually dimorphic manner to extend the healthy lifespan

The second-generation microbial sequencing results suggest that there is sexual dimorphism in the structure of DOE-regenerated microbial communities. Specifically, DOE promoted Acetobacter sp. abundance in females while suppressing it in males, whereas an opposite effect was observed for Lactobacillus sp. This finding was confirmed by selective culture assays. Studies indicate distinct sex-specific metabolic profiles in D. melanogaster: Females predominantly utilize lipid reserves, whereas males are more prone to glucose metabolic dysregulation[32-34]. Correspondingly, the insulin-like peptides Dilp2 and Dilp3 selectively regulate glycogen and lipid metabolism, respectively[33,35,36]. Therefore, we evaluated the expression of key insulin/insulin-like growth factor (IIS) signaling pathway components, including InR, Dilp2, and Dilp3. Inhibiting the IIS pathway has been proven to prolong lifespan in multiple species[37] (Figure 8A). Compared with controls, DOE regulates the increase of InR expression in aging flies, indicating IIS pathway suppression (Figure 8B). DOE also markedly restored Dilp2 and Dilp3 expression to healthier levels, with sex-dependent differences. DOE showed more significant Dilp2 inhibition in male animals (Figure 8C) and more significant Dilp3 inhibition in female animals (Figure 8D). These results suggest that DOE may modulate glucose/Lipid metabolism in a sex-specific manner.

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.

To investigate the roles of Acetobacter pomorum and Lactobacillus plantarum in modulating sex-related metabolic bias, we measured Dilp2 and Dilp3 expression in flies fed with bacteria. Both Acetobacter pomorum and Lactobacillus plantarum supplementation significantly reduced InR, Dilp2, and Dilp3 expression in males and females (Figure 8E-G). Notably, two “sex-microbe” combinations maximized the expression of Dilp2 and Dilp3: “Lactobacillus plantarum in male” downregulated the glycometabolism regulator Dilp2 by 67% (Figure 8F), while “Acetobacter pomorum in female” reduced the lipid metabolism regulator Dilp3 by 62% (Figure 8G). These results suggest that the dominant microbial anchoring of DOE may follow an optimal “sex-microbe” association that is sex-dependent, dynamically adapting to sex-specific glycometabolism/Lipid metabolic demands.

To determine whether the effects of DOE depend on the IIS pathway, we generated Da-gal4 > UAS-InR RNAi flies. We found that interference of InR lifespan extension (Figure 8H and Supplementary Table 15), gut barrier restoration (Figure 8I), ROS accumulation (Figure 8J) and abolished DOE-induced Keap1-Nrf2-ARE signaling activation (Figure 8K). To elucidate the hierarchical relationship between InR and Nrf2, we evaluated related genes in Da-gal4 > UAS-CncC RNAi flies. Key level evidence shows that Nrf2 knockout maintains DOE’s regulation of IIS pathway genes (Figure 8L), ultimately locating InR upstream of Nrf2. To explore the age-specific efficacy of DOE, we measured intestinal InR and Nrf2 expression across ages. We identified a midlife-specific (20-30 days) molecular shift characterized by declining Nrf2 and rising InR (Supplementary Figure 5J and K). These data indicate that there is a middle-aged-specific imbalance in the InR-Nrf2 axis (functional crosstalk between InR and Nrf2). DOE restores its balance by utilizing dominant strains to enhance Nrf2-mediated antioxidant response through InR.

Notably, the in vitro culture experiments demonstrated that DOE directly promoted the growth of both Acetobacter pomorum and Lactobacillus plantarum (Supplementary Figure 6), suggesting that the mutually exclusive abundance pattern of the dominant bacteria regulated by DOE depended on the host microenvironment rather than its direct action. Exogenous supplementation experiments showed that administering Acetobacter pomorum to females reduced Lactobacillus plantarum abundance, while supplementing Lactobacillus plantarum in males decreased Acetobacter pomorum levels (Supplementary Figure 6). These results indicate that DOE may indirectly alter the interaction between dominant bacterial species through sex-specific host metabolic demands. Previous studies have reported that Acetobacter pomorum and Lactobacillus plantarum can engage in either mutualistic or competitive relationships[38,39]. Under DOE intervention, a competitive pattern characterized by opposing abundance shifts was observed; however, this competitive adaptation did not compromise intestinal health or lifespan. The underlying mechanisms warrant further investigation.

Chemical composition analysis and active component screening of DOE

To identify the major bioactive components of DOE, we characterized its primary constituents, including polysaccharides, proteins, and anthocyanins (Supplementary Figures 7 and 8, Supplementary Tables 16 and 17), the latter being consistent with previous phytochemical reports on this species[40]. Polysaccharides constituted the most abundant fraction (20.03% total content), significantly exceeding proteins (5.16%) and anthocyanins (0.27%). Subsequent bioactivity evaluation revealed that DOP demonstrated superior efficacy in lifespan extension, intestinal barrier enhancement, and ROS suppression compared with other components (Supplementary Figure 9A-C and Supplementary Table 18).

Given DOP’s dominance in composition and anti-aging potency, we investigated its mechanistic targets using Da-gal4 > UAS-InR RNAi and da-gal4 > UAS-CncC RNAi models. DOP activity was markedly attenuated following InR or CncC knockdown, paralleling the effects observed with DOE treatment (Supplementary Figure 9D-J, Supplementary Tables 19 and 20). These findings indicate that DOP serves as the primary bioactive component of DOE, counteracting oxidative stress, preserving intestinal homeostasis, and extending the healthy lifespan via modulation of the IIS and Keap1-Nrf2-ARE signaling pathways.

DISCUSSION

D. officinale is a natural anti-aging and antioxidant agent rich in active ingredients and nutrients, and it improves oxidative stress, delays aging, and regulates immunity[23]. However, the material basis, cross-species conservation, optimal time window for intervention, and potential mechanisms underpinning the lifespan extension of DOE have not been fully elucidated. To overcome these knowledge gaps, we explain the possible pathways and material basis for DOE in mediating cross-species longevity, maintaining gut function, and counteracting oxidative stress.

Lehallier et al[41] found that aging is a process characterized by gradual degradation of body systems and tissues, and it does not occur at a uniform rate. Identifying the optimal time or age window for DOE intervention may help yield the greatest benefits. In this context, DOE has been shown to extend the lifespan of D. melanogaster and C. elegans to alleviate senescence in human cellular models, indicating its broad, phylogenetically conserved potential. Importantly, systematic intervention screening revealed that midlife administration delivers optimal efficacy in rejuvenating age-related phenotypes and prolonging the lifespan, outperforming early-life, late-life, or lifelong treatments. This aligns with evidence showing that midlife represents a critical phase during which interventions can bidirectionally modulate health trajectories and restore cellular function[42,43]. Therefore, the anti-aging benefits of DOE are not only applicable across multiple species but also confined to a precise strategic window. These findings provide a data basis for the role of DOE in delaying natural aging and support its translational relevance as a candidate for further preclinical investigation.

Delayed aging manifests mainly as lifespan prolongation and health maintenance, and maintaining functional homeostasis in tissues and organs is the fundamental goal of delayed aging. We demonstrated that DOE can reverse motility decline in naturally aging flies and nematodes and has a significant ability to restore gastrointestinal function. Notably, DOE did not slow aging through common dietary restrictions[44] or reproductive suppression[45]. Salazar et al[46] found that the intestine is an important target organ for regulating lifespan, and proliferation of ISCs is thought to be a causative factor in intestinal damage[47]. In the senescent gut, ISCs strongly upregulate genes in the antigen presentation pathway and overexpress secretory lineage marker genes, which impair stem cell regeneration and result in intestinal ISCs accumulation, leading to loss of regenerative potential and functional decline in the gut[48]. Importantly, DOE prevented age-associated over-proliferation of ISCs and restored intestinal shielding, indicating a strong protective effect in the intestines.

On the other hand, the gut microbiota is a key regulator of intestinal barrier function. The characteristics of the microbiota during the aging process are a decrease in the diversity of beneficial bacteria and an increase in pathogenic species. While DOE significantly increased the abundance of Acetobacter sp. and Lactobacillus sp., which are two common and important probiotics[31,49-51]. Through in vitro cultivation, identification, and purification techniques, we have confirmed that Acetobacter pomorum and Lactobacillus plantarum are the key bacterial strains. However, this promoting effect has significant gender correlation. Regan et al[52] and Shi et al[53] clarified through their research and summary that gender dimorphism has been recorded in age-related diseases, with notable differences observed even in the responses to anti-aging interventions like dietary restriction, pharmacological treatments, and genetic therapies. Specifically, rapamycin enhances autophagy in female intestinal cells through the H3/H4 histone Buchs axis, while it is insensitive to males. Studies in D. melanogaster have revealed sex-specific metabolic strategies. For instance, Hudry et al[34] and Li et al[54] found that females enhance lipogenesis while suppressing lipolysis. In contrast, males prioritize trehalose metabolism to counter metabolic stress, consistent with male-biased carbohydrate processing in specific gut regions. These findings directed our focus to Dilp2 (sugar metabolism regulator) and Dilp3 (lipid metabolism regulator) as potential mediators of the DOE-induced sexual dimorphism[55].

To further elucidate the physiological functions of the gut microbiota, we successfully isolated and expanded two bacterial strains Acetobacter pomorum and Lactobacillus plantarum through in vitro culture, identification, and purification techniques. Sannino and Dobson[56] indicated that inoculation with Lactobacillus plantarum significantly enhances the antioxidant capacity of mango juice, potentially mediated through upregulation of 1,2,3,4,6-O-pentagalloylglucose expression. Consistent with these findings, Allam et al[30] reported that Lactobacillus plantarum effectively ameliorates free radical-induced hepatic damage in murine models through its remarkable antioxidant properties. Furthermore, Sannino and Dobson[56] revealed that Acetobacter pomorum in the D. melanogaster gut efficiently modulates triglyceride metabolic disorders induced by dietary preservatives. Collectively, these findings suggest that these two predominant bacterial strains may play crucial metabolic and oxidative stress regulatory roles in host physiology. Interestingly, DOE does not universally regulate metabolism; rather, it differentially modulates the expression of Dilp2 and Dilp3 by establishing sex-specific optimal microbial associations (Acetobacter pomorum in females and Lactobacillus plantarum in males), thereby suppressing the IIS pathway to align with the inherent metabolic sexual dimorphism of D. melanogaster. Importantly, when Acetobacter pomorum and Lactobacillus plantarum were cultured in vitro, DOE promoted both strains to a similar extent. This demonstrates that the sex-bacteria matching facilitated by DOE depends primarily on differences in the host’s sex-specific internal environment, rather than on a regulatory preference of DOE itself.

Based on the antioxidant potential of dominant bacterial strains and BugBase phenotype prediction, we tested the oxidative stress resistance performance of DOE. Consistent with our speculation, we demonstrated that DOE significantly activates Nrf2 by promoting its dissociation from Keap1 and subsequent nuclear translocation, thereby enhancing Keap1-Nrf2-ARE-mediated gene transcription and antioxidant enzyme production[57,58], and this process is highly dependent on gut microbiota for DOE. These results suggest that DOE play a role in intestinal protection and lifespan extension by activating the Keap1-Nrf2-ARE antioxidant signaling pathways against oxidative stress. The present study, through combined perturbation experiments targeting InR and Nrf2, established the critical hierarchical relationship, with InR functioning as an upstream regulator of Nrf2. Interestingly, we observed a significant midlife-specific shift in the baseline activity of the InR-Nrf2 axis in intestinal tissue. This finding aligns with the previously reported midlife-specific decline in Nrf2 in mouse neural stem cells[59] and midlife-specific activation of InR in D. melanogaster ISCs[60]. The observed window of molecular susceptibility likely explains the organism’s heightened sensitivity to DOE during midlife, with the InR-Nrf2 axis serving as a central regulatory node mediating this window-specific response. However, the precise mechanisms underlying the microbiota adaptive competition mediated by DOE and their intricate interactions with host metabolism remain incompletely understood, and we will further explore them in the next step of research.

It is worth mentioning that Wolbachia sp. was also significantly increased in the flies administered DOE. Wolbachia sp. is a common vertically transmitted intracellular bacterium that is well-known for its ability to destroy the reproductive function of the host[61]. However, Wolbachia sp. may not be just a harmful bacterium. Experiments in flies infected with several arthropod RNA viruses have demonstrated that flies harboring native Wolbachia strains have reduced viral loads and longer lifespans. Wolbachia sp. can also rescue the reproductive phenotype of flies with impaired fertility[62]. The mechanism through which DOE increases the abundance of Wolbachia sp. requires further exploration.

In this study, we identified the abundant DOP as a principal active component of DOE. DOP was characterized by a high content of mannose and glucose, monosaccharides previously linked to potent ROS-scavenging and detoxification activities[63]. In contrast, other components, such as anthocyanins, are recognized as antioxidants[64], but unfortunately only showed subtle effects in our model. This significant difference can be attributed to the lower absolute content of anthocyanins we isolated in DOE, especially compared to the abundant and highly active DOP. In order to evaluate the efficacy of the original DOE components, our administration concentration follows the proportion of each component analyzed by mass spectrometry, while the anthocyanin levels in the experiment may not have reached the highest threshold for their antioxidant effects. On the other hand, the bioavailability of anthocyanins may be limited by gastrointestinal instability, low absorption, and complex plant matrix interactions.

Through comparative intervention studies in D. melanogaster, we demonstrated that DOP alone recapitulates the majority of the anti-aging effects of DOE, largely via the InR-Nrf2 axis. This confirms that DOP is both necessary and sufficient for the core bioactivity of DOE. However, the full therapeutic profile of whole-plant DOE may involve more complex pharmacodynamics. Although DOP appears to be the primary driver, non-polysaccharide constituents could contribute through potential synergistic mechanisms, such as: (1) Activating parallel or complementary longevity pathways (e.g., adenosine 5’-monophosphate-activated protein kinase, mammalian target of rapamycin); (2) Enhancing the bioavailability, stability, or targeted delivery of DOP; and (3) Buffering potential off-target effects to ensure smooth metabolic and functional homeostasis. To conclusively determine whether the complete benefits of DOE require multi-component synergy, future work will utilize a combinatorial intervention strategy. This will involve comparing DOP alone, DOP paired with individual non-polysaccharide fractions, and DOP reconstituted with multiple components to mirror the native DOE profile. Such an approach is critical for fully elucidating the pharmacodynamic material basis of D. officinale’s anti-aging properties.

CONCLUSION

This study demonstrated that DOE extends the healthy lifespan by maintaining intestinal homeostasis and enhancing systemic oxidative stress resistance. Mechanistically, DOE induces sexually dimorphic remodeling of the gut microbiota, enriching Acetobacter pomorum in females and Lactobacillus plantarum in males of D. melanogaster. This microbial shift activates the microbiota-InR-Nrf2 signaling axis, thereby establishing a potentiated endogenous antioxidant defense system (Figure 9 and Video 1). Future work will define the principles of sexually dimorphic microbial selection by DOE and investigate potential synergies between DOP and other constituents, which may provide crucial molecular insights for the development of novel antioxidant and anti-aging nutraceuticals.

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.

ACKNOWLEDGEMENTS

The authors express thanks to all the persons who contributed to this study.