Recent research has identified certain molecules with anti-aging properties in Drosophila, which have also shown similar benefits in mammals (Du et al., 2020; Fang et al., 2019; Wu et al., 2022). Building on these insights, we aimed to identify compounds in vegetables and fruits that could potentially extend lifespan. Several vegetable and fruit extracts have been reported to have such effects on Drosophila. Among these, Kaempferol, a flavonoid-rich in vegetables and fruits, drew our attention due to its well-documented health benefits and disease-preventing capabilities (An and Kim, 2015; Bangar et al., 2023; Dong et al., 2023; Gao et al., 2023; Gidaro et al., 2016; Guo et al., 2015; Han et al., 2021; Holland et al., 2020; Lee and Kim, 2016; Luo et al., 2012; Luo et al., 2011; Shrestha et al., 2021). To evaluate Kaempferol’s potential to inhibit ISC aging, we employed the esg-GFP/CyO reporter line, where green fluorescent protein (GFP) is regulated by the escargot (esg) gene (Korzelius et al., 2014), to assess its impact on ISC proliferation.

Previous research has indicated that in young Drosophila, ISCs either self-renew or differentiate into ECs and EEs (Guo and Ohlstein, 2015) (Figure 1A). With aging, These cells undergo aberrant proliferation and differentiation, leading to the excessive proliferation of ISCs and progenitor cells (Jasper, 2020). To examine this, we first fed Drosophila normal food for 26 days, then supplemented their diet with different concentrations of Kaempferol (0.2, 1.5, 20, 50 μM) for 14 days (Figures 1B, C). We assessed the number of ISCs and progenitor cells marked by esg-GFP⁺, pH3⁺ (a proliferation marker), and Dl⁺ (an ISC marker). Flies treated with 20 μM Kaempferol had fewer esg-GFP⁺, pH3⁺ (Figures 1C–E), and Dl⁺ cells (Figures 1F, G) compared to those fed diets added DMSO. At 50 μM, Kaempferol’s ability to reduce age-related ISC hyperproliferation diminished, suggesting potential side effects at higher concentrations (Figures 1C–E). TUNEL staining confirmed that the reduced ISC proliferation was not due to apoptosis of esg-GFP⁺ cells (Figure 1H). We also examined the impact of Kaempferol on ECs (marked with pros⁺) and EEs (marked with NRE⁺), which showed similar effects in aged flies (Supplementary Figures S1A–D). Overall, our data indicate that Kaempferol exerts a potent inhibitory effect on ISC hyperproliferation and attenuates gut hyperplasia in aged Drosophila.

Previous studies have indicated that Drosophila midguts undergo continuous turnover and can regenerate after tissue damage (Lucchetta and Ohlstein, 2012). Chemical damage, such as from bleomycin (BLM), triggers ISC proliferation. Given Kaempferol’s positive effects on Drosophila gut health (Amcheslavsky et al., 2009), we examined whether it could enhance the gut’s resistance to injury. After a 1-day BLM treatment, the flies were placed on normal food for a 2-day recovery (Figure 2A). Surprisingly, 2 days post-injury, Drosophila treated with Kaempferol showed a more significant reduction in ISC numbers (marked by esg and Dl) and pH3⁺ cells compared to controls, indicating that Kaempferol supports intestinal repair and limits ISC hyperproliferation under stress (Figures 2B–E). Additionally, Paraquat (PQ), known to induce oxidative stress by increasing ROS production, is commonly used to cause intestinal damage in Drosophila (Szafrańska et al., 2016). Kaempferol notably extended the lifespan of both female and male Drosophila exposed to PQ/BLM treatment (Figures 2F, G; Supplementary Figures S2A, B). In conclusion, these findings imply that Kaempferol safeguards ISCs from damage while improving Drosophila’s resilience to stress.

Ensuring the intestinal barrier remains intact is essential for upholding epithelial balance, shielding against pathogens, and supporting immune tolerance to beneficial bacteria (Gervais and Bardin, 2017; Naszai et al., 2015). The decline in ISC functionality and genetic integrity is a key factor in the deterioration of tissue function with aging (Biteau et al., 2008; Gervais and Bardin, 2017; Naszai et al., 2015). Since Kaempferol has shown promise in preventing age-related and injury-induced ISC decline, we further examined its potential to protect intestinal functions in Drosophila during aging. To assess this, we analyzed the copper cell region (CCR) condition in both young and old Drosophila. The CCR’s function decreases with age, disrupting intestinal acid-base balance (Li et al., 2016). In the Drosophila midgut, the CCR secretes acid, which is detected using bromophenol blue, a pH indicator. When intestinal homeostasis deteriorates and acid secretion decreases, the CCR remains blue. In contrast, if acid secretion improves and homeostasis is restored, the CCR turns yellow. Our results indicate that Kaempferol supplementation effectively maintains food intake and intestinal acid-base balance in aged Drosophila (Figures 3A–C). Additionally, Kaempferol improves intestinal function and enhances excretion in these flies (Figures 3D, E). Considering previous research and our findings that highlight the importance of intestinal regeneration for lifespan extension (Biteau et al., 2010), we also investigated Kaempferol’s impact on lifespan. Our data show that Kaempferol feeding extends lifespan in both male and female Drosophila (Figures 3F, G). In summary, Kaempferol supplementation counteracts the decline in intestinal function associated with aging and enhances the lifespan of Drosophila.

The proliferation of ISCs is tightly controlled by gene expression (Guo and Ohlstein, 2015). To understand how Kaempferol influences ISC proliferation, we conducted the RNA-seq on midguts from flies treated with Kaempferol compared to controls. Principal component analysis (PCA) highlighted significant differences between the two groups (Figure 4A). Gene Ontology (GO) enrichment analysis indicated a dramatic increase in genes associated with the unfolded protein response (UPR) in flies treated with Kaempferol (Figure 4B). The UPRER refers to a subset of UPR genes activated by ER stress, which occurs when the ER becomes overwhelmed with misfolded proteins (Rath et al., 2012). To suppress ER stress, the UPR triggers its target genes to alleviate the accumulation of unfolded proteins by enhancing the production of several stress-responsive chaperones (Du et al., 2021; Hetz, 2012). GO enrichment analysis revealed significant differences in UPRER expression between Kaempferol-treated and control flies. We analyzed URPER-related genes and created a volcano plot to highlight the significance and fold changes of differentially expressed genes. The results showed that UPRER genes, such as Hsp23 and HSP68, were upregulated in the midguts of 40-day-old wild-type flies treated with Kaempferol compared to controls (Figure 4C). Heatmap analysis further identified UPR target genes (Hsp70Bbb, Hsp70Ab, HSC70-5, Hsp70Bb, Hsp68, Hsp70Bc, Hsp23, and Hsp70Aa) with markedly higher expression levels in the aged midguts of Drosophila treated with Kaempferol compared to untreated flies (Figure 4D). The UPRER is known to be crucial in regulating ISC hyperproliferation associated with aging (Du et al., 2021). Drawing on prior research and our current data, we propose that Kaempferol addresses aging-related gut hyperplasia through pathways involving UPRER genes. Moreover, we analyzed the expression levels of p-eIF2α, an essential transcription factor in the ATF4 ER stress pathway (Mao et al., 2019). Phosphorylated eIF2α (p-eIF2α) serves as a reliable biomarker for evaluating ER stress levels in cells (Rath et al., 2012), In Drosophila ISCs, p-eIF2α was significantly reduced (Figure 4E). This finding suggests that Kaempferol likely mitigates ISC aging by alleviating ER stress, as evidenced by the increased expression of UPRER target genes in aged flies treated with Kaempferol. Immunostaining further demonstrated that Kaempferol effectively lowered elevated p-eIF2α levels in these cells (Figure 4E). To validate Kaempferol’s effect on ER-stress signaling, we conducted RT-qPCR analyses, which revealed decreased levels of ER-stress-related genes (Ko and Brandizzi, 2024; Kumar and Maity, 2021). ER-stress-related genes (ATF6, ATF4, and XBP1) were reduced in the guts of aged Drosophila treated with Kaempferol (Figure 4F). These results suggest that Kaempferol may affect ISC proliferation by modulating ER-stress signaling.

To further understand Kaempferol’s impact on aging ISCs, we reviewed existing studies, revealing that Kaempferol can inhibit antioxidant activity (Wang et al., 2018; Wang et al., 2020). It also impacts the expression of genes involved in ER-stress signaling (Figures 4B–D, F). ER stress is closely linked to ROS accumulation (Uddin et al., 2021). ER stress can lead to ROS production through mitochondrial DNA damage. Connecting ROS to mitochondrial stress as a consequence of ER signaling (Salminen and Kaarniranta, 2010). To investigate whether Kaempferol’s antioxidant effects are preserved in Drosophila, we measured the expression of antioxidant-related genes via RT-qPCR. Results showed increased levels of Catalase (Cat), Sod1, and Sod2 in aged esg ⁺ cells treated with Kaempferol (Figure 5A). Additionally, Using the fluorescent probe DHE, we evaluated ROS levels in esg ⁺ cells. The results indicated a dramatic decrease in ROS in aged flies that received Kaempferol (Figures 5B, C). To examine Kaempferol’s role in ISC homeostasis, we overexpressed Cat and used Keap1 RNAi in esg ⁺ cells with a temperature-sensitive system (esg ^ts ), as Cat and Keap1 are key regulators of intracellular redox balance (Hochmuth et al., 2011). This approach revealed that Cat overexpression and Keap1 RNAi reduced ROS levels and ISC hyperproliferation, as shown by fewer esg ⁺ and pH3⁺ cells in 40-day-old Drosophila (Figures 5D–F). Importantly, Kaempferol also decreased ISC proliferation in Drosophila with Cat overexpression and Keap1 depletion (Figures 5D–F), suggesting its role extends beyond antioxidant activity to regulating ISC homeostasis.

Kaempferol’s ability to further inhibit ISC proliferation in Drosophila with Keap1 RNAi and Cat overexpression suggests it might engage other pathways in its mechanism. Although lifespan varies greatly among species, fundamental genetic pathways that control longevity are conserved (Green et al., 2022; Guo et al., 2016; Hartl, 2016; Jasper, 2020; Melzer et al., 2020). A well-known example is the insulin/IGF-1 signaling (IIS) pathway (Giannakou and Partridge, 2007; Partridge, 2001). RNA-seq data show that Kaempferol significantly affects insulin signaling (Figure 4B). This pathway contains the insulin/IGF-1 receptor tyrosine kinase, and the serine/threonine kinase AKT (Giannakou and Partridge, 2007; Serrano et al., 2009). To investigate the impact of Kaempferol on this pathway, we measured insulin signaling activity by assessing levels of phosphorylated Akt (pAKT). Immunostaining results indicated that Kaempferol decreased pAKT levels in ISCs of aged flies (Figures 6A, B). Further validation was performed by inhibiting the insulin pathway using either InR RNAi (insulin-like receptor) or a dominant-negative (DN) form of InR, leading to a reduction in esg-GFP⁺ and pH3⁺ cells compared to controls (Figures 6C–E). Kaempferol supplementation also reduced these cell populations in aged flies, likely due to its antioxidant properties related to ER stress. These discoveries indicated that Kaempferol supports ISC homeostasis in aging Drosophila by partially inhibiting the insulin signaling pathway.

As mentioned, ER stress produces ROS in mitochondria through mitochondrial DNA damage (Forgie et al., 2022). This process highlights the link between ER stress and mitochondrial stress. To explore how Kaempferol influences proliferation via ROS and insulin signaling, we conducted experiments involving Keap1 RNAi or Cat overexpression, combined with the expression of a DN form of InR to suppress the insulin signaling pathway in 40-day-old ISCs. The findings indicated a notable reduction in both esg-GFP⁺ and pH3⁺ cells in these flies when compared to the control group (Figures 7A–C). Interestingly, Kaempferol did not increase these effects any further (Figures 7A–C). These results indicated that Kaempferol suppresses age-related hyperproliferation ISC by modulating both the insulin and ROS signaling pathways (Figure 7D).

Any unique or stable reagents and materials discussed in this study can be synthesized using the protocols provided in the Materials and Methods section or can be requested from the lead contact.

Drosophila was maintained at 25°C with a 12-h light-dark cycle in controlled incubators and was provided with standard cornmeal/yeast medium. For conditional expression studies, the flies were initially kept at 18°C. To activate temperature-sensitive transgenes, flies were Switched to 29°C for the necessary period; otherwise, they were aged at 25°C. Only mated females were used for midgut studies.

Dissolution of Kaempferol in Dimethyl sulfoxide (DMSO) and added to the normal food medium. Female flies, collected within 3 days of eclosion, were evenly distributed into tubes containing the diet enriched with Kaempferol. The control diet was prepared with the same volume of DMSO.

A4 paper was cut into strips measuring 3 cm × 5 cm and soaked in a 25 μg/mL solution of bleomycin (Aladdin, B107423). Flies were first hungered for 2 h in empty tubes, then placed in tubes containing the bleomycin solution. After 24 h, the flies were moved to new tubes with food supplemented with 20 μmol/L Kaempferol, while DMSO was used as a control.

All Drosophila genotypes and their sources used in this study are listed in Supplementary Table S4.

In the lifespan study, 100 mated wild-type females and 100 males of the same genetic background were separated 10 days after eclosion and distributed into five tubes, each with about 20 males and 20 females. Each tube contained food supplemented with either 20 mM PQ (Aladdin, M106760) and 5 µg BLM (Aladdin, B107423), added with 20 mM Kaempferol (MCE, HY-14590) or DSMO. Sacrificed flies were counted and recorded every 2 days, and the food in each vial was also replaced every 2 days. The experiment was conducted three times to confirm the results’ reliability and reproducibility.

For the Kaempferol lifespan study, 100 mated wild-type females and 100 males of the same genetic background were placed into five tubes, each containing around 20 males and 20 females, 3 days after eclosion. The control group was fed with food mixed with DMSO, while the experimental group received Kaempferol-mixed food. Sacrificed flies were counted and recorded every 2 days, and the food in each vial was also replaced every 2 days. The experiment was conducted three times to confirm the results’ reliability and reproducibility.

Total RNA was extracted from 30 midguts dissected from wild-type female Drosophila for RNA sequencing. The samples were immediately frozen in liquid nitrogen and processed by Shenzhen Chengqi Biotechnology Co., LTD (China). Sequencing was performed on the Illumina NovaSeq 6000 platform (San Diego, US) with 150 bp paired-end reads, yielding over 20 million reads per sample. Initial quality checks of the raw FASTQ files were done using FastQC (v0.11.9, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The processed reads were aligned to the Drosophila reference genome (Ensembl build BDGP6, https://support.illumina.com/sequencing/sequencing_software/igenome.html), Gene symbols were annotated based on the Drosophila BDGP6 genome from Ensembl. Differential gene expression was assessed using DESeq2 (v1.26.0) with default parameters, focusing on genes with an absolute log2 fold change exceeding 0.5 between modified (e.g., RNAi) and control samples, and a p-value of less than 0.05. Pathway analysis was performed with a cluster Profiler.

One hundred dissected midguts from 100 wild-type female Drosophila were placed in 4°C DEPC-PBS. The samples were then incubated in 1 mg/mL elastase (Sigma, cat. no. E0258) in DEPC-PBS at 25°C, with gentle mixing every 15 min. After incubation, the samples were centrifuged at 600 × g for 15 min at 4°C and resuspended in cold DEPC-PBS. They were then filtered through 70 µm filters and sorted using a FACS Aria II Sorter (BD Biosciences, USA). Three biological replicates were performed. For subsequent analyses, samples were lysed with a lysis buffer, and total RNA was extracted using the Cell Total RNA Isolation Kit (FOREGENE, RE-03111) following the manufacturer’s instructions. Reverse transcription was performed with oligo dT using the PrimeScript RT Reagent Kit (Vazyme, R312-01), and the first-strand cDNA was diluted tenfold with water for real-time PCR analysis (Vazyme, Q711-02). Expression levels were quantified using the 2^−ΔΔCT method and normalized to RpL15, with the control sample’s expression set to Primer sequences for qPCR are listed in Supplementary Table S3.

Dissected midguts of the flies were placed in cold PBS and fixed in 4% paraformaldehyde for 30–35 min. They were then washed twice times with 0.1% PBST (PBS with Triton X-100), each wash lasting 10 min. Apoptosis was detected using the colorimetric TUNEL Apoptosis Assay Kit (Beyotime, C1098) following the instructions.

The midguts of adult female Drosophila should be immersed in PBS after dissection and incubated with 20–30 μM DHE (MCE, HY-D0079) in the dark for 5 min. After washing the samples three times with PBS, they were immediately imaged using confocal microscopy. Signal intensities in the intestinal epithelium were analyzed with LAS-X software, and cells were identified based on fluorescence.

Dissected midguts of adult female Drosophila deposited in cold PBS. After dissection, remove the PBS and add a mixture of 4% paraformaldehyde and n-heptane (1:1) for 30 min at 25°C (room temperature). After fixation, the samples were washed twice with methanol for 5 min each. They were then subjected to three washes with PBS containing 0.1% Triton, each wash lasting 10 min. Then, overnight incubation of the midguts at 4°C was performed using primary antibodies diluted in wash buffer. The primary antibodies used in this study were listed in Supplementary Table S5.

The next day, the midguts were subjected to three washes with 0.1% PBST, each lasting 10–15 min. Subsequently, incubated with secondary antibodies and DAPI (Sigma) for 2–3 h at 25°C (Alexa 488 and Alexa 568, Invitrogen) were used at a 1:2,000 attenuation. Confocal imaging was conducted using a Leica TCS-SP8 microscope, and the results were analyzed by LAS X software.

Confocal microscopy was used to examine immunofluorescence images. Fluorescence intensity was measured from z-stacks using LAS X software. Images were captured with a Leica TCS-SP8 microscope and analyzed using Leica Application Suite X, Adobe Illustrator, Photoshop, and ImageJ.

To categorize Drosophila midguts, 200 µL of 2% Bromophenol blue was first added to the food tubes (using a pipette tip to pierce the food, creating holes to ensure thorough absorption). After a 2-h fasting period, the flies were switched into the tubes for 24 h. The flies were then promptly dissected to capture images.

200 µL of 2% Bromophenol blue was first added to the food tubes (using a pipette tip to pierce the food, creating holes to ensure thorough absorption). A4 paper strips (3 cm × 5 cm) were rolled and placed inside. Flies were hungered for 2 h, then exposed to the solution for 24 h. Afterward, the paper was imaged and analyzed for deposits.

R version 3.5.3 for RNA-seq analysis is available at R Project. The custom ImageJ for quantifying immunofluorescence can be accessed at ImageJ. Prism 7.0 (GraphPad), used for data analysis in this study, is available on the GraphPad website.