Aging-related problems with cell structures that process fats affect intestinal stem cell development

Intestinal aging is characterized by alterations in villus length, crypt size, and cell composition within aged crypts, ultimately leading to the development of epithelial dysplasia and barrier dysfunction. These changes are primarily attributed to the mis-regulation of ISC lineage fidelity [7]. It has been reported that aging results in an increase in the number of secretory lineage cells in mammals [3]. In Drosophila, ISCs give rise to the absorptive enterocyte (EC) and secretory enteroendocrine lineages. Similarly, a continuous accumulation of progenitor cell enteroblasts (EBs) (esg⁺ cells) [27] and pre-ECs (Pdm1⁺ and esg⁺ cells) has also been found in aged Drosophila (Fig 1A and 1C–1E). To screen compounds that can inhibit age-associated differentiation defects within ISCs, we conducted a comprehensive screening of 31 small-molecule anti-aging compounds. And we found that the ratios of progenitor cells and pre-ECs were significantly decreased after Aspirin treatment in aged Drosophila midguts, which demonstrates that Aspirin is an effective anti-aging compound (Fig 1B–1E and S1A Fig).

Previous studies have shown that the differentiation defects of ISCs during aging cause a significant decrease in the digestive functions of Drosophila, including the loss of gastrointestinal acid–base homeostasis, a decline in excretion, and a loss of barrier function [24–26,28]. Since Aspirin administration can promote age-related ISC differentiation, the effects of Aspirin administration on improving the digestive functions of aged flies were further tested. Aspirin treatment starting at an intermediate age significantly alleviated the reduction of excretion (Fig 1F and S1B Fig), prevented the further deterioration of gastrointestinal acid–base homeostasis (Fig 1G and S1C Fig), and increased the intestinal barrier function (Fig 1H and S1D Fig) in aged flies. In addition, our results demonstrated that aspirin prolongs the life span of Drosophila when feeding begins 20 days after eclosion (Fig 1I and S1E Fig). These data suggested that Aspirin could prevent the ISC aging-induced functional decline of gastrointestinal tracks.

To investigate the possibility that Aspirin plays a role in alleviating ISC differentiation defects in mammals, a three-dimensional mouse intestinal crypt organoid culture system was prepared in our study. Colon crypts were isolated from the intestine of both young and aging mice, and treated with or without Aspirin in vitro (Fig 1J). We found that the organoids of aged mice had less budding and organoid-forming abilities, Aspirin treatment increased these abilities (Fig 1K, 1L, S1F and S1G) and enhanced the differentiation of aged ISC, as shown by real-time qPCR analysis (Fig 1M). Furthermore, the aged mice administrated with Aspirin for 3 months also showed alleviation of colon histological symptoms, as decreased crypt width and length (Fig 1N and 1O), which meant that Aspirin also inhibits the ISC differentiation defect and rescue the aged intestinal alterations in vivo. Taken together, these data suggest that Aspirin administration significantly increases aged ISC differentiation and relieves the age-associated intestinal functional decline.

To identify the mechanism by which Aspirin promotes the differentiation of ISCs during aging, RNA sequencing (RNA-seq) was performed on dissected aged Drosophila midguts (40 days under 25 °C) both with and without Aspirin administration (Fig 2A). The transcriptional signature enrichment plot shows that a cluster of peroxisome-related genes was significantly expressed in aged midguts of Drosophila that received Aspirin (Fig 2B and 2C). To determine whether Aspirin regulates peroxisome function to promote ISC differentiation during aging, we first examined the number and function of peroxisomes in ISCs of aged flies. The endogenous peroxisome reporter line Peroxin 10-mCherry (Pex10-mCherry) was generated [15] and showed that the abundance of peroxisomes increased dramatically in aged ISCs (ISCs are indicated by esg-GFP, and peroxisomes are indicated by Pex10-mCherry) (Fig 2D and 2E). However, the peroxisome activity, as indicated by catalase activity assay, was decreased in the ISCs of aged flies (Fig 2F). To test whether the changes to peroxisomes were conserved in mammals, human colon tissues (using the normal para-carcinoma region of young and aged humans) and mouse colons were collected. By performing Western blotting, immunofluorescence staining, and transmission electron microscopy (TEM), we also found a significant abundance with peroxisome (Fig 2G–2K, using PMP70 antibody or “Pex” indicate peroxisomes), and a decrease in peroxisome activity (as indicated by Catalase staining) (Fig 2L) in the crypts of aged mammal colon tissue. In addition, in Aspirin-treated aged mice, we found there was no change in peroxisome abundance, but an increase in peroxisome function in crypts (Fig 2M–2P). These data clearly indicate that the abundance of peroxisomes increases dramatically and that peroxisome function decreases significantly in ISCs during aging, while Aspirin administration may reverse the decline of ISC differentiation by enhancing peroxisomal function.

We further explored how Aspirin would enhance the function of peroxisomes. Firstly, the volcano plots and heatmap from the RNA-seq data revealed that Aspirin can up-regulate a cluster of peroxisome function-related genes in aged Drosophila, including acox1, acat1, acox3, abcd1, and others (Fig 3A and 3B). It is reported that conserved peroxin (Pex) genes are responsible for the formation and maintenance of cellular peroxisome populations. In the volcano plots and heatmap, there are only three Pex genes significantly increased (including Pex1, Pex5, and Pex6) after Aspirin administration; these genes mainly function by importing matrix proteins into the peroxisome (Fig 3A–3C). There are two ways for importing peroxisomal matrix proteins in mammal: PEX5 is the principal import receptor, ferrying most proteins into the peroxisome via the C-terminal peroxisomal targeting signal 1 (PTS1) motif, whereas a minor subset bearing N-terminal PTS2 signals is delivered by PEX7—a pathway that has yet to be confirmed in flies [9,29–32] (Fig 3C). Then, we confirmed that there was a significant increase in the expression of Pex5 in ISCs following Aspirin administration (Fig 3D). Aspirin treatment also increased the expression of luciferase driving by Pex5-Gal4 (S2A and S2B Fig). Consequently, we speculated that Aspirin may regulate the function of peroxisomes by enhancing PEX5-mediated peroxisomal matrix transport.

Next, we analyzed the expression of PEX5 in the human colon during aging. And a significant decrease in PEX5 and increase in PEX7 expression were found in aged human colon (Fig 3E–3G) and mouse crypts (Fig 3H and 3I). Meanwhile, we analyzed the expression of acyl-CoA oxidase 1 (ACOX1), which represents peroxisomal matrix proteins imported by PEX5. Western blot analysis indicated that ACOX1 decreased similarly to PEX5 in aged mouse crypts (Fig 3J). To trace PEX5 endogenous expression within the ISCs of aged Drosophila, the peroxisome reporter line Peroxin 5-HA (Pex5-HA) was generated using the CRISPR-Cas9 knock-in system (S2C Fig). Immunofluorescence analyses also showed that PEX5 decreased significantly in ISCs (indicated by esg-GFP) of aged Drosophila (Fig 3K and 3L). Taken together, these results indicated that the decreased expression of PEX5 in aged ISCs was highly conserved.

The peroxisomal GFP-SKL reporter protein (GFP engineered with a C-terminal S-K-L tripeptide sequence, which serves as a peroxisome targeting signal imported by PEX5) was used to visualize the endogenous PTS1 peroxisomal matrix protein. We found that the intensity of GFP-SKL in per peroxisome was decreased dramatically in the aged ISCs of Drosophila, which demonstrated that the peroxisomal matrix proteins imported by PEX5 were significantly impeded during aging (Fig 3M). However, Aspirin administration increased GFP-SKL in the peroxisome of aged ISCs, which indicates that Aspirin enhances the function of peroxisomes by enhancing PEX5-mediated peroxisomal matrix transport.

In line with findings in Drosophila, aged mice or colonic organoids treated with Aspirin also displayed an increase in PEX5 levels (Fig 3N–3R). To further understand the function of Aspirin on peroxisomal matrix proteins imported by PEX5, mass spectrometry (MS) analysis of peroxisomes was performed in old mouse colon with or without Aspirin treatment (Fig 3S). We showed that the peroxisomal matrix proteins imported by PEX5 significantly increased following Aspirin treatment in aged mice (Fig 3T). Taken together, these findings suggest that PEX5-mediated transport of peroxisomal matrix proteins is impaired in aged ISCs during the aging process; however, Aspirin enhanced this function.

As demonstrated, the transportation of matrix proteins into peroxisomes was significantly impaired in ISCs during the aging process, while Aspirin increased the levels of PEX5-mediated matrix proteins within the peroxisome and restrained ISC mis-differentiation. To further understand PEX5 function, cell-type-specific RNA interference (RNAi) was used. Firstly, we found that peroxisome-related matrix proteins (indicated by GFP-SKL) were uniformly distributed in the cytoplasm of the normal ISCs, while knocking down Pex5 in ISCs resulted in GFP-SKL dispersing throughout the entire cell, including the nucleus, without condensed puncta (Fig 4A). Then, we also found a decreased peroxisomal function (indicated by Catalase and ACOX1) in ISCs after Pex5 knockdown (Fig 4B–4E). Therefore, we conclude that PEX5 downregulation directly leads to peroxisomal impairment. In addition, Pex5 RNAi in ISCs resulted in a significant accumulation in pre-ECs under 29 °C for 20 days, which mimics the phenotype of aged midguts in Drosophila (termed as mimic aging; Fig 4F and 4G). The digestive functions of Pex5 RNAi flies under the mimic aging condition were further investigated, showed a decline in gastrointestinal acid–base homeostasis (Fig 4H–4J) and excretion (Fig 4K). More importantly, Aspirin did not rescue the ISC differentiation defect (Fig 4F and 4G) and the digestive functions of Pex5 RNAi flies (Fig 4H–4K). In addition, to further confirm that aspirin enhances ISC differentiation through PEX5 in mammalian systems, we silenced Pex5 in middle-aged mouse colon organoids (S3A–S3C Fig). Then found that Pex5 knockdown markedly reduced mouse organoid budding, and subsequent aspirin treatment failed to restore its metrics. Therefore, these data indicate that the pro-differentiation effect of aspirin is PEX5-dependent.

Since Aspirin restrains the age-associated ISC differentiation defect by promoting the expression of PEX5, it is reasonable to investigate whether the overexpression of PEX5 in aged flies promotes ISC differentiation, relieves the age-associated functional intestine decline. We found that the overexpression of PEX5 from an old age for 10 days significantly decreased age-associated pre-ECs retained in aged midguts (Fig 5A and 5B) and increased the intensity of GFP-SKL in the peroxisome of aged ISCs (Fig 5C and 5D). Furthermore, overexpression PEX5 significantly prevented the deterioration of gastrointestinal acid–base homeostasis (Fig 5E) and excretion (Fig 5F) in aged flies. We also found that the overexpression of PEX5 improved the life span of flies, while the knockdown of Pex5 shortened its life span when conducted after eclosion of 10 days (Fig 5G and 5H). These results show that overexpression of PEX5 restrains the age-associated ISC differentiation defect and improves the digestive functions of aged flies. Aspirin promotes the differentiation of aged ISCs and enhances digestive functions, specifically through the activation of PEX5-mediated PTS1 signaling.

Fatty acids with a chain length exceeding 20 carbon atoms (referred to as VLCFAs) are predominantly oxidized in peroxisomes. Most enzymes involved in the β-oxidation of VLCFAs in peroxisomes are imported by PEX5; this includes ACOX1, which is the primary rate-limiting enzyme [33] (Fig 6A). To investigate whether VLCFAs play a role in the decreased differentiation function of ISCs with aging, targeted quantitative analysis of free fatty acids (FFAs) was conducted by Gas Chromatography–Mass Spectrometry (GC–MS) using mouse colon. Principal component analysis (PCA) revealed a distinct separation between the young, old, and old Aspirin-treated group (Fig 6B). Furthermore, heatmap enrichment, volcano plot analyses, and the bar charts of FFAs demonstrate a significant enrichment of VLCFAs in old mouse colonic tissue, which were shown to significantly decrease after Aspirin treatment (Fig 6C–6E). As we have demonstrated a decrease in ACOX1 expression in peroxisomes isolated from aged mouse colon crypts (Fig 3J), all these findings provide evidence that VLCFAs also accumulated in aged ISCs. To establish that reduced PEX5 in aged ISCs drives VLCFA accumulation, we isolated ISCs with Pex5 knockdown and quantified FFAs by GC-MS. VLCFAs—namely behenic (C22:0) and lignoceric (C24:0) acids—were markedly elevated, whereas shorter-chain species such as palmitic (C16:0) and stearic (C18:0) acids remained unchanged (S4A Fig). This selective accumulation confirms a strong link between decreased PEX5 and VLCFA buildup in aged ISCs. Collectively, our findings indicate that aspirin restores ISC differentiation by enhancing PEX5-dependent peroxisomal β-oxidation of VLCFAs.

We further investigated whether the accumulation of VLCFAs regulates ISC differentiation upon aging. When feeding flies with behenic acid (BA, C22-0, typical VLCFAs), it significantly led to an ISC differentiation defect in the young midguts of flies as indicated by the Pex5 RNAi phenotype (Fig 7A and 7B). Then, feeding old flies with Bezafibrate [34], which reduces VLCFA synthesis through direct inhibition of fatty acid elongation activity, significantly relieved the aged ISC differentiation defect as indicated (Fig 7C and 7D). In addition, we observed that inhibiting the degradation of VLCFAs in the ISCs of flies by Acox1 RNAi also led to the accumulation of progenitor cell and pre-ECs (Fig 7E and 7F). Reducing VLCFAs generation in aged ISCs by Elov1 (a VLCFA synthase) [33] RNAi can alleviate aged ISC differentiation defects, as indicated by the Pex5 overexpression phenotype (Fig 7G and 7H). Therefore, these data demonstrate that the accumulation of VLCFAs in aged ISCs led to an ISC differentiation defect during aging.

Given that our previous research demonstrated that peroxisomes enhance ISC differentiation by enhancing RAB7-dependent late endosome maturation and JAK-STAT-SOX21A signaling [15], it is logical to investigate whether VLCFAs regulate aged ISC differentiation by influencing the expression of SOX21A and late endosome maturation. To determine the effect of VLCFAs on the expression of SOX21A, which is specifically found in ISCs and EBs [35], we conducted VLCFAs and bezafibrate feeding experiments. Firstly, we found that the expression of SOX21A was decreased in aged ISCs, which was consistent with mis-differentiation of aged ISCs. As expected, the treatment of young Drosophila with VLCFAs resulted in a decrease in SOX21A expression, while bezafibrate treatment in aged Drosophila led to an increase in SOX21A levels (Fig 7I and 7J). Moreover, the forced expression of either SOX21A or RAB7 in ISCs partially corrected the differentiation defects of ISCs in Acox1 RNAi Drosophila, which manifested in a reduction in the accumulation of pre-ECs (Fig 7K and 7L). Therefore, these results indicated that the increased VLCFAs regulated aged ISC differentiation by preventing RAB7-dependent late endosome maturation and JAK-STAT-SOX21A signaling. To further confirm that PEX5 governs aged ISC differentiation by regulating RAB7-dependent late-endosome maturation and JAK-STAT-SOX21A signaling, we specifically knocked down Pex5 in ISCs. This manipulation markedly reduced the levels of SOX21A, 10XSTAT-GFP, and RAB7-GFP, underscoring PEX5’s central role in regulating these (S4B–S4D Fig). Meanwhile, overexpression of either SOX21A or RAB7 ameliorated the differentiation deficiencies caused by Pex5 RNAi (Fig 7M and 7N). Taken together, these findings indicate that the β-oxidation of VLCFAs, which are downstream of PEX5, governs aged ISC differentiation by modulating RAB7-dependent late endosome maturation and SOX21A expression in the midguts of flies (Fig 7O).

All Drosophila stocks used in this study are listed below with complete fly genotypes provided in S1 Table. The following lines were obtained from the Bloomington Drosophila Stock Center (BDSC): w¹¹¹⁸ (BDSC# 3605), UAS-lacZ (BDSC# 8529), UAS-lacZ (BDSC# 3956), UAS-GFP-SKL (BDSC# 28881), UAS-Sox21a (BDSC# 68156), UAS-Rab7-CA (BDSC# 50785), UAS-Rab7-GFP (BDSC# 42706), 10XStat-GFP(BDSC# 26200), CAT-GFP(BDSC# 51546), UAS-Luciferase (BDSC# 35788), Elovl RNAi (BDSC# 50710), ACOX1 RNAi (BDSC# 68109). PEX5 RNAi (TH02247.N) was obtained from the TsingHua Fly Center (THFC). The esg-GFP/CyO and esg-Gal4 were kindly provided by Allan Spradling. The esg^ts-Gal4 line: esg-Gal4, UAS-GFP, tub-Gal80^ts/CyO was a gift from Benjamin Ohlstein. The transgenic Drosophila lines SOX21A-HA, UAS-PEX5-HA, Pex10-mCherry, Pex5-HA, ACOX1-Flag, and Pex5-Gal4 were constructed in our laboratory. Fly lines were backcrossed into the w¹¹¹⁸ background (6 generations) and sibling populations were derived from crosses of w¹¹¹⁸.

Drosophila was raised on a standard cornmeal-agar medium under conditions of 25 °C and 65% humidity, following a 12:12 light and dark cycle, unless otherwise specified. For temperature-sensitive control of Gal4-driven gene expression, flies were first reared at 18 °C to minimize driver activity and then maintained at 29 °C to induce gene activation. The experiments only included mated female flies.

The animal procedures in this study were conducted in accordance with the guidelines provided by the Committee on the Ethics of Animal Experimentation at West China Hospital. Ethical approval was granted (Permit Number: 202203170). Male C57BL/6J mice were used in this study and were purchased from GemPharmatech in Nanjing, China, and Aniphe Biolaboratory Mice of varying ages were utilized in the study, with 3-month-old mice classified as young and 18–21-month-old mice categorized as aged. The specific age of the mice used in each experiment will be delineated in the respective figure legends or methods. The mice were housed in the SPF facility at the Animal Experimental Center of West China Hospital, Sichuan University. They were kept under a 12-hour light-dark cycle, with a relative humidity of 40–50% and a temperature of 22 ± 2 °C. The mice had ad libitum access to food and water, unless otherwise stated. Prior to the experiment, the mice were adaptively fed for 7 days.

Human colon tissues from patients were obtained from the Division of Gastrointestinal Surgery, West China Hospital, Sichuan University. These tissues were harvested from 30 CRC patients and are classified as adjacent normal tissue (refer to Table 1 for a comprehensive list of patient details). These patients were contacted to obtain verbal informed consent. This study protocol adhered to the ethical principles of the 1975 Helsinki Declaration and received approval from the Human Ethics Committee of West China Hospital, Sichuan University, in 2023 (Approval numbers:20231214).

To examine the expression of endogenous PEX5 in Drosophila, we created a PEX5-HA knock-in line. First, we inserted the sgRNA sequence into the PMD18T vector that contains the U6 promoter. Next, we amplified the U6 promoter and sgRNA from the PMD18T vector through PCR. Then, we transferred them into the PCR8 vector using the Golden Gate assembly method. These products were subsequently recombined into the attB vector through LR recombination. We integrated the HA tag into the PEX5 gene through homologous recombination in the PASK vector. Lastly, the Drosophila embryos were microinjected, a procedure performed by Qidong Fungene Biotechnology, in Beijing, China.

We also created the PEX5-RA-Gal4 transgenic lines. Briefly, the pUASTattB-gal4 vector (from Qidong Fungene Biotechnology) which has the attB site and miniwhite marker was used as the backbone. 2,137 bp of DNA immediately upstream of the transcription start site of gene pex5-RA was cloned into Not I/Nde I sites of pUASTattB-gal4. The injection was performed by Qidong Fungene Biotechnology and screening integration flies.

To overexpress PEX5 in Drosophila, we generated UAS-PEX5-HA transgenic lines. First, we cloned cDNAs of Pex5 into the pEntry-3×HA vector and then into the pTW vector. After confirming the plasmids through sequencing, UniHuaii Corporation (Zhuhai, China) performed the injections.

Primers used for these constructions are listed in. S2 Table

Drosophila midguts were dissected in cold PBS and fixed for 30 min using a 4% EM-grade paraformaldehyde solution mixed with n-Heptane. The tissues were then washed twice with methanol for 5 min each. Next, they were permeabilized twice in 0.1% PBST (PBS containing 0.1% Triton X-100), for 10 min each time. The tissues were incubated overnight at 4 °C with primary antibodies. Following this, they were washed three times with 0.1% PBST before incubating with secondary antibodies for 2 hours at room temperature. After the secondary antibody incubation, the guts were washed three times with 0.1% PBST for 10 min each time. Place the tissue on a slide and cover it with Antifade Mounting Medium with DAPI. The dilutions and sources of the antibodies used are listed as follows: anti-GFP (1:1,000; Abcam), anti-HA (1:1,000; Cell Signaling Technology), anti-Armadillo (1:50; DSHB), anti-Delta antibody (1:75; DSHB), anti-mCherry (1:1,000; Invitrogen), anti-Pdm1 (1:200), and Alexa Fluor secondary antibodies (1:2,000). The Leica TCS-SP8 confocal microscope was used to acquire all immunofluorescence images. For each set of experiments, images were acquired as confocal stacks using the same settings.

Behenic acid (BA, Sigma), C22-0, typical VLCFAs, was dissolved and mixed into regular food at a concentration of 2.5% (w/v). The Bezafibrate (MCE) was dissolved in DMSO and then added to the regular food to achieve a concentration of 0.4 µM. Newly eclosed flies were collected and placed into vials containing the regular food for 7 days or 40 days, and then fed with BA or Bezafibrate for 7 days. Aspirin (Aladdin) was dissolved in water and then added to the regular food to achieve a concentration of 5 µM. Newly eclosed flies were collected and placed into vials containing the regular food for 20 days, and then they were transferred to food supplemented with the forementioned drugs for 20 days. The food was changed every two days until the midguts were dissected.

Drosophila guts were dissected in cold DEPC-PBS. After the dissection, the midguts were incubated in a Trypsin-EDTA solution at 25 °C for 40 min with gentle shaking. The dissociated samples were then centrifuged at 3,000 rpm for 20 min, filtered through 40μm cell strainers, and sorted using flow cytometry (FACS Aria III sorter, BD Biosciences, USA). Specifically, esg-GFP⁺ cells from the midgut of flies treated with or without Aspirin were sorted. The fluorescence threshold was set using the w¹¹¹⁸ midgut as a reference for sorting. Approximately 180,000 esg-GFP⁺ cells were collected for each of the three biological replicates.

In accordance with a previously established protocol [50], the Bromophenol blue assay was used to assess the acid–base balance in the gut. 2% Bromophenol blue sodium (pH indicator, Sigma, B5525) was added to the standard food in a vial, and the food’s surface was punctured multiple times with a pipette tip to ensure complete absorption. After a 24-hour feeding period, images of the dissected guts were promptly captured.

After 2 hours of starvation, an assay on fly excretion was conducted. The flies were placed into vials containing food with 2% bromophenol blue sodium, while the interiors of the vials were wrapped with chromatography paper. After observing the flies for 24 hours, the deposits of excretion on the paper were photographed and measured. Each group in the study consisted of 15 flies.

FD&C Blue #1 dye was added to the standard cornmeal-agar food at a concentration of 2.5% (w/v). After 2 hours of starvation, the flies were fed with the FD&C Blue #1 dyed food for 12 hours and then observed. Flies that exhibited a visible blue color outside their digestive tract, known as Smurf (+), were counted.

For the survival tests under normal conditions, we collected 50 mated female or male flies of each treatment (esg^ts> UAS-lacZ, esg^ts> Pex5 RNAi, and esg^ts> UAS-Pex5). These flies were housed at 18 °C for 10 days, and then equally distributed into five vials containing standard food and housed at 29 °C. To test the protective effect of Aspirin, we collected 50 mated female or male flies of each group fed with regular food for 20 days, and then equally distributed them into five vials containing 5 µM Aspirin food, housed at 25 °C. The number of living flies was counted and transported to new vials every 2 days. The experiment was repeated three times independently.

The transcriptional regulation effect of drug treatment on target genes can be assessed by measuring the activity of the luciferase enzyme. To confirm the effect of Aspirin on Pex5, we conducted a luciferase assay using the reporter fly: Pex5-Gal4 > UAS-Luciferase. We compared the luciferase activity in the guts of flies that were fed with Aspirin and those without. The Firefly Luciferase Reporter Gene Assay Kit (Beyotime) was used to measure luciferase activity. Fifteen female midguts were dissected in cold PBS and homogenized in 50 µL lysis buffer. The sample extracts were then centrifuged at 13,000g for 10 min at 4 °C. The supernatant was transferred to a 96-well plate for detection. Equal amounts of firefly luciferase detection reagent were added to each well, and the relative light unitswere measured after incubation.

The fluorescence intensity was quantified using the fluorescence analysis tool in Leica confocal software. For each ISC cell, the total fluorescence intensity of GFP-SKL was measured, and the number of GFP-SKL puncta within the cell was counted. Then, the average fluorescence intensity per GFP-SKL punctum was calculated by dividing the total fluorescence intensity by the number of GFP-SKL puncta.

About 30 female flies for each biological replicate were dissected in cold PBS and frozen immediately on liquid nitrogen. The isothiocyanate-alcoholphenyl-chloroform was used to collect total RNA for midguts. Then, total RNA was sent to Berry Genomics Corporation (Beijing, China) for sequencing on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Quality control was performed using FastQC (version 0.11.8). The raw RNA-seq data with read lengths of 150 bp were aligned to the Drosophila reference genome (Ensembl BDGP6 release-110). The aligned reads (sam files) were transferred to bam files and sorted using feature Counts (version 1.5.1). Gene expression levels in different samples were determined using DESeq2 (version 1.40.2). Differential expression was verified with adjusted <0.05 and log2 (fold change) >1 or log2 (fold change) <−1. The cluster Profiler software package version 4.8.3 was used to perform functional annotation and enrichment analysis of differentially expressed genes. The Complex Heatmap software package version 2.13.1 was used to visualize expression patterns among samples using heatmap representation. The PCA was performed by the statistics function prcomp within R (v4.3.1) (www.r-project.org↗).

Mouse colonic crypts were isolated as described previously with some modifications [51]. Mouse colons were flushed with cold PBS and cut longitudinally. Villi were scraped off gently with a glass slide. The remaining tissue was cut into 1–2 mm sections. Colonic pieces were washed several times until the supernatant was clear and incubated in 5 mM EDTA-PBS for 90 min in the ice box on a rocking platform. The lysis buffer was removed carefully and replaced with cold PBS containing 0.1% bovine serum albumin (BSA, Gibco). The collected crypts were filtered with a 70-µm mesh to remove villous fragments. Finally, filtered crypts were used for real-time qPCR, western blotting analyses, or organoid culture.

For organoid culture, collected crypts were resuspended in Advanced DMEM/F12 medium (Gibco, 12634010), and the quality of the suspensions was assessed using a microscope. Unless otherwise specified, crypts were grown in a mixture of Advanced DMEM/F12 and GlutaMAX (31980030, 12491015) supplemented with N2 (1:100, Thermo 17502048), B27 (1:50, thermo17504044), Wnt3a (1:10, MLB J2-001), Noggin 100 ng/ml (1:1000, Peprotech, 250–38), R-Spondin1,500 ng/ml (1:500, Peprotech, 120–38), N-acetyl-l-cysteine 1 mM (Sigma, A7250), Recombinant Mouse EGF Protein 50ng/ml (1:100,000, Peprotech, 315-09), SB202190 (1:1,000, Sigma, S7067), Y-27632 10 µM (APExBIO, A3008), A8301 0.5 µM (Tocris, 2939) and Matrigel (BD bioscience, 356230) and incubate at 37 °C in a humidified incubator at 5% CO₂. Organoids were exposed to 2 mM Aspirin (Aladdin), and the crypt medium was changed every three days. Images of the organoids were captured under a 20× microscope. Reagents were typically solubilized in dimethyl sulfoxide (DMSO) for experimental use unless otherwise specified. Noggin and R-Spondin1 dried protein powder were dissolved in a 5% alginate solution.

Two days before electroporation, mouse organoids were passaged and cultured in organoid medium. To promote the formation of cystic hyperproliferative crypts, the medium was supplemented with 10 µM Y27632 (HY-10071, Med Chem Express), 10 µM CHIR99021 (HY-10182, Med Chem Express), and 10mM nicotinamide (N108087, Aladdin) [52]. The siRNA sequences were as follows: Pex5 siRNA sense strand, 5′-GGAUCCUAAGCACAUGGAA(dT) (dT)-3′, and antisense strand, 5′-UUCCAUGUGCUUAGGAUCC (dT) (dT)-3′ (antisense). As previously reported methods [53], organoids were dissociated into single cells using 0.25% Trypsin-EDTA (Gibco 25200072) at 37 °C. Then, isolated single cells were resuspended in BTXpress electroporation buffer (BTX) along with the siRNA and subjected to electroporation using the Gene Pulser Xcell Total System (Bio-Rad, 1652660) under parameters optimized as described. Following electroporation, the single cells were cultured in the ENR medium supplemented with 500 ng/mL R-spondin-1 (final concentration, 1 μg/mL), 10μM Jagged-1 peptide (Anaspec, AS-61298), and 10% Afamin-Wnt-3A serum-free conditioned medium (MBL) to facilitate the generation of organoids.

Total RNA from human colon samples, isolated mouse colonic crypts, mouse colonic organoids, or sorted Drosophila esg-GFP⁺ cells was extracted using RNA-easy Isolation Reagent (Vazyme, R701-01) according to the manual. After measuring the RNA concentration and quality, the RNA was reverse transcribed into cDNA using Evo M-MLV RT Mix Kit (AG, AG11728). Real-time qPCR was completed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q712-02). Relative expression of target genes was normalized to Rp49 in Drosophila or Gapdh in mice and human, and estimated with the 2^−ΔΔCT method. Primers used for real-time qPCR are listed in S2 Table.

In the aged group treated with Aspirin, 17-month-old male C57BL/6J mice were fed with drinking water with 1 mg/ml (5.5 mM) Aspirin for 3 months. Conversely, in the control aged group, the same aged male C57BL/6J mice were provided with drinking water without Aspirin for the 3-month period.

The colon tissues were fixed in Neutral Buffered, followed by dehydration, paraffin embedding, and sectioning. Bake the sections in the oven at 65 °C for 30 min and apply biotransparent for 30 min to remove the paraffin. The sections were washed sequentially with a gradient of ethanol, starting with anhydrous ethanol, then 95% ethanol, then 90% ethanol, and finally 80% ethanol, each for 5 min. Following this, a 5-min rinse with double-distilled water (ddH2O) was performed. The sections were incubated with 3% H₂O₂ at room temperature for 30 min, followed by washing with ddH2O. Citrate was used for microwave antigen repair, followed by washing with PBS for 5 min twice. Then blocking with 5% serum for 30 min, primary antibody (anti-PMP70 1:500, anti-catalase 1:500, anti-PEX5 1:400) was added and incubated at 4 °C overnight. After washing with PBS for 10 min, three times, secondary antibodies were added and incubated at room temperature for 90 min. Samples were then washed and imaged. For immunohistochemistry, after washing with PBS for 10 min, three times, biotin-conjugated secondary donkey anti-rabbit or anti-mouse antibodies were added and incubated at 37 °C temperature for 1 h. The Vectastain Elite ABC immunoperoxidase detection kit (Vector Labs, PK-6101) followed by DAB Horseradish Peroxidase Color Development Kit (Beyotime, P0203) was used for visualization.

The colon tissues were fixed in a 10% formalin solution for 24 h, and then underwent a process of dehydration and paraffin embedding to prepare sections. Hematoxylin and eosin (H&E) staining was used to visualize the crypt structures in the colon slices. Statistical analysis was performed to measure the length and width of the colonic crypts.

In order to better investigate the role of peroxisomes in the aging process and the effects of the drug Aspirin on peroxisomes, we utilized the Peroxisome Isolation Kit and a density gradient centrifugation method with iodixanol [OptiPrep] to isolate peroxisomes from mouse colonic crypts.

To further explore the changes in peroxisomes, we subjected the isolated peroxisomes from the intestines of old mice and old mice treated with Aspirin to Shotgun Proteomics analysis. The isolated peroxisomes underwent protein extraction, enzymatic digestion into peptides, and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. Briefly, the samples were lysed using an SDT lysis buffer SDT (4%SDS, 100 mM Tris-HCl, 1 mM DTT, pH7.6) for protein extraction, followed by protein quantification using the BCA kit. Protein digestion was performed by trypsin. The digest peptides of each sample were desalted on C18 Cartridges concentrated by vacuum centrifugation and reconstituted in 0.1% formic acid. Each sample was then separated using an HPLC liquid chromatography system. The mobile phase A was a 0.1% formic acid aqueous solution, and phase B was a 0.1% formic acid acetonitrile solution (with acetonitrile at 84%). The chromatographic column was equilibrated with 95% phase A before the sample was loaded for separation through the analytical column. Post-chromatographic separation, the samples were analyzed using Q Exactive mass spectrometer coupled with an Easy nLC system. The raw MS data were processed for database searching and quantitative analysis using the software MaxQuant. The Shotgun Proteomics analysis was completed by the Institute of New Life Sciences. The protein quantification method is label-free quantification (LFQ). The LFQ values of the samples are normalized by Z-score, and the protein content of the samples is displayed in the form of a heat map.

The targeted FFA assay was performed using gas chromatography–mass spectrometry (GC–MS). Tissue samples from the colons of mice categorized as young, old, and old treated with Aspirin were individually weighed, each approximately 100 mg, for the analysis. Fatty acid extraction from the colonic tissue was performed as previously described [16]. Sample derivatives were analyzed using a GC-EI-MS system (GC, Agilent 8890; MS, 5977B System) with SIM scanning mode. The analytical conditions were the same performed as previously described [16]. The above fatty acid detection tests were performed by MetWare using the Agilent 8890-5977B GC-MS platform. Unsupervised PCA was performed by the statistics function prcomp within R (v4.3.1) (www.r-project.org↗). The data was unit variance scaled before unsupervised PCA. Differential metabolites selected between groups were determined by absolute Log₂ (fold change) and showed by volcano plot.

We assessed catalase enzyme activity in human colon tissue, mouse colonic crypts, and sorted Drosophila esg⁺GFP using the Catalase Assay Kit (Beyotime, Cat# S0051). The above tissues were lysed with RIPA Lysis Buffer (Beyotime, P0013C) to prepare the samples for analysis. Subsequently, the protein content was quantified using the BCA Protein Assay Kit (Beyotime, P0011). We formulated a hydrogen peroxide solution and the chromogenic working solution, added the latter to the samples, and initiated a timed color development reaction. The residual hydrogen peroxide in the samples was measured by absorbance at A520 with a microplate reader, allowing us to calculate the catalase activity based on the protocols provided by the manufacturer.

The human colon was fixed with a 3% glutaraldehyde solution, followed by post-fixed with 1% osmium tetroxide. It was then infiltrated with acetone and resin mixtures, before being embedded in epoxy resin (Epon812) at 60 °C for 36 hours. For microscopic examination, ultrathin sections were prepared and stained with uranyl acetate and lead citrate. These sections were subsequently observed under a JEM1400 TEM. Intestinal stem cells (ISCs) were identified by their small nuclei and their proximity to the basal membrane of the gut epithelium. Meanwhile, peroxisomes were identified through their crystalline morphology and the presence of a granular matrix that stained positively for DAB.

To monitor changes in peroxisome-related proteins at the protein level, we performed western blot experiments on human colon, isolated mouse colonic crypts, mouse colon-isolated peroxisome, and mouse colonic organoids. The above samples were frozen with liquid nitrogen and homogenized with tissue lysate in RIPA lysis buffer containing 1% cocktail and 1% PMSF, then placed on ice for 60 min for thorough lysis. The tissue lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants were collected. Protein mixed with loading buffer was boiled for 5 min and analyzed using 10%–12% SDS-PAGE. Then, the protein was transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked with 5% non-fat milk in TBST for 60 min and then incubated overnight at 4 °C with primary antibodies (anti-PMP70, 1:500; anti-PEX5, 1:1,000; anti-PEX7, 1:1000; anti-ACOX1, 1:1000; anti-ACAA1, 1:1,000; anti-α-Tubulin, 1:1,000). After being washed three times with TBST at room temperature (10 min each), the PVDF membranes were incubated with the secondary antibody at room temperature for 90 min. Detection was performed using an ECL chemiluminescence detection kit (Vazyme, E422-01). Images were captured using the ChemiDoc XRS+ system and analyzed with Image Lab (v5.1, Bio-Rad). The band gray value statistics of Western Blotting were calculated using ImageJ software, following the previous methods [16].

Data analysis for the detection of FFAs, as well as for shotgun proteomics and RNA-seq, is thoroughly detailed in the methods section of the relevant section. Excepting that, all statistical analysis was performed with GraphPad Prism 8. Data are presented as the means ± SD of at least three independent tests unless otherwise noted. The life span assays were tested for significance with a log-rank test. Statistical significance was assessed with the unpaired two-tailed Student’s t test unless otherwise specified. In all cases, a p < 0.05 was used to test for statistical significance. N.S., not significant. All experiments were independently repeated for at least three times.