Ethics statement

The BALB/c mouse and A. japonicus, utilized in this study were commercially bred, and all studies followed the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. The study procedure was authorized by Ningbo University’s Experimental Animal Ethics Committee (No. NBU.ES-2021–11180).

Animals and treatment

Healthy adult A. japonicus (weight 110 ± 10 g) were obtained from the Dalian Pacific Aquaculture Company. They were housed in natural saltwater (salinity 28, temperature 11℃) and fed daily with commercially produced feed for one week before treatment. Following acclimation, A. japonicus were divided into 6 groups, each with three replicates containing 12 individuals. Group 1 served as the control and did not undergo visceral removal; their mesentery was sampled. Groups 2–6 were treated to induce evisceration via intracoelomic injection of 0.35 M KCl (3–5 mL) and were placed in seawater aquaria to allow for intestinal regeneration [9]. At 2, 7, 12, 20, and 28 days post-evisceration (dpe), samples from groups 2–6 were collected sequentially. For analysis, the mesentery and regenerated intestines of 6 A. japonicus (weight 100 mg each) were sampled and placed into centrifuge tubes containing 500 µL of cell lysate buffer (Cat#P0013, Beyotime, Beijing, China) for protein extraction and western blotting (WB). The mesentery and regenerated intestines of 3 additional A. japonicus were wrapped in frozen section compound (OCT, Cat#4583, Sakura, CA, USA) and stored at −80℃ for indirect immunofluorescence assay (IIFA). The remaining 3 A. japonicus had their mesentery and regenerated intestines fixed in a 2.5% glutaraldehyde solution for subsequent transmission electron microscopy (TEM).

To investigate the effect of autophagy inhibition on A. japonicus intestinal regeneration, 243 A. japonicus were eviscerated following an intracoelomic injection of KCl (0.35 M, 3 mL). They were divided into 3 treatment groups: the 3-Methyladenine (3-MA, 5 mM, Cat#5142-23-4, MedChemExpress, New Jersey, USA) group, the Bafilomycin A1 (Baf-A1, 20 nM, Cat#88899-55-2, MedChemExpress, New Jersey, USA) group, and the Dimethyl Sulfoxide (DMSO, Cat#D8371, Solarbio, Beijing, China) group. Each treatment group consisted of 3 replicates, each with 27 A. japonicus. For drug administration, A. japonicus sampled at 2-dpe were individually injected with one of the three reagents 6 h after evisceration. Those sampled at 7-dpe received injections at 6 h post-evisceration, as well as at 2-dpe, 4-dpe, and 6-dpe. Specimens taken at 12-dpe were injected at 6 h post-evisceration and additionally at 2-dpe, 4-dpe, 6-dpe, 8-dpe, and 10-dpe. For sampling, the mesentery and regenerated intestines of 6 A. japonicus (weighing 100 mg each) from the 3 treatment groups at 2-dpe, 7-dpe, and 12-dpe were individually collected. These were washed with phosphate buffered saline (PBS, Sigma-Aldrich, Saint-Louis, USA) and placed into centrifuge tubes for protein extraction and WB. The tissue of an additional 3 A. japonicus was also sampled individually at 2-dpe, 7-dpe, and 12-dpe. This tissue was embedded in a frozen section compound (OCT, Cat#4583, Sakura, USA) for IIFA.

Fig. 1

Autophagy is upregulated in the early phases of intestinal regeneration. A WB was performed to evaluate the expression changes of AjLC3-II/I and Ajp62 proteins in the control (non-regenerating stage mesentery) and various regenerative stages (from 2 to 28-dpe). B The band density of AjLC3-II/I and Ajp62 proteins in (A) was quantified using the ImageJ program. Data are presented as mean ± SD, n = 3 replicates. Statistical analysis was performed using one-way ANOVA and Duncan’s post-hoc test, with p < 0.05 considered statistically significant. Different letters above each bar indicate significant differences. C Schematic diagram of the injection method for DMSO and autophagy inhibitors (Baf-A1) during the intestinal regeneration process. D WB was used to detect the protein expression changes of AjLC3-II/I and Ajp62 in regenerating mesentery and intestine at 2 and 7-dpe post Baf-A1 or DMSO treatment. E The band density of AjLC3-II/I and Ajp62 proteins in (D) was quantified using the ImageJ program. Data are presented as mean ± SD, n = 3 replicates. Statistical significance was determined using the Student’s t-test: *p < 0.05; ***p < 0.001; ****p < 0.0001. F Indirect immunofluorescence was used to detect the expression changes of AjLC3 during the course of intestine regeneration (2-28 dpe). All samples were taken from the same location in different individuals. Blue indicates DAPI-stained nuclei, and red indicates the expression of AjLC3. Bar = 100 μm. G TEM was used to observe mesentery and regenerative primordia in control (non-regenerating stage mesentery) and regenerating intestinal cells at 2-dpe. In the regenerative primordia at 2-dpe, autophagic vacuoles accumulated (red arrowheads). The left panel shows low magnification images, whereas the right panels provide high magnification  views of specific areas. These images are enlargements of specific portions of the images in the left panel. M: mitochondria. L: lysosome. AVi: initial autophagic vacuoles

To clarify the interaction between ROS and autophagy during the regeneration of the intestine in A. japonicus, the following experimental steps were carried out. First, 36 A. japonicus were randomly selected and subjected to evisceration. At various time points—0-dpe, 2-dpe, 7-dpe, 12-dpe, 20-dpe, and 28-dpe—samples of the mesentery and regenerating intestine from 3 A. japonicus (each weighing 50 mg) were collected and processed to measure ROS concentrations. To facilitate this, 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA, 10 µM, Cat#HY-D0940, MedChemExpress, New Jersey, USA) and DAPI (3 µM, Cat#C1002, Beyotime, Beijing, China) were administered 2 h and 30 min before each sampling, respectively. The collected tissues from the 3 A. japonicus were then embedded in a frozen section compound (Cat#4583, Sakura, CA, USA) for fluorescence observation. After establishing a significant association between ROS, autophagy, and intestinal regeneration, Apocynin (APO, 0.1 µM, a common ROS inhibitor, Cat#HY-N0088, MedChemExpress, New Jersey, USA) was introduced to further investigate the role of ROS in autophagy and intestinal regeneration. A total of 54 A. japonicus were randomly divided into 2 groups: the APO group and the DMSO (solvent control) group, with 27 A. japonicus in each. After evisceration, starting at 6 h post-procedure, both groups received intraperitoneal injections of either APO or DMSO every 48 h. At critical time points of 2-dpe, 7-dpe, and 12-dpe, mesentery and regenerating intestine samples from 3 A. japonicus (each weighing 50 mg) were taken and immediately immersed in cell lysate buffer (Cat#P0013, Beyotime, Beijing, China) for protein extraction and WB analysis. Additionally, samples from another 3 A. japonicus were embedded in a frozen section compound (Cat#4583, Sakura, CA, USA) for immunofluorescence experiments. Tissue samples from the remaining 3 A. japonicus were used for ROS content detection.

Phos-tag SDS-PAGE and WB

The total protein from mesentery and regenerated intestine samples in each group was ground at −10℃ using a tissue grinder. These samples were then lysed with cell lysis buffer (Cat#P0013, Beyotime Biotechnology, Shanghai, China), which contains 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, and several inhibitors, including sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, and leupeptin. Nuclear and cytoplasmic proteins were isolated from each group using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Cat#78833, Thermo Scientific, MA, USA) according to the manufacturer’s instructions. The extracted proteins were analyzed for concentration using a BCA Protein Assay Kit (Cat#CW0014S, CWBIO, Beijing, China). A total of 50 µg of protein was separated by 12% or 15% SDS-PAGE in a Tris-Gly buffer system under constant voltage at 120 V. After electrophoresis, wet transfer was performed using a mini-transfer tank (Bio-Rad Laboratories) with NcmBlot Rapid Transfer Buffer (Cat#WB4600, NCM Biotech, Suzhou, China) at 400 mA for 30 min onto PVDF or nitrocellulose membranes (MilliporeSigma). The membranes were then blocked using 5% non-fat milk in TBST buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, pH 7.4) at room temperature for 2 h. Next, the membranes were incubated overnight at 4℃ with relevant primary antibodies (Supplementary Table 1). On the following day, the membranes were washed three times with TBST and incubated at room temperature for 2 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (Supplementary Table 1). The membranes were then washed three additional times with TBST. Finally, the proteins were visualized using a chemiluminescence assay with NcmECL Ultra (Cat#P10200, NCM, Suzhou, China) and imaged with an Aplegen Omega Lum C (Gel Company, San Francisco, CA, USA). The protein band intensities were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The mean values of band intensities from three independent experiments were calculated, and the ratio of target protein intensity to tubulin intensity was determined.

The phosphorylation status of AjFoxO was analyzed using Phosphate-binding tag (Phos-tag™) acrylamide gels, a phospho-affinity SDS-PAGE technique developed by Kinoshita et al. [21]. The separating gel was created through the copolymerization of acrylamide and Phos-tag acrylamide. Phosphorylated proteins migrate more slowly in the gel than their unphosphorylated counterparts due to the reversible trapping of phosphoproteins by Phos-tag during electrophoresis. The Phos-tag SDS-PAGE was performed on gels containing 10% polyacrylamide, incorporating 50 µM Phos-tag Acrylamide AAL-107 (Cat#300–93523, Wako Chemicals, Osaka, Japan) and 100 µM MnCl2, as outlined in reference [22], under a current of 30 mA. This method allows for the differentiation of proteins by observing the mobility shift in phosphoproteins, which appear as distinct bands corresponding to their degree of phosphorylation. After separation, the proteins were blotted onto PVDF membranes using a submerged apparatus. The immunoreaction was carried out according to the protocol described in the previous paragraph.

Tissue immunofluorescence and image analysis

The mesentery and regenerated intestine from both experimental and control groups were embedded in frozen section compound (OCT, Cat#4583, Sakura, CA, USA) and frozen in liquid nitrogen. Slices were made using a Leica CM1900 cryostat at −20℃ and adhered to adhesion microscope slides (Cat#188105, Citoglas, Jiangsu, China). The Sect. (6 μm) were then removed and placed in 4% formaldehyde for 15 min. Permeabilization followed with 0.3% Triton X-100 for 20 min, and incubation with 10% goat serum for 1 h at room temperature. Next, the sections were incubated overnight at 4℃ with rabbit anti-AjLC3 antibody (1:1000 dilution, Cat#EPR18709, Abcam, Cambridge, UK) and/or mouse anti-AjFoxO antibody (1:500 dilution). The following day, the sections were washed three times with PBS containing 0.05% Tween-20 (PBST) and then incubated with Alexa488-conjugated goat anti-rabbit IgG (1:1000 dilution, Cat#A11008, Invitrogen, Carlsbad, CA, USA) at 37℃ for 90 min. Afterward, they underwent three additional washes with PBST and were incubated with Cy3-conjugated goat anti-mouse IgG (1:1000 dilution, Cat#M30010, Invitrogen, Carlsbad, CA, USA) at 37℃ for another 90 min. Following three more washes with PBST, the sections were stained with DAPI (10 mg/ml in PBS, Cat#C1002, Beyotime, Beijing, China) for 10 min at room temperature. Finally, fluorescence microscopy (Olympus, BX51) equipped with DAPI (330–380 nm), Alexa488 (460–490 nm), and Cy3 (510–550 nm) filters, along with objectives offering 5x magnification for broad views of the regenerated intestine and 100x magnification for detailed local structures, was performed for comprehensive examination. This setup enabled precise observation of fluorescence-positive signals and facilitated high-quality imaging of the sections.

Fluorescence signals and the area size of regenerated tissues across different regeneration periods and treatment groups were measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). To quantify fluorescence signal intensity, fluorescence images were initially converted to an 8-bit grayscale format. Regions of interest (ROIs) were defined using the Rectangle or Ellipse selection tools. Mean grayscale intensity values were measured for each ROI. This procedure was repeated for all ROIs within the image. Consistent settings were maintained during image capture and analysis to ensure accuracy and reliability of the results. The freehand selection tool in ImageJ was used to capture and measure the area of the regenerated intestine. The average area of these intestinal rudiments was calculated from at least three discontinuous tissue sections.

Transmission electron microscopy

TEM is considered the gold-standard method for detecting autophagy by analyzing morphological structures [23]. To directly observe the number, morphology, and structure of autophagosomes in regenerated intestinal tissue cells, the mesentery and regenerated intestine of A. japonicus from the control and 2-dpe groups were sampled. These samples were treated with 1% osmium tetroxide for 2 h at 25 °C and washed three times with 0.1 M PBS. The tissues were then dehydrated through a series of increasing ethanol concentrations and incubated in pure propylene oxide. During dehydration, the tissues were stained with 1% uranyl acetate (Serva, Heidelberg, Germany) in 70% ethanol. The tissues were subsequently embedded in a mixture of propylene oxide (Electron Microscopy Sciences, PA, USA) and Epon resin (Serva, Heidelberg, Germany), followed by pure Epon resin. After polymerization at 60 °C, 70 nm thick sections were cut using a Leica ultramicrotome (Leica UC7, Germany) and collected on TEM copper grids (Ted Pella, CA, USA). Electron micrographs were obtained using a transmission electron microscope (HITACHI HT7800, Japan) at 5000–10,000x magnification. Three optimal fields of view from each control/treatment group were selected for photography, and the number of autophagosomes in the cells of each group was counted for statistical analysis.

ROS probe-based chemiluminescence and fluorescence microscopy assay

We investigated the dynamic changes and tissue distribution of ROS in the mesentery and regenerating intestines of A. japonicus at different time points during the intestinal regeneration process in the Control, DMSO, and MK2206 (a novel allosteric inhibitor of Akt, 2 µM, Cat# SF2712, Beyotime, Shanghai, China) groups. Briefly, the entire length of the mesentery and regenerative primordia (30 mg) from the end of the esophagus to the cloaca of 6 A. japonicus from the control (non-regenerating stage mesentery), 2-dpe, 7-dpe, 12-dpe, 20-dpe, and 28-dpe groups were collected, ground into a homogenate in a −10°C cryogrinder, with 3 replicates per group. The intracellular ROS concentrations in these tissues were measured using the Tissue ROS Test Kit (DHE) (Cat# HR8821, Baiaolaibo, Beijing, China) according to the manufacturer’s instructions. Fluorescence intensity absorbance was detected using a microplate reader (Thermo Varioskan Flash 3001, USA) at 610 nm. Additionally, the distribution of ROS in A. japonicus was assessed in the control, 2-dpe, 7-dpe, 12-dpe, 20-dpe, and 28-dpe groups using 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA, HY-D0940, MedChemExpress, New Jersey, USA). Three A. japonicus were sampled from each group. At each sampling time point of intestinal regeneration, H2DCFDA (10 µM) and DAPI (3 µM) were intraperitoneally injected 30 min and 2 h in advance, respectively. The samples were then embedded in frozen section compounds (OCT, Cat# 4583, Sakura, CA, USA), frozen in liquid nitrogen, and cut into 6 μm sections using a Leica CM1900 low-temperature cryostat. Images were captured using a fluorescence microscope (Olympus BX51). For co-localization of ROS and AjLC3 fluorescence, we first used the free radical-permeable H2DCFDA to visualize ROS localization. Indirect immunofluorescence staining was then performed as described in the section “Tissue Immunofluorescence and Image Analysis” to visualize AjLC3 localization. Images of ROS probe fluorescence and AjLC3 fluorescence were taken sequentially from the same location in the same tissue and merged using ImageJ software.

Recombinant expression and antibody preparation

To obtain a recombinant Escherichia coli Rosetta (DE3) strain capable of expressing A. japonicus FoxO (AjFoxO) protein, the plasmid vector pET28a(+)-AjFoxO was constructed. First, the pET28a(+) plasmid was prepared by digestion with BamHI and XhoI. Next, the AjFoxO sequence (1704 bp, A. japonicus genomic Bioproject: PRJNA354676: MRZV01000610.1:105711|106124, MRZV01000610.1:122773|124062) was amplified from cDNA of A. japonicus using the primers F: 5’-AGCAAATGGGTCGCGGATCCATGGATGAAATAGATCCTGATT-3’ (homology arm underlined) and R: 5’-GGTGGTGGTGGTGCTCGAGTTTAGTGCACCCAATTGGTTC-3’ (homology arm underlined), then purified using a PCR product purification kit (Cat#B610363-0050, Sangon Biotech, Shanghai, China). The PCR products were ligated to the pET28a(+) plasmid with homologous arms at both ends using a pEASY®-Basic Seamless Cloning and Assembly Kit (Cat#CU201, TransGen Biotech, Beijing, China) to generate the recombinant pET28a(+)-AjFoxO vector in E. coli DH5α cells. The structure of pET28a(+)-AjFoxO was confirmed by DNA sequencing (Sangon Biotech). Following successful sequencing confirmation, the recombinant pET28a(+)-AjFoxO vector was transformed into E. coli Rosetta (DE3) and induced with isopropyl-β-D-thiogalactopyranoside at 18 °C overnight. The expressed recombinant AjFoxO protein (rAjFoxO) was purified using Ni-NTA Agarose (QIAGEN, Germany) and validated by SDS-PAGE. The purified rAjFoxO was used as an immunogen to prepare mouse anti-AjFoxO polyclonal antisera. Six BALB/c mice were immunized with rAjFoxO protein in four separate doses. The immunization procedure was as follows: 50 µg of rAjFoxO protein suspension was mixed with Complete Freund’s Adjuvant (Cat#HY-153808, MedChemExpress, New Jersey, USA) in a 1:1 ratio, and 0.2 mL of the mixture was intraperitoneally injected into the mice. Two weeks later, the first booster immunization was performed, using a mixture of rAjFoxO protein suspension and Incomplete Freund’s Adjuvant (Cat#HY-153808 A, MedChemExpress, New Jersey, USA) in a 1:1 ratio, injected intraperitoneally. Subsequently, immunizations were strengthened weekly with 0.2 mL of adjuvant-free rAjFoxO protein suspension injected into the tail vein. On the 7th day after the last booster, blood was collected from the mice by eyeball exsanguination. The collected blood was incubated at room temperature for 2 h, then left overnight at 4 °C. The next day, the serum was separated by centrifugation at 10,000 rpm for 5 min. Mouse IgG was purified using a protein G-agarose rapid flow column (Sigma, USA) and characterized by enzyme-linked immunosorbent assay (ELISA) and WB.

Enzyme-linked immunosorbent assay

ELISA was used to detect the potency titer of the anti-AjFoxO antibody. Specifically, 100 µL of 100 µg/mL rAjFoxO protein was coated onto 96-well flat-bottom microplates (Costar, USA) and incubated overnight at 4 °C. The wells were then washed three times with PBST and blocked with 200 µL of 3% bovine serum albumin (BSA) solution in PBS for 1 h at 37 °C. After washing, the wells were incubated with 100 µL of serially diluted anti-AjFoxO polyclonal antibodies in PBS at concentrations of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000 for 1 h at 37 °C. Following additional washes with PBST, 100 µL of AP-conjugated goat anti-mouse IgG (Cat#ZY2044, Ziyunbao, Shanghai, China) at a 1:1000 dilution in PBS was added and incubated for 1 h at 37 °C. After the final wash, 100 µL of 0.1% (w/v) p-nitrophenyl phosphate (pNPP, Sigma, USA) in pNPP buffer (1% diethanolamine, 0.5 mM MgCl2, pH 9.8) was added to each well and incubated for 30 min at 37 °C in the dark. The reaction was stopped by adding 50 µL of 2 M NaOH per well, and the absorbance was read at 450 nm using an automatic ELISA reader (Molecular Devices, USA). Serum from a non-immunized mouse served as the negative control. Each experimental step was replicated three times to ensure data reliability. The potency of the polyclonal antibody was determined when the ratio (P/N) of the OD 450 value of the polyclonal antibody well to the negative control well exceeded 2.1, with the highest dilution representing the antibody potency.

Cell immunofluorescence staining

Immunofluorescence staining was performed to examine the subcellular distribution and translocation of AjFoxO in the mesentery and regenerating primordium during intestinal regeneration. The mesentery and regenerated intestine from the DMSO, Apocynin, and APO + MK2206 groups were minced and digested in a mixture of PBS containing 0.2% Trypsin (Cat#25200-072, Gibco, Grand Island, NY, USA) and 0.2% Collagenase IV (Cat#17104019, Gibco, Grand Island, NY, USA) at 4 °C for 30 min to form a single-cell suspension. The suspension was washed twice with PBS containing 10% fetal bovine serum (FBS, Cat#10270-106, Gibco, Grand Island, NY, USA) and twice with PBS alone. The suspension was then dropped onto adhesion microscope slides (Cat#188105, Citoglas, Jiangsu, China) and allowed to settle for 1 h. After removing the liquid, the slides were air-dried, and a circle was drawn around the dried surface using a PAP pen to prevent fluid loss. The slides were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.3% Triton-X-100 in PBS for 15 min, and blocked with 5% BSA at room temperature for 1.5 h. The slides were then incubated overnight at 4 °C with anti-AjFoxO mouse antibody (1:500 dilution), followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse IgG (1:1000 dilution, Cat#A0516, Beyotime, Beijing, China) for 1 h at room temperature. The cell membrane was stained with DiL (red) (Cat#C1991S, Byotiome, Beijing, China) and the nuclei were stained with DAPI (Cat#C1002, Byotiome, Beijing, China). Images were captured using a confocal laser scanning microscope (LSM 880; ZEISS, Germany) with a 40× objective lens.

RNA interference (RNAi) and inhibitor assays

The specific siRNA (Supplementary Table 2) targeting AjFoxO (siAjFoxO-1 and siAjFoxO-2) and a control siRNA (Negative control, NC) were designed and synthesized by GenePharma Company. Before the formal experiment, the interference reagents were tested at various concentrations (0 µM, 10 µM, 20 µM, 40 µM) to evaluate their interference ability and off-target effects. After determining the optimal concentration and specificity of the interfering primers, the experimental and control siRNAs were dissolved in RNase-free water to prepare 20 µM (optimal interference concentration) stock solutions of siAjFoxO-1/siAjFoxO-2 or siNC. The interference reagent mixture for each A. japonicus was prepared in 100 µL total volume: 10 µL of Lipo6000 transfection reagent (Cat#C0526, Byotiome, Shanghai, China), 10 µL of siAjFoxO-1/siAjFoxO-2 (20 µM) or siNC (20 µM), and 80 µL of PBS. This mixture was intraperitoneally injected into the coelom of A. japonicus every 48 h throughout the intestinal regeneration process. The first injection was administered 6 h post-evisceration, with the last injection given at 6-dpe. Mesentery and regenerating intestine samples were collected at 2-dpe and 7-dpe and stored at −20 °C for subsequent RT-qPCR (AjFoxO mRNA levels) and WB (AjFoxO, AjLC3-II/I, Ajp62 protein levels).

All chemical inhibition treatments were performed in vivo. Control groups were treated with DMSO dilution. The inhibitors applied in this study are listed in Supplementary Table S1.

Quantitative Real-Time RT-PCR

Total RNA was extracted from 100 mg of intestinal regeneration tissues (6 A. japonicus) at different stages of regeneration (0-, 2-, 7-, 12-dpe) and from the regenerated tissues of the AjFoxO interference and control groups at 2-dpe, following the manufacturer’s instructions using Trizol reagent (Cat#T9108, Takara, Otsu, Japan). RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific), and RNA integrity was assessed by 1.5% agarose gel electrophoresis. To remove genomic DNA (gDNA), 1 µg of total RNA was treated with 1 unit of DNase I for 15 min at 37 °C. cDNA was synthesized using the PrimeScript™ RT Reagent Kit (Cat#RR047A, Takara, Otsu, Japan). The cDNA from each sample was diluted 10-fold and used as a template for RT-qPCR on an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, USA) with TB Green Premix Ex Taq™ II mix (Cat#CN830A, Takara, Otsu, Japan). Oligonucleotide primers for AjLC3, AjATG4, and AjFoxO were designed using Primer 5 software, based on sequences from the A. japonicus genomic (NCBI accession number: MRZV00000000.1) and transcriptome (NCBI accession number: SAMN29388289) databases [24]. The amplification efficiency and specificity of the primers were evaluated using the standard curve, amplification plot, and melt curve (Supplementary Figure S1). For validation of the housekeeping gene expression stability during intestinal regeneration, the expression stability of 8 candidate housekeeping genes (β-actin, β-Tubulin, Elongation factor-1 (EF1α), 40 S ribosomal protein S9 (RPS9), RPS18, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), NADH dehydrogenase (NADH) and 60 S ribosomal protein L18a (RPL18A)) was evaluated using four algorithms: geNorm (qbase+, version 3.1) [25], NormFinder (version 0.953) [26], BestKeeper (version 1) [27], and RefFinder (https://blooge.cn/RefFinder/?type=reference). The amplification efficiency and specificity of these 8 housekeeping genes were previously reported [28], and their expression stability is shown in Supplementary Figure S2. Amplification was conducted in a 20 µL reaction volume containing 10 µL of SYBR Green I Master, 2 µL of diluted cDNA, 0.4 µL of each forward and reverse primer (10 µM) (Supplementary Table 2), 0.4 µL of ROXII (Takara), and 6.8 µL of RNase-free water. The reaction was carried out at 95 °C for 30 s, followed by 45 cycles of denaturation at 95 °C for 5 s and extension at 60 °C for 30 s [29]. Each sample was run in triplicate, and the experiments were repeated three times using the Ajβ-tubulin gene as the endogenous control. Data were analyzed relative to the Ajβ-tubulin gene using the 2−△△Ct method [30].

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Transcriptomic analysis of intestinal regeneration after DMSO/APO treatment in A. japonicus

The mesentery and regenerated intestines from 9 A. japonicus in the APO group and the corresponding control (DMSO) group were sampled at 2-dpe and stored at −80 °C for transcriptome sequencing. Total mRNA was extracted as described above. RNA integrity was evaluated using the RNA Nano 6000 Assay Kit (Agilent Technologies). RNA-seq libraries were constructed using the NEBNext Ultra RNA Library Prep Kit, following the manufacturer’s guidelines. Libraries were subjected to paired-end sequencing with a read length of 150 bp on the Illumina NovaSeq 6000 platform (Illumina, USA). Adapter sequences and low-quality reads were filtered using Fastp (v0.19.7, parameters: -g -q 5 -u 50 -n 15 -l 150), and the remaining clean reads were aligned to the A. japonicus genome (NCBI accession number: MRZV00000000.1) using HISAT2 software (v2.0.5) [31]. Potential novel transcripts were identified using StringTie and CPC2 (version 1.0.1) [32]. Gene expression levels were quantified using RSEM (v1.2.15) software [33]. Differentially expressed genes (DEGs) between the APO and DMSO groups were identified using the DESeq2 package (Bioconductor, v1.14.1, p < 0.05 and |log2(FoldChange)| > 1) [34]. DEGs were further analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (R software 4.2.1) and heatmap analysis (heatmap.2 in R package v3.6.0).

Cell culture

Human embryonic kidney epithelial cells (HEK 293T) were cultured in DMEM medium (Cat#10013102, Corning, NY, USA) supplemented with 10% FBS. The cells were maintained in a humidified incubator at 37 °C with 5% CO2. Petri dishes and culture plates were purchased from Jet Bio-Filtration Co., Ltd. (Guangzhou, China) and NEST Biotechnology Co., Ltd. (Wuxi, China).

Chromatin immunoprecipitation (ChIP) assay

To systematically investigate the downstream genes regulated by AjFoxO and their specific binding sites during the sea cucumber intestine regeneration process, we selected the mesentery and regenerated intestine at 2-dpe and prepared DNA fragments following the protocol of a commercial ChIP kit (Cat# P2078, Beyotime, Beijing, China). Specifically, 200 mg of mesentery and regenerated intestine tissues were chopped into small pieces and incubated in RBI buffer (10 mM KCl, 5 mM MgCl2, 5 mM EGTA pH 8, 5 mM Na pyrophosphate, 1 mM PMSF, 1X Complete™ protease inhibitors from Roche Diagnostics, Basel, Switzerland) on ice for 10 min. The samples were then centrifuged at 1000 g for 5 min and resuspended in ice-cold PBS containing 1X Complete™ protease inhibitors (PBS-C) and 1% formaldehyde (FA, Cat#28906, Thermo Fisher Scientific, Waltham, USA) for 10 min at 37 °C under agitation. Reactions were stopped by adding 1X glycine and incubating for 5 min at room temperature whereas agitating. Tubes were centrifuged for 5 min at 1000 g, and the tissues were washed three times with ice-cold PBS-C. The samples were then ground using an ice-cold mortar and pestle until homogeneous and incubated for 10 min on ice with 1 mL of ice-cold Lysis Buffer (10 mM Tris-HCl pH 8, 5 mM EDTA pH 8, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, 1X Complete™ protease inhibitors). The tissue lysate was centrifuged at 5000 g for 5 min, and the cellular pellets were resuspended in 500 µL of SDS Lysis Buffer (50 mM Tris-HCl pH 8, 10 mM EDTA pH 8, 1% SDS, 10% glycerol, 1X Complete™ protease inhibitors) and incubated for 10 min. The tubes were vortexed for 20 s, incubated on ice for 10 min, and vortexed again for 20 s. Ultrasonic treatment was performed using an Ultrasonic Homogenizer (Cat# JY92-IIN, Scientz, Ningbo, China) set at a frequency of 15 W (10 s on, 10 s off, 8–10 cycles). Then, 0.2 mL of the ultrasonically treated sample was transferred to a centrifuge tube, 8 µL of 5 M NaCl solution was added, and the mixture was thoroughly mixed. The sample was incubated at 65 °C for 4 h. DNA purification was carried out using the Macherey Nagel NucleoSpin® Gel and PCR Clean-up kit (Macherey Nagel, Duren, Germany), and DNA smears were visualized by migration on a 1% agarose gel electrophoresis stained with Gel Red (Biotium, Fremont, USA).

ChIP experiments were conducted on samples with qualified ultrasonic treatment, which achieved the desired fragment size (200–1000 bp). The samples were centrifuged at 12,000–14,000 g for 5 min. A volume of 0.2 mL of the supernatant was transferred into a 2 mL centrifuge tube, and 1.8 mL of ChIP Dilution Buffer containing 1 mM PMSF was added and mixed thoroughly. A 20 µL aliquot was set aside as the “input” for subsequent testing. To the remaining 2 mL of sample, 70 µL of Protein A + G Agarose was added, and the mixture was gently rotated at 4 °C for 30 min. The samples were then centrifuged at 4 °C and 1000 g for 1 min, and the supernatant was carefully transferred to a fresh 2 mL centrifuge tube. Subsequently, 10 µL of AjFoxO antibody was added, and the mixture was incubated overnight at 4 °C with rotation. After incubation, 60 µL of Protein A + G Agarose was added, and the mixture was rotated or shaken at 4 °C for 60 min to precipitate the protein or corresponding complex recognized by the AjFoxO antibody. The solution was then centrifuged at 1000 g for 1 min at 4 °C. The precipitate was washed sequentially with Low Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer, and TE Buffer. Each wash used 1 mL of solution, with the precipitate gently rotated at 4 °C for 3–5 min after each addition. After the final wash, the liquid was carefully removed to avoid contact with the sediment. The precipitate was sent to Cloud-Seq Biotech (Shanghai, China) for ChIP-seq sequencing. Sequencing libraries were prepared using the NEB Next Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, USA). The process involved 10 ng of immunoprecipitated (IP) DNA and 100 ng of input DNA, with PCR amplification cycles totaling 7 for IP DNA and 4 for input DNA. DNA fragments of approximately 200 bp were selected using Agencourt AMPure XP beads (Beckman Coulter, Brea, USA). Library concentrations were quantified using Qubit fluorometry and stored at − 80 °C prior to sequencing. Sequencing was performed in single-end 50-base format on an Illumina 4000 system by the IGBMC GenomEast Platform (Illkirch, France).

For bioinformatics analysis, sequencing quality was checked with FastQC. Low-quality reads were removed using cutadapt software (v1.9.3) [35], and the remaining clean reads were aligned to the A. japonicus genome using Bowtie2 (v2.2.6.2) [36]. Peak detection was performed using Epic (v0.1.23), a SICER rework, with the following settings: fragment-size 50 -gaps-allowed 2 -false-discovery-rate-cutoff 0.05 [37]. AjFoxO regions were identified per sample. To account for read count differences, 40 million unique reads were randomly selected for analysis. Common peaks were identified by intersecting the results, and peak visualization was done using the Integrative Genomics Viewer. For BigWig files, the mapped reads were determined using flagstat with 40 million reads, and input data was scaled to AjFoxO data using genomeCoverageBed, then converted to bedGraph. AjFoxO files were converted directly to bedGraph without scaling. BigWig files were generated and normalized [log2(AjFoxO/input)] using bigwigCompare (default settings except for –pseudocount 0.1 and –bs 1) [37].

Dual luciferase reporter assay

To verify the regulatory effect of AjFoxO transcription factor on AjLC3 and AjATG4 genes, fluorescent reporter vectors for wild-type and mutant-type AjLC3 and AjATG4 were constructed. The steps are as follows: First, the pGL3 dual enzyme digestion vector was prepared by digestion with XhoI and HindIII. Then, the promoter sequences containing the conserved FoxO protein binding site (TGTTT) on AjLC3 and AjATG4 (Supplementary Fig. 19, underlined sections) were amplified from A. japonicus genomic DNA using the primers listed in Supplementary Table 2 (homology arm underlined) and purified using a PCR product purification kit (Cat# B610363-0050, Sangon Biotech, Shanghai, China). The PCR products were ligated to the pGL3 plasmid using a pEASY®-Basic Seamless Cloning and Assembly Kit (Cat# CU201, Transgene, Beijing, China) to generate the recombinant pGL3-AjLC3-promo-WT and pGL3-AjATG4-promo-WT vectors. The structure of these vectors was confirmed by DNA sequencing (Sangon Biotech). After confirming the sequencing results, the vectors were transformed into E. coli DH5α for large-scale amplification. These amplified plasmids served as templates for further mutation. Using mutant primers detailed in Supplementary Table 2 (homology arm underlined) and the Mut Express® II Fast Mutagenesis Kit (Cat# C214-01, Vazyme, Nanjing, China), PCR amplification was carried out. The mutant primers were designed via a website (https://crm.vazyme.com/cetool/singlepoint.html). Upon completion of amplification, the products were treated with Dpn I enzyme to digest methylated plasmid templates, ensuring that only mutated sequences were retained. The amplified products were then recombined with homologous arms at both ends and transformed into Trans1-T1 cell (Cat# CD501-02, Transgene, Beijing, China). Single colonies were selected and sequenced for validation. After verification, large-scale plasmid amplification was performed to yield pGL3-AjLC3-promo-MUT and pGL3-AjATG4-promo-MUT vectors. Next, the pcDNA3.1-Flag vector was prepared by double enzyme digestion using EcoRI and XhoI. The AjFoxO sequence was amplified from A. japonicus cDNA using primers from Supplementary Table 2 (homology arms underlined), designed to match the homologous arms of the double-digested plasmid. The recombinant pcDNA3.1-AjFoxO-Flag plasmid was obtained following the homologous recombination method.

For the Dual-Luciferase Reporter assay, 20 ng of pRL-TK vector (containing the Renilla luciferase gene as an internal control), 300 ng of pcDNA3.1-AjFoxO-Flag or pcDNA3.1-Flag, and 200 ng of pGL3 or one of the constructed pGL3 vectors were transfected into HEK-293T cells in 24-well plates using Lipofectamine 6000 (Cat# C0526, Beyotime, Beijing, China). After 48 h of transfection, cells were harvested, and both firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit (Cat# E1910, Promega, WI, USA) and a Tecan Spark 10 M multimode microplate reader. Relative luciferase activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity. The results represent the average of three independent assays.

DNA electrophoretic mobility shift assay (EMSA)

Biotin-labeled wild-type and mutant-type AjLC3 and AjATG4 probes were synthesized by Youkang Biotech (Hangzhou, China) (Supplementary Table 2). These probes contained the conserved AjFoxO binding site “CTGTTT” or its mutated sequence, along with 20–30 bases upstream and downstream to ensure the GC content was between 40% and 60%. The sequence information was submitted to Youkang Biotech for probe synthesis, HPLC purification, 3’ biotin labeling, and annealing to obtain biotin-labeled complementary double-stranded probes. DNA EMSA experiments were performed following the manufacturer’s instructions from the LightShift™ Chemiluminescent DNA EMSA Kit (Thermo Fisher Scientific, USA) [38].

Biotin-labeled probes (wild-type and mutant-type) and purified rAjFoxO (500 ng) were combined and incubated in a binding buffer at room temperature for 20 min. For super-shift tests, rAjFoxO was pre-incubated with AjFoxO antibodies for 20 min before incubation with biotin-labeled probes for 20 min. DNA-protein complexes were separated on a 6% non-denaturing polyacrylamide gel at 90 V for 1 h. After electrophoresis, wet transfer was performed using a mini-transfer tank (Bio-Rad Laboratories, Inc.) in pre-cooled 0.5× TBE buffer (Cat# R0223, Beyotime, Beijing, China) at 300 mA for 30 min at 4 °C onto a nylon membrane. The membrane was crosslinked with UV light (254 nm) for 15 min, blocked with 15 mL blocking solution (Cat# GS009B, Beyotime, Beijing, China) for 15 min, and treated with horseradish peroxidase-linked streptavidin (dilution 1:2000) in blocking solution for 15 min at room temperature. After three washes with ddH2O, the membrane was treated with NcmECL Ultra (Cat# P10200, NCM, Suzhou, China) and imaged using an Aplegen Omega Lum C imaging system (Gel Company, San Francisco, CA, USA).

Statistical analysis

All data are expressed as the mean ± SD from three independent replicate studies. The Student’s t-test was used to compare results between two groups, including protein expression levels, ROS content, regenerated intestinal area, and fluorescence intensity between the control group and other drug treatment groups at the same regeneration stage. One-way ANOVA followed by Duncan’s post-hoc test was used to detect statistical significance among three or more groups. For example, the analysis of protein expression grayscale values, regenerated intestinal area, and fluorescence intensity at different stages (control, 2-dpe, 7-dpe, 12-dpe, 20-dpe, 28-dpe) of intestinal regeneration, or protein grayscale analysis, ROS content analysis, tissue fluorescence intensity analysis, and regenerated area analysis of different drug treatment groups (DMSO, APO, APO + MK2206) at the same time point. A p-value < 0.05 was considered statistically significant. Statistical analyses were conducted using Prism version 7.0 (GraphPad Software).