Ethics approval and consent to participate

The study was approved by the Ethical Committee of Jiangsu University (2012258). Animal experiments were carried out following the U.K. Animals (Scientific Procedures) Act, 1986, and associated guidelines.

Cell culture

HucMSCs were obtained from newborn umbilical cords after securing ethical approval and maternal consent and identified following the previous method (Qiao et al. 2008; Kestendjieva et al. 2008). HucMSCs were cultured in α-MEM medium (Invitrogen, Grand Island, NY, USA). Mouse mononuclear macrophage leukemia cells RAW264.7 were purchased from Beiner Biotechnology Company (Beijing, China) and cultured in DMEM high glucose medium (Invitrogen) containing 10% fetal calf serum (FBS; Excell, Uruguay). Human myeloid leukemia mononuclear cells THP-1 were cultured in RPMI 1640 medium (Invitrogen) containing 10% FBS, which was also purchased from Beiner Biotechnology Company. All the above cells were cultured at 37 °C in humid air with 5% CO2. Cell morphology was observed using a general light microscope (Nikon, Japan).

Extraction and identification of exosomes

Exosomes were extracted by ultra-centrifugation method as previously described (Yuan et al. 2021; Yang et al. 2021). hucMSCs surface markers CD34, CD45, CD11b, CD73, CD105, and CD29 were identified by using the FACS Calibur (Beckman Coulter, USA). A transmission electron microscope (Philips, Amsterdam, The Netherlands) was used to observe the morphology of exosomes. Nanoparticle Tracking Analysis (NTA) was used to analyze the size distribution and concentration of exosomes by ZetaView (Malvern Panalytical, Malvern, UK). In addition, Western-blot was used to detect the expression of marker proteins Calnexin, CD9, CD81, HSP70, and Alix.

In vitro experiment

An in vitro experiment was conducted with LPS (Sigma Aldrich, USA), stimulating the inflammation of RAW264.7 and THP-1 cells. THP-1 cells were induced to macrophage-like phenotype by adding 50 ng/ml PMA (Sigma Aldrich) for 16 h before the experiment. The LPS group received only LPS stimulation, whereas, in the hucMSC-Ex group, hucMSC-Ex was added to the culture system following LPS stimulation. The concentrations of LPS and hucMSC-Ex used in this experiment were 1 μg/ml and 200 μg/ml, respectively.

In vivo experiment

Male BALB/c mice (20 ± 3 g) were purchased from the Animal Research Center of Jiangsu University (Zhenjiang, China). Mice were randomly allocated into three groups (n = 5/group): the negative control (NEG) group, the DSS-induced colitis (DSS) group, and the hucMSC-Ex-treated colitis (hucMSC-Ex) group. Mice in the NEG group were fed autoclaved water during the experiment, while mice in the DSS and hucMSC-Ex groups were fed autoclaved water containing 3% DSS (MP Biomedicals, USA). A total of 1 mg of hucMSC-Ex was administered by caudal vein to mice in the hucMSC-Ex group on the 3rd, 6th, and 9th day of modeling, while mice in other groups were injected with PBS. All mice were sacrificed when severe hematochezia and weight loss were observed in the DSS group. The disease progression of mice in the different groups was evaluated by weight change, DAI score, colorectal length, and weight ratio. After the mice were sacrificed, the general view of the spleen and colorectum of the mice was observed. The colorectal mucosa and spleen tissues of the mice in each group were separated, and RNA and protein of the tissues were extracted for subsequent experiments.

CCK8

RAW264.7 cells (5000 cells per well) were seeded in a 96-well plate for CCK8 assay. 1 μg/ml LPS and 200 μg/ml hucMSC-Ex were cultured with cells for 12 h. CCK8 reagent (Vazyme, Nanjing, China) was then added to each well and incubated away from light for 30 min. Absorbance was measured at 450 nm with a microplate reader (Thermo Fisher Scientific).

RNA extraction and qRT-PCR

RNA from the tissues and cells was obtained via TRIzol reagent (Invitrogen, USA) and chloroform extractions, followed by reverse transcription using HiScript® III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme). qRT-PCR was performed using AceQ® Universal SYBR qPCR Master Mix (Vazyme). mRNA expression was normalized to the internal control β-actin. The qRT-PCR primers are shown in Table 1.

Table 1 Primer sequences for qRT-PCRWestern-blot

Cell lysates were obtained using RIPA lysate (Thermo Fisher Scientific, MA, USA). Protein concentration was determined using the BCA protein detection kit (Vazyme). The sample (20 μg protein) was separated by 10% SDS-PAGE, then transferred to a PVDF membrane (Millipore, Billerica, USA), and incubated in a closed buffer solution (5% skim milk powder dissolved in Tris-buffered brine (TBS) with 0.1% Tween20) for 1 h. The membrane was then treated at 4 °C overnight with anti-Calnexin (1:500; Affinity, China), anti-CD9 (1:500; Affinity, China), anti-CD81 (1:1000; abcam, UK), anti-HSP70 (1:500; Affinity, China), anti-Alix (1:500; Affinity, China), anti-METTL3 (1:1000; abcam, UK), anti-YTHDF1 (1:1000; proteintech, China), Anti-PCNA (1:1000; abcam, UK), anti-Occludin(1:500; proteintech, China), anti-Claudin-1 (1:1000; proteintech, China), anti-SLC37A2 (1:500; proteintech, China) and β-actin (1:8000; Abclonal, Boston, MA, USA) antibodies. Secondary incubation of the membrane was performed using goats against rabbits/mice (1:10000; proteintech, China) at room temperature for 1 h. A chemical gel imaging system (GE Healthcare Life Sciences China, Beijing, China) was used to visualize protein bands and generate images.

Nitric Oxide (NO) measurement

The supernatant of cultured RAW264.7 cells was collected and centrifuged at 600 g for 5 min to remove dead cells for subsequent detection. According to the instructions provided by the manufacturer (Beyotime, China), NO production in cells was measured using the Griess method.

Immunofluorescence (IF)

RAW264.7 cells were fixed with 4% paraformaldehyde for 20 min, followed by 0.1% Triton X-100 for 20 min to break the membrane, then 5% bovine serum albumin (BSA) for blocking non-specific binding. The target antibodies were diluted (CD86, 1:300, Novus; CD206, 1: 200, CST; METTL3, 1:1000, Abcam; YTHDF1, 1:1000, Proteintech), and incubated with cells at 4℃ overnight, followed by the corresponding fluorescent secondary antibodies (FITC and Carolight594,1:200, Proteintech) at room temperature for 2 h. The nuclei were stained with DAPI (1:500, Sigma) at room temperature for 10 min away from light. The fluorescence was observed and photographed with a laser confocal microscope (Nikon, Japan).

Flow cytometry

RAW264.7 cells were collected into sterile EP tubes, centrifuged at room temperature at 500 g for 5 min, then the cell precipitates were re-suspended in 100 μl PBS. First, 0.5 μl CD86 antibody (Novus) was added to each tube and incubated at 4℃ for 30 min away from light. Then, 100 μl PBS and 100 μl IC Fixation Buffer (Thermo Fisher) were added, respectively. After incubating at 4℃ for 20 min away from light, the cells were suspended in 40 μl washing buffer, then 1 μl CD206 antibody (Biolegend) was added to each tube and incubated at 4℃ for 45 min-1 h. Finally, the cells were centrifuged and suspended in 200 μl PBS in each tube for immediate detection.

Immunohistochemical staining (IHC)

Paraffin-embedded mouse colorectal tissues were exposed to 3% hydrogen peroxide at room temperature for 30 min after dewaxing and then steamed in a citric acid buffer for 30 min to repair antigens. Non-specific antigens were blocked by covering the tissues with a 5% bovine serum albumin (BSA) solution. The primary antibody (METTL3, 1:1000, Abcam; YTHDF1, 1:1000, Proteintech; Slc37a2, 1:300, Proteintech) was incubated overnight at 4℃, followed by the goat and mouse secondary antibody (Bost Biological, Wuhan)) at 37 °C for 30 min. Then Strept Avidin Biotin Complex (SABC) was added and incubated at 37 °C for 30 min. Finally, 3,3′-Diaminobenzidine (DAB) substrate was applied to the sections and then re-stained with hematoxylin for observation.

m6A content analysis

As mentioned above, total RNA was extracted using TRIzol lysate (Invitrogen). RNA quality was assessed by NanoDrop (Thermo Fisher Scientific, USA). The m6A modification level of total RNA was examined via EpiQuik m6A RNA methylation quantitative kit (Epigentek Group Inc., USA). In simple terms, the assay well was coated with 200 ng RNA and an m6A standard, followed by the application of antibody solution and antibody detection solution. The m6A level was quantified through colorimetry by reading the absorbance of each well at 450 nm.

Methylated RNA immunoprecipitation sequencing (MeRIP-Seq)

The m6A-IP-Seq service was provided by CloudSeq Inc. (Shanghai, China). Total RNA was subjected to immunoprecipitation with the GenSeq® m6A-IP Kit (GenSeq Inc.) by following the manufacturer’s instructions. Briefly, RNA was randomly fragmented to ~ 200 nt by RNA Fragmentation Reagents. Protein A/G beads were coupled to the m6A antibody by rotating the sample at room temperature for 1 h. The RNA fragments were incubated with the bead-linked antibodies and rotated at 4 °C for 4 h. After incubation, the RNA/antibody complexes were washed several times, and then, the captured RNA was eluted from the complexes and purified. RNA libraries for IP and input samples were then constructed with GenSeq® Low Input Whole RNA Library Prep Kit (GenSeq, Inc.) by following the manufacturer’s instructions. Libraries were qualified using an Agilent 2100 bioanalyzer and then sequenced in a NovaSeq platform (Illumina).

mRNA stability measurement

The transcription of RAW264.7 cells was blocked by adding Actinomycin D with a concentration of 5 μg/ml into the cell well and incubating for 0 h, 2 h, and 4 h, respectively. Cells were collected, RNA extracted, and mRNA levels measured by qRT-PCR.

MeRIP

The m6A modification of Slc37a2 was determined using the Magna EpiQuik CUT&RUN m6A RNA Enrichment (MeRIP) kit (Epigentek, USA) according to the manufacturer’s instructions. In brief, 10 μg total RNA was taken for m6A immunoprecipitation, and 1/10 was retained as the input control group. First, an immune capture solution containing m6A antibody, non-immune IgG, affinity beads, immune capture buffer, and RNA samples was prepared and swirled at room temperature for 90 min to immunologically capture m6A RNA. The RNA was then cleaved using a lyase mixture, and the m6A-containing RNA was purified by adding protease K and RNA purification solution to the immunoprecipitation complex to remove excess proteins. Finally, the immunoprecipitated m6A RNA was recovered with an elution buffer and its level was detected by qRT-PCR.

RNA immunoprecipitation (RIP)

RNA immunoprecipitation kit (Geneseed, Guangzhou, China) was used to detect the combination of Scl37a2 and YTHDF1 according to the manufacturer’s instructions. Briefly, 3 × 107 RAW264.7 cells were collected, cleaved with RIP lysis buffer, and incubated with magnetic beads conjugated with antibodies against IgG (proteintech, China) or YTHDF1(proteintech, China). The coprecipitated RNA was detected by qRT-PCR.

Dual-luciferase reporter assay

The luciferase reporter vectors of Slc37a2 3’UTR m6A modification sites were constructed, and gene fragments carrying mutant or wild type of Slc37a2 m6A modification sites were cloned into pGL3 alkaline vector (Fenghbio, China). It was co-transfected into RAW264.7 cells with METTL3 overexpressed vector or simulated vector. Cells were harvested 48 h later and firefly and Renera luciferase activity were measured using the Dual-Luciferase Reporting and Detection System Kit (Vazyme).

Cell transfection

si-RNA specifically targeting METTL3 (si-METTL3) and the corresponding non-targeting control siRNA (si-NC) were purchased from Genepharma, China. Transfections of siRNAs were performed using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. The si-METTL3 sequence is shown in the table below (Table 2).

Table 2 Sequences of si-METTL3Statistical analysis

All data were shown as mean ± standard deviation (SD). Each experiment was repeated three times. Statistical analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). Comparisons between multiple groups were assessed by student t-test or one-way ANOVA with the Bonferroni post hoc test. P < 0.05 was considered significant.