Animals and the IBD mannequin
All mouse experiments on this examine had been accredited by the Committee on the Use of Stay Animals in Educating and Analysis on the Southern Medical College (approval quantity: SMUL2022002) and had been carried out following the Tips for Care and Use of Laboratory Animals of Southern Medical College. Particular pathogen-free (SPF) wholesome male C57BL/6J mice (6–8 week-old) had been procured from Guangdong Zhiyuan Biomedical Know-how Co., Ltd. and housed within the SPF animal facility of Southern Medical College. The mice had been maintained beneath fixed temperature, humidity, and 12/12 h gentle/darkish cycle circumstances and supplied advert libitum entry to meals and water.
After 1 week of adaptation, the mice within the IBD mannequin group had been allowed free entry to a 3% DSS aqueous resolution (MB5535, meilunbio) for 7 days. Mice had been handled with 300 µL of PBS or different interventions comparable to E. coli (109 CFU/mL), E. coli-EVs (30 µg), L. plantarum (109 CFU/mL), L. plant-EVs (30 µg) and watermelon supernatant every day because the management or experimental teams.
A mix of vancomycin hydrochloride (10 mg/mL, 200 µL, V820413, Macklin), metronitridin (10 mg/mL, 200 µL, M813526, Macklin), neomycin (20 mg/mL, 200 µL, XY-K1226, X-Y Biotechnology) and ampicillin (20 mg/mL, 200 µL, A830931, Macklin) was used for 7 days to clear the intestine microbiota of the mice.
Cell tradition
Human regular colonic epithelial (NCM 460) cells had been cultured in Dulbecco’s modified Eagle medium (DMEM, C11995500BT, Gibco) supplemented with 10% fetal bovine serum (FBS, 164210-50, Procell), 10 U/mL penicillin (PB180120, Procell), and 0.1 mg/mL streptomycin (PB180120, Procell). The cells had been maintained at 37 °C with 5% CO2 in an incubator.
Cell scratching assay
NCM 460 cells had been plated at 2.5 × 105/properly in a 12-well plate. After the cells had adhered in a single day, 2 scratches per properly had been made with a 200 µL pipet tip. Then, the cells had been washed twice with PBS. Subsequent, 1 mL of serum-free medium containing 3% DSS and 10 µg/mL L. plant-EVs or E. coli-EVs was added to every properly. Cell migration was evaluated at 0 h, 12 h and 24 h with a Nikon inverted microscope, and the scratch areas had been quantified with ImageJ software program.
Western blot
NCM 460 cells had been plated at 3.5 × 105/properly in a 6-well plate. After that they had allowed to stick in a single day, the cells had been washed twice with PBS. Subsequently, 2 mL of serum-free medium containing 3% DSS and completely different concentrations of L. plant-EVs (5, 10, 15, 20 µg/mL) was added to every properly. The cells had been cultured with L. plant-EVs for 0 h, 12 h and 24 h. Then, protein samples from NCM 460 cells had been obtained by way of RIPA lysis buffer (WB3100, NCM Biotech) containing 1% protease and phosphatase inhibitor cocktail phenylmethanesulfonyl fluoride (P002, NCM Biotech). The whole protein focus was decided utilizing the BCA Protein Assay Equipment (FD2001, Fdbio Science). Equal quantities of proteins (30 µg per group) had been loaded for 4-20% Protein Pre made Gel (ET15420Gel, ACE Biotechnology) and subjected to electrophoresis. After transferring protein to polyvinylidene difluoride (PVDF) membranes, the blots had been blocked with 5% Bovine Serum Albumin (BSA, FD0030, Fdbio Science) for two h at room temperature. The membranes had been incubated with main antibodies in a single day at 4 °C, after which with the corresponding secondary antibodies for two h at room temperature. All main antibodies had been obtained from Affinity Biosciences. All secondary antibodies had been obtained from Fdbio science. GAPDH (ab201822, Abcam) and β-tublin (AF7011, Affinity Biosciences) was used for normalization. The bands had been quantified utilizing ImageJ software program.
Bacterial DNA extraction
DNA extraction from all fecal samples was carried out by way of QIAamp Quick DNA Stool Mini Equipment (51604, Qiagen) with a modified protocol following the producer’s directions particular for the fecal samples. The amount and purity of all of the extracted DNA had been checked with a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific) after which saved at − 20 °C.
Quantitative real-time PCR (qRT-PCR)
The whole mobile RNA was extracted through the use of TRIzol agent (15596026, Invitrogen) and synthesized into cDNA by way of a reverse transcription package (RR036A, Takara). Subsequently, the SYBR® Inexperienced Professional Taq HS Premix (AG11701, Correct Biology) was added for gene expression evaluation. The primers used for qRT-PCR are described Desk S1.
Annexin V/PI staining and cell cycle detection
NCM 460 cells had been plated at 3.5 × 105/properly in a 6-well plate. After that they had allowed to stick in a single day, the cells had been washed twice with PBS. Subsequently, 2 mL of serum-free medium containing 3% DSS, completely different concentrations of L. plant-EVs (5, 10, 15, 20 µg/mL) and PBS was added to every properly. The cells had been cocultured with L. plant-EVs for 0 h, 12 h, 24 h.
An Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Equipment (40302ES50, Yeasen Biotechnology) was used to establish apoptotic and necrotic cells. Following the producer’s directions, 1-5 × 105 dwell cells had been harvested and stained (10 µL PI and 5 µL Annexin V-FITC) for 15 min at nighttime at room temperature, earlier than evaluation by way of the BD Fortessa multidimensional high-definition move cytometer. Within the ensuing dot plots, the X-axis represents the depth of inexperienced fluorescence (Annexin V), and the Y-axis represents the depth of purple fluorescence (PI). The cell inhabitants distribution evaluation was divided into 4 quadrants: Annexin V-/PI-, Annexin V+/PI-, Annexin V-/PI+, and Annexin V+/PI + represented the traditional state, early apoptosis, necrosis, and late apoptosis, respectively.
Enzyme-linked immunosorbent assay
Protein samples from NCM 460 cells had been obtained utilizing RIPA lysis buffer containing 1% protease and phosphatase inhibitors. MPTP protein ranges had been measured through the use of the Human MPTP ELISA Equipment (DG12601H, Dogesce).
Bacterial development situation
E. coli was grown at 37 ℃ in Luria-Bertani (LB) Broth (HB0128, Hopebio) with steady shaking at 160 rpm for 18 h. L. plantarum was grown anaerobically at 37 ℃ in de Man, Rogosa and Sharpe (MRS) broth (HB0384-1, Hopebio) supplemented with 1% D-(+)-Maltose monohydrate (D813131, Macklin) and 1% yeast extract (050090, HuanKai Biology) for 12 h.
Fruits supernatant promoted bacterial development
The MRS broth was supplemented with 1‰ cysteine and 1% PBS, or the supernatant of fruits comparable to pitaya, peach, and watermelon. L. plantarum was inoculated into the medium, and after 2, 4, 8, 12, and 24 h of tradition, the bacterial tradition was centrifuged at 3,000 × g for 10 min. After eradicating the supernatant, an equal quantity of PBS was added to resuspend the micro organism, and the OD600 values had been measured.
Watermelon supernatant promoted the discharge of BEVs
The MRS broth was supplemented with 1% cysteine, 1% PBS, or watermelon supernatant. L. plantarum was then inoculated into the medium and cultured for 8 h. L. plant-EVs had been remoted by ultracentrifugation, and protein quantification was carried out utilizing the BCA Protein Assay Equipment.
Isolation of BEVs
BEVs had been remoted from the bacterial tradition. The micro organism had been first eliminated by centrifugation at 300 × g for 10 min, then 3,000 × g for 20 min, and eventually 12,000 × g for 30 min at 4 ℃. The supernatants had been additional filtered with a 0.22 μm pore-sized filter, eradicating residual micro organism and mobile particles. BEVs had been remoted by ultracentrifugation of the filtrate at 135,000 × g for 70 min at 4 ℃ and resuspended in PBS. BEV samples had been saved at -80 ℃ for subsequent research. The protein quantification of BEVs was carried out with the BCA Protein Assay Equipment.
Characterization of BEVs
The morphology and the scale distribution of L. plant-EVs had been noticed by Transmission electron microscope (H-7650, Hitachi) and Nanoparticles monitoring evaluation (ZetaView PMX-120, Particle Metrix). The floor marker LTA (MA1-7402, Thermo Fisher Scientific) of L. plant-EVs was characterised by Western blot.
16 S rRNA gene sequencing
16S rRNA sequencing was carried out at Novogene (Beijing, China) utilizing NovaSeq 6000 system. Complete DNA was extracted from human fecal samples and saved at − 80°C. Later, the content material of the extracted whole DNA was quantified by Nanodrop, and the standard was checked by 1% agarose gel electrophoresis. For 16S amplicon sequencing, the variable V4 area of bacterial 16S rRNA was amplified with the primers V4F (5’-GTGTGYCAGCMGCCGCGGTAA-3’) and V4R (5’-CCGGACTACNVGGGTWTCTAAT-3’) and the PCR merchandise had been purified with a GeneJET Gel Extraction Equipment (K0691, Thermo Scientific). Sequencing libraries had been generated utilizing Illumina TruSeq DNA PCR-Free Library Preparation Equipment (Illumina) and index codes had been added. Eventually, the library was sequenced on an Illumina NovaSeq platform and 250 bp paired-end reads had been generated. Sequence denoising, or operational taxonomic unit (OTU) clustering, was carried out consistent with the QIIME2 dada2 evaluation course of or the evaluation means of Vsearch software program. On the amplicon sequence variant (ASV)/OTU degree, the space matrix of every pattern was calculated, and the variations in range and the importance of variations amongst completely different samples (teams) had been measured by numerous unsupervised sorting and clustering strategies, mixed with the corresponding statistical assessments comparable to PERMANOVA, ANOSIM, and PERMDISP.
RNA sequencing and evaluation
RNA samples from NCM 460 cells had been obtained utilizing AG RNAex Professional Reagent (AG21102, Correct Biotechnology). Complete quantities and integrity of the RNA had been assessed utilizing the RNA Nano 6000 Assay Equipment of the Bioanalyzer 2100 system (Agilent Applied sciences, CA, USA). After the library was certified, the completely different libraries had been pooling based on the efficient focus and the goal quantity of knowledge off the machine, then being sequenced by the Illumina NovaSeq 6000. The tip studying of 150 bp pairing was generated. The picture information measured by the high-throughput sequencer had been transformed into sequence information (reads) by CASAVA base recognition. We downloaded the reference genome and gene mannequin annotation information from the GENCODE database (https://www.gencodegenes.org). Index of the reference genome was constructed by way of HISAT2 (v2.0.5) and paired-end clear reads had been aligned to the reference genome by way of HISAT2 (v2.0.5). FeatureCounts (v1.5.0-p3) was used to depend the reads numbers mapped to every gene. Differential expression evaluation was carried out utilizing the DESeq2 R bundle (1.20.0). Gene Ontology (GO) enrichment evaluation of the differentially expressed genes was carried out by the clusterProfiler R bundle (3.8.1).
Medical samples
Wholesome donors and colitis sufferers
The pattern assortment protocol was accredited by the Ethics Committee of Nanfang Hospital, Southern Medical College (approval quantity: NFEC-2022-001). Wholesome donors and colitis sufferers had been chosen from Nanfang hospital based on normal pointers [31]. All of the stool samples had been saved at − 80 ℃.
Volunteers
Volunteers had been requested to eat quick fruit for 3 days and stool samples had been collected. Then, they had been assigned to eat recent watermelon for 7 days and stool samples had been collected once more. The feces earlier than and after watermelon consumption had been paired and saved at − 80 ℃. The pattern assortment was accredited by the Ethics Committee of Nanfang Hospital, Southern Medical College (approval quantity: NFEC-2022-001).
Statistical evaluation
All information had been offered as imply ± normal deviation (SD). One-way evaluation of variance (ANOVA) was used for comparisons amongst a number of teams. Pupil’s t-test was used for comparisons between two teams. When the information didn’t cross the normality and lognormality assessments, the Mann Whitney check was used for comparisons between two teams, whereas the Kruskal-Wallis check was used for comparisons amongst a number of teams. All of the statistical analyses had been performed utilizing GraphPad Prism 9.0. P worth of < 0.05 was thought of statistically vital. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
