Animal experiments
All animal experimental procedures were carried out in accordance with the National Institutes of Health guidelines and were approved by the local Animal Care and Use Committee of the Nanfang hospital of Southern Medical University (Guangzhou, China). Six- to eight-week-old specific pathogen-free male C57BL/6 mice were purchased from the animal center of Nanfang Hospital of Southern Medical University (Guangzhou, China). TLR2-/- and IL-10-/- mice were purchased from Shanghai Model Organisms Center, Inc. All mice were housed under controlled temperature and humidity conditions with a 12-hour light-dark cycle, had free access to food and water and were fasted overnight before the experiment.
The model of intestinal I/R was established as described in our previous study [13]. Briefly, mice were anesthetized with 4% isoflurane, and a non-invasive microvascular artery clip was placed on the superior mesenteric artery (SMA) for 60 minutes (min) followed by clip removal for reperfusion of 120 min or 5 days. During the study period, body temperature was maintained at 37 °C with a heating pad and liquid resuscitation was performed by injecting 0.5 ml of physiological saline subcutaneously, just after reperfusion.
Extraction and culture of organoids and the establishment of hypoxia-reoxygenation (H/R) models in vitro
The extraction and culture of small intestinal organoids was performed as previously described [14, 15]. The separated intestinal crypts were fixed onto the bottom of the dish with Matrigel (STEMCELL Technologies Inc., Shanghai, China) drops and covered with IntestiCult medium (STEMCELL Technologies Inc.). For the establishment of the organoid H/R model, the organoids were placed in a humid, anaerobic environment at 37 °C for 12 hours and then placed in an aerobic environment containing 5% CO2 in a 37 °C incubator for 4 hours.
Patient Samples
From 2019 September to 2020 January, we recruited consecutive patients who underwent elective cardiac valve replacement or coronary artery bypass graft under cardiopulmonary bypass (CPB) at the Department of Cardiac Surgery, and healthy volunteers who underwent physical examination at the Department of Health Management, in Southern Medical University Nanfang Hospital, Guangzhou, China. Participants were not included if they (1) aged <18 or >75 years, (2) had chronic kidney disease, (3) had chronic digestive system diseases, previous gastrointestinal surgery, or confirmed or suspected intestinal ischemia/necrosis, (4) used antidiarrheals, laxatives or prebiotics within one week, or used antibiotics within 3 months. A total of 26 participants were enrolled, including 20 patients who underwent CPB and 6 healthy volunteers. There was no significant difference between the patient group and the healthy group in terms of demographic characteristics. The study protocol was approved by the Ethical Committee of Nanfang hospital, Southern Medical University (approval number NFEC-202009-k2-01).
Fecal and blood samples: Blood samples were collected at preoperatively (T0) and at 0 h (T1), 2 h (T2), 6 h (T3), 12 h (T4) and 24 h (T5) after surgery in EDTA plasma tubes as well as in serum separator tubes for analyses of intestinal fatty acid-binding protein (IFABP) and citrulline respectively. Fecal samples were collected at preoperatively, and the relative abundance of L. murinus was quantified by real-time PCR. IFABP and citrulline in the plasma samples were measured at multiple time points, to allow for of (T3-T0) concentration differences, respectively by means of a human IFABP ELISA Kit (Bio-Swamp, Wuhan, China) and Citrulline ELISA Kit (USCN, Wuhan, China), following the manufacturer's instructions. The gastrointestinal complication score of the patient on the seventh day after surgery was performed according to the acute gastrointestinal injury (AGI) standard described previously[16]. The detection of L. murinus, IFABP, citrulline and AGI scores were performed by researchers blinded to the group allocation.
Microbe analysis
Feces were collected with sterilized 1.5 ml tubes before intestinal I/R and frozen at − 80 °C until DNA extraction. All extracted DNA was stored in − 20 °C until further test. The extracted fecal DNA concentration was diluted to 10 ng/μl and quantitative real-time polymerase chain reaction (PCR) was processed according to 16S rRNA primers, Firmicutes primers and Bacteroidetes primers, the primers were listed in Table S1. Moreover, 16S rRNA abundance from blood was normalized to host 18S.
16S rRNA gene sequencing
Microbial DNA from preoperative fecal samples of mice were extracted using the classical phenol-chloroform extraction method as previously described [17, 18]. Next, the V4 region of 16S rDNA was amplified using barcode specific primers (V4F, 5′-GTGTGYCAGCMGCCGCG GTAA-3′ and V4R, 5′-CCGGACTACNVGGG TWTCTAAT-3′), and the amplified products were quantified using QuantiFluorTM and mixed in equal amounts.
All samples were paired-end sequenced on the Illumina Hiseq PE2500 sequencing platform. Low-quality reads were filtered after quality control, and high-quality reads were assigned to operational taxonomic units (OTUs) of ≥ 97 % similarity using UPARSE pipeline [19]. QIIME was applied to analyze the alpha and beta diversities, based onweighted and unweighted UniFrac distances successively Metastasis (version 20090414) and Linear discriminant effect size (LEfSe) software[20] (version 1.0) were used to explore biomarker features in each group. The KEGG pathway analysis of the OTUs was performed using Tax4Fun[21] (version 1.0) and was performed using the OmicShare tools, a free online platform for data analysis (www.omicshare.com/tools). The calculated p-value was gone through FDR Correction, taking FDR ≤ 0.05 as a threshold.
Bacterial Strains and Growth Conditions
L. murinus freeze-dried powder (BN, Beijing, China) was dissolved with 0.5 ml MRS medium (HKM, Guangdong, China), then the bacteria liquid was coated on the blood plate, and bacterial colonies appeared after about 24 h. Single colonies were picked into MRS medium, were incubated at 37 °C under anaerobic conditions, and OD600=0.6-0.7 of cultures was measured until mid-log phase after 12-16 h of growth, at which time the colony count was 6.8×108 CFU/mL by plate count. Frozen stocks of L. murinus (in MRS medium with 25% glycerol) were prepared, stored at -80 °C for further experiments. 50 µl frozen stocks of L. murinus were added to 5 ml MRS medium and incubated at 37 °C under aerobic conditions for 12 h, and then used for gavage of mice. In order to evaluate the total amount of L. murinus DNA in cecum and stool samples, was quantified by quantitative real-time PCR using the following primers: L. murinus[22], LactoM-F (5’-TCGAACGAAACTTCTTTATCACC-3’) and LactoM-R (5’-CGTTCGCCACTCAACTCTTT-3’).
Experimental design
L. murinus pretreatment experiment: As shown in Additional file 1a, mice were randomly divided into 3 groups: (1) Sham group; (2) I/R group; (3) I/R+L. murinus group. The sham group of mice were gavaged daily for 7 days with 200 µl MRS medium and then intestinal I/R was performed without SMA occlusion. The I/R group of mice were gavaged daily for 7 days with 200 µl MRS medium, and intestinal SMA was occluded for 60 min followed by 120 min reperfusion. The I/R+ L. murinus group of mice were gavaged daily for 7 days with 200 µl 6.8×108 CFU/ml L. murinus prior to establishing intestinal I/R. In addition, to explore a single strain of L. murinus alleviates intestinal I/R-induced intestinal injury, mice were allocated randomly to 2 groups: Group ABX+I/R, in which antibiotics (ABX) was administered 1w before intestinal ischemia; Group ABX+I/R+L. murinus, treatment of mice with ABX for a week, and then mice were gavaged daily for 7 days with 200 µl 6.8×108 CFU/ml L. murinus prior to establishing intestinal I/R.
Macrophage depletion experiment: Mice were allocated randomly to 4 groups: (1) I/R group; (2) PBS+Lipo+I/R group; (3) Clodronate-Lipo+I/R group; (4) Clodronate-Lipo+L. murinus+I/R group. I/R group of mice, in which intestinal SMA was occluded for 60 min followed by 120 min reperfusion was established as described previously; PBS+Lipo+I/R group of mice, which were intraperitoneally injected with 200 µl empty liposomes (Yeasen, Shanghai, China) 24 h before establishing I/R; Clodronate-Lipo+I/R group of mice, which were intraperitoneally injected with 200 µl clodronate liposomal (Yeasen, Shanghai, China) 24 h before establishing I/R model; Clodronate-Lipo+L. murinus+I/R group of mice, which were given treatment of L. murinus and clodronate liposomal injection before establishing I/R (Additional file 1b).
To explore L. murinus alleviates intestinal injury through TLR2 on the surface of macrophages, wild type (WT) mice were randomly divided into the following groups (Additional file 1c): (1) Sham; (2)I/R; (3)I/R+L. murinus. TLR2-/- mice were randomly divided into the following groups: (1)I/R; (2)I/R+L. murinus.
To explore L. murinus promotes the release of IL-10 from macrophages through TLR2 to alleviate intestinal I/R injury, wild-type (WT) mice were randomly divided into the following groups (Additional file 1d): (1)I/R; (2)I/R+L. murinus. IL-10-/- mice were randomly divided into the following groups: (1)I/R; (2)I/R+L. murinus.
To explore L. murinus promotes the release of IL-10 by macrophages through TLR2 to reduce organoid H/R injury, we established the transwell co-culture system of macrophages separately extracted from WT, or TLR2-/- or IL-10-/- mice and small intestinal organoids extracted from WT mice. In our experiment, the transwell chamber (0.4 μm pore size polyester membrane; Corning, Inc.) were placed in a 6-well culture plate, small intestinal organoids were planted in the upper chamber, and macrophages were planted in the lower chamber. The 200 µl supernatant of L. murinus stimulates the upper chamber to further confirm the L. murinus may require the participation of macrophages to improve the intestinal injury in vitro.
Fecal microbiota transplantation
Fecal microbiota transplantation (FMT) was performed according to the modified method described previously [18]. Briefly, 6-8-week-old male C57BL/6 mice were given antibiotics (vancomycin, 100 mg/kg; neomycin sulfate 200 mg/kg; metronidazole 200 mg/kg; and ampicillin 200 mg/kg) intragastrically once each day for 1 week to deplete the gut microbiota (receptor mice). Preoperatively collected feces of sensitive and resistant mice or low L. murinus patient feces and high L. murinus patient feces (donor mice) were resuspended in PBS at 0.125 g /ml. An amount of 0.1 ml of the solution was administered to mice in the corresponding groups orally via gastric gavage tube 1 week. All the mice had free access to food and water, and mice were performed intestinal ischemia 1 h and reperfusion 2 h surgery after1 weeks of transplantation, and then blood, ileum, kidney, lung and liver samples were harvested in a sterile manner for further examination.
Gene expression analysis
A reverse transcript enzyme (TOYOBO, Tokyo, Japan) was applied to prepare cDNA according to the manufacturer’s protocol. The real-time PCR reaction was performed using the ABI Q5 real-Time PCR System with SYBR Green detection protocol (TOYOBO, Tokyo Japan). The expression of target genes in mice were normalized to house-keeping gene 18S using the 2-^^CT method. The target genes primers were listed in Additional file 2
Protein expression and biochemical analysis
The endotoxin level was measured via commercial kit (GenScript, Nanjing, China). Plasma AST was determined manually with a commercial kit (KeyGene, Nanjing, China). The protein level of IL-10 in the ileum was measured by IL-10 ELISA kit (ABdonal, Wuhan,China).
Histological staining
Ileum, liver, lung and kidney samples tissue were collected and fixed in 4% paraformaldehyde. Then, the samples were embedded in paraffin, 5-μm-thick sections were sliced and were stained with hematoxylin-eosin (HE) according to the experimental protocol. The degree of ileum injury after reperfusion was evaluated using a modified Chiu method [23] according to changes of the intestinal mucosa villus and glands. Liver, lung and kidney tissue histological damage were assessed according to previously described scoring system [24, 25, 26]. Images were captured at 200 X with Olympus fluorescence microscope. (Olympus, Japan).
Immunofluorescence
Paraffin section samples were generated as above, blocked for1 h, and incubated overnight at 4℃ with anti-ZO-1 antibody (Abcam, Cambridge, MA, USA) and anti-Occludin antibody (Abcam, Cambridge, MA, USA). Tissues were then washed, stained with DAPI for 10 min, and Images were captured at 200 X with fluorescent microscopy (Olympus, Japan). Quantification of the fluorescence intensity of ZO-1 and Occludin staining were performed by automated image analysis in five randomly chosen 200 X fields of each sample.
Peritoneal Macrophage Collection
Injection of 4 ml of normal saline solution into the peritoneum was used for peritoneal macrophage collection, and the mice's abdomen was gently rubbed for 2 min to make the liquid flow in the abdominal cavity. The peritoneal fluid was sucked out and transferred into a centrifugal tube with a glue-head dropper. The amount of each suction was about 4 ~ 5 ml. The collected peritoneal lavage fluid was centrifuged at 1000 r / min for 10 min and the supernatant was removed, then further flow experiment was carried out.
Flow Cytometry and Antibodies
Peritoneal macrophages were washed and stained with 0.25 µg of anti-mouse F4/80 Antigen FITC (Invitrogen eBioscience, USA), PE-Cy7 anti-mouse CD45 (BD Bioscience, USA), APC anti-mouse CD206 (Invitrogen eBioscience, USA), anti-mouse CD11C (Invitrogen eBioscience, USA), BV421 anti-mouse CD282(TLR2)(BD Bioscience, USA) and BV510 anti-mouse IL-10 (BD Bioscience, USA). F4/80+CD45+ served as a macrophage marker, with F4/80+CD45+CD206+CD11C- cells considered to be M2 macrophages and F4/80+CD45+CD206-CD11C+ cells considered to be M1 macrophages. Flow cytometric analysis was performed on FACS Calibur (BD Biosciences, USA) instruments and analyzed using FlowJo software (Tree Star Inc.).
Detection of organoid injury by lactate dehydrogenase (LDH) assays
The LDH kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to detect the level of LDH in the culture medium to assess organoid damage. The detection of LDH was carried out based on the manufacturers’ protocols.
Statistical analysis
All analyses were performed using GraphPad Prism software (version 7.0). Data are presented as means ± standard error of mean (SEM). Statistical analyses were performed using two-sided Student’s t tests or by one-way analysis of variance (ANOVA) as indicated in the figure legends. p values <0.05 were considered statistically significant.