The male Sprague-Dawley rats (10–12 weeks) were purchased from The Fourth Military Medical University (Xian, Shaanxi, China). The rats were housed in a pathogen‐free rat colony (temperature, 25 ± 2°C; relative humidity, 45%‐60%; lighting cycle, 12 h/day, 08:00‐20:00 for light) and had free access to food and drinking water. The normal chow diet was purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China). After superovulation of rats by an intraperitoneal injection of 10 IU PMSG (Sigma, St Louis, MO, USA), the female rats were mated. The pregnant rats were selected for experiments. The animal experiments were all approved by the Research Ethics Committee of Northwest A&F University (2011-31101684).
For the pregnant and lactation rat experiments (Fig. 1A), a total of 30 pregnant rats were randomly divide into 3 groups, including control, melatonin (Mel) and FMT group. Control and FMT groups: rats received a basal diet and normal drinking water; Mel group: a basal diet and drinking water containing melatonin (0.2 g/L; M5250, Sigma, MO, USA). After 3 days of melatonin supplementation, the fecal contents of rats were collected and diluted with phosphate buffer saline (PBS) through modified procedure according to precious method [3,23,24]. Briefly, fresh fecal samples were collected from Mel group in sterile PBS (100 mg/mL). The suspension was homogenized by vortex for 1 min and centrifuged at 1000 g for 3 min. The resulting supernatant was collected into new sterile tubes and used for oral gavage. The fecal contents dilution (0.1mL/rat) was feed into rats in FMT group each day until the day 5 before delivery and the day 3 after delivery, respectively. Five rats were sacrificed to collect duodenum, jejunum, ileum and colon in each group. These intestinal tissues were fixed in 2.5% glutaraldehyde solution and 2.5% paraformaldehyde solution and saved at -80°C. The 30 fresh fecal samples on the day 5 before delivery and the day 3 after delivery were used for bacterial 16S rDNA gene amplicon sequencing analysis were stored at -80°C before genomic DNA extraction.
For the weaned rat experiments (Fig. 3A), a total of 80 suckling rats at 11 days of age (weaned at 21 days of age) were randomly selected for four groups including control, Mel, FMT and FC group. Control and FMT groups: suckling rats fed a basal diet and normal drinking water; Mel group: suckling rats fed a basal diet and drinking water containing melatonin (0.2 g/L); FC group: lactating rats received a basal diet and drinking water containing melatonin (0.2 g/L). After 3 days of melatonin supplementation, the fecal contents dilution (0.1mL/rat) was feed into suckling rats in FMT group each day until 24 days of age. Similarly, five rats were sacrificed to collect serum, duodenum, jejunum, ileum and colon in each group. The 20 fresh fecal samples on the day 3 after weaning were used for bacterial sequencing analysis.
Hematoxylin and eosin staining
Hematoxylin and eosin (H&E) staining was performed as previously described . Briefly, the rat intestinal tissue, including duodenum, jejunum, ileum and colon, were fixed with 4% paraformaldehyde-PBS overnight, and then dehydrated and embedded in paraffin blocks. The 5 μm sections were deparaffinized, hydrated and stained with H&E. Intestinal villus height and crypt depth were measured using ImageJ software.
Intestinal antioxidant capacity
The levels of malondialdehyde (MDA) and total antioxidant capacity (TAOC) in intestinal tissues were measured by spectrophotometric methods according to manufacturer instructions of assay kits (Nanjing Jiancheng, Nanjing, China). All samples were measured by a UV/visible spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan).
ROS generation assay
The fresh intestinal tissues were collected and frozen at -80°C. The samples were sliced into 5-10 μm pieces and stained with DHE at 37°C for 30 min. The sections were washed on time with PBS for 10 minutes and stained with DAPI. Likewise, intestinal epithelial cells were washed 3 times with PBS and incubated with 10 μM DCFH-DA (Invitrogen, Eugene, OR, USA) for 30 min, and then washed with PBS and stained with DAPI. The sections and cells were captured with a Zeiss 710 laser scanning confocal microscope. The ROS assay kit (Beyotime Institute of Biotechnology, Shanghai, China) were used to measure ROS concentration according to the manufacturer’s instructions. Briefly, the crushed intestinal tissues were incubated with 10 μM DCFH-DA for 30 min, and then were measured by UV/visible spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan).
Transmission electron microscopy
The fresh ileum tissues or the rat intestinal epithelial cell line IEC6 cells were fixed in 2.5% glutaraldehyde solution at 4°C overnight and incubated with PBS containing 2% OsO4 for 4 h. The ileum tissues then were dehydrated in a graded series of ethanol (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 10 min at each step for 2 times and then transferred into mixture of alcohol and spurr resin (v:v = 1:1, v:v = 1:2, v:v = 1:3) for 12 h and spurr resin for 24 h for 2 times. Ultrathin sections were obtained using a diamond knife and stained with uranyl acetate, and then specimens were observed by TEM (JEM-1011, JEOL, Japan).
Immunohistochemistry stains were detected using IHC kit (MaiXin, China). Briefly, the tissues were cut into 3-10 μm long pieces and blocked with 1% w/v BSA for 1 h, and then anti-NF-κB antibody were incubated overnight at 4°C. Next, the samples were dehydrated in an ethanol (30-100%) gradient and treated with xylene to increase the transparency of slides.
The levels of IL-6, IL-1β, IL-10 and TNF-α were determined using ELISA kits (Raybiotech, GA, USA) according to the manufacturer’s instructions.
Intestinal permeability analysis
Intestinal permeability was assessed using the FITC-dextran (4-kDa) permeability assay. On 24 days of age, the weaned rats were starved for 12 h, then feed with FITC-dextran (4.4 mg/kg BW) by oral gavage. After 5 h, whole blood was collected and serum was isolated. Serum was diluted with equal volume of PBS and tested in duplicate. The pictures of FITC-dextran were captured with fluorometer.
Western blotting analysis
Total protein was isolated from intestinal tissues and cells by incubating in RIPA buffer containing protease inhibitor cocktail for 30 min and then centrifuged at 12,000 ×g for 10 min at 4°C to remove the precipitate. The total protein content was determined using the BCA Protein Assay Kit (Pierce), and 30 μg proteins were separated by a reducing SDS-PAGE electrophoresis on 10% or 15% Bis-Tris gels, transblotted onto nitrocellulose membranes and probed with different primary antibodies: GPx5 (18731-1-AP, 1 : 1000, Proteintech), SOD1 (10269-1-AP, 1 : 1000, Proteintech), SOD2 (13194S, 1 : 1000, CST), CAT (14097S, 1 : 1000, CST), HSP72, NF-κB (8242S, 1 : 1000, CST), LC3B (3868S, 1 : 1000, CST), P62 (23214S, 1 : 1000, CST), ATG7 (2631S, 1 : 1000, CST), Beclin1 (11306-1-AP, 1 : 1000, Proteintech), ZO-1 (ab221547, 1 : 1000, Abcam), Occludin (27260-1-AP, 1 : 1000, Proteintech), Claudin-1 (13050-1-AP, 1 : 1000, Proteintech), Bcl-2 (ab194583, 1 : 1000, Abcam), Bcl-XL (26967-1-AP, 1 : 500, Proteintech), Cleaved capsase-3 (ab49822, 1 : 1000, Abcam), PARP (13371-1-AP, 1 : 1000, Proteintech), Cytochrome C (10993-1-AP, 1 : 1000, Proteintech), β-actin (4967S, 1 : 1000, CST) and COX IV (ab202554, 1 : 500, Abcam). The gray values of the bands were measured by ImageJ software and then normalized to β-actin or COX IV content.
Cell culture and treatment
The rat intestinal epithelial cell line IEC-6 were purchased from the American Type Culture Collection (ATCC, USA). The cells were cultured in DMEM-F12 medium supplemented with 10% FBS (Life Technologies) in a humidified 5% CO2 incubator at 37°C. For H2O2 and melatonin treatments, cells were pretreated with melatonin (10 μM) for 24 h, followed by H2O2 (200 μM) stimulation for 2 h before they were used for the next assay.
Cell viability assay
IEC-6 viability was measured using Cell Counting Kit-8 (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer’s instructions.
Evaluation of autophagosome
IEC-6 were seeded in 24-well plates with 1 cm coverslips, and the cells grown to 80% confluence prior to transient transfection with the GFP-MAP1LC3B (Beyotime Institute of Biotechnology, Shanghai, China). After 24 h cultured, the cells were treated with 10 μM melatonin and 200 μM H2O2. Finally, the cells were washed with PBS for 3 times, and observed under a laser-scanning confocal microscope (Zeiss LSM 710 META, Oberkochen, Germany).
For immunofluorescence analysis, IEC6 were fixed in 4% paraformaldehyde for 10 min and were permeabilized in 0.5% Triton X-100 for 20 min at room temperature and blocked in 1% BSA for 30 min. Next, the cells were incubated with primary antibodies against Cleaved caspase-3 overnight at 4℃. After that, the samples were washed three times with PBS for 10 min each time and incubated with secondary antibodies for 2 h. The nuclei were stained with DAPI for 10 min and then washed one time with PBS for 15 min. Immunofluorescent images were captured with a Zeiss 710 laser scanning confocal microscope.
Gut microbiota analysis
Total genomic bacterial DNA of fecal mixture of the duodenum, jejunum, ileum and colon was extracted using an E.Z.N.A.® Stool DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions. PCR amplification of bacteria DNA in the present study was performed. Briefly, the V3‐V4 regions of the bacteria 16S ribosomal RNA gene were amplified by PCR as following: 95°C for 2 min, followed by 25 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. The primers used are 338F 5′‐ACTCCTACGGGAGGCAGCA‐3′ and 806R 5′‐GGACTACHVGGGTWTCTAAT‐3′. PCRs were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer 5 μM, 0.4 μL of FastPfu DNA polymerase, and 10 ng of template DNA.
Amplicons were extracted from 2% agarose gel, purified by the AxyPrep DNA gel extraction kit (Axygen Biosciences, Union City, CA, USA), and quantified by QuantiFluorTM-ST (Promega, Madison, WI, USA) according to the manufacturer’s protocols. Then purified amplicons were pooled in equimolar amounts and paired-end sequenced on an Illumina MiSeq platform according to the standard protocols by a commercial company (Novogene, Beijing, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: SUB2623584).
Raw data were firstly conjuncted by using the FLASH analysis tool (V1.2.7, http://ccb.jhu.edu/software/FLASH/), and then filtered with the Quality Control software package (V1.7.0, http://qiime.org/scripts/split_libraries_fastq.html). To obtain the effective tags, the UCHIME algorithm (http://www.drive5.com/usearch/manual/uchime_algo.html) was used to remove the chimera sequences. The reads were picked to form distinct OTUs using Uparse software (http://drive5.com/uparse/) at 97% of sequence similarity, and then were classified to different levels by comparing to GreenGenes database using PyNAST software (V1.2).
For detection of SCFAs, fecal samples were measured using the previous method [22,26]. Briefly, the fecal mixture samples were treated as follows: 0.3 g of feces was added to 1.2 mL sterile saline, evenly dispersed by stirring with a glass rod, and left to stand for 5 min; 600 μL 50% sulfuric acid (Sigma Aldrich) and 1.2 mL ether were added, standing for 10 min with intermittent shaking. The supernatant was obtained by centrifugation at 5000 rpm for 30 min and used for chromatographic analysis. The content of SCFA were measured by GC‐MS (Agilent, USA).
Statistical analyses were performed using the SPSS 23.0 statistical software package (SPSS Inc, Chicago, IL, USA). The variables were first tested for normality (Shapiro–Wilk test) and homoscedasticity (Levene’s test) and followed with Student’s t test. The data were compared by analysis of variance (ANOVA) and differences located with Duncan’s multiple range test. Correlation analysis between microbiota and SCFAs was conducted by Pearson correlation analysis. All data of each group were presented as mean ± SEM and statistical significance was set at P < 0.05.