香京julia种子在线播放

TMC3115 was kindly provided by Hebei Inatural Biotech Co. Ltd. (Hebei, PR China). Freeze-dried living TMC3115 (2.12 × 10¹¹ colony-forming unit [CFU]/g) was dissolved in sterile saline to prepare bacterial suspensions of 5 × 10⁹ CFU/mL. The daily intake of living TMC3115 was estimated as 10⁹ CFU/mouse, 0.2 mL/d, once a day.

Fourteen pregnant specific pathogen-free BALB/c mice at gestational day 10–13 (Approval number: SCXK2015-0001) were purchased from Liaoning Changsheng Biotechnology Co., Ltd (Liaoning, PR. China), and kept in individually ventilated plastic cages at an ambient temperature of 23°C ± 1°C and humidity of 50%–70% under a 12 h light/dark cycle with free access to water and food.

The study design is shown in Figure 1A . Pregnant mice were randomly divided into two groups: a Control group and a TMC3115 group (n = 7 per group). Mice in the Control group were gavaged with saline from 1 week before and until delivery, while mice in the TMC3115 group were gavaged with a TMC3115 suspension. The organs, feces, vaginal douches, and blood of dams were collected at and after delivery day (ADD) 0 and 21, and a proportion of pups at postnatal day (PND) 0, 7, 14, and 21 were euthanized to collect blood, stomach contents (PND 0, 7, and 14), spleen, intestinal contents (PND 0, 7, and 14), colon and feces (PND 21). Then, half of the remaining pups in the two groups were intraperitoneally sensitized with ovalbumin (OVA) while the other half of the pups were not injected with any substance. All the remaining pups were sacrificed at PND49, and their organs, blood, and feces were collected.

Feces of dams were collected at ADD 0 and 21 and pups at PND 21 and 49. Plastic boxes were wiped with 75% ethanol, the mice were placed into these boxes, and more than 200 mg of naturally excreted feces were collected from each mouse within several hours.

Vaginal douches and blood of dams at ADD 0 were collected within 12 hours after the dams gave birth. Dams were placed into a 50 mL centrifuge tube, and their tails were exposed to the outside of the centrifuge tube and secured with tape. Then, the skin near the vagina and the tails of dams were wiped twice with 75% ethanol, with the dams ventral side up. A pipette was used to aspirate 50 μL of sterile saline, and the tip was carefully inserted into one side of the dams’ vagina, and then pipetting was repeated several times, avoiding saline leakage and contamination, and then the rinsing fluid was sucked out and placed in a 1.5 mL centrifuge tube. Vaginal douches were collected from the other side of the dams’ vaginas using the same method. A sterile surgical blade was used to make a small incision 2–3cm from the base of the tail, and 100 μL of blood was collected in a 1.5 mL centrifuge tube. Finally, a cotton ball was pressed on the wound for two minutes to stop the bleeding and keep the dams alive.

The blood of dams at ADD 21 and of pups at PND 21 and PND 49 were collected by removing the eyeballs after the mice were euthanized by inhalation of carbon dioxide. The blood of pups at PND 0, 7, and 14 was collected by beheading after they were euthanized by inhalation of carbon dioxide. The colon and spleen of mice were collected under sterile conditions after the mice were sacrificed.

The stomach and intestinal contents of pups were collected under sterile conditions at PND 0, 7, and 14, after the pups were sacrificed. The stomach contents were considered to be a proxy for breast milk (Nyangahu et al., 2018), because it was difficult to collect milk from the nipples of the dams, while avoiding contamination. The intestinal contents was considered to be a proxy for feces, because the pups were too young for the collection of. Briefly, the whole stomach was collected using sterile scissors and tissue forceps, and placed into a sterile 1.5 mL centrifuge tube. Then, the stomach tissue was bluntly dissected using tissue forceps, and the stomach contents of the pups were left in the tubes. The whole intestine of pups were collected under sterile conditions and placed in a sterile plastic plate. The intestinal contents were expelled from the intestinal lumen using a sterile curved forceps along the entire intestinal tract. Two hundred microliters of sterile PBS was added to the plate, and the viscous intestinal contents were collected by repeatedly pipetting and rinsing with a pipette. The resulting mixture was placed into a 1.5 mL centrifuge tube. The collected intestinal contents mixture actually contained the contents in the small intestine and colon, thus the microbiota found in the intestinal contents was named as small intestinal and colonic microbiota in the following text.

The OVA sensitization procedure was performed as previously described, with some modifications (Cheng et al., 2019). Briefly, pups were injected intraperitoneally with 40 μg of OVA and 4 mg of Imject Alum (Sigma-Aldrich, St. Louis, MO, USA) suspension on PND 21, 35, and 42.

Total RNA was extracted from spleen and colon using a commercial kit (Foregene, Chengdu, PR. China) in accordance with the manufacturer’s instructions. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed as previously described, with minor modifications (Cheng et al., 2020). Briefly, complementary DNA (cDNA) was synthesized using iScript™ cDNA synthesis kits (Bio-Rad, Hercules, CA, USA), and the quantification of cDNA was performed using SsoAdvanced™ Universal SYBR^® Green Supermix (Bio-Rad). The quantitative PCR reaction system consisted of 5 μL of 1x SsoAdvanced™ Supermix, 0.5 μL of 100 μM forward primer, 0.5 μL of 100 μM reverse primer, 1 μL of cDNA (100 ng to 100 fg), and 3 μL of double-distilled water (ddH2O). The cycling conditions were as follows: initial denaturation at 98°C for 30 s, followed by 40 cycles of denaturation at 98°C for 15 s, and annealing/extension at 60°C for 30 s. β-actin was used as the reference gene, and 2^−△△ct calculation was used to determine the relative levels of the mRNA of the targeted gene. Primer sequences are listed in Table S1 . All primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China PR).

Colons were collected from pups at PND 21. One centimeter of each sample was cut and fixed with 10% neutral phosphate-buffered saline formalin for 24 h, and then stained with hematoxylin-eosin (HE). Optical microscopic images were inspected by a pathologist blinded to the experimental design. As previously described (Cheng et al., 2017), an optical microscope (Olympus Corporation, Tokyo, Japan) was used to image the intestinal tissues. The fields of view were randomly selected, and included non-necrosis, hemorrhage, and non-specific staining marginal areas. A charged coupled device was used to collect high-quality images. Ten areas were measured from each of the upper, lower, left, right, and middle of each slice, and the parameters of 50 colonic crypts in total on each slice were measured.

The levels of IgG, IgM, IgA, sIgA, IgG1, IgG2a, IgG2b, and IgG3 in serum and the supernatant of stomach contents, and sIgA from the supernatants of feces and intestinal contents were detected using enzyme-linked immunosorbent assay (ELISA) with commercial kits (Elabscience, Wuhan, PR. China). Aliquots of 10 mg of stomach contents were added to 90 μL of sterile PBS and centrifuged for 3 min at 6000 g to obtain supernatants. Feces (50 mg) were mixed with 200 μL of sterile PBS, vortexed, and then centrifuged for 3 min at 6000 g to obtain supernatants. Serum IgE was determined via ELISA using mouse IgE ELISA kits (Invitrogen, Thermo Fisher Scientific, Inc., MA, USA). Serum OVA-specific IgE and IgG1 were detected using Anti-Ovalbumin IgE (mouse) ELISA kits and IgG1 (mouse) ELISA kits (Cayman Chemical, MI, USA). The serum cytokines IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, IL-17A, and IFN-γ were measured using Luminex (R&D Systems Inc., MN, USA). Assays were performed according to the manufacturer’s instructions and read using a Multiskan™ GO microplate spectrophotometer (Thermo Fisher Scientific, Inc., MA, USA) and a Luminex 200™ multiplexing instrument (Merck Millipore, MA, USA).

Total DNA was extracted from mouse intestinal contents and feces (100 mg) of offspring at PND 0, 7, 14, 21, and 49, maternal feces at ADD 0 and 21, stomach contents at PND 0, 7, and 14, and maternal vaginal douches at ADD 0, using the TIANamp Stool DNA Kit (Tiangen Biotech Co. Ltd., Beijing, PR China) in accordance with the manufacturer’s instructions.

The 5’ ends of the primers were tagged with barcodes specific to each sample and sequencing universal primers. PCR amplification was performed in a total volume of 25 μL of reaction mixture containing 50 ng of DNA, 12.5 μL of Phusion Hot start flex 2X Master Mix (NEB, M0536L), 2.5 μL of each of forward primer 338F 5’-ACTCCTACGGGAGGCAGCAG-3’ and reverse primer 806R 5’-GGACTACHVGGGTWTCTAAT-3’ (16S V3-V4 primer), and ddH₂O was used to adjust the volume. The PCR conditions were 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 54°C for 30 s, and 72°C for 45 s, and then 72°C for 10 min. The PCR products were separated using 2% agarose gel electrophoresis, purified with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA), and then quantified using a Qubit (Invitrogen, USA). Amplicon pools were prepared for sequencing, and the size and quantity of the amplicon library were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and the Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), respectively. A PhiX Control library (v3) (Illumina) was combined with the amplicon library (expected at 30%). The libraries were sequenced on an Illumina MiSeq Instrument (Illumina Inc., San Diego, CA, USA) using the 300-base pair (bp) paired-end protocol with the standard Illumina sequencing primers.

Briefly, raw FASTQ files were generated using Illumina’s bcl2fastq (v1.8.4) software. Trimmomatic (v0.36) was used to remove the barcode and primer sequences, and paired-end reads were then merged using FLASH (v1.2.11). The two-terminal valid and high-quality sequences were then spliced using the QIIME2-DADA2 process, which required sequence overlap of at least 10 bp and allowed a maximum of two base mismatches. The Amplicon Sequence Variants (ASVs) were constructed and taxonomic assignment was performed based on the SILVA Living Tree Project v138 (LTPv138) database (16S V3-4). Multiple sequences were aligned and a phylogenetic tree was constructed. The phylogenetic tree, ASVs abundance table, alpha diversity (ACE, Chao1, observed features, Shannon, Simpson), and beta diversity (Bray–Curtis, weighted and unweighted UniFrac) were calculated using QIIME2. The pairwise difference between the mean values species richness and the diversity index was measured using the Wilcox test (R program). Microbiota community composition was shown at phylum and genus level. All plots were visualized within the R program (v3.4.1).

Fecal SCFA (acetic, propionic, and butyric acid) were detected using gas chromatography. Feces (100 mg) were mixed with 400 μL of ether, 100 μL of internal standard (4-methyl valeric acid), and 100 μL of 15% phosphoric acid, vortexed, and then centrifuged for 10 min at 4°C at 13400 g. Sample supernatant (1 μL) was analyzed using an Agilent 7890A (Agilent Technologies, Inc.).

Statistical analysis was performed using GraphPad Prism 8.0.1 (GraphPad Software, San Diego, California USA, www.graphpad.com). Data are expressed as the mean ± standard error of the mean. Student’s t-tests or Mann–Whitney U tests were used for pairwise comparisons. A probability (P) value < 0.05 was considered significant. All statistical tests were two-tailed.

After delivery day 0, there was a significant decrease in the levels of serum IgG1, IgG3, and fecal sIgA in dams treated with TMC3115 in comparison with those in the Control group, while the levels of serum IgM tended to decrease ( Figure S1 ). After delivery day 21, the levels of serum immunoglobulin and fecal sIgA of dams treated with TMC3115 were similar to those of the Control group ( Figure S2A ). However, compared with those in the Control group, the mRNA expression of splenic IL-8, IL-10, and IL-17A in dams treated with TMC3115 was significantly increased; moreover, the expression of TNF-α and TGF-β1 showed a tendency to be increased ( Figures S2B–H ).

After delivery day 0, the alpha diversity of dams treated with TMC3115 did not significantly change in comparison with that of the Control ( Figure S3A ). However, the fecal microbiota community was altered to some extent, and Principal Coordinates Analysis (PCoA) analysis showed two clear clusters ( Figures S3B–D ). At the phylum and genus level, the relative abundances of Proteobacteria, Deferribacteres, Helicobacter, Mucispirillum, and Prevotella in the TMC3115 group were significantly lower than those in the Control group, while the relative abundance of Rikenella in the TMC3115 group was significantly higher ( Table S2 ). The level of fecal acetic acid from dams treated with TMC3115 was significantly decreased compared with that in the Control group ( Figure S3E ).

After delivery day 21, the Simpson index of the TMC3115-treated group was significantly lower than that of the Control group, while the Shannon index of the TMC3115-treated group tended to decrease ( Figure S3F ). The fecal microbiota composition was altered by TMC3115 treatment during pregnancy, as demonstrated by significantly decreased abundance of Alistipes and increased abundances of Lactobacillus, Roseburia, and Staphylococcus; PCoA also showed two clusters separated by the axis, with several overlapping areas ( Figures S3G–I and Table S2 ). The fecal acetic and propionic acid levels in the TMC3115-treated group were significantly lower than those in the Control group ( Figure S3J ).

On the day after dams gave birth, vaginal douches were collected and sequenced to observe changes to the vaginal microbiota. The ACE, Chao1, and observed OTUs of the TMC3115 group were significantly lower than those of the Control group, while the Shannon and Simpson indexes also tended to decrease ( Figure S4A ). The Bray–Curtis distance was used to compare the beta diversity between groups, and two clusters were separated using PCoA analysis ( Figure S4B ). At the phylum level, the relative abundances of Firmicutes and Desulfobacterota in the TMC3115 group were significantly decreased, while the abundance of Proteobacteria was significantly increased when compared with those of the Control group ( Figure S4C and Table S2 ). At the genus level, the relative abundances of Oscillibacter A2, Lachnospiraceae UCG.006, Clostridia vadin BB60 group, Desulfovibrio, Lachnospiraceae NK4A136 group, Citrobacter undefined, Lachnospiraceae, and Lactobacillus in the TMC3115 group were significantly decreased when compared with those in the Control group; however, the abundances of Escherichia, Shigella, Bifidobacterium, and Psychrobacter were significantly increased ( Figure S4D and Table S2 ).

Between PND 0 and PND 14, the level of IgG and associated subclasses, and the IgM level in stomach contents gradually decreased, while the levels of IgA and sIgA gradually increased ( Figures 1B–I ). At PND 0, the IgG content of the stomach contents in the TMC3115 group was significantly higher than that in the Control group ( Figure 1B ). The IgG2a content at PND 7 and PND 14, and the IgM content at PND 14 in the TMC3115 group were significantly lower than those in the Control group ( Figures 1C, G ). The IgA and sIgA contents at PND 14 in the TMC3115 group tended to be lower than those in the Control group ( Figures 1D, E ).

In the Control group, the alpha diversity at PND 0 was significantly higher than those at PND 7 and PND 14 ( Figure 2A ). Similarly, in the TMC3115 group, the alpha diversity, including ACE, Chao1, observed OTUs, and Shannon index at PND 0 were significantly higher than those at PND 7; the Shannon index at PND 14 was also significantly higher than that at PND 7 ( Figure 2B ). At PND 0 and PND 7, the Shannon and Simpson indexes of the TMC3115 group were significantly lower than those of the Control group; however, there were no significant differences in alpha diversity at PND 14 between the groups ( Figure 2C ).

At PND 0, Firmicutes, Actinobacteriota, and Proteobacteria were the three predominate phyla, and Lactobacillus, Staphylococcus, Streptococcus, and Corynebacterium were the four predominate genera in the stomach contents. However, the relative abundances of Actinobacteriota, Staphylococcus, Streptococcus, and Corynebacterium gradually decreased, while the relative abundances of Firmicutes and Lactobacillus largely increased. Therefore, Firmicutes and Proteobacteria became the predominate phyla and Lactobacillus became the only predominate genus in stomach contents at PND 7 and PND 14 ( Figures 2D, E ). Only the abundances of Bacteroidota and Lactobacillus in the TMC3115 group at PND 0 were significantly lower than those in the Control group ( Figure 2F ). No significant differences in abundance were found between the groups at PND 7 and PND 14. Consistently, PCoA analysis indicated that the microbiota community was similar at PND 7 and PND 14 between the Control and TMC3115 groups, which were gathered into one cluster but clearly separated from samples at PND 0. The samples in the Control and TMC3115 groups were gathered into two clusters with several overlapping areas ( Figure 2G ).

The levels of serum immunoglobulin and fecal sIgA dynamically changed with age. At PND 0, the serum IgG and sIgA levels in the TMC3115-treated group were significantly higher than those in the Control group, while the IgG3 levels were lower than those in the Control group ( Figures 3A, E, I ).

At PND 7, the levels of serum IgG3 in the TMC3115 group were significantly higher than those in the Control group while those of IgE and IgG2b only tended to be higher ( Figures 3C, H, I ). Compared with that in the Control group, the level of mRNA of splenic TGF-β1 was significantly decreased, and the mRNA level of colonic occludin was significantly increased ( Figures 3K, L ).

At PND 14, the levels of serum IgE and IgG2b in the TMC3115 group tended to be lower, while that of sIgA tended to be higher than those in the Control group ( Figures 3C, E, H ). The mRNA expression of splenic IL-17A tended to decrease when compared with that of the Control group ( Figure 3M ).

At PND 21, the levels of serum IgG and IgE and fecal sIgA in the TMC3115 group were significantly lower than those in the Control group, while the levels of serum IgG1 levels tended to be higher ( Figures 3A, C, F, J ). Colonic Ki67 and occludin mRNA expression and the colonic depth were significantly increased compared with those in the Control group ( Figures 3N–P ).

At PND 49, the levels of serum IgG, IgM, IgA, sIgA, IgG2a, IgG2b, and IgG3 in the TMC3115 group were significantly lower, and levels of serum IgE and fecal sIgA were significantly higher than those in the Control group ( Figures 3A–J ). The splenic mRNA expression of IL-10, IL-17A, and ROR-γt, and levels of serum IL-10 and IL-17A in the TMC3115 group were significantly increased compared with those in the Control group, while the splenic GATA-3 mRNA expression in the TMC3115 group tended to be increased ( Figures 3Q–V ).

In the Control group, the alpha diversity (including ACE, Chao1, Observed OTUs, Shannon, and Simpson index) gradually but significantly increased with age. However, at PND 7, the alpha diversity was the lowest among the time points, and the Shannon index was significantly lower than at other time points ( Figure 4A ). Similarly, in the TMC3115 group, the alpha diversity gradually but significantly increased with age ( Figure 4B ). There were no significant differences found in alpha diversity between groups at any of the time points, although the Shannon index in the TMC3115 group tended to be lower than that in the Control group ( Figure 4C ).

In the Control group at PND 0, the phyla predominating in the small intestinal and colonic microbiota were Proteobacteria, Firmicutes, and Actinobacteriota, while the predominate genera were Lactobacillus, Corynebacterium, Streptococcus, and Ammoniphilus. In the TMC3115-treated group, the two phyla predominating the small intestinal and colonic microbiota were Proteobacteria and Firmicutes, while Acinetobacter and Rodentibacter were the two predominate genera ( Figures 4D, E ). With age, the relative abundances of Proteobacteria and Actinobacteriota gradually decreased, while the abundance of Bacteroidota gradually increased in the two groups. The relative abundances of the predominate genera at PND 0 gradually decreased, and several newly colonized genera predominated with increasing age. At PND 7, the two predominate phyla in both groups were Proteobacteria and Firmicutes, while the three predominate genera were Lactobacillus, Escherichia-Shigella, and Rodentibacter. At PND 14, the phyla Bacteroidota, Proteobacteria, and Firmicutes and genera Lactobacillus, Escherichia-Shigella, and Bacteroides predominated in both groups. At PND 21 and 49, the phyla Bacteroidota and Firmicutes and genera Lactobacillus, Muribaculaceae, and Lachnospiraceae NK4A136 group were predominate in both groups.

At PND 0, the relative abundances of Proteobacteria, Acinetobacter, and Rodentibacter in the TMC3115 group were significantly increased compared with those in the Control group, whereas those of Firmicutes, Bacteroidota, Lactobacillus, Corynebacterium, and Staphylococcus were significantly decreased, and the abundances of Actinobacteriota and Streptococcus were slightly decreased. At PND 21, the relative abundances of Patescibacteria and Prevotellaceae UCG.001 were significantly decreased, and those of Bacteroidota, Alistipes, and Muribaculaceae tended to be decreased, whereas the relative abundances of Firmicutes, Desulfobacterota, Lactobacillus, and Enterorhabdus tended to be increased. At PND 49, the relative abundances of Actinobacteriota, Bacteroidota, Rikenellaceae RC9 gut group, Clostridia UCG 014, and Muribaculaceae were significantly increased, whereas those of Desulfobacterota, Firmicutes, undefined Lachnospiraceae, Lachnospiraceae NK4A136 group, Eubacterium siraeum group, and Colidextribacter were significantly decreased ( Table 1 ).

There were four clusters separated via PCoA analysis based on Bray–Curtis distance. These groups included samples taken at PND 0, 7, 14, 21, and 49. Samples in the two groups at PND 0 and PND 49 were clearly separated, while samples in the two groups at PND 7, 14, and 21 overlapped ( Figure 4F ). The levels of fecal acetic and propionic acid at PND 21 and of propionic acid at PND 49 in the TMC3115 group were significantly lower than those in the Control group ( Figures 4G, H ).

After sensitization by OVA, there were no significant changes in the levels of serum IgE and OVA-specific IgE of offspring at PND 49 in the TMC3115 group compared with the Control group ( Figures 5A, B ). However, the levels of serum OVA-specific IgG1 in the TMC3115 group tended to be decreased in comparison with those in the Control group ( Figure 5C ). Moreover, the level of serum IgM in the TMC3115 group was significantly higher than that in the Control group, while that of serum sIgA also tended to be higher ( Figures 5D, E ). The colonic mRNA expression of Ki67, Muc2, ZO-1, claudin-1, claudin-2, and occludin in the TMC3115 group was significantly higher than that in the Control group ( Figures 5F–K ).

After sensitization by OVA, the ACE, Chao1 index, and observed OTUs of the TMC3115 group tended to be higher than those of the Control group, while the Simpson index of the TMC3115 group was significantly lower ( Figure 6A ). Samples in the two groups were clearly separated into two clusters via PCoA ( Figure 6B ). The phyla Bacteroidota, Firmicutes, and genera Lactobacillus, Muribaculaceae, and Lachnospiraceae NK4A136 group predominated in both groups ( Figures 6C, E ). Compared with the Control group, the relative abundances of the phyla Actinobacteriota and Firmicutes, and the genera Blautia and unidentified Lachnospiriaceae in the TMC3115 group were significantly increased, while the abundance of the Lachnospiraceae NK4A136 group tended to be increased. However, the relative abundances of the phylum Bacteroidota and the genera Muribaculum, Muribaculuceae, and Odoribacter in the TMC3115 group were significantly decreased ( Figures 6D, F ). The fecal acetic, propionic, and butyric acid levels in the TMC3115 group were significantly higher than those in the Control group ( Figure 6G ). All the main findings of this manuscript have been summarized in Table 2 .