L. casei ATCC 393 and ETEC K88 strain were kept in our laboratory. PMMCs were purchased from Saiqi (Shanghai) Biological Engineering Co., Ltd (Cat # CBR-131443). Man, Rogosa and Sharpe (MRS) broth (Cat # CM1153B) was purchased from Oxoid (Basingstoke, UK). Luria-Bertani (LB) broth (Cat # 12780052) were purchased from Gibco-Invitrogen. The reagents for cell culture were purchased from Invitrogen/Gibco (Carlsbad, CA, USA). Enzyme-linked immunosorbent assay (ELISA) Kits for porcine tumor necrosis factor-α (TNF-α, Cat# JL13203), porcine and mouse interferon-γ (IFN-γ, Cat# JL11792), porcine interleukin-6 (IL-6, Cat# JL21880), porcine interleukin-8 (IL-8, Cat#JL45446), porcine granulocyte-macrophage colony stimulating factor (GM-CSF, Cat#JL21931), porcine β-hexosaminidase (Cat# JL45717), porcine tryptase (Cat# JL17996), and porcine histamine (Cat#JL10076), and mouse TNF-α (Cat#JL10484), mouse IFN-γ (Cat#JL10967), mouse IL-6 (Cat#JL20268), mouse IL-1β (Cat#JL18442), mouse β-hexosaminidase (Cat#JL20214), mouse tryptase (Cat#JL20445), mouse histamine (Cat#JL10420), and mouse MPO (Cat#JL10367) were purchased from Jianglaibio Co., Ltd (Shanghai, China). The bicinchoninic acid (BCA) protein assay kit (Cat#P0012S) was purchased from Beyotime Biotechnology (Shanghai, China). VIP and VIP receptor antagonist (VIP_6-28, aVIP) were synthesized by Shanghai Qiangyao Biological Co.,Ltd (Shanghai, China). Lipopolysaccharides (LPS, Cat#L4391) from Escherichia coli O111:B4 was purchased from Sigma Aldrich Company (Saint Louis, MO, USA). Primary antibodies for MUC2, Occludin, ZO-1, TLR4, NF-κB, p-NF-κB, MyD88 and β-actin were purchased from ABclonal Company (Wuhan, China).

PMMCs were cultured in high glucose Dulbecco’s modified Eagles’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic mixture (100 U/mL of penicillin and 100 μg/mL streptomycin) in an incubator at 37°C in a humidified atmosphere with 5% CO₂.

L. casei ATCC 393 was incubated in MRS broth at 37°C for 24 h without shaking. ETEC K88 was cultured in LB broth with shaking at a speed of 120 rpm at 37°C overnight. Bacteria pellets were collected by centrifuging at 5,000 × g at 4°C for 10 min, and then washed with phosphate-buffered saline (PBS). The obtained bacteria were suspended in FBS-free cell culture medium and diluted to different concentrations. The supernatants of 1×10⁸ CFU/mL L. casei ATCC 393 and 1×10⁸ CFU/mL of ETEC K88 culture medium were separately harvested by centrifugation at 5000 × g at 4°C for 10 min. The bacterial supernatants and bacterial resuspension solution were collected and used for subsequent experiments.

PMMCs were seeded at a concentration of 4×10⁵ cells/mL in 24-well cell culture plates and cultured at 37°C for 24 h. Normal control cells were exposed to 1 mL of FBS-free DMEM. For the L. casei ATCC 393 treatment group, 1 mL of 1×10⁸ CFU/mL of L. casei ATCC 393 bacterial culture supernatants was added to each well. Other groups received 1 mL of FBS-free DMEM. VIP and aVIP treatment groups were administrated with 1 mL of FBS-free DMEM containing 0.1 μM VIP and/or 0.1 μM aVIP, respectively. The plates of all groups were incubated at 37°C for 12 h. After the above treatments, supernatants of cell culture medium supernatants were collected, and the concentration of β-hexosaminidase, tryptase, and histamine were determined using the corresponding ELISA kits according to the manufacture’s instruction.

PMMCs were seeded at a concentration of 4×10⁵ cells/mL in 24-well cell culture plates and cultured at 37°C for 24 h. The cells were divided into four groups and treated accordingly: the normal control group received 1 mL of FBS-free DMEM; VIP and aVIP treatment group received 1 mL of FBS-free DMEM containing 0.1 μM VIP or/and 0.1 μM aVIP, respectively, and cultured at 37°C for 12 h. Afterwards, the cell culture medium supernatants were collected, and the concentration of β-hexosaminidase, tryptase, and histamine were determined using corresponding ELISA kits.

PMMCs were inoculated at a density of 4×10⁵ cells/mL in sterile 24-well cell culture plate and cultured at 37°C for 24 h until the cell confluence reached higher than 90%. Normal control cells received 1 mL of FBS-free DMEM. The VIP alone group was treated with 1 mL of FBS-free DMEM containing 0.1 µM VIP for 12 h. The L. casei ATCC 393 alone group was exposed to 1 mL of 1×10⁸ CFU/mL L. casei ATCC 393 culture supernatant for 3 h. The VIP and L. casei ATCC 393 co-cultured cells were first administered with 1 mL of 1×10⁸ CFU/mL of L. casei ATCC 393 culture supernatant for 3 h, then changed to 1 mL of FBS-free DMEM containing 0.1 µM VIP and culured for 12 h. Afterwards, the cell culture supernatants were collected, and the concentration of β-hexosaminidase, tryptase and histamine were determined using the corresponding ELISA kits.

PMMCs were seeded at a density of 4×10⁵ cells/mL in sterile 24-well cell culture plates and cultured at 37°C for 24 h. First, normal control cells were exposed to 1 mL of FBS-free DMEM, the VIP alone treatment group was given 1 mL of FBS-free DMEM containing 0.1 µM VIP, and the aVIP treatment group received 1 mL of FBS-free DMEM containing 0.1 µM aVIP, and the aVIP-VIP co-treatment group received 1 mL of FBS-free DMEM containing 0.1 µM aVIP and 0.1 µM VIP. Then all group cells were cultured at 37°C for 12 h. Control groups were exposed to 1 mL of FBS-free DMEM, other experimental groups were changed to 1 mL of 1×10⁸ CFU/mL ETEC K88 culture supernatant or 1 mL of FBS-free DMEM containing 0.1 µM of LPS. After additional incubation for 2 h, cell culture medium supernatants were collected. The concentration of β-hexosaminidase, tryptase, histamine, IL-6, IL-8, TNF-γ and GM-CSF were determined by the corresponding ELISA kits.

Animals can produce endogenous VIP, but whether it mediates the regulation of the L. casei ATCC 393 effect on intestinal barrier function remains unclear. We hypothesized that L. casei ATCC 393 can regulate the effect of pathogenic bacteria such as ETEC K88-induced intestinal mucosal MC activation and inhibit the release of MCs released mediators, regulate inflammatory responses, and further regulate intestinal barrier function through the binding of VIP and VIP receptors on the surface of intestinal mucosal MCs. To verify the above hypothesis, we conducted experiments to assess the effect of a VIP receptor antagonist (aVIP) in C57BL/6 mice challenged by ETEC K88. This animal experimental protocol was approved by the Laboratory Animal Welfare and Ethics Committee of Northwestern Polytechnical University and the experiment was conducted strictly in accordance with the International Laboratory Animal Assessment and Accreditation Committee guidelines for the care and use of laboratory animals. The 50 healthy male C57BL/6 mice (20 ± 2 g) used in the experiment were purchased from the Experimental Animal Center of Xi’an Jiaotong University (Xi’an, Shaanxi, China). The entire feeding experiment was conducted at the Experimental Animal Center of Northwestern Polytechnical University. After an adaptive period of 7 days, the mice were randomly divided into five groups with 10 mice per group: normal control group, ETEC K88 infected group, L. casei ATCC 393 protective group, aVIP + ETEC K88 treatment group, L. casei ATCC 393 + aVIP + ETEC K88 treatment group. The living conditions were as follows: relative humidity of 55 ± 5%, ambient temperature of 22 ± 5 °C, and under a 12 h light and dark cycle. The experimental scheme is depicted in Figure 3A . The mice in the L. casei ATCC 393 protective group were orally administered with 200 µL of 1×10⁸ CFU/mL of L. casei ATCC 393 resuspension solution per day for 14 days. The other groups were orally given the same volume of MRS broth. On days 1, 3, 5, 7, 9, and 11, mice in the ETEC K88-infected group were orally given 100 µL of 1×10⁸ CFU/mL ETEC K88 resuspension solution, and the other groups were given the same volume of LB broth. On days 0, 5 and 10, the aVIP treatment groups were intraperitoneally (i.p.) injected with aVIP (10 nmol/kg/BW). Other groups were i.p. injected with the same volume of normal saline. The body weight, diarrhea and mental status were observed and recorded daily. After the above treatments, mice were anesthetized with ether. Peripheral blood was drawn from mice and centrifuged immediately at 1,500×g for 15 min at 4°C to obtain serum. Serum samples were stored at -80°C until analyzed. Then, immediately the mice were dissected, the tissues of interest (the duodenum and ileum) were collected.

The duodenum is an important intestinal segment of the digestive tract for nutrient digestion and absorption. Its histology and morphology are closely related to intestinal function. The tissues samples of the proximal duodenum were fixed in 10% neutral buffer formalin, dehydrate, and paraffin-embedded. Then, the sections prepared from the paraffin-embedded tissue blocks were stained with hematoxylin-eosin (H&E) and observed under a phase-contrast microscope for histological and morphological characterization of the duodenum. To further evaluate the changes in intestinal barrier function, the expression level of MUC2, Occludin, and ZO-1 proteins in duodenum were detected by immunofluorescence and Western Blot respectively. The expression levels of MUC2 and Reg3g genes were detected by q-PCR. Primer sequences are shown in Table 1 .

Intestinal mucosal MCs are key modulators of barrier function and homeostasis, which widely distributed in ileum. Therefore, the numbers of MCs and the degranulated MCs in proximal ileum were detected by toluidine blue (TB.) staining. Serum β-hexosaminidase, tryptase and MPO activities, as well as VIP, sIgA, histamine, TNF, IFN-γ, IL-6 and IL-1β concentrations were determined by the corresponding ELISA kits according to the manufacture’s instruction.

To investigate the changes of intestinal microbiota, we detected the cecal contents of mice by 16S rRNA amplicon sequencing. As in previous research methods, whole-genome DNA was extracted and sequenced for analysis (23). The sequences with similarity ≥97% were clustered. OTUs were assigned to each representative sequence in the cluster by searching against the GreenGene Database.

To further explore the mechanism of the effect on intestinal immune barrier function, we detected the protein expression levels of MyD88, TLR4, NF-κB (p65), and p-NF-κB (p-p65) by Western Blot.

All experimental data were statistically analyzed using Graphpad Prism 5.0 statistical software (GraphPad Software Inc., San Diego, CA, USA) and are presented as the mean ± standard error of mean (S.E.M.). The statistical significance was calculated by one-way analysis of variance (ANOVA) or Student’s t-test. Differences were considered significant at P<0.05. All assays were performed in at triplicate three independent experiments.

As shown in Figure 1A , compared with the normal control group, exposure to L. casei ATCC 393 or VIP significantly induced the activation of PMMCs, and promoted the release of MCs-related mediators including β-hexosaminidase, tryptase and histamine. However, aVIP had no effect on PMMCs.

As shown in Figure 1B , compared with the normal control group, treatment with VIP significantly increased the concentration of β-hexosaminidase, tryptase and histamine in the supernatant of PMMCs. Exposure to aVIP alone had no effect on PMMCs. However, administration of aVIP significantly inhibited VIP-induced activation of PMMCs.

As shown in Figure 1C , compared with the normal control group, both VIP and L. casei ATCC 393 treatments increased the β-hexosaminidase, tryptase and histamine concentration in the supernatant of PMMCs. However, L. casei ATCC 393 pretreatment significantly inhibited the VIP-induced activation of PMMCs and the release of MCs-related mediators.

As shown in Figure 2 , compared with the normal control group, both ETEC K88 and LPS induced a significant increase of the TNF-α, IFN-γ, IL-6, IL-8, GM-CSF, β-hexosaminidase, tryptase and histamine levels in PMMCs supernatant. VIP significantly inhibited the increase of inflammatory factors, histamine, β-hexosaminidase and tryptase concentration in the supernatant of ETEC K88- and LPS-induced PMMCs. Administration with aVIP alone had no effect on ETEC K88- and LPS-induced activation of PMMCs. However, aVIP pretreatment significantly inhibited the regulatory effect of VIP on ETEC K88- and LPS-induced activation of PMMCs.

The change of body weight of the mice during the whole experiment is shown in Figure 3B . According to the overall trend, except for the ETEC K88-infected group and L. casei ATCC 393 protection group, the body weight of the other groups remained relatively stable. On the days 6, from days 8 to 13, the body weight of mice in the ETEC K88-infected group was significantly lower than that in the normal control group. The groups that were orally administered L. casei ATCC 393, aVIP and L. casei ATCC 393 + aVIP all showed a significant alleviation of the reduction of body weight caused by ETEC K88. Moreover, the body weight of mice treated with L. casei ATCC 393 + ETEC K88+aVIP were significantly higher than that of mice in the L. casei ATCC 393 + ETEC K88 co-treated group.

As shown in Figures 4A, B , compared with the normal control group, infection by ETEC K88 caused a significant increase in crypt depth (CD), a significant decrease in villus height (VH) and VH/CD of duodenum. Orally administered L. casei ATCC 393 significantly alleviated the above phenomenon. However, compared with the ETEC K88-infected group, the administration of aVIP alone had no effect on the VH, CD and VH/CD in mice exposed to ETEC K88. In addition, administration of aVIP significantly inhibited the effects of L. casei ATCC 393 on the VH, CD and VH/CD. As shown in Figure 4C , ETEC K88 significantly reduced the expression levels of ZO-1 and Occludin. L. casei ATCC 393 significantly alleviated the occurrence of the above phenomenon. aVIP showed antagonistic effect with L. casei ATCC 393 and inhibited the protective effect of L. casei ATCC 393 on intestinal barrier function. As shown in Figure 4D , compared with the control group, the expression levels of MUC2 protein in ETEC K88 group was significantly increased. Administration of L. casei ATCC 393 significantly inhibited the above phenomenon. However, aVIP had antagonistic effect on the regulatory effect of L. casei ATCC 393. As shown in Figure 4E , L. casei ATCC 393 significantly alleviated the increase of MUC2 and Reg3g mRNA expression levels induced by ETEC K88. However, the regulatory effects of L. casei ATCC 393 on MUC2 expression was inhibited by aVIP under ETEC K88 challenge.

TB staining of MCs in the proximal ileum of mice reveals, as shown in Figures 5A, B , that the number of total degranulated MCs in the proximal ileum of ETEC K88-infected mice was significantly higher than that in the normal control mice. Also, L. casei ATCC 393 or (and) aVIP treatment significantly attenuated the ETEC K88-induced increase in the number of total degranulated MCs in the proximal ileum. However, compared with the L. casei ATCC 393 treatment group, i.p. administration of aVIP significantly inhibited the regulatory effect of L. casei ATCC 393 on ileal mucosal MCs in mice infected by ETEC K88. As shown in Figures 5C, D , compared with the normal control group, infection by ETEC K88 significantly increased serum TNF-α, IL-6, IL-1β, IFN-γ, VIP, sIgA and histamine levels, as well as the β-hexosaminidase, tryptase and MPO activities. However, pretreatment with L. casei ATCC 393 or (and) aVIP significantly inhibited the increase of TNF-α, IL-6, IL-1β, IFN-γ levels, as well as the MPO activities induced by ETEC K88. L. casei ATCC 393 significantly inhibited the increase of sIgA and VIP levels induced by ETEC K88. Moreover, aVIP exhibited significantly antagonistic effect on the regulation of the release of TNF-α, IL-6, IL-1β, IFN-γ, VIP, sIgA and MCs-related mediators by L. casei ATCC 393.

As shown in Figure 6A , compared with the other groups, administration of aVIP significantly increased the α-diversity of cecum microbiome. As for the ACE, Chao1 and Shannon index, there was no significant difference between each experimental group. As shown in Figure 6B , there was no significant difference for β-diversity in the genus level among each experimental group. In Figure 6C , there was no significant difference among those groups in the phylum level. However, as shown in Figures 6D, E , compared with the control, ETEC K88 significantly reduced the abundance of norank_f_Bacteroidales_S24-7_group. However, L. casei ATCC 393 and aVIP interventions alleviated the reduce of norank_f_Bacteroidales_S24-7_group. As shown in Figure 6F , LEfSe analysis showed that g_Lachnospiraceae_NK4A136_group and g_Ruminococcus_1 were dominant species in mice challenged by ETEC K88, and g_unclassified_f_Peptostreptococcaceae and f_Peptostreptococcaceae were dominant species in mice administered with L+ETEC K88.

As shown in Figures 7A, B , compared with the control group, ETEC K88 challenge improved the expression levels of MyD88, p-NF-κB and TLR4. However, administration of L. casei ATCC 393 significantly inhibited the upregulation of MyD88, p-NF-κB and TLR4 induced by ETEC K88. Moreover, aVIP intervention exhibited antagonistic effects on L. casei ATCC 393.