The Role of Vasoactive Intestinal Peptide and Mast Cells in the Regulatory Effect of Lactobacillus casei ATCC 393 on Intestinal Mucosal Immune Barrier

Vasoactive intestinal peptide (VIP) plays an important role in the neuro-endocrine-immune system. Mast cells (MCs) are important immune effector cells. This study was conducted to investigate the protective effect of L. casei ATCC 393 on Enterotoxigenic Escherichia coli (ETEC) K88-induced intestinal mucosal immune barrier injury and its association with VIP/MC signaling by in vitro experiments in cultures of porcine mucosal mast cells (PMMCs) and in vivo experiments using VIP receptor antagonist (aVIP) drug. The results showed that compared with the ETEC K88 and lipopolysaccharides (LPS)-induced model groups, VIP pretreatment significantly inhibited the activation of MCs and the release of β-hexosaminidase (β-hex), histamine and tryptase. Pretreatment with aVIP abolished the protective effect of L. casei ATCC 393 on ETEC K88-induced intestinal mucosal immune barrier dysfunction in C57BL/6 mice. Also, with the blocking of VIP signal transduction, the ETEC K88 infection increased serum inflammatory cytokines, and the numbers of degranulated MCs in ileum, which were decreased by administration of L. casei ATCC 393. In addition, VIP mediated the regulatory effect of L. casei ATCC 393 on intestinal microbiota in mice. These findings suggested that VIP may mediate the protective effect of L.casei ATCC 393 on intestinal mucosal immune barrier dysfunction via MCs.


INTRODUCTION
The intestinal barrier is essential for maintaining intestinal homeostasis and health. It prevents the loss of water and electrolytes and the invasion of antigens and microorganisms (1,2). Therefore, the integrity of the intestinal barrier is particularly important for human and animal health. One of the main causes of gastrointestinal diseases such as necrotizing enterocolitis, irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) is the impairment of intestinal epithelial barrier integrity (3,4). The intestinal mucosal immune barrier is mainly composed of intestinal mucosal epithelial cells and intestinal associated lymphoid tissues. Intestinal mucosal immune cells mainly include lymphocytes, goblet cells, mast cells, etc. The intestinal microbiome as a key component of the intestinal barrier system are strongly associated with the host health (5). Lactobacillus casei (L. casei) strains are commonly added to yogurt and fermented dairy products to improve their health benefits (6). L. casei could relieves constipation and diarrhea (7,8). L. casei has also shown promise in preventing or alleviating human IBDs (9). However, the attenuation of colitis by L. casei BL23 was found to be dependent on the dairy delivery matrix (10). The impact of L. casei on the cecal microbiome and innate immune system is strain specific (11), and dose and time-dependent (12). L. casei expressing internalins A and B significantly reduced Listeria monocytogenesinduced cell cytotoxicity and epithelial barrier dysfunction (13). Moreover, L. casei ATCC 393 alleviated ETEC K88-induced intestinal barrier dysfunction via the toll-like receptors (TLRs)/ mast cells (MCs) pathway (14).
MCs are an important natural immune effector cells, which participate in mucosal immunomodulatory process by releasing cytokines. The activation and degranulation of MCs is involved in the regulation of variety of physiological and pathological conditions in various settings (15). The reversal of ion permeation and transmembrane transport of macromolecules by MCs stabilizers was demonstrated in animal studies (16). MCs serve as a line of defense against antigens entering the body, and contribute to maintain the homeostasis of the immune system (17). MCs express various receptors including pathogenassociated molecular patterns (PAMPs), vasoactive intestinal peptide receptors (VPACs), NOD-like receptors (NLRs) as well as TLRs, all of which are involved in MC activation and immune response (18). A variety of endogenous and exogenous drugs can stimulate MCs to release mediators. The impairment of the intestinal barrier is related to the increase of intracavity antigens entering the mucosa, which further promotes the activation of MCs in the mucosa inflammatory response and changes in MC-enteric nerve interaction (19). Vasoactive intestinal peptide (VIP) plays a crucial role in the neuroendocrine-immune system. Some studies have demonstrated the role of VIP in intestinal permeability regulation (20). Other studies have shown that MCs and VIP regulate the ileal barrier of healthy people and the stress response in rats through the VPAC1/VPAC2 receptors on the surface of MCs (21). An increase in plasma VIP levels was found in IBS patients and animal models. Also, intestinal epithelial permeability appears to be a positively correlated with mucosal MCs (22). However, the association of the protective effect of L. casei ATCC 393 on intestinal barrier function with VIP/MCs remains unclear.
This study was aimed to investigate the role of VIP in the protective effect of L. casei ATCC 393 on intestinal barrier dysfunction in mice challenged by ETEC K88 and its association with MCs. The ETEC K88 and LPS-induced VIPmediated regulatory effect on MCs activation, and its association with the protective effect of L. casei ATCC 393 on intestinal epithelial barrier function were evaluated through in vitro coculture experiments of porcine intestinal mucosal mast cells (PMMCs) with L. casei ATCC 393 and in vivo experiments using VIP receptor antagonist (aVIP) drug.

Cell Culture Conditions
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 mg/mL streptomycin) in an incubator at 37°C in a humidified atmosphere with 5% CO 2 .
Bacterial Culture Conditions 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 1×10 8 CFU/mL L. casei ATCC 393 and 1×10 8 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.

Effect of VIP, aVIP and L. casei ATCC 393 on PMMCs Activation
PMMCs were seeded at a concentration of 4×10 5 cells/mL in 24well 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 1×10 8 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 mM VIP and/or 0.1 mM 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 b-hexosaminidase, tryptase, and histamine were determined using the corresponding ELISA kits according to the manufacture's instruction.

Effect of VIP and aVIP Interaction on the Activation of PMMCs
PMMCs were seeded at a concentration of 4×10 5 cells/mL in 24well 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 mM VIP or/and 0.1 mM aVIP, respectively, and cultured at 37°C for 12 h. Afterwards, the cell culture medium supernatants were collected, and the concentration of bhexosaminidase, tryptase, and histamine were determined using corresponding ELISA kits.

Effect of L.casei ATCC 393 on VIP-Mediated Activation of PMMCs
PMMCs were inoculated at a density of 4×10 5 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 1×10 8 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 1×10 8 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 b-hexosaminidase, tryptase and histamine were determined using the corresponding ELISA kits.

Effect of VIP and aVIP on ETEC K88 or LPS-Induced PMMCs Activation
PMMCs were seeded at a density of 4×10 5 cells/mL in sterile 24well 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 1×10 8 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 bhexosaminidase, tryptase, histamine, IL-6, IL-8, TNF-g and GM-CSF were determined by the corresponding ELISA kits.

Animal Experimental Design
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 1×10 8 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 K88infected group were orally given 100 µL of 1×10 8 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.

Histological Analysis of Duodenum and Evaluation of Intestinal Barrier Function
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 phasecontrast 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.

Detection of Intestinal Immune Responses
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 bhexosaminidase, tryptase and MPO activities, as well as VIP, sIgA, histamine, TNF, IFN-g, IL-6 and IL-1b concentrations were determined by the corresponding ELISA kits according to the manufacture's instruction.

Intestinal Microbiota Analysis
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.

Effects of aVIP and L. casei ATCC 393 on TLRs/MyD88/NF-kB Signaling Pathway
To further explore the mechanism of the effect on intestinal immune barrier function, we detected the protein expression levels of MyD88, TLR4, NF-kB (p65), and p-NF-kB (p-p65) by Western Blot.

Statistical Analysis
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.

Effect of VIP, aVIP and L. casei ATCC 393 on PMMCs Activation
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 b-hexosaminidase, tryptase and histamine. However, aVIP had no effect on PMMCs.

Effect of Interaction Between VIP and aVIP on PMMCs Activation
As shown in Figure 1B, compared with the normal control group, treatment with VIP significantly increased the concentration of b-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.
Effect of VIP Mediated L. casei ATCC 393 on Activation of PMMCs As shown in Figure 1C, compared with the normal control group, both VIP and L. casei ATCC 393 treatments increased the b-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.

Effects of VIP and aVIP on ETEC K88-and LPS-Induced Activation of PMMCs
As shown in Figure 2, compared with the normal control group, both ETEC K88 and LPS induced a significant increase of the TNF-a, IFN-g, IL-6, IL-8, GM-CSF, b-hexosaminidase, tryptase and histamine levels in PMMCs supernatant. VIP significantly inhibited the increase of inflammatory factors, histamine, bhexosaminidase 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 Effects of aVIP and/or L.casei ATCC 393 on the Body Weight of Mice 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.
Effects of aVIP and/or L. casei ATCC 393 on Intestinal Morphology and Intestinal Barrier Function in Mice Challenged by ETEC K88 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 . *P < 0.05, **P < 0.01, ***P < 0.001. VIP means the PMMCs were exposed to VIP. L means the PMMCs were exposed to L. casei ATCC 393. aVIP means the PMMCs were exposed to aVIP. aVIP-VIP means the PMMCs were exposed to VIP and aVIP. L-VIP means the PMMCs were exposed to L. casei ATCC 393 and VIP. 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  (n=6). *P < 0.05, **P < 0.01, ***P < 0.001. VIP means the PMMCs were exposed to VIP. aVIP means the PMMCs were exposed to aVIP. aVIP-VIP means the PMMCs were exposed to VIP and aVIP. As shown in Figure 6A, compared with the other groups, administration of aVIP significantly increased the a-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 b-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.

DISCUSSION
The intestinal mucosal immune barrier plays a critical role in maintaining host homeostasis (24,25). Studies have shown that intestinal barrier dysfunction is closely associated with the occurrence and development of various diseases, such as inflammatory bowel disease (IBD), chronic kidney disease, type II diabetes, fatty liver and heart disease (26,27). Intestinal symbiotic bacteria and exogenous probiotics work together to maintain the integrity of the intestinal barrier, meaning that probiotics protect the intestinal barrier function (28). Lactobacilli MTCC 5690, LrhS3, Lp9, Lp4 and Lr120 can improve intestinal barrier function through toll-like receptor 2 (TLR2)-and toll-like receptor 4 (TLR4)-mediated mechanism and regulation of the expression of mucin 2 (MUC2) and tight junction proteins (29). The mixed probiotics of bifidobacteria, Lactobacillus acidophilus and Enterococcus faecalis can reduce the dextran sodium sulfate salt (DSS)-induced intestinal inflammation, improve multiple barrier functions, increase mucosal integrity, enhance transepithelial electrical resistance, reduce the permeability of the epithelium and endothelium to macromolecules, and increase the abundance of bifidobacteria, lactobacillus and bacteroides (30). In this study, we found that oral administration of L. casei ATCC 393 effectively protected the intestinal histomorphology and intestinal barrier function, and improve the ETEC K88-induced cecum microbiome dysbiosis. MCs are fundamental elements of the intestinal barrier (18), MCs as important immunological effector cells play a key regulatory role in adaptive and innate immunity (31). In pathological conditions, MCs release pro-inflammatory compounds, including cytokines (32). Our preliminary research indicated that L. casei ATCC 393 can relieve ETEC K88-induce intestinal barrier dysfunction via the TLRs/MCs pathway (14). However, the regulatory mechanism of the intestinal barrier function mediated by MCs has not been elucidated. According to the recent studies, it may be associated with the release of neurotransmitters (such as VIP, substance P), and inflammatory mediators (such as cytokines) (33,34). MCs not only senses the stimulation of harmful substances through the numerous receptors on their surface, including Fc receptor, complement receptor, TLRs, neuropeptide receptors, such as VPACs and antimicrobial peptide receptor, but also can synthesize and release transmitters to act on mucosal epithelium, nerve and other immune cells (35). As an extremely  important neuroendocrine immunomodulatory peptide, VIP may be strongly associated with the regulation of intestinal barrier function (19). VIP can have a good therapeutic effect on necrotizing enterocolitis by reducing the inflammatory response and the destruction of TJ proteins (36). Furthermore, DSS induced colitis was associated with VIP and VPAC1 receptors (37). Moreover, VIP regulates a variety of immune cells, including MCs (38). VIP protects testis from torsion injury  by inhibiting the activation of MCs (39). Additionally, VIP can also play a protective role on septic mice by regulating the activation of MCs (40). In this study, we found that VIP promoted the activation of PMMCs, and inhibited the activation of PMMCs exposed to ETEC K88 or LPS. These results suggested that the regulatory effect of VIP on PMMCs activation was strongly associated with the physiological and pathological conditions of cells. However, L. casei ATCC 393 or the aVIP can inhibit the VIP-induced activation of PMMCs.
MCs are important mediators of allergic responses on host surfaces including the intestine. When MCs is challenged by an external stimulus, it may respond by degranulation. In this process, a number of powerful preformed inflammatory "mediators" are released, including cytokines, histamine, serglycin proteoglycans, and several MC-specific proteases: chymases, tryptases, and carboxypeptidase A (41). In this study, the high numbers of degranulated MCs were observed in mice exposed to ETEC K88. MCs not only senses the stimulation of harmful substances through many receptors on the surface (including Fc receptors, complement receptors, TLRs, neuropeptide receptors, antimicrobial peptide receptors, etc.), but also can synthesize and release transmitters to act on mucosal epithelium, nerve, and other immune cells (35). Intestinal flora not only produces ligands for pattern recognition receptors (PRRs) (42), but also releases neurotransmitters and neuromodulators that target specific nervous systems on the brain-gut axis (43). If a TLR on MCs is activated, myeloid differentiation factor 88 (MyD88) and MyD88-adaptor like (MAL)/Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) associate and promote nuclear factor kappa-B (NF-kB) translocation to the nucleus resulting in cytokines transcription (44). TLR4 can be activated by LPS from Gram-negative bacteria (45).
Antimicrobial peptides themselves have the function of protecting epithelial barrier and adjusting intestinal microbial balance (46). Probiotic L. rbamnosus Lc705 and L. rbamnosus GG could diminish mast cell activation (47). In this study, we observed that orally given L. casei ATCC 393 significantly inhibited the activation of ileal mucosal MCs in mice infected by ETEC K88. However, administration of aVIP by intraperitoneal injection abolished the regulatory effect of L. casei ATCC 393 on MCs activation in ileum, reduced the serum  and also the mast cell stabilizer doxantrazole (21). The above research results support the functional observations in the current study. The role of VIP in regulating antiinflammatory-proinflammatory balance and intestinal barrier function has been demonstrated in humans and mice (20,48,49). The regulation of VIP on MC function may be bidirectional. It has been reported that VIP can affect the secretory activity of MCs, but the results are contradictory (38,50). Therefore, the roles of VIP and mast cells in the regulatory effect of probiotics on intestinal barrier function require confirmation in further experiments.

CONCLUSIONS
The regulatory effect of VIP on MCs activation may be related to the physiological and pathological conditions of cells. L. casei ATCC 393 inhibited the ETEC K88-and LPS-induced activation of intestinal mucosa MCs, and alleviated the intestinal mucosal injury in mice challenged by ETEC K88. VIP receptor antagonist abolished the protective effect of L. casei ATCC 393 on barrier function. Therefore, we speculated that the mechanism of L. casei ATCC 393 regulating intestinal mucosal injury may be related to VIP/MCs-mediated signaling pathway. However, the roles of VIP/MCs in the regulatory effects of probiotics on intestinal mucosal immune barrier function need further confirmation. This study is useful because it revealed the interaction of probiotics with enteric neuro-immunity and intestinal barrier function. L. casei ATCC 393 may be investigated as a potential and promising microecological food or feed additives in order to modulate intestinal barrier dysfunction.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The animal study was reviewed and approved by Laboratory Animal Welfare and Ethics Committee of Northwestern Polytechnical University.