Antimicrobial Peptide MPX Against Escherichia Coli O157:H7 Infection and Inhibiting in ﬂ ammation, Enhancing Epithelial Barrier and Promoting Nutrient Absorption in the Intestine

Background: Escherichia coli can cause intestinal diseases in humans and livestock, destroy the intestinal barrier, exacerbate systemic inammation, and seriously threaten human health and animal husbandry development. The antimicrobial peptide MPX is extracted from venom and possesses good antibacterial activity against gram-negative bacteria. The aim of this study was to investigate whether MPX could be effective against E. coli infection. Results: In this study, the CCK-8 and lactic dehydrogenase results showed that MPX exhibited no toxicity in IPEC-J2 cells even at a concentration of 128 µg/mL. Furthermore, MPX notably suppressed the levels of IL-2, IL-6, TNF-α, myeloperoxidase and LDH induced by E. coli and reduced inammation by inhibiting the p-p38-, TLR4- and p-p65-dependent pathways. In addition, MPX improved the expression of ZO-1, occludin, and claudin and enhanced the wound healing ability of IPEC-J2 cells. The therapeutic effect of MPX was evaluated in a murine model, and the results showed that MPX could protect mice against lethal infection with E. coli, improve the survival rate of the mice, and reduce the colonization of E. coli in organs and feces. H&E staining showed that MPX increased the length of villi and reduced the inltration of inammatory cells into the jejunum, and the effect of MPX was better than that of enrooxacin. The SEM and TEM results showed that MPX effectively ameliorated the damage caused by E. coli to the jejunum and increased the number and length of microvilli. In addition, real-time PCR revealed that MPX decreased the expression of IL-2, IL-6, and TNF-α in the jejunum and colon. Furthermore, immunohistochemistry and immunouorescence studies revealed that MPX could reduce the expression of p-p38 and p-p65 in the jejunum, thereby reducing the secretion of inammatory factors. Moreover, MPX increased the mRNA and protein expression of ZO-1, occludin and MUC2 The length of jejunal villi, the depth of crypts, and the ratios of villi length to crypt depth were detected by ipwin32 software. # P < 0.05; ## P < 0.01; ### P < 0.001 E. coli vs control; *P < 0.05; **P < 0.01; ***P < 0.001 MPX and Enro treatment vs E. coli.

Due to the alarming increase in pathogen resistance to conventional antibiotics and threats to public health worldwide, exploring new drugs to treat infections with antimicrobial-resistant pathogens is urgently needed [5]. Antimicrobial peptides are a class of small molecular peptides produced by the innate immune system of the body that can resist pathogenic infection. The functions of antimicrobial peptides include antibacterial, antiviral, anti-in ammatory and immune regulatory function [6,7], and antimicrobial peptides are currently considered the best alternatives to antibiotics. The antimicrobial peptide MPX is extracted from venom, contains both acidic and basic residues, including three basic residues, and possesses a net charge of 4; MPX has good antibacterial activity against gram-positive and gram-negative microorganisms [8]. At present, Henriksen et al found that modulating peptide hydrophobicity by introducing an unnatural amino acid with an octyl side chain via amino acid substitution at positions 1, 8 and 14 could increase the bactericidal potency of the antimicrobial peptide MPX [9]. Our previous laboratory studies showed that MPX had good antimicrobial activities against A. pleuropneumoniae infections, reduced the colonization of A. pleuropneumoniae in the lungs, alleviated the symptoms of pneumonia and improved the survival rate of mice [10]. However, the effect of MPX against E. coli infections and in the regulation of E. coli-induced in ammation and intestinal disruption remains unknown.
In this study, the effects of MPX on E. coli -induced intestinal in ammation and barrier dysfunction were investigated in vitro and in vivo. In IPEC-J2 cells, the results showed that MPX exhibited no cytotoxicity and reduced the mRNA expression of IL-2, IL-6 and TNF-α by inhibiting the phosphorylation of p38 and p65. In addition, MPX increased the E. coli-induced expression of the tight junction proteins ZO-1 and occludin and promoted the healing of damage to intestinal epithelial cells. These results were further con rmed in vivo. The results showed that MPX could protect against lethal infection with E. coli, improve the survival rate of mice, alleviate intestinal in ammation in the jejunum and colon by reducing the expression of in ammatory factors, and increase the expression of tight junction proteins and the number of microvilli, thereby improving intestinal barrier function. These ndings suggest a promising protective role of MPX in preventing E. coli infections, laying the foundation for the development of alternatives to conventional antibiotics.

Ethics statement
All the animal experiments were approved by the Animal Ethics Committee of Zhengzhou University in accordance with the guidelines of the Animal Welfare and Research Ethics Committee.

Peptides synthesis
MPX (H-INWKGIAAMAKKLL-NH2) was synthesized and puri ed by Ji er sheng hua (Shanghai, China). The purity of MPX was higher than 98%, as determined by high-performance liquid chromatography (HPLC) and mass spectrometry. MPX was dissolved in ddH 2 O and stored at -20 °C.
Preparation of E.coli strain E. coli was obtained from the China Institute of Veterinary Drug Control (Beijing, China). The E. coli strain was seeded on LB (Solarbio, China) agar to obtain single pure colonies. Then, a single colony of E. coli was inoculated into LB broth and incubated overnight at 37 °C and 180 rpm/min. 100 µL were transferred to 10 mL fresh LB broth and incubated at 37 °C and 180 rpm/min for 4 h. Then, 1 mL cultures were collected and washed twice with phosphate buffer (pH = 7.4) at 8000 rpm/min for 5 min. Then, ten times serial dilutions were performed and seeded on LB agar to obtain single pure colonies, and 4.5 × 10 7 CFU/mL bacteria were used.
Cell culture IPEC-J2 cells were cultured in DMEM with 10% fetal bovine serum and 1% antibiotics (penicillin and streptomycin) and incubated in an environment at 37 °C with 5% CO 2 . At 90% con uence, the cells were digested by trypsin and cultured in six-well cell plates (Solarbio, China). When they reached 80-90% con uence, the cells were cultured in DMEM medium without 1% antibiotics and then treated with MPX (10 µg/mL) for 2 h. Then, the cells were further cultured with E. coli (MOI = 10) for 12 h.

Cytotoxicity studies
Cell viability was determined by the CCK-8 kit (Meilunbio, China). The experiments were carried out in accordance with the instructions. First, IPEC-J2 cells were cultured in 96-well plates at 1 × 10 4 cells/well and treated with the indicated concentration (2-512 µg/mL) of MPX for 24 h. Then, the IPEC-J2 cells were incubated with 10 µL CCK-8 per well in a cell culture incubator for 2 h. Then, a microplate reader was used to detect the absorbance of each well at 450 nm (Dynatech Laboratories, USA). Three replicates per sample were assessed.
The LDH release assay (Nanjing Jiancheng, China) was used to determine whether MPX caused IPEC-J2 cell membrane damage. The experiments were carried out in accordance with the instructions. Brie y, IPEC-J2 cells were cultured in 96-well plates at 1 × 10 4 cells/well and treated with the indicated concentration (2-512 µg/mL) of MPX in an environment at 37 °C with 5% CO 2 for 24 h. In addition, IPEC-J2 cells were pretreated with MPX (10 µg/mL) for 2 h and infected with E. coli (MOI = 10) for different times (3 h, 6 h, 12 h, 24 h). The optical densities were measured at 450 nm using a microplate reader (VARIOSKAN FLASH, USA). Three replicates per sample were assessed.

Real-time PCR
The primer sequences for real-time PCR are shown in Table 1. Total RNA was extracted using RNA extraction kit reagent (Soliabao, China). The quantity and quality of the RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scienti c, USA). cDNA was obtained using a reverse transcription kit (Thermo Scienti c Molecular Biology, USA). Each reaction (10 µL) volume included 5 µL SYBR Green Master Mix (QuantiNova, China), 0.5 µL forward primer (10 µM), 0.5 µL reverse primer (10 µM), 0.5 µL cDNA and 3.5 µL ddH 2 O. The thermocycler reaction included 2 min at 95 °C and 40 cycles Page 6/19 of 20 s at 95 °C and 30 s at 60 °C, and melt curves were added. The housekeeping gene was GAPDH. The 2 −ΔΔCt method was used to calculate the relative mRNA expression [11]. Three replicates per sample were assessed. Table 1 The primer sequences for real-time PCR Genes Sequence

Western blotting
Total protein was extracted with RIPA lysate buffer (KeyGEN, China). The protein concentration was determined using a BCA protein content kit (Biyuntian, China). Then, the proteins in the lysate supernatants were separated by 10% SDS-PAGE and transferred onto a PVDF membrane. After blocking with 5% bovine serum albumin (BSA) for 2 h, the membrane was incubated with primary antibodies overnight at 4 °C and washed with TBST 5 times for 7 min each time. The membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. The membranes were washed with TBST 5 times for 7 min each time [12]. The bands were detected using ECL (Solaibao, China). ImageJ software was used to quantify the band intensities. Primary antibodies against β-actin (Abcam, USA) and occludin (Abcam, USA) were used in this study.

Transmission Electron Microscopy
The tight junctions (TJs) and microvillus morphology between the intestinal epithelial cells of mice were observed by TEM [13]. Mouse jejunum specimens were obtained with a scalpel and xed in 2.5% glutaraldehyde for 12 h at 4 °C. Then, the jejunum of the mice was treated with osmic acid and embedded in epon. Ultrathin sections were acquired using a diamond knife and then stained with uranyl acetate and lead citrate before being observed by TEM (7610plus/FEI Apreo, Japan).

Scanning Electron Microscopy
The morphology of the jejunum villi and microvilli in mice was observed by SEM [14]. Jejunum tissue from the mice was xed with 2.5% glutaraldehyde overnight at 4 °C and then incubated with 1% OsO4 for 1 h. The jejunum specimens from the mice were then dehydrated with an ethanol gradient (30%, 50%, 70%, 80%, 90%, 95% and 100%) for 15 min at each step and then treated with a mixture of alcohol and isoamyl acetate (v:v = 1:1) for 30 min. Then, isoamyl acetate was added for 1 h. Then, the dehydrated specimens were coated with gold-palladium and visualized using a Philips Model SU8010 FASEM (HITACHI, Japan).

ELISA
The serum levels of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) were determined using ELISA kits (Biolegend, USA). In addition, the serum level of myeloperoxidase (MPO) was detected using an ELISA kit (Multi Science, China). All the samples were measured according to the manufacturers' instructions. Three replicates per sample were assessed.

Histopathology and immunohistochemistry
The jejunum and colon tissues of the mice were xed in 4% paraformaldehyde for 24 h and embedded in para n. Hematoxylin and eosin (H&E) staining was used to stain the jejunum. Images were obtained using a DM3000 microscope (PHASE CONTRAST, Japan). Image-Pro software (Media Cybernetics, USA) was used to measure the villous height and crypt depth, and then, the ratio of villous height to crypt depth was calculated [15].
For immunohistochemistry (IHC), 5-µm sections of the jejunum and colon tissues were embedded in para n. The sections were incubated in sodium citrate buffer (pH = 6.0) to repair the antigens and placed in 3% hydrogen peroxide solution to block endogenous peroxidase after dewaxing and rehydration. To block nonspeci c binding sites, the sections were incubated with 3% BSA for 30 min. Then, the sections were incubated with anti-p-p38, anti-p-pERK, and anti-p-pJNK antibodies overnight at 4 °C. Then, HRPconjugated secondary antibodies were added to the sections and incubated for 50 min. Next, the sections were counterstained with hematoxylin after development with DAB buffer [16].
IPEC-J2 cells were cultured in special plates for confocal laser microscopy, pretreated with MPX (10 µg/mL) for 2 h and then incubated with E. coli (MOI = 10) at 37 °C for 12 h. Then, the IPEC-J2 cells were washed with PBS (pH = 7.4) 3 times and then xed with 2.5% glutaraldehyde for 30 min. The IPEC-J2 cells were washed 3 times in PBS (pH = 7.4), blocked with 5% bovine serum albumin at room temperature for 1 h, incubated with primary rabbit antibodies overnight at 4 °C (anti-occludin, anti-p-p38, and anti-p-p65), incubated with the secondary antibody (Alexa Fluor 594-anti-rabbit) for 1 h and stained with DAPI for 15 min. Tight junction proteins and in ammatory proteins were observed under a confocal laser microscopy (EVOS M7000, USA) [17].

Animals and sample collection
A total of 50 BALB/c mice (6 to 8 weeks old, body weights of 18 to 20 g, female) were purchased from Zhengzhou University. The animals were random divided into 4 experimental groups, including the control, E. coli, E. coli + MPX, and E. coli + Enro groups, with 10 mice per group. The each BALB/c mice were challenged with intraperitoneal injection of E. coli(4.5 × 10 7 CFU/mL) [18]. The mice were treated with intraperitoneal injection of normal saline (control), MPX (20 mg/kg), or Enro (20 mg/kg) once a day for 3 d after infection with E. coli for 2 h. The clinical symptoms of the mice, including the state of the hair, body weight change, mental state, and appetite, were recorded every day. The speci c scoring criteria were as follows: no clinical signs as 0; slight as 1; moderate as 2; severe as 3. The mice were sacri ced at 96 h after E. coli infection. The serum was used for in ammatory factor analysis. The liver, spleen, lung and intestines were collected and xed with 4% paraformaldehyde for H&E staining, immunohistochemistry and immuno uorescence analysis. Glutaraldehyde (2.5%) was used to x the jejunum to observe the changes in the intestinal villi and microvilli by scanning electron microscopy and transmission electron microscopy.

MPX reduces the release of LDH and inhibits the expression of in ammatory cytokines
The cytotoxicity of MPX was tested since the aim of this study was to develop this peptide as a safe alternative to antibiotics. A CCK-8 kit was used to determine the viability of IPEC-J2 cells after treatment with MPX at different concentrations (2-512 µg/mL) for 24 h. Compared with the control treatment, MPX increased the cell viability ( Fig. 1a, P < 0.001). The effect on cultured cells was not signi cant, even at a high concentration of 128 µg/mL. Interestingly, the results showed that low concentrations of MPX could promote the growth of IPEC-J2 cells (P < 0.05). The release of LDH was examined to further determine the toxicity of MPX. Compared with the control group, treatment with different concentrations (2-512 µg/mL) of MPX for 24 h did not signi cantly increase the release of LDH, even at a concentration of 128 µg/mL (Fig. 1b). Moreover, the LDH release from IPEC-J2 cells was notably reduced after pretreatment with MPX for 2 h prior to infection with E. coli (Fig. 1c, p < 0.05). These results indicated that MPX maintained the cellular membrane integrity of IPEC-J2 cells.
The increased expression of in ammatory factors, such as IL-2, IL-6 and TNF-α, is closely related to the in ammatory response [19]. To evaluate the anti-in ammatory effects of MPX after E. coli infection, the expression of IL-2, IL-6 and TNF-α was determined by real-time PCR. Compared with E. coli alone, treatment with MPX signi cantly inhibited the E. coli-induced mRNA expression of IL-2, IL-6 and TNF-α (Fig. 1d, e and f, p < 0.05). In addition, the confocal laser microscopy results showed that E. coli infection signi cantly increased the expression of p-p38, p-p65 and TLR4, while pretreatment with MPX signi cantly decreased the expression of p-p38, p-p65 and TLR4 (Fig. 1g), indicating that MPX could inhibit the release of in ammatory cytokines by reducing the phosphorylation of p38 and the activation of p65 and TLR4.  [20]. To evaluate the effects of MPX after E. coli infection, the expression of ZO-1, occludin, and claudin-1 was determined by real-time PCR. As shown in Fig. 2A, the E. coli-induced mRNA expression of ZO-1 and occludin in IPEC-J2 cells was signi cantly increased after MPX treatment (p < 0.05), while the expression of claudin-1 was not signi cantly altered (p > 0.05). Interestingly, in a wound healing assay, the wound width was signi cantly reduced at 48 h in IPEC-J2 cells treated with MPX (Fig. 2b,   dose of E. coli, and the survival rate of the mice was 90%; this effect of MPX was better than that of Enro. However, the mice infected with E. coli without MPX treatment all died within 60 h (Fig. 3a). The observation of clinical symptoms revealed that E. coli infection caused severe diarrhea, lack of energy, loss of appetite, clustering, and messy back hair, while these symptoms were signi cantly alleviated after MPX treatment; these effects of MPX were superior to the effects of the same dose of Enro (Fig. 3b). The weight of the mice infected with E. coli was notably reduced (Fig. 3c, p < 0.01), but it was signi cantly increased after MPX and Enro treatment (Fig. 3c, p < 0.01) and not signi cantly different from the control (Fig. 3c, p > 0.05). The weights of the livers and spleens in the E. coli infection group were notably heavy (Fig. 3d, e, p < 0.05) and signi cantly decreased after MPX treatment; after MPX treatment, these weights were not signi cantly different from those in the control group (Fig. 3d, e, P > 0.05). The weight of the lung was not signi cantly changed after E. coli infection (Fig. 3f, P > 0.05). The colonization of E. coli in the liver, spleen, lung and feces of the mice was examined by counting on LB agar plates. The results showed that the number of bacteria colonizing the spleens of the E. coli group was greater than that colonizing the liver and lung, and this number was signi cantly decreased after MPX and Enro treatment (Fig. 3g, P < 0.05). The number of bacteria colonizing the feces was signi cantly lower after MPX and Enro treatment than after infection with E. coli alone (Fig. 3h, P > 0.05). These results indicated that MPX exerted good antibacterial effects in vivo and protected against lethal infection with E. coli in mice. injected with E. coli. # P < 0.05; ## P < 0.01; ### P < 0.001 E. coli vs control; *P < 0.05;**P < 0.01; ***P < 0.001 MPX and Enro treatment vs E. coli. MPX reduces the levels of in ammatory cytokines and improves intestinal morphology MPO activity is an index of neutrophil in ltration and in ammation, and MPO can produce speci c oxidative species [21]. To evaluate the effect of the MPX-mediated anti-in ammatory response after E. coli infection, the levels of IL-2, IL-6, TNF-α and MPO were detected by ELISA. As shown in Fig. 4a, the levels of the in ammatory factors IL-2, IL-6, TNF-α and MPO were signi cantly increased after E. coli infection, while MPX signi cantly reduced the secretion of IL-6 (p < 0.01), IL-2, TNF-α and MPO (p < 0.05). H&E staining was used to explore the effect of MPX on the intestinal morphology of the jejunum in mice infected with E. coli; the results showed that infection with E. coli caused typical intestinal in ammation and barrier damage, shortened villi, necrosis, large amounts of in ammatory cell in ltration into the jejunum and disrupted intestinal villi, while MPX treatment increased villous height and goblet cell counts and decreased the in ltration of leukocytes into the jejunum, and these levels were not signi cantly different compared with those observed in control group (Fig. 4b). Moreover, compared with E. coli infection alone, MPX treatment increased the villi length in the jejunum in the mice, decreased the crypt depth, and increased the ratio of villi height to crypt depth, and these effects of MPX were better than those of Enro (Fig. 4c, p < 0.05). These results suggest that MPX effectively reduced in ammatory factor secretion and improved intestinal morphology and integrity in mice infected with E. coli.  (Fig. 5a); this effect of MPX was better than that of Enro, which was consistent with H&E staining. We further evaluated the effect of MPX on the microvilli and tight junction proteins of intestinal epithelial cells by TEM. The results showed that E. coli infection caused microvilli to fall off, decreased the number of microvilli, and damaged the tight junction structure of the intestinal epithelial cells, while MPX treatment signi cantly increased the quantity of the microvilli in intestinal epithelial cells; this effect of MPX was better than that of Enro, and the results were not signi cantly different compared with those observed in the control group (Fig. 5b). These results indicate that MPX could protect against E. coli-induced damage to jejunal villi and microvilli in intestinal epithelial cells.

MPX suppresses intestinal in ammation by downregulating the expression of p-p38 and p-p65
Previous studies showed that MPX treatment could reduce the serum levels of in ammatory factors after E. coli infection. To further investigate the anti-in ammatory effect of MPX on the intestine, the mRNA expression of IL-2, IL-6 and TNF-α in the jejunum and colon was detected using real-time PCR. E. coli infection led to a signi cant increase in the expression of the in ammatory factors IL-2, IL-6 and TNF-α in the jejunum and colon (Fig. 6a, p < 0.01), while MPX and Enro treatment signi cantly inhibited the mRNA expression of IL-2, IL-6 and TNF-α in the jejunum (p < 0.01) and colon (p < 0.05); after MPX and Enro treatment, the levels were not signi cantly different from those observed in the control group (P > 0.05). Mitogen-activated protein kinases (MAPKs), including JNK, ERK1/2 and p38, are a group of serine/threonine proteins and the nal step of cytoplasmic signal transduction pathways that are activated by multiple extracellular signal pathways. These proteins play a role in the activation of nuclear transcription factor p65, regulating gene expression and participating in cytokine secretion and apoptosis after activation [22]. Immunohistochemistry and immuno uorescence were used to further explore the mechanism by which MPX inhibits the secretion of in ammatory factors. The immunohistochemistry results showed that MPX notably reduced the expression of p-p38 in the crypts of the jejunum, and this effect was superior to the effects of the same dose of Enro (Fig. 6b). However, the expression of p-pJNK and p-pERK in the jejunum was not signi cantly changed after treatment with MPX and Enro (Fig. 6b), indicating that MPX had no signi cant effect on the expression of p-pJNK and p-pERK and mainly regulated the p-p38 signaling pathway, decreasing the secretion of in ammatory factors and thereby reducing the in ammatory response. The results were consistent with Fig. 1g. In addition, the activation of p65 was analyzed by immuno uorescence, and the results showed that MPX signi cantly decreased the phosphorylation of p65 compared with E. coli infection alone (Fig. 6c). These results indicated that MPX could inhibit the release of in ammatory cytokines by reducing the phosphorylation of p38 and the activation of p65. Mucin is the key matrix-forming component of mucus, which is an innate protective barrier that protects the host from pathogenic attack [23]. The IPEC-J2 cells results showed that MPX could improve tight junction protein expression after E. coli infection. Immuno uorescence and real-time PCR were used to further investigate the effects of MPX on E. coli-induced tight junction protein and MUC2 expression in the jejunum and colon. The results showed that E. coli infection decreased the expression of ZO-1, occludin and MUC2, while MPX treatment signi cantly increased the expression of ZO-1, occludin and MUC2 in the jejunum and colon; this effect of MPX was superior to that of Enro (Fig. 7a, P < 0.05). However, none of the groups showed a signi cant effect on the expression of the tight junction protein claudin-1 (Fig. 7a, P > 0.05). Immuno uorescence was used to further study the effect of MPX on tight junction protein and MUC2 expression after E. coli infection. The results showed that E. coli infection reduced the expression of ZO-1, occludin and MUC2 in the jejunum and colon of mice, while the expression of ZO-1, occludin and MUC2 was improved after treatment with MPX. The effect was better than that of Enro, and the expression levels were not signi cantly different from those observed in the control group (Fig. 7b, c). Collectively, these results indicate that MPX treatment could signi cantly improve the expression of tight junction proteins and mucin in the jejunum and colon.

Discussion
Antimicrobial peptides are considered to be the best substitutes for antibiotics due to their bene cial antibacterial, anti-in ammatory, and immune regulatory effects and have become a focus of research in recent years [24]. MPX belongs to the mastoparan family, contains 14 amino acids, has a high concentration in wasp venom, and has good antimicrobial activity against bacteria, indicating that MPX can be used as a substitute for antibiotics in the treatment of bacterial infection [9]. In this study, we Previous studies have found that the morphological integrity of villi and microvilli, which plays a key role in the absorption of intestinal nutrients, is an important indicator of the performance and health of the host [25]. Yoon et al investigated the effect of the addition of the antimicrobial peptide cLFchimera (20 mg/kg) to the diet of broiler chickens in the context of necrotic enteritis (NE) challenge and found that cLFchimera ameliorated intestinal lesions and changes to villus morphology in the jejunum [26]. Liang et al found that bovine antimicrobial peptide-13 (APB-13) has good antiviral activity against transmissible gastroenteritis virus (TGEV) and signi cantly reduced the piglet diarrhea induced by TGEV, improving intestinal villus morphology [27]. Previous studies have found that the antimicrobial peptide MccJ25 could protect against ETEC infection and signi cantly alleviate the destruction of intestinal morphology and changes in villus morphology in mice infected with ETEC [3]. Wang et al investigated the effect of the antimicrobial peptide JH-3 on the intestinal in ammation induced by Salmonella CVCC541 and found that JH-3 could effectively alleviate the pathological damage to the duodenum and jejunum, reduce the loss of intestinal villi and improve the morphology of intestinal villi [28]. In this study, the results showed that MPX could signi cantly improve the pathological damage to the intestinal caused by E. coli, reduce the loss of intestinal villi, and maintain the morphology of intestinal villi. The effects of MPX on the intestinal epithelial cell microvilli were further con rmed using TEM, and MPX not only improved the morphology of intestinal villi but also increased the number of microvilli, thereby increasing nutrient absorption in the intestine. These results indicated that MPX could effectively alleviate intestinal damage and maintain villi and microvilli morphology, promoting nutrient absorption in the intestine.
Antimicrobial peptides, as an important part of the natural immune system, possess good antiin ammatory activity [29]. Long-term and excessive production of proin ammatory cytokines may lead to intestinal damage and high energy requirements [30]. Atikan Wubulikasimu et al found that the antimicrobial peptide AKK8 possessed good antibacterial activity against drug-resistant strains of C. albicans, signi cantly reducing the levels of IL-6, IL-1β and TNF-α in the serum of mice infected with C. albicans [31]. Ding et al evaluated the effect of the antimicrobial peptide microcin J25 against ETEC infections in a murine model and found that microcin J25 decreased the secretion of in ammatory factors by inhibiting the activation of the MAPK and NF-κB signaling pathways, thereby alleviating the intestinal in ammatory response induced by ETEC [3]. Min Kyoung Shin et al investigated the effect of the antimicrobial peptide Lycotoxin-Pa4a on the LPS-induced in ammatory response in RAW264.7 cells and found that Lycotoxin-Pa4a signi cantly reduced the expression of the in ammatory cytokines IL-1β and TNF-α by inhibiting the activation of the MAPK pathway, thereby inhibiting the LPS-induced in ammatory response in RAW264.7 cells [32]. In this study, we found that MPX signi cantly reduced E. coli-mediated expression of the in ammatory factors IL-2, IL-6 and TNF-α by inhibiting the activation of p-p38 and p-p65 in vitro and in vivo, thereby attenuating intestinal in ammation. These results indicated that MPX exerts good anti-in ammatory effects and can be a bene cial agent for alternative antibiotics.
The intestinal barrier mainly includes the intestinal epithelial barrier, immune barrier, chemical barrier and biological barrier. In addition, the intestinal epithelial barrier is the rst barrier that can prevent bacteria, antigens and other toxic and harmful substances from entering the submucosa of the intestine and blood [33]. The tight junction (TJ) structure is composed of TJ proteins, such as ZO-1, occludin and claudins, which are an important part of the intestinal epithelial barrier and play an important role in intestinal epithelial cells [34]. Lin et al investigated the effect of the antimicrobial peptide gloverin A2 (BMGlvA2) on ETEC-induced intestinal barrier disruption in mice and found that BMGlvA2 clearly improved the expression of the tight junction protein ZO-1 in the intestine after ETEC infection [35]. Zhang [37]. In this study, we found that MPX improved the expression of ZO-1 and occludin in IPEC-J2 cells. Furthermore, MPX enhanced the expression of the tight junction proteins ZO-1, occludin and MUC2 in the jejunum and colon in mice, indicating that MPX attenuates intestinal barrier dysfunction by improving tight junction protein and mucin expression. Surprisingly, MPX is more effective in increasing tight junction protein and mucin expression than Enro. The reason may be due to the antibacterial properties of antibiotics, which control the balance of microorganisms in the intestine.

Conclusion
In summary, as shown in Fig. 8, we demonstrated that MPX could reduce E. coli growth, attenuate the in ammatory response and intestinal damage, inhibit E. coli-induced TLR4 expression, and decrease the IL-2, IL-6 and TNF-α levels by blocking the activation of the p65 and p38 in ammatory pathways in vitro and in vivo. In addition, MPX improved intestinal barrier function and increased the expression of the tight junction proteins ZO-1, occludin and mucin. These ndings suggest that MPX can be an excellent antimicrobial or anti-in ammatory agent to protect against pathogen infections, laying a foundation to develop MPX as a substitute for conventionally used antibiotics or drugs.