**1. Introduction**

Anti-infective drugs play important roles in the prevention and treatment of inflammatory bowel disease (IBD) in humans and animals. IBD is a complex gastrointestinal disease, mainly induced by infection with gram-negative bacteria such as *Escherichia coli* (*E. coli*) and *Salmonella* [1]. In recent decades, an increasing prevalence of antibiotic resistance has threatened human health [2]. Finding effective alternatives to antibiotics has become an increasingly urgent task. Among the potential alternatives, antimicrobial peptides (AMPs) are particularly important, due to their

broad spectrum antibacterial activity and decreased likelihood of inducing antibiotic resistance. Currently, more than 2800 AMPs have been found in animals, plants or microorganisms [3]. In animals, AMPs play important roles in host defense and are crucial in the immune system [4]. The activities of AMPs vary greatly due to their different sequences and structures. In addition to antimicrobial activity, some AMPs also have wound healing abilities through promoting cell proliferation, reducing inflammation or enhancing intestinal barrier function [5,6].

Cecropins are a group of peptides with an α-helical structure and were initially found in insects. Currently, there are more than 30 records of cecropins in the Antimicrobial Peptide Database (APD), including naturally discovered and artificially synthesized cecropins [3]. Cecropin A is one of the earliest discovered cecropins by Steiner et al. from *Hyalophora cecropia* [7]. Over the past decades, the antibacterial mechanisms of cecropin A have been extensively researched [8,9]. In addition, cecropin A is also a commonly used template for peptide molecular hybrids to enhance the antibacterial activity of AMPs [10]. Although the antibacterial activity of cecropin A has been demonstrated for decades, to our best knowledge, the effect of cecropin A on intestinal barrier function is still unknown.

IBD is caused by pathogenic bacterial infection and intestinal mucosal barrier disruption. Intestinal mucosal surfaces consist of epithelial cells, such as absorptive cells, endocrine cells and Paneth cells [11]. The epithelial cells form a selectively leaky barrier, which is crucial for nutrient substance exchange and host defense [12–15]. These functions depend on intact intestinal epithelial cell layers, which are composed of cell–cell attachments at the cell lateral membrane by tight junctions (TJs) and subjacent adherens junctions [13]. The TJs consist of transmembrane proteins such as claudins, occludin and junctional adhesion molecules (JAMs). These proteins are clustered and stabilized by cytoplasmic scaffolding proteins called zonula occludens (ZOs) and cytoskeletons such as F-actin. Different tight junction proteins play various roles in barrier function. Claudins and occludin are located at apical and basal positions of the lateral membrane, respectively [16]. ZOs, such as ZO-1, can interact with cytoskeleton, claudin-1 and occludin [17]. To summarize, the TJ-cytoskeleton structure is essential for the intestinal barrier.

The regulation of TJ expression and membrane distribution is complex. The mitogen-activated protein kinase (MAPK) pathways, which contain three downstream pathways including extracellular signal-regulated kinase (ERK), p38 and c-jun, are responsible for cell proliferation, proliferation and immune reaction in the gastrointestinal tract [18]. ERK, which may be activated by mitogen-activated protein kinase (MEK), is one of the most important pathways for maintaining gastrointestinal tract homeostasis and regulating the intestinal barrier. However, according to previous studies, the effect of MEK/ERK on intestinal barrier function is controversial. Piegholdt et al. [19] showed that biochanin A and prunetin may improve epithelial function through downregulation of ERK, while Wang et al. [20] showed an improvement of the intestinal epithelial barrier through upregulation of the ERK pathway by polyphenol-rich propolis extracts. The regulatory effect of the MEK/ERK signaling pathway on the intestinal barrier is unclear.

In this study, we evaluated the effects of cecropin A on intestinal barrier function in an IPEC-J2 cell monolayer model. We also detected the TJ protein level and membrane distribution by using Western blotting and cell immunofluorescence, respectively. Finally, the changes in the MEK/ERK signaling pathway were detected to reveal the regulatory mechanism of cecropin A on the barrier function.

#### **2. Results**

#### *2.1. The Antibacterial Activity of Different AMPs*

Seven AMPs were selected from the APD database, antibacterial activities including minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were tested by using 11 specific bacterial strains (Table S1) and the information of origin source, peptide length, net charge were also described (Table S2). The results showed that cecropin A possessed the best antibacterial activity (MIC and MBC between 1.5 and 6.25 μg/mL) to gram-negative bacterial strains, such as *E. coli*, *Salmonella* and *Pseudomonas aeruginosa*.

#### *2.2. Cytotoxicity to IPEC-J2 Cells*

The cytotoxicity of AMPs to the pig intestinal epithelium cell IPEC-J2 was evaluated by using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Figure S1A–G). The concentration was 1.5–100 μg/mL. The results showed that IPEC-J2 cell viability was not reduced after treatment with cecropin A (1.5–12.5 μg/mL, Figure S1C) for 8 h compared to that of the control group. Six other AMPs reduced cell viability in a dose-dependent manner.

#### *2.3. Cecropin A Inhibits E. coli Adherence and Ameliorates Inflammation*

IPEC-J2 cells pretreated with 3.125, 6.25, and 12.5 μg/mL cecropin A displayed reduced *E. coli* adherence in a dose-dependent manner (Figure 1A). In addition, after coculture with *E. coli*, the TNF-α, IL-6, and IL-8 mRNA expression in IPEC-J2 cells was also downregulated after cecropin A treatment for 48 h, compared to that in the non-treated cells (Figure 1B). The results suggested that the defense capability of IPEC-J2 cells against bacteria was increased.

**Figure 1.** Cecropin A inhibited bacterial adherence and reduced the expression of inflammatory factors. The porcine jejunum epithelial cells (IPEC-J2) cells were pretreated with cecropin A (3.125, 6.25, and 12.5 μg/mL) for 48 h, and the CFU of adherent bacteria were counted ((**A**), *n* = 3). The relative mRNA expression of *TNF*-*α*, *IL*-*8*, and *IL*-*6* were tested by using qPCR ((**B**), *n* = 6). Control: control group; cecropin A: cells were pretreated with cecropin A; cecropin A + *E. coli*: cells were pretreated with cecropin A and then cocultured with *E. coli*; *E. coli*: cells cocultured with *E. coli*. The results were confirmed by three independent experiments per treatment. Representative results of the three independent experiments are shown. Data (mean ± SEM) were analyzed with one-way ANOVA. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.
