*2.4. Cecropin A Increases the TER and Decreases the Paracellular Diffusion of FITC-Dextran through the IPEC-J2 Monolayer*

The integrity of the intestinal barrier may increase the defense capability of the host and reduce the adherence of pathogenic bacteria. We examined whether cecropin A could enhance the intestinal monolayer barrier function. Transepithelial electrical resistance (TER) values were assessed at 24, 48 and 72 h after cecropin A treatment. Our data showed that, compared to those of the control cells, the TER values of cecropin A-treated cells were significantly increased at 48 h and 72 h (Figure 2A; *p* < 0.01). In addition, we measured the permeability of large solutes by using 4 kDa FITC-dextran as a tracer. The data showed that, after cell incubation with FITC-dextran, the concentrations of FITC-dextran in the basal compartment were significantly higher than those in cecropin A-treated cells at 48 h and 72 h (Figure 2B, *p* < 0.05).

**Figure 2.** Cecropin A enhances the transepithelial electrical resistance (TER) and reduces the permeability of the IPEC-J2 cell monolayer. The TER ((**A**), *n* = 6) and permeability ((**B**), *n* = 6) of the cell monolayer were tested after treatment with cecropin A for 24 h, 48 h, and 72 h. 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 the Student's *t*-test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### *2.5. Cecropin A Regulates TJ Protein Expression Levels, Membrane Distribution and F-Actin Polymerization*

To elucidate how cecropin A increases the TER value and decreases paracellular permeability, we measured the protein levels of ZO-1, claudin-1 and occludin. The results showed that the protein levels of TJs were significantly upregulated (Figure 3A,B). We also detected TJ membrane distribution by using cell immunofluorescence. The results showed that ZO-1, claudin-1 and occludin were much more polymerized at the cell–cell boundary in the cecropin A group than in the control group. (Figure 3C). The integrity of the intestinal monolayer barrier is coordinated by the connection between TJs and the cytoskeleton. Representative F-actin staining indicated that the cecropin A group had more extensive F-actin than the control group (Figure 3C). In addition, F-actin was much more polymerized at the cell–cell boundary in the cecropin A group than in the control group. In accordance with the TJ distribution, the results suggested that the better organized F-actin-TJ structure may help increase the monolayer barrier function and the defense capability against the adherence of *E. coli*.

#### *2.6. Cecropin A Regulates the Intestinal Barrier by Downregulating the MEK/ERK Pathways*

To elucidate how cecropin A regulates the TER and TJs, the MEK/ERK signaling pathway was detected using Western blotting. The data showed that phosphorylation of MEK and ERK in control group cells was significantly higher than that in the cecropin A group cells (*p* < 0.01, Figure 4). Caudal type homeobox 2 (CDX2), a transcriptional factor that regulates the differentiation of intestinal cells, was also detected (*p* < 0.001). The results showed that the CDX2 protein level was upregulated in the cecropin A group compared with that in the control group. In addition, the results suggested that cecropin A may regulate TJ expression through regulating the phosphorylation of MEK and ERK and the expression of CDX2.

**Figure 3.** The effect of cecropin A on tight junction (TJ) protein expression, membrane distribution and F-actin polymerization. Western blotting analysis of zonula occludens-1 (ZO-1), occludin and claudin-1 expression were upregulated by cecropin A treatment ((**A**), *n* = 3); quantification of ZO-1, occludin, and claudin-1 protein expression was shown ((**B**), *n* = 3); cell immunofluorescence (**C**) (400×) showed that the cecropin A induced the TJs polymerized at the cell–cell boundary (**a**–**f**) and indicated F-actin polymerization (**g**,**h**) in cells ((**c**), *n* = 3). 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 the Student's *t*-test. Cell nuclei were stained by 4 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI) and are shown in blue. Claudin-1, ZO-1 and occludin are shown in red and pointed out by white arrow heads. F-actin is shown in green. Scale bar is 50 μm. \*\*\* *p* < 0.001.

**Figure 4.** Cecropin A downregulates mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation and upregulates CDX2 expression, *n* = 3. Western blotting analysis of p-MEK, MEK, p-ERK(1/2), ERK (1/2) and CDX2 showed that cecropin A downregulated the MEK/ERK pathway and increased CDX2 protein level (**A**,**B**). 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 the Student's *t*-test. Control: control group; Cecropin A: cells treated with cecropin A group. \*\* *p* < 0.01, \*\*\* *p* < 0.001.

### *2.7. Inhibition of the MEK/ERK Pathway Increases TER, TJ Expression, Membrane Distribution and F-Actin Polymerization*

To confirm the regulatory effect of the MEK/ERK pathway on the intestinal barrier and TJs, a specific inhibitor (PD184352) of ERK 1/2 was used (Figure 5A). After treatment with the inhibitor for 48 h, the adherence of *E. coli* was reduced compared to that in the cecropin A group (Figure 5B). The TER values of the cecropin A-treated group were significantly higher than those of the control group, while the TER values of the PD184352 + cecropin A group were higher than those of the cecropin A group (Figure 5C, *p* < 0.01). In addition, protein levels of CDX2, ZO-1, claudin-1, and occludin in the PD184352 + cecropin A group were significantly higher than those in the cecropin A group (*p* < 0.001, Figure 6A,B). Moreover, the TJs and F-actin in the PD184352 + cecropin A group were distributed at the cell-cell adjacent position (Figure 6C). The results showed that the inhibition of MEK/ERK may increase intestinal barrier function by increasing the TJ expression level and membrane distribution.

**Figure 5.** The effect of inhibiting ERK phosphorylation on TER and *E. coli* adherence. TER was upregulated ((**B**), *n* = 3) through inhibition of ERK phosphorylation ((**A**), *n* = 3). IPEC-J2 cells were treated with the ERK-specific inhibitor PD184352, cecropin A and cecropin A + PD184352 for 24 and 48 h, and the CFUs of adherent *E. coli* were decreased after the PD184352 treatment for 48 h (*n* = 3). Data (mean ± SEM) were analyzed with the Student's *t*-test (**A**) and one-way ANOVA (**B**,**C**). The results were confirmed by three independent experiments per treatment. Representative results of the three independent experiments are shown. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

**Figure 6.** The inhibitory effects of MEK/ERK on TJ protein expression, membrane distribution and F-actin polymerization. Western blotting analysis of ZO-1, occludin and claudin-1 expression ((**A**,**B**), *n* = 3); cell immunofluorescence ((C), *n* = 3, 400×) showed the membrane distribution (**a**–**i**) and F-actin polymerization (**j**–**l**). 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 the Student's *t*-test. Cell nuclei were stained by DAPI and are shown in blue. Claudin-1, ZO-1 and occludin are shown in red and pointed out by white arrow heads. F-actin is shown in green. Scale bar is 50 μm. \*\* *p* < 0.01, \*\*\* *p* < 0.001.

#### **3. Discussion**

In recent decades, thousands of AMPs have been found in organisms. The antibacterial activities of AMPs differ greatly from each other because of their different sequences and structures [21,22]. Although several antibacterial theories for AMPs have been put forward, their potential mechanisms are still unclear. According to previous studies, most of the AMPs have hemolytic activity or cytotoxicity when used at high concentrations [23]. In this study, we have shown that cecropin A had better antibacterial activity and lower cytotoxicity compared to those of other AMPs (PG-1, LL-37, etc.) selected from the APD3 database. Interestingly, we also found that the antibacterial activity of cecropin A is much higher against *E. coli*, *Salmonella typhimurium* and *Pseudomonas aeruginosa* than *Staphylococcus aureus*, which may suggest that cecropin A was more effective against gram-negative bacteria.

In addition to the antibacterial activities, we also showed that cecropin A may enhance the barrier function of the IPEC-J2 monolayer and increase the defense capability against pathogenic bacteria. In this study, we showed that after treatment with cecropin A for 48 h, adherent *E. coli* were significantly reduced and the mRNA levels of TNF-α, IL-6 and IL-8 were downregulated, which indicates that inflammation may be alleviated. The integrity of intestinal barrier function and morphology may affect the colonization of harmful bacteria and play an important role in protecting hosts from microorganism infection and inflammatory diseases induced by pathogenesis [24,25]. To evaluate the intestinal barrier function, TER and cell monolayer permeability experiments were performed. The data showed that cecropin A could significantly increase TER and reduce permeability. The TJs (ZOs, occludin, claudins, etc.) are the most important parts of the epithelial barrier and are essential for cell–cell adhesion [13]. Our results showed that protein expression levels of ZO-1, occludin and claudin-1 were increased. In addition, immunofluorescence showed that the TJs we detected were distributed on the cell membranes after cecropin A treatment. ZOs (mainly ZO-1) are connected to the cytoskeleton (F-actin). On the other hand, ZO-1 also connected to the intracellular loops of claudins and occludin. Claudins and occludin are responsible for adjacent cell connections. The cytoskeleton-TJ structure

may prevent pathogenic bacterial adherence or invasion into cells. The function of TJs depends on the protein expression level and membrane distribution [26]. In this study, we evaluated the expression and location of claudin-1, ZO-1, and occludin by using cell immunofluorescence. The results showed that the membrane distribution of claudin-1, ZO-1 and occludin was significantly increased by cecropin A stimulation, which suggests that cecropin A may regulate the barrier function through regulation of TJ expression and membrane expression.

To elucidate the mechanism of cecropin A regulating the intestinal monolayer barrier, the MEK/ERK signaling pathway was detected. The results showed that cecropin A may enhance the barrier function, regulate TJ expression and membrane distribution by downregulating the MEK/ERK pathway. The MEK/ERK pathway is conserved among eukaryotes, and one of the most important roles of MEK/ERK is to regulate cell proliferation and inhibit differentiation in epithelial cells and tumor cells [18,21]. In the MEK/ERK signaling pathway, ERK 1/2 may be activated by MEK, and then ERK 1/2 may regulate downstream transcriptional factors and widely regulate cell physiological processes, such as proliferation, differentiation, migration and apoptosis [18]. In addition, previous studies have also showed that MEK/ERK inhibition may induce upregulation of TJs in undifferentiated cells, such as embryonic stem cells, intestinal epithelial cells (IEC-6, caco-2) or tumor cells [27,28]. Similarly, ERK activation may induce blood–brain barrier injury [29]. The related physiological processes are regulated by the transcriptional factors downstream of ERK. CDX2, a caudal-related homeobox gene, is an essential regulator of gene transcription and tumor suppression in gastrointestinal tract development and homeostasis [30,31]. Previous studies have shown that CDX2 may play important roles in cell differentiation and proliferation and that it is regulated by the MEK/ERK signaling pathway [27]. In addition, CDX2 also plays an important role in TJ regulation in the intestinal epithelium. Previously, studies showed that in rat intestinal epithelium-derived line, IEC-6, caco-2 and colorectal carcinoma cells, downregulation of CDX2 by the MEK/ERK signaling pathway may decrease the protein expression levels of claudin-1, occludin and ZO-1 [27,32,33]. Consistent with this, our results showed that cecropin A may downregulate MEK and ERK phosphorylation, upregulate CDX2 expression, and upregulate protein levels of ZO-1, claudin-1 and occludin. Interestingly, previously studies showed that LL-37 and human beta defensin-3 (hBD-3) may activate phosphatidylinositide 3 kinases (PI3K)-Protein kinase B (Akt), PKC (protein kinase C) and Glycogen synthase kinase 3β (GSK-3β) and upregulate TJ expression and membrane distribution in human skin cells [26,34], which may be involved in the cell adherence and junction remodeling pathway, suggesting that more than one pathway exists to regulate TJ expression. Overall, in this study, we found that cecropin A enhanced the barrier function in the IPEC-J2 cell monolayer model by upregulating the TJ protein level (ZO-1, claudin-1 and occludin) and membrane polymerization, which was negatively regulated by the MEK/ERK signaling pathway. Our results suggested that cecropin A has the potential to replace antibiotics in the treatment of IBD due to its antibacterial activity on gram-negative bacteria and enhancement effect on intestinal barrier function.
