Cathelicidin-BF, a Novel Antimicrobial Peptide from Bungarus

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Cathelicidin-BF, a Novel Antimicrobial Peptide from Bungarus fasciatus, Attenuates Disease in a Dextran Sulfate Sodium Model of Colitis Haiwen Zhang,† Xi Xia,† Feifei Han, Qin Jiang, Yili Rong, Deguang Song, and Yizhen Wang* Key Laboratory of Animal Nutrition and Feed Science (Hua Dong), Ministry of Agriculture College of Animal Sciences, Zhejiang University, Hangzhou 310058, China S Supporting Information *

ABSTRACT: Antimicrobial peptides are molecules of innate immunity. Cathelicidin-BF is the first cathelicidin peptide found in reptiles. However, the immunoregulatory and epithelial barrier protective properties of C-BF have not been reported. Inflammatory bowel diseases, including ulcerative colitis and Crohn’s disease, can lead to colon cancer, the third most common malignant tumor. The objective is to develop the new found cathelicidin-BF as a therapeutic to patients of ulcerative colitis. The morphology of the colon epithelium was observed by H&E staining; apoptosis index and infiltration of inflammatory cells in colonic epithelium were measured by TUNEL and immunohistochemistry; the expression level of endogenous mCRAMP was analyzed by immunofluorescence; and phosphorylation of the transcription factors c-jun and NF-κB in colon were analyzed by Western blot. Our results showed that the morphology of the colon epithelium in the C-BF+DSS group was improved compared with the DSS group. Apoptosis and infiltration of inflammatory cells in colonic epithelium were also significantly attenuated in the C-BF+DSS group compared with the DSS group, and the expression level of endogenous mCRAMP in the DSS group was significantly higher than other groups. DSS-induced phosphorylation level of c-jun and NF-κB while C-BF effectively inhibited phosphorylation of NF-κB (p65). The barrier protective effect of C-BF was still excellent. In conclusion, C-BF effectively attenuated inflammation and improved disrupted barrier function. Notably, this is the first report to demonstrate that C-BF attenuates DSS-induced UC both through the regulation of intestinal immune and retention of barrier function, and the exact pathway was through NF-κB. KEYWORDS: antimicrobial peptides, cathelicidin-BF, ulcerative colitis, intestinal immunity, epithelial barrier function

1. INTRODUCTION Inflammatory bowel disease (IBD) includes the intestinal diseases ulcerative colitis and Crohn’s disease. The etiology of IBD remain unclear; however, it is established as a relapsing remitting inflammation of the intestinal tract.1 Recently, studies have shown that various immune, genetic, and environmental factors affected both the initiation and progression of colitis.2−4 Historically, IBD is more common in Western countries than other regions. However, the incidence is increasing in Asia possibly because of the influence of high-fat diet.5 Ulcerative colitis is a chronic gastrointestinal disorder caused by inflammation of the surface of the colon mucosa.6 Notably, ulcerative colitis increases the risk of colorectal cancer, which is the third most common malignancy in humans globally.7 There are two main large antimicrobial peptide families, cathelicidin and β-defensin, both of which have been widely studied due to their antimicrobial functions in innate immune responses.8 Furthermore, antimicrobial peptides (AMPs) originated from cathelicidin have been reported to possess antibacterial, antiviral, and antifungal functions,9−11 these © 2015 American Chemical Society

include the widely studied cathelicidin peptides human LL-37 and mCRAMP (mouse cathelicidin-related antimicrobial peptide).12−14 Injection of LL-37 into rats decreased mortality caused by Gram-negative bacterial infection, and low concentration of LL-37 in plasma was associated with increased risk of infection and mortality in patients undergoing renal hemodialysis,15 indicating that cathelicidin peptides like LL-37 have an intrinsic role in preventing pathogen infection. As mentioned above, LL-37 exerts various functions, it has been proven to modulate the activity of immune and inflammatory cells,16,17 promote healing of human skin wounds,18 and attenuate inflammation by neutralizing the pro-inflammatory effects of lipopolysaccharide (LPS).19−23 Studies of the influence of rat derived cathelicidin peptide on ulcer healing in a rat gastric ulcer model showed that cathelicidin induced Received: Revised: Accepted: Published: 1648

January 22, 2015 March 20, 2015 March 25, 2015 March 25, 2015 DOI: 10.1021/acs.molpharmaceut.5b00069 Mol. Pharmaceutics 2015, 12, 1648−1661

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Molecular Pharmaceutics

chromatography (RP-HPLC). C-BF was prepared in PBS at a concentration of 2 mg/mL and was stored at −80 °C until use. Dextran sulfate sodium was purchased from MP (USA) and was dissolved in the sterile water at a concentration of 3% DSS. Induction of UC like Enteritis and the Observation of the Effect of C-BF through DAI Score. After 1 week of adaptation, the experimental period lasted for 1 week and there remained two treatments (Table 1): the first was to drink

proliferation of gastric epithelial cells through TGF-α-dependent transactivation of epidermal growth factor receptors and related signaling pathways.24 Cathelicidins are secreted primarily from epithelial cells and macrophages in the intestinal tract;25 after treatment with dextran sulfate sodium (DSS), the expression level of cathelicidin was significantly increased.26 Interestingly, the latest study has revealed that the expression of cathelicidin is increased in UC, but not CD patients.27 It was also demonstrated that endogenous cathelicidin could attenuate DSS-induced intestinal inflammation using mCRAMP knockout mice.28 Additionally, intracolonic administration of mCRAMP to wild-type mice treated with DSS ameliorated colitis.29 Therefore, it could be deduced that there are close links between antimicrobial peptides and UC. Cathlicidin-BF (C-BF) is a novel antimicrobial peptide isolated from Bungarus fasciatus (B. fasciatus), which has broadspectrum antibacterial activity, especially against gram-negative bacteria, and may be an excellent therapeutic for acne vulgaris.30,31 The researches about C-BF were mainly focused on the antibacterial function so far; however, the immunoregulatory and epithelial barrier protective functions of C-BF were rarely studied. Based on the studies of cathelicidin derived peptides, we speculated that exogenous cathelicidin-BF might influence the development and healing of mucosal damage and inflammation that occurs in UC. Therefore, the present study examined the effects of cathelicidin-BF on regulating DSSinduced colitis using both in vivo and in vitro models; DSSinduced UC mouse model, Caco-2 cells based epithelium model, and inflammatory model of LPS stimulated RAW264.7 cells were set up to evaluate the possible immunoregulatory and epithelial barrier protective functions of C-BF. We found that colonic administration of exogenous cathelicidin-BF could significantly reduce inflammation and intestinal barrier damage caused by DSS. Combining the in vivo and in vitro results, we could further conclude that cathelicidin-BF attenuated DSSinduced inflammation by inhibiting activation of the NF-κB pathway in response to DSS. Currently, the main treatment for IBD is confined to alleviation of the inflammatory response temporarily, while sustained inflammation in the colon increases the risk of colorectal cancer.32 Therefore, a safer and more efficient therapy is urgently needed for the treatment and prevention of IBDs, with the antimicrobial peptide cathelicidin-BF showing great potential.

Table 1. Experimental Design with Different Treatment Groupsa groupsb

control c

treatment 1

d

treatment 2

distilled water PBS

DSS

C-BF

DSS solution PBS

distilled water C-BF

C-BF+DSS DSS solution C-BF

a

Abbreviations: DSS, dextran sulfate sodium; C-BF, cathelicidin-BF; PBS, phosphate buffer saline. bForty-eight C57/BL6 male mice weighing 18−21 g (6−8 weeks old) were randomly distributed to four groups. Animals were individually housed and maintained on a 12:12 h light−dark cycle under specific pathogen free conditions. The dosage of C-BF was 5 mg/kg mouse weight, the concentration of C-BF was 2 mg/mL, and the dosage of DSS was 3% (m/v). cTreatment 1: mice were supplied with distilled water or DSS solution for 7 days. d Treatment 2: mice got rectal injection of PBS or C-BF in PBS solution for 7 days.

sterile water (Control and C-BF groups) or DSS dissolved water (DSS and C-BF+DSS groups) freely for 1 week, the second was to conduct rectal injection of C-BF (dissolved in PBS) for C-BF and C-BF+DSS groups or PBS alone for Control and DSS groups; the injection was continued for 1 week and the frequency was every other 24 h, the dosage of CBF was 5 mg/kg mouse weight, the total injection volume was 50 μL. Body weight, presence of rectal bleeding, and stool formation were recorded and graded according to standard protocol.33 The scoring system for the comparative analysis of DAI are seen in Table 2. The dynamic DAI score is the average score of these three indices. Table 2. Scoring System for the Comparative Analysis of DAI

2. MATERIALS AND METHODS Animals. Forty-eight C57/BL6 male mice weighing 18−21 g (6−8 weeks) were obtained from Zhejiang Provincial Center for Disease Control and Prevention (Zhejiang CDC, Hangzhou, China). Animals were individually housed and maintained on a 12:12 h light−dark cycle under specific pathogen-free conditions. All animals were provided with food and water ad libitum during the experimental period (1 week) according to the protocol approved by the Committee on the Ethics of Animal Experiments of Zhejiang University. Preparation of C-BF and DSS. Cathelicidin-BF (C-BF) was synthesized and purified by the Chinese Peptides Company (Hangzhou, China) and its molecular weight was confirmed via matrix-assisted laser desorption/ionization them-of-flight mass spectroscopy (MALDI-TOF MS, Model Auto flex, Broker Daktronics Inc., USA) using α-cyano-4-hydroxycinnamic acid as the matrix. Purity of C-BF was determined at more than 95% with analytical reverse phase high performance liquid

score

body weight

occult

stool formation

0 1 2 3

no change decrease by 1% decrease by 3% decrease by 5%

negative positive positive, visible traces in stool positive, bleeding

normal soft but formed very soft diarrhea

Assessment of Histopathology Changes. Intestinal tissues of the middle ileum and colon from five individuals in each group were examined. Full thickness sections of the middle ileum and colon were excised, opened longitudinally, immediately fixed in 4% paraformaldehyde solution, and embedded in paraffin. Samples were sliced (direction) and stained with hematoxylin−eosin (H&E). The epithelial morphological characteristics were observed with Leica NEWDM 4500BR microscope under varying magnifications. Immunofluorescence and Immunohistochemistry Staining of Colon Tissue. The distal colons were fixed in 4% paraformaldehyde solution, embedded in paraffin and cut into slices longitudinally, subsequently permeabilized, stained, and fixed. For immunofluorescence analysis of mCRAMP (Abcam USA) and ZO-1 (Abcam USA), nonspecific binding 1649

DOI: 10.1021/acs.molpharmaceut.5b00069 Mol. Pharmaceutics 2015, 12, 1648−1661

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production of PGE2 and NO were determined by analyzing PGE2 and NO levels with EIA kit (R&D Systems USA) and Griess reagent (Beyotime China Beijing) respectively, as described previously.34 Briefly, after being treated with LPS for 3 h, cell supernatant was collected and stored at −80 °C for further tests. The secretion of PGE2 was tested according to the instruction of the PGE2 parameter assay kit (R&D Systems USA). Generation of nitric oxide was assayed using a NO measurement kit (Beyotime China Beijing) and by the colorimetric Griess’ method. Optical density was measured at 450 nm for PGE2 and 550 nm for NO with the ELISA reader (Bio-Rad 680, USA). Electrophysiology Measurements. Transepithelial electrical potential was measured using a multichannel voltage− current clamp (model VCC MC6, Physiologic Instruments) following previously described protocols.35,36 Colon tissues were excised from mice, immersed in oxygenated Krebs’s buffer, and mounted onto Ussing chambers (World Precision Instruments; Narco Scientific, Mississauga, Ontario, Canada). The Ussing chambers were equipped with two pairs of Ag/ AgCl electrodes connected to the chambers via 3 M KCl/3.5% agar bridges, to measure the potential difference (PD) and passing current (I). The experiment was performed under open circuit conditions, and the current was set to zero, so the natural transepithelial PD could be recorded. For each measurement, the PD value was clamped to 20 mV, and the necessary current was recorded. Electrical resistance was determined according to Ohm’s law [R = (20 mV − PD)/I]. The fluid resistance measured prior to mounting the tissue in the diffusion chamber system was subtracted, and the net electrical resistance of the colonic epithelium was multiplied by the apparent exposed tissue area (0.07 cm2) to yield TEER value. To ensure comparability of TEER measurements of each group, the differences between the effective exposed area of the epithelium and the apparent exposed tissue area were normalized by presenting all measurements as a percentage of the TEER value at the end of the equilibration period for each tissue insert. Caco-2 cells were cultured in transwell dishes at a density of 5 × 105, at the temperature of 37 °C, with 5% CO2 and 95% humidity. The medium was changed every other day. The resistance value was tested by Millicell ERS-2 epithelial volt− ohm meter (Millipore MERS00002, America) every day. Until the resistance value was stabilized, the follow-up experiment was conducted Then, 0.5 and 1.5 mL of fresh medium were added to the upper layer and basal layer of transwell equipment, respectively; C-BF was added to the upper layer at a final concentration of 6.87 μmol/L and incubated with the cells for 24 h; C-BF was removed with D-hanks; and DSS was added with fresh medium at a final concentration of 3% (m/v) for another 24 h. Cells were cultured in the incubator, and the resistance per hole was tested at 2 h intervals. Western Blot Analysis. Distal colons were grinded and lysed with a total protein extract kit (Keygen China Nanjing) with a complete protease inhibition cocktail (PIC). The protein content of each tissue was quantified and diluted, and 30 μg of protein was loaded in each lane of a 7.5%/10% SDS-PAGE gel. Cell samples were lysed with the same total protein extraction kit. The protein was separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with 5% (w/v) defatted milk in TBS and incubated with a primary antibody (NF-κB p65, p-p65, c-jun, p-c-jun, ZO-1, Claudin-1 (Santa cruze, USA); Occludin (Abcam, U.K.); AKT, p-AKT,

sites were blocked with PBS containing 1% w/v BSA for 30 min. Anticathelidicin at a ratio of 1:1000 or anti-ZO-1 at a ratio of 1:500 was added to the samples to incubate overnight at 4 °C. Slices were washed with PBS five times, and TRITCconjugated goat antirabbit IgG (JIR USA) was added at a ratio of 1:100 and left to incubate at room temperature for 1 h in darkness. DAPI was used to stain the nucleus. Glycerol was used to mount the samples onto slides. Images were taken under a fluorescence microscope immediately. For immunohistochemical analysis of CD177 and F4/80, nonspecific binding sites were blocked with PBS containing 1% w/v BSA for 30 min. Anti-CD177 and F4/80 antibodies (Santa USA) were added at a dilution of 1:100 and incubated overnight at 4 °C. Samples were washed five times in PBS and treated with HRP-conjugated rabbit antigoat IgG (JIR USA) at a ratio of 1:100. Samples were incubated at 4 °C for 1 h and washed with PBS three times. DAB (50−100 μL) was added (DAKO USA), then hematoxylin was used to counterstain the slices. Finally, the samples were dewatered with gradient alcohol (70−100%), and xylene was used to increase the transparency of slides. A neutral balsam was applied for mounting. To evaluate the apoptosis level of colon epithelium, paraffin sections were dewaxed with water, and antigen retrieval was executed. TdT and dUTP (Roche USA) were mixed at a ratio of 1:9 and incubated at 37 °C for 60 min. The endogenous peroxidase was blocked, and the slides were allowed to dry. The tissue was then covered with converter-POD (Roche USA) and incubated at 37 °C for 30 min and washed with PBS three times. DAB was added to the slices, and distilled water was used to stop color development. Finally, the cell nucleus was stained, and the slices were dehydrated and mounted. Measurement of Cytokines, FITC-Dextran Concentration in Serum, and Enzyme Level of β-N-Acetyl Glucosaminidase (NAG), Myeloperoxidase (MPO), and Eosinophil Peroxidase (EPO) in Colon. To determine the concentration of TNF-α, IL-6, IL-8, and IL-10 in serum, an ELISA Kit (Boster China Wuhan) was used. The kit assays were carried out according to the manufacturer’s instructions. To evaluate the permeability of the intestinal lining, 200 μL of FITC-dextran was injected into the colon 4 h before sampling. Serum from the orbit vein was obtained and added into a 96-well plate, avoiding light. Fluorescence intensity was detected with a microplate reader (Spectrumax M5Molecular devices USA), at excitation and emission wavelength of 488 and 525 nm, respectively. The concentration of enzyme MPO, EPO, and NAG in colon were tested by ELISA Kits (R&D USA), and the kit assays were carried out according to the manufacturer’s instructions. Determination of Enzyme Level of INOS and COX-2 in Colon. To analyze the enzymatic concentration of INOS and COX-2 in colon, the corresponding INOS and COX-2 ELISA kits (Bio Value, U.K.) were used. Total protein from colon tissues were extracted with RIPA Lysis Buffer (Boster China Wuhan), and the quantification of protein was assayed via a BCA kit (Keygen China Nanjing). The kit assays was carried out according to the manufacturer’s instructions. Determination of the Concentration of PGE2 and NO in Cell Cultural Supernatant. RAW264.7 cells were cultured in DMEM basic medium. After reaching a density of 1 × 106 cells/mL, cells were pretreated with C-BF (6.87 μmol/L) for 4 h and further incubated with LPS (1 μg/mL) for 3 h. The 1650

DOI: 10.1021/acs.molpharmaceut.5b00069 Mol. Pharmaceutics 2015, 12, 1648−1661

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Figure 1. Body weight gain (A), length (B), weight (C), and length/weight ratio (D,E) of the colon in UC mice. Disease activity index over the whole inducing period (F). The body weight gain was calculated as the net plus or minus of weight of 7 days from an average of 12 mice (A). The whole colon was removed and the length measured, and the result shown represents the average value of 12 mice (B). The corresponding colons were weighed (C) and the ratio of length/weight calculated and analyzed by t-test (D). The final values were the means ± SEM, and values without the same letters are significantly different (P < 0.05). The ratios of length/weight were calculated, and multiple comparison tests were carried out by Tukey’s HSD and one way analysis of variance (ANOVA) (E). The final values were the means ± SEM, and values without the same letters are significantly different (P < 0.05). The change in body weight, rectal bleeding, and stool formation were recorded everyday (n = 12/group) and graded as the standard of the nature protocol (F); the dynamic DAI score was the average value of these three indices. The values are expressed as means ± SEM.

Figure 2. H&E staining of colon tissue (100×, 400×). The distal colons of at least six mice per group were removed, opened longitudinally, immediately fixed in 4% paraformaldehyde solution, embedded in paraffin, and stained with hematoxylin and eosin. The epithelial morphological characteristics were viewed under a Leica NEWDM 4500BR microscope at magnifications of 100× and 400×. (A) Control; (B) C-BF; (C) DSS; (D) C-BF+DSS. Scale bar = 200 μm (100×) or 50 μm (400×). The black arrow (single sided) indicates morphological changes of mucous layer, and the black arrow (double sided) indicates edema status in submucosa.

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DOI: 10.1021/acs.molpharmaceut.5b00069 Mol. Pharmaceutics 2015, 12, 1648−1661

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Figure 3. H&E staining of ileum tissues (40×, 200×). The middle ileum of at least six mice per group were removed, opened longitudinally, immediately fixed in 4% paraformaldehyde solution, embedded in paraffin, and stained with H&E. The epithelial morphological characteristics were viewed under a Leica NEWDM 4500BR microscope at magnifications of 40× and 200×. (A) Control; (B) C-BF; (C) DSS; (D) C-BF+DSS. Scale bar = 400 μm (40×) or = 100 μm (200×). The black arrow indicates a truncated intestinal villus.

IκB-α, p-IκB-α (Epitomic, USA)) overnight at 4 °C. The manufacturer’s instructions were referred to when determining the appropriate dilution for each antibody. HRP-conjugated secondary antibody (1:1000) was applied before the proteins were visualized via chemiluminescence. RNA Isolation and qRT-PCR Determination. RNA samples were extracted from the distal colon or samples of Caco-2 and RAW264.7 cells. Total RNA isolation and cDNA synthesis by reverse transcription were conducted using Trizol reagent (Invitrogen Corporation, Carlsbad, CA, USA) and MMuLV reverse transcriptase kit (Fermentas, EU, Glen Burnie, MD, USA), respectively. The mRNA levels of individual genes were measured by real-time PCR using the SYBR Premix Ex Taq Kit (Takara Biotechnology Co. Ltd., Otsu, Shiga, Japan) in the ABI Step One Plus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Data was analyzed according to the comparative threshold cycle (Ct) method and normalized to an endogenous reference, GAPDH. The primers used in this experiment were listed in Supplementary Table 3. Relative expression levels of inflammation related (TNF-α, IL-6, IL-8, IL-10) and barrier function related genes (ZO-1, ZO-2, claudin-1, occluding, mucin-1, mucin-2) were analyzed on colon tissues. The expression levels of ZO-1, claudin-1, and occluding were analyzed on Caco-2 cells, and the level of TNF-α was analyzed on RAW264.7 cells. Statistical Analysis. Multiple comparison test were carried out by Tukey’s HSD and one way analysis of variance (ANOVA) with SPSS 16.0; a value of p < 0.05 was considered as significant. Results are expressed as mean ± SEM or mean ± SD.

3. RESULTS Effect of DSS and Cathelicidin-BF on Body Weight and Disease Activity Index (DAI). During the experimental period, photographs of the anus of control and DSS groups were taken at day 5 to demonstrate that the model was established successfully. From the photos we can see the DSS group had bloodstains at the anus, while the control was normal (Supplementary Figure 1). Furthermore, the large intestine (cecum, colon, rectum) of these mice contained significant bleeding (Supplementary Figure 1). From the data of body weight gain and the ratio of colon length/weight (Figure 1A− D), we concluded that the model was set up successfully as we found DSS significantly decreased the ratio of colon length/ weight. Notably, C-BF improved the symptoms of DSStreatment nearly to the level of the controls (Figure 1E). The disease activity index (DAI, a composite grade of stool formation, colon bleeding, and body weight) of DSS group increased daily, while the C-BF+DSS group had attenuated progression (Figure 1F). Histopathological Analysis of DSS-Induced Colitis. From the H&E staining of distal colon tissue (100×, Figure 2), we found the structure of the mucous layer was destroyed in the DSS treated group, with the substratum of the mucous layer showing pathological changes including obvious edema (Figure 2C). In contrast, the administration of C-BF altered the appearance of the mucosa, reverting the structure of the mucous layer closer to the control group and decreasing the thickness of edema (Figure 2D). Furthermore, under the magnification of 400×, we could clearly see the same effect on the mucous layer. The middle ileum was also stained with H&E to analyze the structural changes, we also observed the structural characteristics of the middle ileum under 40× and 200× magnification (Figure 3), and the same conclusion was 1652

DOI: 10.1021/acs.molpharmaceut.5b00069 Mol. Pharmaceutics 2015, 12, 1648−1661

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Figure 4. Concentration of cytokines in the serum of colitis mice. For the detection of cytokines of TNF-α, IL-6, IL-8, and IL-10 in serum, the ELISA kits were applied. The determination of cytokine concentration was carried out as per the manufacturer’s instructions, and the OD value was recorded at 450 nm. There were at least six repetitions in each group, the average values were calculated, and multiple comparison test were carried out by Tukey’s HSD and one way analysis of variance (ANOVA) with SPSS 16.0. The final values were the means ± SEM, and values without the same letters are significantly different (P < 0.05).

made that the DSS group had more dramatically truncated intestinal epithelium (Figure 3C) than other groups (Figure 3A,B,D). Immunofluorescence Analysis of mCRAMP Expression. The expression of mCRAMP in colon and ileum were investigated by immunofluorescence. The expression abundance of mCRAMP in the colon of the DSS treated group was significantly elevated (Figure 4C), while C-BF treatment lowered its expression (Figure 4D). However, no changes showed significance in ileum (data not shown). Analysis of Circulating Cytokines in Serum and Cytokine Gene Expression in Colon. The serum of mice was collected for the analysis of cytokine secretion. The concentration of the classical proinflammatory cytokines TNFα, IL-6, and IL-8 and anti-inflammatory cytokine IL-10 were tested by ELISA. Simultaneously, total RNA was extracted from colon tissue and used for qRT-PCR analysis. The results consistently showed that the level of all cytokines in the DSS treated group were significantly higher than other groups (Figure 4 and Supplementary Figure 2). In contrast, the CBF +DSS group had reduced cytokine expression (Figure 4 and Supplementary Figure 2), with the level of TNF-α, IL-8, and IL-10 being closer to the control group, while the concentration of IL-6 in serum remained higher than control group (Figure 4B). Evaluation of Macrophage and Neutrophil Infiltration of Intestinal Mucosa. Antibodies against surface markers

specific for phagocytic neutrophils and macrophages, CD177 and F4/80, respectively, were used to investigate the infiltration of the intestinal mucosa by inflammatory cells through immunohistochemistry. Invasion of intestinal mucosa by inflammatory cells in the colon of the DSS group were significantly higher than other groups (Figure 5C,G), while infiltration status in the C-BF+DSS group was significantly attenuated (Figure 5D,H). Enzymes that are specifically secreted by phagocytic neutrophils and macrophages, MPO and NAG, respectively, were analyzed for concentration. The concentration of MPO demonstrated the result of immunohistochemistry indirectly (Figure 5I). Level of Apoptosis in Colon Epithelial Tissue. TUNEL (terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling) staining was performed on sections of colon tissue to analyze alterations in the level of apoptosis. The apoptosis index of the DSS treated group was significantly higher than other groups (Figure 6C). Notably, the C-BF+DSS group had significantly attenuated apoptosis (Figure 6D), although the apoptosis index was still significantly higher than the control group (Figure 6A). Analysis of COX-2 and INOS Concentration and c-jun and NF-κB p65 Phosphorylation in Colon Tissue. The two classical signaling pathways related to inflammation are MAPK and NF-κB, which control the transcription level of certain cytokines and inflammation related enzymes including INOS and COX-2. We first tested the INOS and COX-2 1653

DOI: 10.1021/acs.molpharmaceut.5b00069 Mol. Pharmaceutics 2015, 12, 1648−1661

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Figure 5. Immunohistochemistry staining of neutrophils (CD177) (A−D) and macrophages (F4/80) (E−H) in colitis (400×). Concentrations of MPO, NAG, and EPO in colon tissues (I−K). The microphotographs were analyzed by Image pro-plus 6.0 software (USA), and four slices of each picture were taken under 400× magnification, and the background light was kept consistent between images. Five random duplicates in each group were analyzed and the integrated optical density (IOD) collected according to the positive reaction of cells,. The average values were calculated, and multiple comparison tests were carried out by Tukey’s HSD and one way analysis of variance (ANOVA) with SPSS 16.0. The final values were the means ± SD, and values without the same letters are significantly different (P < 0.05). (A,E) Control; (B,F) C-BF; (C,G) DSS; (D,H) C-BF+DSS. Scale bar = 50 μm. To test the concentration of MPO (I), EPO (J), and NAG (K) in the colon in colitis, the ELISA kits (R&D USA) were applied. The determination of enzymatic concentration was carried out as per the manufacturer’s instructions, and the OD value was recorded at 40 nm. There were at least six repetitions in each group. The average values were calculated, and multiple comparison test were carried out by Tukey’s HSD and one way analysis of variance (ANOVA) with SPSS 16.0. The final values were the means ± SEM, and values without the same letters are significantly different (P < 0.05).

determine ZO-1 expression; (4) Total RNA of colon tissue was extracted and used for qRT-PCR to determine the expression of tight junction related genes. The fluorescence intensity of FITC in the DSS group was significantly higher than other groups (Figure 8A). The same result was found in TEER value (Figure 8B), while C-BF treatment improved the trends. The expression of ZO-1 in colon tissue was significantly lower in the DSS group than the other groups (Figure 8E). Furthermore, the relative expression levels of ZO-1, ZO-2, occludin, claudin1, mucin-1, and mucin-2 in the DSS group were all significantly lower than in the other groups. Notably, the addition of C-BF reduced the trends (Supplementary Figure 4). Evaluation of the Barrier Protective Function of C-BF in a DSS-Induced Caco-2 Cell Injury Model. To further verify the protective effect of C-BF on epithelial barrier function, the DSS stimulated Caco-2 cell line model was established. Caco-2 cells were cultured in transwell chambers for 21 days until the value of transepithelial electric resistance was kept stable. Millicell ERS-2 epithelial volt−ohm meter (Millipore MERS00002, USA) was used to test the resistance value of the cell monolayer. The resistance value increased to a

enzymatic concentration in colon tissue and found the concentration of both INOS and COX-2 rose significantly compared with other groups, which was decreased by the addition of C-BF (Figure 7A,B). Additionally, total protein was extracted from colon tissue to analyze the phosphorylation level of c-jun and p65 by Western blot. Results showed that DSS increased the phosphorylation level of c-jun and p65, while the C-BF+DSS group had reduced phosphorylation levels of p65 but not c-jun (Figure 7C). Therefore, DSS activates both the cjun and NF-κB signaling pathways, while C-BF may exert an anti-inflammatory effect by decreasing the phosphorylation level of p65, but not c-jun. Comprehensive Analysis of Epithelial Barrier Function in the Colon. To assess the effect of C-BF on epithelial barrier function in the colon, four experiments were carried out: (1) Injecting FITC-dextran directly into the colon 4 h before sampling, then the FITC concentration in serum was determined; (2) 1 cm2 living colon tissue was obtained to analyze the transepithelial electrical resistance (TEER) value using the Ussing chambers; (3) the colon tissue was fixed with 4% paraformaldehyde and immunofluorescence analysis used to 1654

DOI: 10.1021/acs.molpharmaceut.5b00069 Mol. Pharmaceutics 2015, 12, 1648−1661

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Figure 6. TUNEL staining of colon epithelial tissues in colitis (400×). The images were analyzed by Image pro-plus 6.0 software (USA), with at least four views of each image taken under 400× magnification, and the background light kept consistent between images. Five random duplications in each group were analyzed, and the number of apoptotic cells counted according to the positive color of brown and the average calculated. The final values were the means ± SD, and values without the same letters are significantly different (P < 0.05). Scale bar = 50 μm. (A) Control; (B) C-BF; (C) DSS; (D) C-BF+DSS.

constant of approximately 3500 Ω (Supplementary Figure 5), then a final concentration of 6.87 μmol/L C-BF was added to the upper layer and incubated for 24 h before treatment with 3% DSS for another 24 h. The DSS group demonstrated a lower TEER value, reflecting damage to the intestinal barrier function by DSS. In contrast, C-BF pretreatment elevated the TEER value to above 80% of the control (Supplementary Figure 6). The cells were collected for RNA and protein extraction to analyze the expression of ZO-1, claudin-1, and occludin at the gene and protein levels. The gene expression level of these three tight junction proteins in the DSS treated group was significantly lower than in the other three groups, demonstrating that DSS could damage the epithelial barrier function. Notably, the C-BF returned the expression level of claudin-1 and occludin to a normal state in DSS treated cells, while the expression level of ZO-1 was elevated but not significantly different from the DSS group (Figure 8G−I) . At the protein level, DSS treatment significantly decreased the level of ZO-1, claudin-1, and occludin compared with the control group, while the addition of C-BF markedly improved the protein expression level (Figure 8J).

Evaluation of the Mechanism of C-BF Immunomodulation in LPS-Stimulated RAW264.7 Cells. Finally, RAW264.7 cells were cultured in six well plates and C-BF added at a final concentration of 6.87 μmol/L for 4 h before stimulation with l μg/mL LPS (O55:B5, Sigma) for 3 h. The cell culture supernatant was collected for analysis of the concentrations of PGE2 and NO. The cells were collected to extract RNA and protein for the analysis of TNF-α gene expression and phosphorylation level of NF-κB related signaling pathways. After LPS treatment alone, the level of PGE2, NO, and TNF-α were significantly elevated, while C-BF pretreatment attenuated the expression (Figure 9A−C). This is consistent with the enhanced enzymatic activities of COX-2 and INOS in DSS treated mice. From the phosphorylation level analysis of NF-κB related signaling pathway (Figure 9D), we could speculate that the possible mechanism by which C-BF attenuates inflammation may be based on the reduced phosphorylation level of NF-κB p65.

4. DISCUSSION Cationic AMPs were originally defined as having antibacterial and antifungal properties.37 However, researchers found not 1655

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Figure 7. Concentration of INOS and COX-2 in colon tissues (A,B). Phosphorylation level of p65 and c-jun in colon tissue (C). For the analysis of the concentration of INOS (A) and COX-2 (B) in colon, INOS and COX-2 ELISA kits were used. The determination of enzymatic concentration was carried out as per the manufacturer’s instructions, and the OD value was recorded at 450 nm. There were at least six repetitions in each group, the average values were calculated, and multiple comparison test were carried out by Tukey’s HSD and one way analysis of variance (ANOVA) with SPSS 16.0. The final values were the means ± SEM, and values without the same letters are significantly different (P < 0.05). For the analysis of the phosphorylation level of p65 and c-jun in colon tissues (C), distal colons were ground and lysed by total protein extract kit with complete protease inhibition cocktail. The protein content of each sample was quantified and diluted to ensure that 30 μg of protein was loaded in each lane of gel.

only that the function of these short amphiphilic molecules was restricted to antibacterial but also that they had immunomodulatory and wound repairing functions.8,24 In this study, we demonstrated that C-BF could prevent DSS-induced colitis in mice, and the protective effect may be due to ameliorating inflammation status and maintaining the integrity of the intestinal mucosal barrier. The possible mechanism of attenuating inflammation may be mediated via the inhibition of NF-κB mediated transcriptional activation of inflammatory gene expression. Meanwhile, we also proved that C-BF could modulate tight junction proteins in colonic tissue and retain the normal function of intestinal epithelium tissues through comprehensive analysis. As the fact that the pathogenesis and molecular mechanisms of ulcerative colitis are still not yet fully understood, the results would provide experimental evidence that C-BF could be a possible prevention treatment for the pathogenesis of UC. The immunomodulatory properties of human derived cationic antimicrobial peptide, LL-37, have been widely investigated. It was reported that LL-37 could up-regulate the expression of chemokines and chemokine receptors without increasing the expression of the proinflammatory cytokine TNF-α.22 A systems biology approach was used to analyze the immune response activated by LL-37 in human mononuclear

cells, and the results showed that several transcription factors including NF-κB, AP-1, AP-2, SP-1, E2F1, and EGR were regulated by LL-37.38 It was also found that LL-37 could regulate the inflammatory response to LPS and other TLR agonists by a complex mechanism with multiple points of intervention including the inhibition of LPS-induced translocation of NF-κB p50 and p65, selective modulation of gene transcription, and direct triggering of MAPK cascades that induce pro-inflammatory pathways.39 In our study, we found CBF could inhibit phosphorylation level of NF-κB p65 but not cjun (Figure 9D), which was different from LL-37. Some antimicrobial peptides could interact directly with immune cells to modulate the inflammatory process and innate defenses.37,40 Cathelin-related antimicrobial peptide (CRAMP) derived from mice, a homologue of LL-37, increased the migration of leukocytes through formyl peptide receptor-like 1 (FPRL1). Additionally, the simultaneous administration of OVA with CRAMP to mice enhanced both humoral and cellular Agspecific immune reactions. It was suggested that mouse derived CRAMP played dual roles as both a leukocyte chemoattractant and a promoter of the adaptive immune response.41 Additionally, antimicrobial peptides such as rCRAMP can promote ulcer healing in the gastric mucosa through enhanced epithelial cell proliferation and angiogenesis, possibly through TGF-α related 1656

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Figure 8. Concentration of FITC-dextran in serum (A). Dynamic change (0−60 min) of TEER value in colon epithelium (B). Immunofluorescence staining of ZO-1 in colon tissue (200×) (C−F). Gene expression level of barrier function related proteins (G−I). Protein level of three tight junction proteins (ZO-1, claudin-1, occludin) (J). For evaluating the permeability of the intestinal tract indirectly (A), 200 μL of FITC-dextran was injected into the colon 4 h before serum was obtained from the orbital vein and added into the 96-well plates, then the fluorescence intensity was detected with a microplate reader with excitation and emission wavelengths of 488 and 525 nm, respectively. There were at least eight repetitions in each group, the average values were calculated, and multiple comparison tests were carried out by Tukey’s HSD and one way analysis of variance (ANOVA) with SPSS 16.0. The final values were the means ± SEM, and values without the same letters are significantly different (P < 0.05). Transepithelial electrical potential was measured using a modified Ussing chamber (VCC MC6, PI) as described previously (B). There were at least five repetitions in each group, the average values were calculated, and multiple comparison test were carried out by Tukey’s HSD and one way analysis of variance (ANOVA) with SPSS 16.0. The final values were the means ± SEM, and values without the same letters are significantly different (P < 0.05). For immunofluorescence staining of ZO-1 in colon tissue (200×) (C−F), the stained slides were observed under a fluorescence microscope immediately at a magnification of 200×. (C) Control; (D) C-BF; (E) DSS; (F) C-BF+DSS. Scale bar = 50 μm. Gene (G−I) and protein (J) expression levels of epithelial barrier function related proteins. After the pretreatment of Caco-2 cells with 6.87 μmol/L C-BF and incubated with 3% (m/v) DSS for another 24 h, total RNA and protein were isolated, respectively. There were at least six repetitions in each group, the average values were calculated, and multiple comparison test were carried out by Tukey’s HSD and one way analysis of variance (ANOVA) with SPSS 16.0. The final values were the means ± SEM, and values without the same letters are significantly different (P < 0.05).

transactivation of EGFR.24 It was reported that FPRL-1 was the receptor for LL-37 that induced angiogenesis,42 and it was further found that the expression of hCAP18 (the precursor of LL-37) was significantly elevated in healing skin epithelium.43 PR-39, which is mainly secreted by macrophages, was reported to accelerate the formation of vascular structures in vitro and increase myocardial vasculature in vivo by inhibiting the ubiquitin-proteasome-dependent degradation of hypoxia-inducible factor-1 protein.44 Cathelicidin-BF, purified from the snake venom of Bungarus fasciatus, was the first identified cathelicidin antimicrobial peptide in reptiles. The precursor of cathelicidin-BF has a cathelin-like domain at the N terminus and contains a mature cathelicidin-BF at the C terminal. In particular, it has an atypical acidic fragment insertion between the cathelin-like domain and the C-terminus, and the acidic fragment is similar to acidic domains of amphibian antimicrobial precursors. The secondary structure of cathelicidin-BF is determined as an amphipathic αhelical conformation, and it was found possessing strong antibacterial activities against Gram-negative bacteria, especially Propionibacterium acnes.30 In our study, C-BF was reported to reduce the expression level of pro-inflammatory cytokines in colon tissues (Supplementary Figure 2) and serum (Figure 4) caused by colitis. From the results of LPS-stimulated cell

model, the effect of anti-inflammation was also demonstrated (Figure 9). The most important potential was the safety and stability of C-BF: no hemolytic and cytotoxic activities were observed at the dose of 400 μg/mL, and it could keep stable in the plasma of the mice for at least 2.5 h.30,31 It was also reported that C-BF could have potential therapeutic value for the prevention and treatment of burn and wound infections.45 BF15, which was made up of 15 amino acids and derived from the C-terminal amidate part of C-BF, was found to exert broad antimicrobial activity and reduced hemolytic activity. The possible mechanism was the structure of amphipathic α-helix and the enhancement of cytoplasmic membrane permeability of bacteria.46 Two mutants of C-BF were reported to have effective antimicrobial activity against drug-resistant strains, and the underlined mechanism path was through binding to DNA after penetrating the cytoplasmic membrane and enabling the leakage of β-galactosidase.47 In this study, DSS-induced UC mouse model, Caco-2 cell model, and LPS stimulated RAW264.7 cell model were set up to evaluate the possible immune regulation and barrier protection function of C-BF. It was the first research on the action of C-BF except for the antibacterial feature. In this study, we set up a DSS-induced UC like model. The etiology of this disease is still unknown, although the inferred 1657

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Figure 9. Concentration of PGE2 (A) and NO (B) in LPS-stimulated RAW264.7 cell culture supernatant, and gene expression level of TNF-α (C) in LPS-stimulated RAW264.7 cell. Phosphorylation level of the NF-κB related pathway (D) in LPS-stimulated RAW264.7 cell. RAW264.7 cells were pretreated with C-BF at a final concentration of 6.87 μmol/L for 4 h, then the culture medium was changed to fresh without C-BF, and cells were incubated with l μg/mL LPS for another 3 h. The cell culture supernatant was collected for analysis of PGE2 and NO concentration (A,B). Total RNA and protein were isolated for the analysis of the TNF-α expression level (C) and the phosphorylation level of the NF-κB related pathway (D).

finding that provided a new mechanistic insight into colitis, pioneering a novel therapeutic approach in experimental colitis. Compared with widely studied LL-37, C-BF showed the obvious advantage of stability,30,49,50 safety,30,50−55 antibacterial activity,30,31,54,56 and other pharmacological potential.57 Our study on comparing the stability, safety, and antibacterial activity of C-BF and LL-37 under the same condition demonstrated that both of them are linear cationic antimicrobial peptides and have amphipathic α-helical conformation (Supplementary Figure 7); however, they do have differences in some biochemical properties: (1) stability, our study showed that antimicrobial peptide C-BF was much better than LL-37 in resistance to artificial gastric juice, artificial intestinal fluid, and serum from mice (Supplementary Figure 8); (2) safety, we found C-BF showed much safer characteristic than LL-37 in human peripheral blood mononuclear cells (PBMCs) and porcine red blood cells (Supplementary Figure 9); (3) antibacterial activity, we compared the antibacterial activity between LL-37 and C-BF. It can be concluded that, under the same concentration, C-BF showed much higher antibacterial activity in general, and to some bacteria, its antibacterial effect was even better than antibiotics tested (Supplementary Table 4). Furthermore, the importance of new findings in this study are underlined by the fact that the pathogenesis and molecular mechanisms of ulcerative colitis are still not yet fully understood, and the current anti-inflammatory and biological therapies could not cure the disease but induce a long-term

factors are possible immunologic and enteric microflora abnormalities.2,48 We found that after treatment with 3% DSS for 7 days the expression level of cathelicidin was significantly increased (Supplementary Figure 3C), consistent with the previous report,26 and the level of mCRAMP in C-BF+DSS group decreased significantly compared with the DSS treated group (Supplementary Figure 3D), indicating the lower inflammatory status after being treated with C-BF. It was reported that intrarectal administration of endogenous mCRAMP ameliorated DSS-induced colitis and significantly reduced the increased number of fecal microflora in colitis animals. Administration of mCRAMP could also upregulate the expression of mucin genes and suppressed DSS-induced apoptosis and neutrophil infiltration.28 Surprisingly, from our results, we found exogenous peptide C-BF could upregulate mucin genes such as mucin1 and mucin2 disrupted by DSS (Supplementary Figure 4E,F). The apoptosis index of colonic epithelium in C-BF+DSS group was significantly lower than DSS treated group (Figure 6), the infiltration status of inflammatory cells such as neutrophils and macrophages were also tested. In accordance with the result of endogenous mCRAMP, C-BF could significantly attenuate the infiltration condition caused by DSS (Figure 5D,H). Recent study showed that localized gene therapy with intrarectal administration of a mCRAMP-encoding plasmid prevented DSS-induced colitis.29 This was the first successful use of cathelicidin gene therapy in colitis and was an important 1658

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also exist. Furthermore, we found that C-BF regulated inflammation through NF-κB signaling pathway and verified the in vivo results with the LPS stimulated cell model. The DSSinduced barrier function disruption model was also established to demonstrate the intestinal barrier protective function of CBF in UC mice. It was deduced that C-BF protects the intestinal barrier mainly through the enhanced expression of tight junction proteins and mucins, although the pathway leading to up-regulation of the expression of these proteins remained to be elucidated. The successful prevention of UC with C-BF indicated its potential as a novel next generation therapeutic in IBD. Thus, this study has laid the foundation for developing C-BF as a specific UC therapeutic and also provides a template to study other antimicrobial peptides with similar functions to those of C-BF.

remission in less than 34% of the patients. All these studies above indicated that other factors, for example, dysregulation of cathelicidin expression/function, may have important roles in the pathomechanism of ulcerative colitis.58,59 It was previously reported that cathelicidin expression localized to mucosal macrophages in inflamed colon tissue in mice, and intracolonic administration of bacterial DNA to wildtype mice also induced the expression of cathelicidin in the colons of healthy mice and mice with DSS-induced colitis, possibly via the activation of TLR9-ERK signaling. Compared with normal mice, CRAMP knockout mice developed a more severe form of DSS-induced colitis, especially after intracolonic administration of E. coli DNA.26 As reported above, the antimicrobial effect of C-BF was excellent, especially to Gramnegative bacteria; thus, it was possible that C-BF reduced bacterial infiltration of the DSS-injured gut epithelium and in turn alleviated inflammation and associated syndrome. However, this required further investigation of the microbial infiltration to the epithelium. In another infection model, it was reported that intracolonic administration of mCRAMP to C. dif f icile-infected WT mice showed significantly reduced colon damage, apoptosis, MPO, and TNF-α levels, and significantly reduced toxin A-induced TNF-α expression through inhibition of NF-κB phosphorylation. These effects were similar to C-BF in UC mice (Figures 2, 4A, 5I, 6, 7C, and 9C,D, and Supplementary Figure 2A). The study also indicated that endogenous cathelicidin may be insufficient to modulate C. dif f icile/Toxin A-mediated intestinal inflammation.60 Through analysis of the phosphorylation level of the transcription factors downstream of MAPK and NF-κB in colon tissue, c-jun and p65 (Figure 7C), it was demonstrated that DSS increased the phosphorylation level of both p65 and c-jun and that C-BF treatment down-regulated the phosphorylation level of p65 alone. This could explain why INOS was not as sufficiently down-regulated by C-BF as COX-2 (Figure 7A,B), for c-jun could also regulate the expression of INOS directly.61,62 From the results above, it could be deduced that the attenuation of inflammation by C-BF may be related to the decreased phosphorylation of NF-κB p65. To verify the possible mechanism of C-BF attenuation of inflammation in the UC mouse model, a LPS-stimulated inflammation cell model was set up. The phosphorylation level of key factors in NF-κB related signaling pathway were determined. The LPS treated group had significantly increased phosphorylation level of AKT, IκB-α, and p65, while C-BF pretreatment downregulated the phosphorylation level of these three factors (Figure 9D). In light of the combined in vivo and in vitro results, we could conclude that the attenuation of inflammation by CBF may be correlated with the regulation of NF-κB related signaling pathway and thus improved the tight junction function, or that C-BF could have a direct effect on barrier function (Figure 8G−J). The exact mechanism involved still needed to be proved further.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Address: College of Animal Science, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China. E-mail: [email protected]. Tel: +8657188982815. Fax: +8657188982650. Author Contributions †

Haiwen Zhang and Xi Xia contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Science Fund for Distinguished Young Scholars of China (Grant No. 31025027), the National Science Fund for China (Grant No. 31172213), and the earmarked fund for Modern Agro-industry Technology Research System (CARS-36).

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DOI: 10.1021/acs.molpharmaceut.5b00069 Mol. Pharmaceutics 2015, 12, 1648−1661

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DOI: 10.1021/acs.molpharmaceut.5b00069 Mol. Pharmaceutics 2015, 12, 1648−1661