The antimicrobial peptide Abaecin alleviates colitis in mice by regulating inflammatory signaling pathways and intestinal microbial composition

The antimicrobial peptide Abaecin alleviates colitis in mice by regulating inflammatory signaling pathways and intestinal microbial composition | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The antimicrobial peptide Abaecin alleviates colitis in mice by regulating inflammatory signaling pathways and intestinal microbial composition Zhineng Liu, Xinyun Qin, Keyi Nong, Xin Fang, Bin Zhang, Wanyan Chen, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3540117/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective The purpose of this study was to determine the effect of Abaecin on dextran sulfate sodium (DSS) -induced ulcerative colitis in mice and to explore its related mechanisms. Methods Twenty-four mice with similar body weight were randomly divided into 4 groups. 2.5% DSS was added to drinking water to induce colitis in mice. Mice were executed after Abaecin administration treatment, and mouse serum and tissues were collected. We measured the concentration of serum inflammatory cytokines in mice and produced colon tissue sections to observe the damage to the colonic structure. Then, we assessed the integrity of the intestinal barrier by the expression of intestinal tight junction proteins. In addition, we determined the phosphorylation levels of NF-κb/MAPK inflammatory signaling pathway proteins and the microbial composition of the intestinal flora to preliminarily investigate the alleviation mechanism of ulcerative colitis by Abaecin. Results The results showed that Abaecin significantly alleviated histological damage and intestinal mucosal barrier damage caused by colitis, reduced the concentration of pro-inflammatory cytokines and the phosphorylation of NF-κB / MAPK inflammatory signaling pathway proteins, and improved the composition of intestinal microorganisms. Conclusion These findings suggest that Abaecin may have potential prospects for the treatment of ulcerative colitis. Abaecin Ulcerative colitis NF-κB MAPK Intestinal microorganisms Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Ulcerative colitis (UC) is an inflammatory process that is confined to the mucosa and submucosa of the colon. Patients with UC show a variety of clinical symptoms, including abdominal pain, blood in the stool, and weight loss. It is often recurrent and of unknown aetiology, and it is now generally accepted that it is influenced by a variety of factors (including genetic, environmental, immunological, physiological, psychological, and the gut microbiome) and their interactions [ 1 ]. Due to the common adverse reactions of common drugs for the treatment of UC, the use of natural extracts with nontoxic side effects and good therapeutic effects as drugs for the treatment of UC is becoming a new trend [ 2 – 3 ]. Antimicrobial peptide (AMP) is a kind of small molecular polypeptide that is naturally present in animals. Most antimicrobial peptides have broad-spectrum antimicrobial activity [ 4 ]. In addition, some antimicrobial peptides also have antiviral [ 5 ], anti-inflammatory [ 6 ], antitumor [ 7 ], immune regulation [ 8 ] and other biological activities. A large number of studies have shown that antimicrobial peptides can affect inflammation through a variety of channels. For example, some peptides not only attract neutrophils, but also block their apoptosis and enhance the phagocytic function of neutrophils [ 9 ]. AMPs can also enhance the Th17 response. In short, AMPs effectively attract Th17, which mainly secretes pro-inflammatory cytokines IL-17A, IL-21F, IL-22 and IL-17, and is responsible for establishing mucosal defense against pathogenic microorganisms in the respiratory tract or intestine [ 10 ]. On the other hand, AMPs can also act on host-microbiome interactions. In this case, AMPs can be used as a buffer to neutralize LPS and LTA released by the microbiota to maintain homeostasis and prevent environmental disorders [ 11 ]. In addition, the immunomodulatory capacity of AMPs is mainly mediated by extensive binding to the membrane and interaction with intracellular receptors, thus stimulating downstream signaling pathways and producing the corresponding regulatory effects on inflammation [ 12 ]. Studies have found that the promoter regions of α-and β-defensin genes contain binding sites for the major cellular transcription factors, especially nuclear factor κB (NF-κB). It is worth noting that NF-κB also plays a crucial role in the pathogenesis of inflammatory bowel disease (IBD). Abaecin is a proline-rich natural antimicrobial peptide from bees. Together with Apidaecin, Hymenoptaecin, and Defensin, it is an important part of the innate humoral immunity of bees and has a broad spectrum of antibacterial ability [ 13 ]. Abaecin enters cells in an insoluble manner, interacts with bacterial cells, and hinders protein synthesis [ 14 ]. Interestingly, Abaecin actually has a weak transmembrane ability, so it needs to be combined with other pore-forming peptides to enhance the effect. For example, Abaecin has a good inhibitory effect on parasites in synergy with other insect antimicrobial peptides [ 15 ]. In addition, the expression of Abaecin in honey bees is also closely related to gut bacteria, and disruption of the gut bacterial community due to excessive use of antibiotics causes a decrease in the expression of the gene encoding abaecin in honey bees [ 16 ]. Currently, there are few reports on the anti-inflammatory effect and preliminary mechanism of Abaecin in bee antimicrobial peptides. In this study, the DSS-induced acute colitis model in mice was used to explore whether Abaecin has a mitigating effect on UC and to further explore the preliminary mechanism of Abaecin in alleviating inflammation to provide a theoretical basis for the development of specific drugs for the treatment of UC with Abaecin as the main material. Materials and Methods Materials and reagents Abaecin (purity 96%) was purchased from GL Biochem (Shanghai) Ltd. DSS (Dextran sulfate sodium; M.W., 36–50 kDa) was obtained from MP Biomedicals (Santa Ana, CA). Mouse tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), interleukin-1β (IL-1β), interleukin-10 (IL-10) and interferon-γ (IFN-γ) ELISA kits were provided by Nanjing Jiancheng Institute of Bioengineering (Nanjing, China). Lipopolysaccharides (LPS), D-lactate (D-LA), and diamine oxidase (DAO) activity ELISA kits were supplied from Shanghai Enzyme-linked Biotechnology Co. Anti-F4/80, CD177 antibody was purchased from Wuhan Servicebio Technology CO., Ltd. Anti-p65, p-p65, p38, p-p38, JNK, p-JNK, ERK, p-ERK, occludin, claudin-1, and ZO-1 antibodies were purchased from Bioss Antibodies Co., Ltd., Beijing, China. All other chemicals were of the highest grade available. Experimental animal Male C57BL/6J mice (7–8 weeks old) were supplied by the Laboratory Animal Center of Guangzhou University of Chinese Medicine, Guangzhou, China. Mice were given one week to acclimate to the new environment before the start of the experiment (temperature of 25 ± 12°Cand 12hlight/dark cycle). The animal experiment was approved by the Animal Care and Use Committee of Hainan University. Experimental design Twenty-four mice were randomly divided into four groups (n = 6/group) after 1 week of acclimatization: control group (Con), colitis group (Mod), colitis plus Abaecin group (Ab + Mod) and Abaecin group (Ab). 2.5% DSS (w/v) was added to drinking water to induce acute ulcerative colitis for seven days. The Ab and Ab + Mod groups were then treated with rectal administration of Abaecin (5mg/kg), and the Con and Mod groups were injected rectally with equal amounts of PBS. The weight was recorded every day. The DAI scores were performed according to previous research criteria [ 17 ]. On the eighth day of the experimental period, all mice were sacrificed. Collect serum, spleen. The colon was removed and measured. Colon samples were fixed in 10% formalin for hematoxylin and eosin (H & E) examination. Histological scoring was performed according to the criteria described in Table 1 . Measurement of inflammatory cytokine and oxidative stress indicators Serum samples were prepared by centrifugation after blood was collected and equilibrated at room temperature for 1 hour. The concentrations of IL-1 β, IL-6, TNF-α, IFN-γ, IL-10, DAO, D-LA and LPS in serum were determined by ELISA kits, which were operated according to the instructions of the kits. Apoptosis index assay Tunel staining was used to determine the apoptosis index. Endogenous peroxidase was blocked after antigen repair of the paraffin sections. After room temperature repair, Streptavidin-HRP reaction solution was added, DAB coloration, nucleus restaining, and finally dehydrated and mounted. Histopathological evaluation and immunohistochemical staining Colon tissues collected were fixed in 4% paraformaldehyde overnight and then dehydrated and transparent in turn. The tissues were then embedded in paraffin sections. The number of goblet cells was assessed by Alcian Blue Periodic Acid Schiff (AB-PAS) staining. Subsequently, immunohistochemical staining was used to observe the degree of infiltration of inflammatory cells. Briefly, sections were incubated with anti-CD177 antibody and anti-F4/80 antibody at 4°C overnight, respectively. And then the sections were incubated with HRP-conjugated rabbit anti-goat IgG in a 1:100 ratio for 1 hour. Image J software was used to visualize the results of the sections. Immunofluorescence staining Immunofluorescence staining was used for the analysis of tight junction proteins of the colon. Paraffin sections were dewaxed and antigen repair was performed with EDTA antigen repair solution. Sections were blocked with 3% BSA (Wuhan Servicebio Technology CO., Ltd.) and then incubated in anti-ZO-1 tight junction protein antibody (1:200, Wuhan Servicebio Technology CO., Ltd.) at 4°C overnight. The corresponding secondary antibody was added and incubated for 50 minutes under light avoidance conditions before adding the autofluorescence quencher B solution. Finally, the slides were sealed with an antifluorescence quenching sealer and the observed results were quantified by image J software. Western Blot analysis. The total protein in the colon was extracted using a total protein extraction kit (Solarbio, Beijing, China) according to the kit manufacturer’s instructions. The protein was separated on a SDS-PAGE gel and transferred to the PVDF membrane. After blocking the membrane with a blocking solution (Wuhan Servicebio Technology CO., Ltd.), the membrane was incubated with the corresponding primary antibody (p65, p-p65, p38, p-p38, JNK, p-JNK, ERK, p-ERK, occludin, claudin-1, ZO-1(Bioss, Beijing, China)) at 4°C overnight and then the membrane was placed in the HRP labeled secondary antibody (1:20,000, Bioss, Beijing, China). The protein band was observed under the action of the ECL hypersensitive luminescent liquid (Biosharp,Beijing). Finally, the gray value of the protein band was quantified by image J software. 16SrRNA Sequencing Analysis The PCR reaction system was configured with 30 ng of acceptable quality genomic DNA samples samples and the corresponding fusion primers, and PCR amplification was carried out by setting the PCR reaction parameters, and the PCR amplification products were purified using Agencourt AMPure XP magnetic beads, dissolved in Elution Buffer, labeled, and then completed the construction of the library. The fragment range and concentration of the libraries were analyzed by Agilent 2100 Bioanalyzer. The library that passed the test was selected for sequencing on the HiSeq platform according to the size of the inserted fragments. Filter the data, the remaining high-quality Clean data for later analysis; through the overlap relationship between the reads to reads spliced into Tags; Tags clustered into OTUs and compared with the database, species annotations; based on the OTUs and annotated results of the sample species complexity analysis, intergroup species difference analysis, as well as correlation analysis and model prediction, and so on. Finally, in order to further explore the relationship between intestinal flora and inflammation-related indicators, we performed spearman analysis and created a cluster-related heat map of genera and indicators using the Omicstudio tool. Statistical analysis GraphPad Prism 8 is used for data processing. IBM SPSS Statistics 27 was used for statistical analysis. One-way ANOVA and Tukey’s multiple comparison test were used for the difference analysis. All experiments were repeated at least three times and all results were expressed as mean ± standard deviation (SD). * p < 0.05, * * p < 0.01. Table 1 Histological scoring criteria Score Inflammatory cells infiltration Depth of invasion Crypt injury Pathological range (%) 0 none none none 0 1 ± Mucosa layer 1/3 1–25 2 + Mucosa and submucosa 2/3 26–50 3 ++ Full Full 51–75 4 +++ 76–100 Result Prediction of Abaecin structure As shown in Fig. 1, we performed MS analysis and HPLC analysis of Abaecin, respectively, and found that the molecular weight and purity of Abaecin were 3878.94 kDa and 96.79%, respectively. On this basis, we predicted the tertiary structure of Abaecin by website https://zhanggroup.org/I-TASSER/ , and also predicted the helical wheel structure of Abaecin to determine hydrophilicity and hydrophobicity. Effects of Abaecin on inflammatory symptoms A large number of studies have shown that DSS-induced ulcerative colitis in mice can lead to significant weight loss, significantly reduced colon length, abnormal organ index, diarrhea, hematochezia, and other symptoms. These symptoms were also observed in this experiment. As shown in Fig. 2, rectal administration of Abaecin significantly increased colon length and body weight of mice (Fig. 2b-d, p < 0.01), and significantly reduced the DAI score and the spleen index of mice (Fig. 2e-f). Effect of Abaecin on serum concentrations of oxidative stress-related factors and inflammatory cytokines In this study, we found that DSS-induced colitis caused a significant increase in serum concentrations of DAO, D-LA, LPS, IL-1β, IL-6, TNF-α, and IFN-γ(Fig. 3a-g, p < 0.01), as well as a significant decrease in the concentration of the inflammation suppressing cytokine, IL-10(Fig. 3h, p 0.05). Effect of Abaecin on histological injury and inflammatory cell infiltration HE staining was used to visualise histological damage to colon tissue and the Mod group had altered colon morphology with a large infiltration of inflammatory cells (p < 0.01), which was significantly ameliorated by Abaecin administration (p < 0.01). In addition, colitis usually causes a decrease in the number of goblet cells (p < 0.01), an increase in the apoptotic index (p < 0.01), and massive infiltration of inflammatory cells (p < 0.01), all of which were significantly ameliorated by rectal administration of Abaecin. Effect of Abaecin on the expression of tight junction proteins Immunofluorescence staining was used to determine the expression of the tight junction protein ZO-1 in the colon (Fig. 5a). In this study, we found that DSS-induced ulcerative colitis significantly reduced the expression of ZO-1, which was significantly restored by Abaecin treatment (Fig. 5b, p < 0.01). Similarly, we found that Abaecin treatment significantly restored the reduced expression of the tight junction proteins occludin, claudin-1, and ZO-1 induced by colitis in mice by Western Blot analysis (Fig. 5c-e, p < 0.01). Effect of Abaecin on the NF-κB/MAPK Signaling Pathway As shown in Fig. 6, western blot analysis showed that NF-κB signaling pathway and the MAPK signaling pathway were activated under the influence of colitis. Relative expression levels of p-p65, p-ERK, p-JNK, and p-p38 were significantly increased (p < 0.01). However, Abaecin treatment significantly reversed these changes(p < 0.01). Effect of Abaecin on the composition of intestinal microorganisms 16SrRNA analysis was used to determine changes in intestinal microbial composition. We obtained petal plots by comparing OTUs between samples or between subgroups (Fig. 7a). The OTU rank curves and the species accumulation curves adequately indicated that the number of samples was sufficient for the prediction of species richness (Fig. 7b-c). The coverage indices for all taxa were close to 1, indicating that the sequencing data covered about 100% of the well-covered microorganisms. In the Partial Least Squares Discriminant Analysis (PLS-DA) plot (Fig. 7d), the Mod group and the Ab + Mod group are separated in the direction of the vertical axis, suggesting that Abaecin, represented by the vertical coordinate, is the main factor responsible for the separation of the two groups. In the Alpha diversity box plot (Fig. 7e), the Chao1 Alpha diversity value was significantly lower in the Mod group compared to the Con group (p < 0.05). And a significant difference was also found in beta diversity between the four groups in the Beta group difference box plot (Fig. 7f, p < 0.05). In order to explore changes in the composition of the intestinal flora, a bar chart of the species composition was made at the genus level. It can be seen in Fig. 7g that DSS-induced colitis caused an abnormal increase in the abundance of Bacteroides , Barnesiella and Escherichia , and the administration of Abaecin inhibited this increase. In addition, colitis in mice also led to a decrease in the abundance of Lactobacillus and Desulfovibrio , while Abaecin restored the abundance of these bacteria. Furthermore, we explored the correlation between intestinal flora composition and indicators related to inflammation using spearman analysis. As shown in Fig. 7h, Bacteroides and Escherichia were significantly positively correlated with IL-1β (p < 0.01), IL-6 (p < 0.01), TNF-α (p < 0.01),IFN-γ (p < 0.01), D-LA (p < 0.01), LPS (p < 0.01), DAO, (p < 0.01),DAI (p < 0.01), Neutrophilic granulocyte invasion index (p < 0.01),Macrophage invasion index (p < 0.01), Cell apoptotic index (p < 0.01), Spleen index (p < 0.01) and Histological score (p < 0.01) and were significantly negatively correlated with IL-10(p < 0.01),colon length(p < 0.01) and Body weight(p < 0.01). On the contrast, Lactobacillus and Desulfovibrio were significantly negatively correlated with IL-1β (p < 0.05,p < 0.01,respectively), IL-6 (p < 0.01), TNF-α (p < 0.01),IFN-γ(p 0.05,P < 0.05,respectively), LPS (p < 0.01), DAO (p < 0.05,p < 0.01,respectively),DAI (p < 0.01), Neutrophilic granulocyte invasion index (p < 0.01),Macrophage invasion index (p < 0.01), Cell apoptotic index (p < 0.01), Spleen index (p < 0.01) and Histological score (p < 0.01) and were significantly positively correlated with IL-10(p < 0.01),colon length(p < 0.01) and Body weight(p < 0.01). Figure 1 (a) MS analysis of Abaecin. (b) HPLC analysis of Abaecin. (c) Prediction of the tertiary structure of Abaecin and its sequence. (d) Analysis of the characteristic of the helical wheel of Abaecin. Yellow and green represent nonpolar amino acids, while other colors represent polar amino acids. Figure 2 (a)Overview of the experimental design. Effects of Abaecin on (b-c) mouse colon length, (d) mouse body weight, (e) DAI scores, and (f) spleen indices. Data represent means ± S.D. (n = 6). *p < 0.05 and **p < 0.01 vs Con group on the same day; #p < 0.05 and ##p < 0.01 vs Mod group. Figure 3 Effect of Abaecin on serum concentrations of (a)LPS, (b)D-LA, (c)DAO, (d)IL-1β, (e)IL-6, (f)TNF-α, (g)IFN-γ, and (h)IL-10. All values present means ± S.D. (n = 6). *p < 0.05 and **p < 0.01. Figure 4 (a) Histological section examination of the colon in mice (100×). (b) Histological scoring. (c)Ration of the AB-PAS positive cell. (d)Cell apoptotic index. (e) Neutrophilic granulocyte invasion index. (f)Macrophage invasion index. All data present means ± S.D. (n = 6). *p < 0.05 and **p < 0.01. Figure 5 (a) Immunofluorescence staining of ZO-1 (20×) and (b) relative fluorescence expression intensity of ZO-1. (c) Western blot bands of three tight junction proteins. Determination of the levels of (d) occludin, (e) claudin-1, and (f) ZO-1 proteins in colonic tissues by Western blotting. Values are presented as means ± SD (n = 6). *p < 0.05, **p < 0.01. Figure 6 (a) Western blot bands of NF-κB / MAPK signaling pathway protein. Relative expression of (b) p-p65 to p65, (c) p-ERK to ERK, (d) p-JNK to JNK, and (e) p-p38 to p38. Values are presented as means ± SD (n = 6). * p < 0.05, ** p < 0.01. Figure 7(a) Petaline graphs. The middle circle represents the number of OTUs shared by the sample or group, and the ellipse outside the middle circle represents the number of OTUs unique to the sample or group. (b) OTU Rank Curve. The richness of the species in the sample is reflected in the length of the horizontal axis of the curve. The wider the curve, the richer the species composition in the sample. The uniformity of the species in the sample is reflected in the shape of the longitudinal axis of the curve. The flatter the curve is, the higher the uniformity of the species composition in the sample is. (c) Curves of species accumulation. The rising trend at the end of the curve tends to be gentle, indicating that the sample amount is sufficient. (d) PLS-DA plot. Points of different colors or shapes represent sample groups under different environments or conditions, and abscissa and ordinate represent the suspected influencing factors of differences in the microbial composition in each group. (e) Alpha diversity difference map between groups. (f) The Beta diversity box-plot. (g) A bar chart of species composition at the genus level. (H) Spearman correlation analysis heat map. Values are presented as means ± SD (n = 6). * p < 0.05, ** p < 0.01. Discussion Abaecin is a proline-rich antimicrobial peptide produced by bees when they are infected by microorganisms or other exogenous substances, and has broad-spectrum antibacterial activity against Gram-negative bacteria [ 18 ]. Antimicrobial peptides are classified into lytic antimicrobial peptides and nonlytic antimicrobial peptides [ 19 ]. The former kills bacteria by destroying bacterial cell membranes and causing cell lysis. Abaecin belongs to the latter, which prevents bacterial growth by acting on the bacterial plasma membrane and on intracellular targets. It is worth noting that many proline-rich antimicrobial peptides (PrAMPs) act on ribosomes [ 20 ]. In our study, predicting the tertiary structure of Abaecin, we found that Abaecin showed an obvious randon coil. And the prediction of the helix structure found that the hydrophobic and hydrophilic residues were distributed symmetrically on both sides, indicating that Abaecin may exhibit excellent amphiphilic properties, this structure is considered related to the antibacterial activity of Abaecin [ 21 ]. A large number of studies have shown that DSS-induced colitis mouse models can lead to a significant decrease in colon length and body weight, a significant increase in disease activity index (DAI) and severe damage to intestinal structure [ 22 – 24 ]. This is consistent with the results observed in this study. Fortunately, rectal injection of Abaecin significantly increased colon length and body weight and decreased disease activity index (DAI) in mice. At the same time, through histological observation, we found that Abaecin significantly reduced infiltration of inflammatory cells, significantly increased the number of goblet cells, and alleviated apoptosis of intestinal cells. These results suggest that Abaecin can alleviate tissue damage caused by ulcerative colitis and protect intestinal health. The intestinal epithelial barrier is an important line of defense against pathogenic microorganisms and toxins in the intestinal lumen [ 25 ]. Tight junction (TJ) protein is an essential factor for a complete intestinal barrier. Once the tight junction protein is reduced or destroyed, it will lead to infiltration of toxic intestinal substances into the intestinal lamina propria and further aggravate the inflammatory response [ 26 ]. A number of reports have shown that the number of TJ in the intestinal tract of IBD patients is reduced, resulting in defects in intestinal barrier function [ 27 – 28 ]. Thus, TJ may be a key factor in the treatment of ulcerative colitis. In this study, immunofluorescence staining and Western blot analysis were used to determine the expression of TJ. The results showed that the expression levels of ZO-1, claudin-1 and occludin in colitis mice were increased significantly after Abaecin administration. In addition, we also found that the concentrations of D-LA, DAO, and LPS in the serum of mice with DSS-induced ulcerative colitis increased significantly, and the intervention of Abaecin significantly reduced the concentrations of these three substances. This suggests that Abaecin can protect the integrity of the intestinal barrier by regulating the expression of TJ in mice, preventing toxic substances from entering the body, and thus alleviating ulcerative colitis. Both NF-κB and MAPK are the main signal messengers that regulate the transcription of pro-inflammatory genes during inflammation [ 29 – 30 ].NF-κB protein is usually composed of p65 and p50 to form a homologous / heterologous dimer, which binds to the inhibitor protein IkB to form a trimer complex and is in an inactive state [ 31 ]. When the upstream signal factor is activated, it will lead to the phosphorylation of IκB protein and the release of the complex p50 / p65, which enters the nucleus and rapidly activates the transcription of various cytokines and chemokines [ 32 ]. In the current results, the concentrations of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IFN-γ in the serum of DSS-induced colitis mice increased significantly, and rectal administration of Abaecin significantly reduced the concentration of these pro-inflammatory cytokines. IL-10 is an anti-inflammatory cytokine, and its concentration in mouse serum is reduced under the DSS treatment. However, Abaecin did not significantly improve the reduction of IL-10 concentration. To further investigate the mechanism associated with Abaecin alleviation in mice, we measured the expression of two signaling pathway proteins, NF-κB and MAPK, and found that the phosphorylation levels of P38, JNK, ERK1/2, and p65 increased significantly in the mouse model with DSS-induced colitis, while Abaecin decreased significantly the levels of phosphorylation of these proteins. It has been reported that p38, JNK and ERK1/2 are crucial regulators of inflammation, autoimmune, apoptosis, cell proliferation and differentiation, and are closely related to the occurrence and development of inflammation [ 33 – 34 ]. This indicates that Abaecin can alleviate DSS-induced colon inflammation and reduce the secretion of pro-inflammatory cytokines by regulating the expression of NF-κB / MAPK signaling pathway. The intestinal immune system maintains a state of tolerance to the diversity and beneficial symbiotic intestinal microorganisms while responding to pathogenic microorganisms, invasive pathogens, and microbial products. These two states achieve balance and maintain intestinal health [ 35 ]. Changing the intestinal microbial community will break this immune balance, leading to disease infection or immune disorders [ 36 ]. The results of 16SrRNA analysis showed that the abundance of Bacteroides , Barnesiella and Escherichia in the intestine increased abnormally. Bacteroides are usually “friendly” symbionts in the gut, but they tend to become opportunistic pathogens when they are hosted elsewhere [ 37 ]. It is reported that Bacteroides Bfr is the main promoter and promoter of human colorectal cancer [ 38 ]. Barnesiella and Escherichia are Gram-negative bacteria. The main component of their outer membrane is LPS, which has strong pro-inflammatory activity [ 39 ]. Therefore, the excessive increase in the abundance of these two bacteria is closely related to the aggravation of intestinal inflammation. Interestingly, the results of the spearman correlation analysis also showed that Bacteroides , Barnesiella , and Escherichia were positively correlated with histological damage, pro-inflammatory cytokines, and indicators related to intestinal permeability, which means that the high abundance of these three genera is related to colon tissue damage, increased inflammation, intestinal barrier damage, and weakening of antioxidant status. Administration of Abaecin inhibited the abnormal increase in the abundance of these three genera. On the contrary, the abundance of Lactobacillus and Desulfovibrio in the intestine of colitis mice decreased significantly. Lactobacillus has been reported to exhibit enzymatic activity and, by producing bile salt hydrolases, Lactobacillus plays a crucial role in bile acid metabolism [ 40 ]. Furthermore, studies have shown that Lactobacillus and other probiotic species can effectively treat and alleviate Crohn's disease and ulcerative colitis [ 41 ]. The results of the Spearman correlation analysis also showed that Lactobacillus and Desulfovibrio were positively correlated with body weight, colon length and anti-inflammatory cytokines in mice, and Abaecin restored the abundance of these bacteria. Therefore, the results of this study indicate that Abaecin can maintain intestinal health by regulating the composition of intestinal flora. In summary, ulcerative colitis can cause infiltration of inflammatory cells, structural intestinal damage, and imbalance of intestinal flora. Unbalance intestinal flora will lead to aggravation of the inflammatory reaction and will form a vicious circle. In this study, Abaecin alleviated these injuries, balanced the intestinal flora, and prevented a vicious cycle. Conclusion In summary, Abaecin can alleviate tissue damage caused by colitis, improve intestinal barrier damage, and excessive secretion of pro-inflammatory cytokines by regulating inflammatory signaling pathways and improving intestinal flora composition. Therefore, Abaecin can be used as a potential natural substance for the development of specific drugs for ulcerative colitis. Declarations Funding This work was funded by the Hainan Provincial Natural Science Foundation High-Level Talents Project (320RC463), the National Natural Science Foundation of China (32260857), and the Innovation and entrepreneurship training project of college students (202310589071). The authors declare no competing financial interest. References Tavakoli P, Vollmer-Conna U, Hadzi-Pavlovic D, Grimm MC. A Review of Inflammatory Bowel Disease: A Model of Microbial, Immune and Neuropsychological Integration. Public Health Rev. 2021;42:1603990. https://doi: 10.3389/phrs.2021.1603990. 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Fucoidan Ameliorated Dextran Sulfate Sodium-Induced Ulcerative Colitis by Modulating Gut Microbiota and Bile Acid Metabolism. J Agric Food Chem. 2022;70(47):14864-76. https://doi: 10.1021/acs.jafc.2c06417. Yang J, Miao L, Xue Y, Wang X. Yiyi Fuzi Baijiang Powder Alleviates Dextran Sulfate Sodium-Induced Ulcerative Colitis in Rats via Inhibiting the TLR4/NF-κB/NLRP3 Inflammasome Signaling Pathway to Repair the Intestinal Epithelial Barrier, and Modulating Intestinal Microbiota. Oxid Med Cell Longev. 2023;2023:3071610. https://doi: 10.1155/2023/3071610. Xue HH, Li JJ, Li SF, Guo J, Yan RP, Chen TG, et al. Phillygenin Attenuated Colon Inflammation and Improved Intestinal Mucosal Barrier in DSS-induced Colitis Mice via TLR4/Src Mediated MAPK and NF-κB Signaling Pathways. Int J Mol Sci. 2023;24(3). https://doi: 10.3390/ijms24032238. Turner JR. Intestinal mucosal barrier function in health and disease. Nat Rev Immunol. 2009;9(11):799-809. https://doi: 10.1038/nri2653. Turner JR. 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Sorafenib inhibits LPS-induced inflammation by regulating Lyn-MAPK-NF-kB/AP-1 pathway and TLR4 expression. Cell Death Discov. 2022;8(1):281. https://doi: 10.1038/s41420-022-01073-7. Rai A, Kapoor S, Singh S, Chatterji BP, Panda D. Transcription factor NF-κB associates with microtubules and stimulates apoptosis in response to suppression of microtubule dynamics in MCF-7 cells. Biochem Pharmacol. 2015;93(3):277-89. https://doi: 10.1016/j.bcp.2014.12.007. He X, Wei Z, Zhou E, Chen L, Kou J, Wang J, et al. Baicalein attenuates inflammatory responses by suppressing TLR4 mediated NF-κB and MAPK signaling pathways in LPS-induced mastitis in mice. Int Immunopharmacol. 2015;28(1):470-6. https://doi: 10.1016/j.intimp.2015.07.012. Pua LJW, Mai CW, Chung FF, Khoo AS, Leong CO, Lim WM, et al. Functional Roles of JNK and p38 MAPK Signaling in Nasopharyngeal Carcinoma. Int J Mol Sci. 2022;23(3). https://doi: 10.3390/ijms23031108. Wen X, Jiao L, Tan H. MAPK/ERK Pathway as a Central Regulator in Vertebrate Organ Regeneration. Int J Mol Sci. 2022;23(3). https://doi: 10.3390/ijms23031464. Broom LJ, Kogut MH. Inflammation: friend or foe for animal production? Poult Sci. 2018;97(2):510-4. https://doi: 10.3382/ps/pex314. Kogut MH, Lee A, Santin E. Microbiome and pathogen interaction with the immune system. Poult Sci. 2020;99(4):1906-13. https://doi: 10.1016/j.psj.2019.12.011. Murphy EC, Mörgelin M, Cooney JC, Frick IM. Interaction of Bacteroides fragilis and Bacteroides thetaiotaomicron with the kallikrein-kinin system. Microbiology (Reading). 2011;157(Pt 7):2094-105. https://doi: 10.1099/mic.0.046862-0. Zamani S, Taslimi R, Sarabi A, Jasemi S, Sechi LA, Feizabadi MM. Enterotoxigenic Bacteroides fragilis: A Possible Etiological Candidate for Bacterially-Induced Colorectal Precancerous and Cancerous Lesions. Front Cell Infect Microbiol. 2019;9:449. https://doi: 10.3389/fcimb.2019.00449. Cani PD, Amar J, Iglesias MA, Poggi M, Knauf C, Bastelica D, et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes. 2007;56(7):1761-72. https://doi: 10.2337/db06-1491. Kumar R, Grover S, Batish VK. Hypocholesterolaemic effect of dietary inclusion of two putative probiotic bile salt hydrolase-producing Lactobacillus plantarum strains in Sprague-Dawley rats. Br J Nutr. 2011;105(4):561-73. https://doi: 10.1017/s0007114510003740. Bibiloni R, Fedorak RN, Tannock GW, Madsen KL, Gionchetti P, Campieri M, et al. VSL#3 probiotic-mixture induces remission in patients with active ulcerative colitis. Am J Gastroenterol. 2005;100(7):1539-46. https://doi: 10.1111/j.1572-0241.2005.41794.x. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3540117","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":246062808,"identity":"40641b86-0fba-4b75-82e0-445eb6459eb7","order_by":0,"name":"Zhineng Liu","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Zhineng","middleName":"","lastName":"Liu","suffix":""},{"id":246062809,"identity":"54e46896-f22c-4596-8e55-a98d47d51d59","order_by":1,"name":"Xinyun Qin","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Xinyun","middleName":"","lastName":"Qin","suffix":""},{"id":246062810,"identity":"1a240f3c-e3cf-44cb-9573-9c983174e38b","order_by":2,"name":"Keyi Nong","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Keyi","middleName":"","lastName":"Nong","suffix":""},{"id":246062811,"identity":"28108c18-f7dd-4d39-9171-ce27a512d12c","order_by":3,"name":"Xin Fang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Fang","suffix":""},{"id":246062812,"identity":"2ccbcd9e-16e1-4ee9-96ed-1c730f5c8b38","order_by":4,"name":"Bin Zhang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Zhang","suffix":""},{"id":246062813,"identity":"4996c0a9-3b52-4f77-9350-010a58a6fbff","order_by":5,"name":"Wanyan Chen","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Wanyan","middleName":"","lastName":"Chen","suffix":""},{"id":246062816,"identity":"95b1e3aa-07ee-47be-8af6-87fa32e38ea8","order_by":6,"name":"Zihan Wang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Zihan","middleName":"","lastName":"Wang","suffix":""},{"id":246062820,"identity":"ab4a64a2-b0ad-4dec-8d7d-64672b1d831e","order_by":7,"name":"Yijia Wu","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Yijia","middleName":"","lastName":"Wu","suffix":""},{"id":246062822,"identity":"fc7c933d-937f-47de-9849-17839938b88e","order_by":8,"name":"Huiyu Shi","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Huiyu","middleName":"","lastName":"Shi","suffix":""},{"id":246062823,"identity":"e595dc2e-a1c8-4422-b81a-680eb8459821","order_by":9,"name":"Xuemei Wang","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Xuemei","middleName":"","lastName":"Wang","suffix":""},{"id":246062828,"identity":"d43578ed-50bd-46d8-8887-f688050752a6","order_by":10,"name":"Youming Liu","email":"","orcid":"","institution":"Yibin Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Youming","middleName":"","lastName":"Liu","suffix":""},{"id":246062829,"identity":"533fd487-6513-4dd4-8ef1-19403e6641c8","order_by":11,"name":"Haiwen Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIie3PMUvEMBTA8YRAu7yS1YAf4oGDCEf7VVoCujj4ARwChedSnXtf5E63HIGbxK4HcTkE55YD4QbFKhzqYHQUzH95b3g/QhiLxf5giZC9eFuQcTOOCUhpwkSmBj+T433V2jBRjd2R99wETRkmuCr15uz8Pp+njvaG6w6QWd4Pp0FiD9rlo75pKlLTWw+Hwgg1nYVIZTQkTqOtyGfk4cjYRGRBopmDl5F0a/LPdAdoyzBRzZLXGbl8fI48J/szkWkjRHblSlytL7aXpEG1izr4l0SA2MCTK7A7ecAt5YWU9aIfAmRXZT52br67+lLxq6tYLBb7n70ChDhZ2dNTPbwAAAAASUVORK5CYII=","orcid":"","institution":"Hainan University","correspondingAuthor":true,"prefix":"","firstName":"Haiwen","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-11-02 03:44:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3540117/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3540117/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":46053395,"identity":"83aea02c-acb3-4db2-ab6c-f564ae5f9ec0","added_by":"auto","created_at":"2023-11-08 00:17:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1118782,"visible":true,"origin":"","legend":"\u003cp\u003e(a) MS analysis of Abaecin. (b) HPLC analysis of Abaecin. (c) Prediction of the tertiary structure of Abaecin and its sequence. (d) Analysis of the characteristic of the helical wheel of Abaecin. Yellow and green represent nonpolar amino acids, while other colors represent polar amino acids.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3540117/v1/a89a04739a2a9ac49578aa20.png"},{"id":46053400,"identity":"9e3d8f2c-e1be-4d0d-85c8-eb5a81bab6ec","added_by":"auto","created_at":"2023-11-08 00:17:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3491971,"visible":true,"origin":"","legend":"\u003cp\u003e(a)Overview of the experimental design. Effects of Abaecin on (b-c) mouse colon length, (d) mouse body weight, (e) DAI scores, and (f) spleen indices. Data represent means ± S.D. (n = 6). *p \u0026lt; 0.05 and **p \u0026lt; 0.01 vs Con group on the same day; #p \u0026lt; 0.05 and ##p\u0026lt;0.01 vs Mod group.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3540117/v1/4cdb26ba7a04e5d35932e9a9.png"},{"id":46053394,"identity":"5cd47b35-8b60-4405-a786-ca898ccb6948","added_by":"auto","created_at":"2023-11-08 00:17:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1832820,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Abaecin on serum concentrations of (a)LPS, (b)D-LA, (c)DAO, (d)IL-1β, (e)IL-6, (f)TNF-α, (g)IFN-γ, and (h)IL-10. All values present means ± S.D. (n = 6). *p \u0026lt; 0.05 and **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3540117/v1/759f8eaea064000900f0fea7.png"},{"id":46053397,"identity":"7c5ae355-a33c-45ed-b77e-ba95c399fc8c","added_by":"auto","created_at":"2023-11-08 00:17:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3966627,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Histological section examination of the colon in mice (100×). (b) Histological scoring. (c)Ration of the AB-PAS positive cell. (d)Cell apoptotic index. (e) Neutrophilic granulocyte invasion index. (f)Macrophage invasion index. All data present means ± S.D. (n = 6). *p \u0026lt; 0.05 and **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3540117/v1/15bf29e637fe2d7fdb33d261.png"},{"id":46053399,"identity":"3c804748-3199-4a43-84c7-39f9535ad202","added_by":"auto","created_at":"2023-11-08 00:17:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3759520,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Immunofluorescence staining of ZO-1 (20×) and (b) relative fluorescence expression intensity of ZO-1. (c) Western blot bands of three tight junction proteins. Determination of the levels of (d) occludin, (e) claudin-1, and (f) ZO-1 proteins in colonic tissues by Western blotting. Values are presented as means ± SD (n = 6). *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3540117/v1/a0085712fa0c8b9349a78f51.png"},{"id":46053401,"identity":"c58e0b33-091d-4409-9095-570a79cb618f","added_by":"auto","created_at":"2023-11-08 00:17:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1573141,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Western blot bands of NF-κB / MAPK signaling pathway protein. Relative expression of (b) p-p65 to p65, (c) p-ERK to ERK, (d) p-JNK to JNK, and (e) p-p38 to p38. Values are presented as means ± SD (n = 6). * p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3540117/v1/838a3946b91bff603889db8e.png"},{"id":46053830,"identity":"9b38513a-abb0-4721-8c40-fadc0e0fe98a","added_by":"auto","created_at":"2023-11-08 00:25:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1111601,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Petaline graphs. The middle circle represents the number of OTUs shared by the sample or group, and the ellipse outside the middle circle represents the number of OTUs unique to the sample or group. (b) OTU Rank Curve. The richness of the species in the sample is reflected in the length of the horizontal axis of the curve. The wider the curve, the richer the species composition in the sample. The uniformity of the species in the sample is reflected in the shape of the longitudinal axis of the curve. The flatter the curve is, the higher the uniformity of the species composition in the sample is. (c) Curves of species accumulation. The rising trend at the end of the curve tends to be gentle, indicating that the sample amount is sufficient. (d) PLS-DA plot. Points of different colors or shapes represent sample groups under different environments or conditions, and abscissa and ordinate represent the suspected influencing factors of differences in the microbial composition in each group. (e) Alpha diversity difference map between groups. (f) The Beta diversity box-plot. (g) A bar chart of species composition at the genus level. (H) Spearman correlation analysis heat map. Values are presented as means ± SD (n = 6). * p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-3540117/v1/37e2e5c1d07c57daf547491c.png"},{"id":46245337,"identity":"493868da-beb4-4e48-80f7-05ba8f119bfc","added_by":"auto","created_at":"2023-11-10 19:07:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2782405,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3540117/v1/86c1b035-4a7d-4db4-ad65-ad29d8b128db.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The antimicrobial peptide Abaecin alleviates colitis in mice by regulating inflammatory signaling pathways and intestinal microbial composition","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUlcerative colitis (UC) is an inflammatory process that is confined to the mucosa and submucosa of the colon. Patients with UC show a variety of clinical symptoms, including abdominal pain, blood in the stool, and weight loss. It is often recurrent and of unknown aetiology, and it is now generally accepted that it is influenced by a variety of factors (including genetic, environmental, immunological, physiological, psychological, and the gut microbiome) and their interactions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Due to the common adverse reactions of common drugs for the treatment of UC, the use of natural extracts with nontoxic side effects and good therapeutic effects as drugs for the treatment of UC is becoming a new trend [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntimicrobial peptide (AMP) is a kind of small molecular polypeptide that is naturally present in animals. Most antimicrobial peptides have broad-spectrum antimicrobial activity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, some antimicrobial peptides also have antiviral [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], anti-inflammatory [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], antitumor [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], immune regulation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and other biological activities. A large number of studies have shown that antimicrobial peptides can affect inflammation through a variety of channels. For example, some peptides not only attract neutrophils, but also block their apoptosis and enhance the phagocytic function of neutrophils [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. AMPs can also enhance the Th17 response. In short, AMPs effectively attract Th17, which mainly secretes pro-inflammatory cytokines IL-17A, IL-21F, IL-22 and IL-17, and is responsible for establishing mucosal defense against pathogenic microorganisms in the respiratory tract or intestine [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. On the other hand, AMPs can also act on host-microbiome interactions. In this case, AMPs can be used as a buffer to neutralize LPS and LTA released by the microbiota to maintain homeostasis and prevent environmental disorders [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, the immunomodulatory capacity of AMPs is mainly mediated by extensive binding to the membrane and interaction with intracellular receptors, thus stimulating downstream signaling pathways and producing the corresponding regulatory effects on inflammation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Studies have found that the promoter regions of α-and β-defensin genes contain binding sites for the major cellular transcription factors, especially nuclear factor κB (NF-κB). It is worth noting that NF-κB also plays a crucial role in the pathogenesis of inflammatory bowel disease (IBD).\u003c/p\u003e \u003cp\u003eAbaecin is a proline-rich natural antimicrobial peptide from bees. Together with Apidaecin, Hymenoptaecin, and Defensin, it is an important part of the innate humoral immunity of bees and has a broad spectrum of antibacterial ability [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Abaecin enters cells in an insoluble manner, interacts with bacterial cells, and hinders protein synthesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Interestingly, Abaecin actually has a weak transmembrane ability, so it needs to be combined with other pore-forming peptides to enhance the effect. For example, Abaecin has a good inhibitory effect on parasites in synergy with other insect antimicrobial peptides [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, the expression of Abaecin in honey bees is also closely related to gut bacteria, and disruption of the gut bacterial community due to excessive use of antibiotics causes a decrease in the expression of the gene encoding abaecin in honey bees [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, there are few reports on the anti-inflammatory effect and preliminary mechanism of Abaecin in bee antimicrobial peptides. In this study, the DSS-induced acute colitis model in mice was used to explore whether Abaecin has a mitigating effect on UC and to further explore the preliminary mechanism of Abaecin in alleviating inflammation to provide a theoretical basis for the development of specific drugs for the treatment of UC with Abaecin as the main material.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials and reagents\u003c/h2\u003e \u003cp\u003eAbaecin (purity 96%) was purchased from GL Biochem (Shanghai) Ltd. DSS (Dextran sulfate sodium; M.W., 36–50 kDa) was obtained from MP Biomedicals (Santa Ana, CA). Mouse tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), interleukin-1β (IL-1β), interleukin-10 (IL-10) and interferon-γ (IFN-γ) ELISA kits were provided by Nanjing Jiancheng Institute of Bioengineering (Nanjing, China). Lipopolysaccharides (LPS), D-lactate (D-LA), and diamine oxidase (DAO) activity ELISA kits were supplied from Shanghai Enzyme-linked Biotechnology Co. Anti-F4/80, CD177 antibody was purchased from Wuhan Servicebio Technology CO., Ltd. Anti-p65, p-p65, p38, p-p38, JNK, p-JNK, ERK, p-ERK, occludin, claudin-1, and ZO-1 antibodies were purchased from Bioss Antibodies Co., Ltd., Beijing, China. All other chemicals were of the highest grade available.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animal\u003c/h2\u003e \u003cp\u003eMale C57BL/6J mice (7–8 weeks old) were supplied by the Laboratory Animal Center of Guangzhou University of Chinese Medicine, Guangzhou, China. Mice were given one week to acclimate to the new environment before the start of the experiment (temperature of 25 ± 12°Cand 12hlight/dark cycle). The animal experiment was approved by the Animal Care and Use Committee of Hainan University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eTwenty-four mice were randomly divided into four groups (n = 6/group) after 1 week of acclimatization: control group (Con), colitis group (Mod), colitis plus Abaecin group (Ab + Mod) and Abaecin group (Ab). 2.5% DSS (w/v) was added to drinking water to induce acute ulcerative colitis for seven days. The Ab and Ab + Mod groups were then treated with rectal administration of Abaecin (5mg/kg), and the Con and Mod groups were injected rectally with equal amounts of PBS. The weight was recorded every day. The DAI scores were performed according to previous research criteria [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. On the eighth day of the experimental period, all mice were sacrificed. Collect serum, spleen. The colon was removed and measured. Colon samples were fixed in 10% formalin for hematoxylin and eosin (H \u0026amp; E) examination. Histological scoring was performed according to the criteria described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of inflammatory cytokine and oxidative stress indicators\u003c/h2\u003e \u003cp\u003eSerum samples were prepared by centrifugation after blood was collected and equilibrated at room temperature for 1 hour. The concentrations of IL-1 β, IL-6, TNF-α, IFN-γ, IL-10, DAO, D-LA and LPS in serum were determined by ELISA kits, which were operated according to the instructions of the kits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis index assay\u003c/h2\u003e \u003cp\u003eTunel staining was used to determine the apoptosis index. Endogenous peroxidase was blocked after antigen repair of the paraffin sections. After room temperature repair, Streptavidin-HRP reaction solution was added, DAB coloration, nucleus restaining, and finally dehydrated and mounted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological evaluation and immunohistochemical staining\u003c/h2\u003e \u003cp\u003eColon tissues collected were fixed in 4% paraformaldehyde overnight and then dehydrated and transparent in turn. The tissues were then embedded in paraffin sections. The number of goblet cells was assessed by Alcian Blue Periodic Acid Schiff (AB-PAS) staining. Subsequently, immunohistochemical staining was used to observe the degree of infiltration of inflammatory cells. Briefly, sections were incubated with anti-CD177 antibody and anti-F4/80 antibody at 4°C overnight, respectively. And then the sections were incubated with HRP-conjugated rabbit anti-goat IgG in a 1:100 ratio for 1 hour. Image J software was used to visualize the results of the sections.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was used for the analysis of tight junction proteins of the colon. Paraffin sections were dewaxed and antigen repair was performed with EDTA antigen repair solution. Sections were blocked with 3% BSA (Wuhan Servicebio Technology CO., Ltd.) and then incubated in anti-ZO-1 tight junction protein antibody (1:200, Wuhan Servicebio Technology CO., Ltd.) at 4°C overnight. The corresponding secondary antibody was added and incubated for 50 minutes under light avoidance conditions before adding the autofluorescence quencher B solution. Finally, the slides were sealed with an antifluorescence quenching sealer and the observed results were quantified by image J software.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern Blot analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe total protein in the colon was extracted using a total protein extraction kit (Solarbio, Beijing, China) according to the kit manufacturer’s instructions. The protein was separated on a SDS-PAGE gel and transferred to the PVDF membrane. After blocking the membrane with a blocking solution (Wuhan Servicebio Technology CO., Ltd.), the membrane was incubated with the corresponding primary antibody (p65, p-p65, p38, p-p38, JNK, p-JNK, ERK, p-ERK, occludin, claudin-1, ZO-1(Bioss, Beijing, China)) at 4°C overnight and then the membrane was placed in the HRP labeled secondary antibody (1:20,000, Bioss, Beijing, China). The protein band was observed under the action of the ECL hypersensitive luminescent liquid (Biosharp,Beijing). Finally, the gray value of the protein band was quantified by image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e16SrRNA Sequencing Analysis\u003c/h2\u003e \u003cp\u003eThe PCR reaction system was configured with 30 ng of acceptable quality genomic DNA samples samples and the corresponding fusion primers, and PCR amplification was carried out by setting the PCR reaction parameters, and the PCR amplification products were purified using Agencourt AMPure XP magnetic beads, dissolved in Elution Buffer, labeled, and then completed the construction of the library. The fragment range and concentration of the libraries were analyzed by Agilent 2100 Bioanalyzer. The library that passed the test was selected for sequencing on the HiSeq platform according to the size of the inserted fragments. Filter the data, the remaining high-quality Clean data for later analysis; through the overlap relationship between the reads to reads spliced into Tags; Tags clustered into OTUs and compared with the database, species annotations; based on the OTUs and annotated results of the sample species complexity analysis, intergroup species difference analysis, as well as correlation analysis and model prediction, and so on. Finally, in order to further explore the relationship between intestinal flora and inflammation-related indicators, we performed spearman analysis and created a cluster-related heat map of genera and indicators using the Omicstudio tool.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8 is used for data processing. IBM SPSS Statistics 27 was used for statistical analysis. One-way ANOVA and Tukey’s multiple comparison test were used for the difference analysis. All experiments were repeated at least three times and all results were expressed as mean ± standard deviation (SD). * p \u0026lt; 0.05, * * p \u0026lt; 0.01.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHistological scoring criteria\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInflammatory cells infiltration\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDepth of invasion\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCrypt injury\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePathological range (%)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003enone\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e±\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMucosa layer\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1/3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1–25\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMucosa and submucosa\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2/3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26–50\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFull\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFull\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51–75\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e76–100\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" type=\"Results\" class=\"Section2\"\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Result","content":"\u003ch2\u003ePrediction of Abaecin structure\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;1, we performed MS analysis and HPLC analysis of Abaecin, respectively, and found that the molecular weight and purity of Abaecin were 3878.94 kDa and 96.79%, respectively. On this basis, we predicted the tertiary structure of Abaecin by website \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zhanggroup.org/I-TASSER/\u003c/span\u003e\u003cspan address=\"https://zhanggroup.org/I-TASSER/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, and also predicted the helical wheel structure of Abaecin to determine hydrophilicity and hydrophobicity.\u003c/p\u003e\u003ch2\u003eEffects of Abaecin on inflammatory symptoms\u003c/h2\u003e\u003cp\u003eA large number of studies have shown that DSS-induced ulcerative colitis in mice can lead to significant weight loss, significantly reduced colon length, abnormal organ index, diarrhea, hematochezia, and other symptoms. These symptoms were also observed in this experiment. As shown in Fig.\u0026nbsp;2, rectal administration of Abaecin significantly increased colon length and body weight of mice (Fig.\u0026nbsp;2b-d, p \u0026lt; 0.01), and significantly reduced the DAI score and the spleen index of mice (Fig.\u0026nbsp;2e-f).\u003c/p\u003e\u003ch2\u003eEffect of Abaecin on serum concentrations of oxidative stress-related factors and inflammatory cytokines\u003c/h2\u003e\u003cp\u003eIn this study, we found that DSS-induced colitis caused a significant increase in serum concentrations of DAO, D-LA, LPS, IL-1β, IL-6, TNF-α, and IFN-γ(Fig.\u0026nbsp;3a-g, p \u0026lt; 0.01), as well as a significant decrease in the concentration of the inflammation suppressing cytokine, IL-10(Fig.\u0026nbsp;3h, p \u0026lt; 0.01).Intervention of Abaecin restored abnormally elevated serum concentrations of DAO, D-LA, LPS, IL-1β, IL-6, TNF-α, and IFN-γ concentrations, but could not restore IL-10 concentrations(p \u0026gt; 0.05).\u003c/p\u003e\u003ch2\u003eEffect of Abaecin on histological injury and inflammatory cell infiltration\u003c/h2\u003e\u003cp\u003eHE staining was used to visualise histological damage to colon tissue and the Mod group had altered colon morphology with a large infiltration of inflammatory cells (p \u0026lt; 0.01), which was significantly ameliorated by Abaecin administration (p \u0026lt; 0.01). In addition, colitis usually causes a decrease in the number of goblet cells (p \u0026lt; 0.01), an increase in the apoptotic index (p \u0026lt; 0.01), and massive infiltration of inflammatory cells (p \u0026lt; 0.01), all of which were significantly ameliorated by rectal administration of Abaecin.\u003c/p\u003e\u003ch2\u003eEffect of Abaecin on the expression of tight junction proteins\u003c/h2\u003e\u003cp\u003eImmunofluorescence staining was used to determine the expression of the tight junction protein ZO-1 in the colon (Fig.\u0026nbsp;5a). In this study, we found that DSS-induced ulcerative colitis significantly reduced the expression of ZO-1, which was significantly restored by Abaecin treatment (Fig.\u0026nbsp;5b, p \u0026lt; 0.01). Similarly, we found that Abaecin treatment significantly restored the reduced expression of the tight junction proteins occludin, claudin-1, and ZO-1 induced by colitis in mice by Western Blot analysis (Fig.\u0026nbsp;5c-e, p \u0026lt; 0.01).\u003c/p\u003e\u003ch2\u003eEffect of Abaecin on the NF-κB/MAPK Signaling Pathway\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;6, western blot analysis showed that NF-κB signaling pathway and the MAPK signaling pathway were activated under the influence of colitis. Relative expression levels of p-p65, p-ERK, p-JNK, and p-p38 were significantly increased (p \u0026lt; 0.01). However, Abaecin treatment significantly reversed these changes(p \u0026lt; 0.01).\u003c/p\u003e\u003ch2\u003eEffect of Abaecin on the composition of intestinal microorganisms\u003c/h2\u003e\u003cp\u003e16SrRNA analysis was used to determine changes in intestinal microbial composition. We obtained petal plots by comparing OTUs between samples or between subgroups (Fig.\u0026nbsp;7a). The OTU rank curves and the species accumulation curves adequately indicated that the number of samples was sufficient for the prediction of species richness (Fig.\u0026nbsp;7b-c). The coverage indices for all taxa were close to 1, indicating that the sequencing data covered about 100% of the well-covered microorganisms. In the Partial Least Squares Discriminant Analysis (PLS-DA) plot (Fig.\u0026nbsp;7d), the Mod group and the Ab + Mod group are separated in the direction of the vertical axis, suggesting that Abaecin, represented by the vertical coordinate, is the main factor responsible for the separation of the two groups. In the Alpha diversity box plot (Fig.\u0026nbsp;7e), the Chao1 Alpha diversity value was significantly lower in the Mod group compared to the Con group (p \u0026lt; 0.05). And a significant difference was also found in beta diversity between the four groups in the Beta group difference box plot (Fig.\u0026nbsp;7f, p \u0026lt; 0.05). In order to explore changes in the composition of the intestinal flora, a bar chart of the species composition was made at the genus level. It can be seen in Fig.\u0026nbsp;7g that DSS-induced colitis caused an abnormal increase in the abundance of \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eBarnesiella\u003c/em\u003e and \u003cem\u003eEscherichia\u003c/em\u003e, and the administration of Abaecin inhibited this increase. In addition, colitis in mice also led to a decrease in the abundance of \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eDesulfovibrio\u003c/em\u003e, while Abaecin restored the abundance of these bacteria.\u003c/p\u003e\u003cp\u003eFurthermore, we explored the correlation between intestinal flora composition and indicators related to inflammation using spearman analysis. As shown in Fig.\u0026nbsp;7h, \u003cem\u003eBacteroides\u003c/em\u003e and \u003cem\u003eEscherichia\u003c/em\u003e were significantly positively correlated with IL-1β (p \u0026lt; 0.01), IL-6 (p \u0026lt; 0.01), TNF-α (p \u0026lt; 0.01),IFN-γ (p \u0026lt; 0.01), D-LA (p \u0026lt; 0.01), LPS (p \u0026lt; 0.01), DAO, (p \u0026lt; 0.01),DAI (p \u0026lt; 0.01), Neutrophilic granulocyte invasion index (p \u0026lt; 0.01),Macrophage invasion index (p \u0026lt; 0.01), Cell apoptotic index (p \u0026lt; 0.01), Spleen index (p \u0026lt; 0.01) and Histological score (p \u0026lt; 0.01) and were significantly negatively correlated with IL-10(p \u0026lt; 0.01),colon length(p \u0026lt; 0.01) and Body weight(p \u0026lt; 0.01). On the contrast, \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eDesulfovibrio\u003c/em\u003e were significantly negatively correlated with IL-1β (p \u0026lt; 0.05,p \u0026lt; 0.01,respectively), IL-6 (p \u0026lt; 0.01), TNF-α (p \u0026lt; 0.01),IFN-γ(p \u0026lt; 0.01), D-LA (p \u0026gt; 0.05,P \u0026lt; 0.05,respectively), LPS (p \u0026lt; 0.01), DAO (p \u0026lt; 0.05,p \u0026lt; 0.01,respectively),DAI (p \u0026lt; 0.01), Neutrophilic granulocyte invasion index (p \u0026lt; 0.01),Macrophage invasion index (p \u0026lt; 0.01), Cell apoptotic index (p \u0026lt; 0.01), Spleen index (p \u0026lt; 0.01) and Histological score (p \u0026lt; 0.01) and were significantly positively correlated with IL-10(p \u0026lt; 0.01),colon length(p \u0026lt; 0.01) and Body weight(p \u0026lt; 0.01).\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;1 (a) MS analysis of Abaecin. (b) HPLC analysis of Abaecin. (c) Prediction of the tertiary structure of Abaecin and its sequence. (d) Analysis of the characteristic of the helical wheel of Abaecin. Yellow and green represent nonpolar amino acids, while other colors represent polar amino acids.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;2 (a)Overview of the experimental design. Effects of Abaecin on (b-c) mouse colon length, (d) mouse body weight, (e) DAI scores, and (f) spleen indices. Data represent means ± S.D. (n = 6). *p \u0026lt; 0.05 and **p \u0026lt; 0.01 vs Con group on the same day; #p \u0026lt; 0.05 and ##p \u0026lt; 0.01 vs Mod group.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;3 Effect of Abaecin on serum concentrations of (a)LPS, (b)D-LA, (c)DAO, (d)IL-1β, (e)IL-6, (f)TNF-α, (g)IFN-γ, and (h)IL-10. All values present means ± S.D. (n = 6). *p \u0026lt; 0.05 and **p \u0026lt; 0.01.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;4 (a) Histological section examination of the colon in mice (100×). (b) Histological scoring. (c)Ration of the AB-PAS positive cell. (d)Cell apoptotic index. (e) Neutrophilic granulocyte invasion index. (f)Macrophage invasion index. All data present means ± S.D. (n = 6). *p \u0026lt; 0.05 and **p \u0026lt; 0.01.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;5 (a) Immunofluorescence staining of ZO-1 (20×) and (b) relative fluorescence expression intensity of ZO-1. (c) Western blot bands of three tight junction proteins. Determination of the levels of (d) occludin, (e) claudin-1, and (f) ZO-1 proteins in colonic tissues by Western blotting. Values are presented as means ± SD (n = 6). *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;6 (a) Western blot bands of NF-κB / MAPK signaling pathway protein. Relative expression of (b) p-p65 to p65, (c) p-ERK to ERK, (d) p-JNK to JNK, and (e) p-p38 to p38. Values are presented as means ± SD (n = 6). * p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;7(a) Petaline graphs. The middle circle represents the number of OTUs shared by the sample or group, and the ellipse outside the middle circle represents the number of OTUs unique to the sample or group. (b) OTU Rank Curve. The richness of the species in the sample is reflected in the length of the horizontal axis of the curve. The wider the curve, the richer the species composition in the sample. The uniformity of the species in the sample is reflected in the shape of the longitudinal axis of the curve. The flatter the curve is, the higher the uniformity of the species composition in the sample is. (c) Curves of species accumulation. The rising trend at the end of the curve tends to be gentle, indicating that the sample amount is sufficient. (d) PLS-DA plot. Points of different colors or shapes represent sample groups under different environments or conditions, and abscissa and ordinate represent the suspected influencing factors of differences in the microbial composition in each group. (e) Alpha diversity difference map between groups. (f) The Beta diversity box-plot. (g) A bar chart of species composition at the genus level. (H) Spearman correlation analysis heat map. Values are presented as means ± SD (n = 6). * p \u0026lt; 0.05, ** p \u0026lt; 0.01.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAbaecin is a proline-rich antimicrobial peptide produced by bees when they are infected by microorganisms or other exogenous substances, and has broad-spectrum antibacterial activity against Gram-negative bacteria [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Antimicrobial peptides are classified into lytic antimicrobial peptides and nonlytic antimicrobial peptides [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The former kills bacteria by destroying bacterial cell membranes and causing cell lysis. Abaecin belongs to the latter, which prevents bacterial growth by acting on the bacterial plasma membrane and on intracellular targets. It is worth noting that many proline-rich antimicrobial peptides (PrAMPs) act on ribosomes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In our study, predicting the tertiary structure of Abaecin, we found that Abaecin showed an obvious randon coil. And the prediction of the helix structure found that the hydrophobic and hydrophilic residues were distributed symmetrically on both sides, indicating that Abaecin may exhibit excellent amphiphilic properties, this structure is considered related to the antibacterial activity of Abaecin [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA large number of studies have shown that DSS-induced colitis mouse models can lead to a significant decrease in colon length and body weight, a significant increase in disease activity index (DAI) and severe damage to intestinal structure [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This is consistent with the results observed in this study. Fortunately, rectal injection of Abaecin significantly increased colon length and body weight and decreased disease activity index (DAI) in mice. At the same time, through histological observation, we found that Abaecin significantly reduced infiltration of inflammatory cells, significantly increased the number of goblet cells, and alleviated apoptosis of intestinal cells. These results suggest that Abaecin can alleviate tissue damage caused by ulcerative colitis and protect intestinal health.\u003c/p\u003e \u003cp\u003eThe intestinal epithelial barrier is an important line of defense against pathogenic microorganisms and toxins in the intestinal lumen [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Tight junction (TJ) protein is an essential factor for a complete intestinal barrier. Once the tight junction protein is reduced or destroyed, it will lead to infiltration of toxic intestinal substances into the intestinal lamina propria and further aggravate the inflammatory response [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A number of reports have shown that the number of TJ in the intestinal tract of IBD patients is reduced, resulting in defects in intestinal barrier function [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Thus, TJ may be a key factor in the treatment of ulcerative colitis. In this study, immunofluorescence staining and Western blot analysis were used to determine the expression of TJ. The results showed that the expression levels of ZO-1, claudin-1 and occludin in colitis mice were increased significantly after Abaecin administration. In addition, we also found that the concentrations of D-LA, DAO, and LPS in the serum of mice with DSS-induced ulcerative colitis increased significantly, and the intervention of Abaecin significantly reduced the concentrations of these three substances. This suggests that Abaecin can protect the integrity of the intestinal barrier by regulating the expression of TJ in mice, preventing toxic substances from entering the body, and thus alleviating ulcerative colitis.\u003c/p\u003e \u003cp\u003eBoth NF-κB and MAPK are the main signal messengers that regulate the transcription of pro-inflammatory genes during inflammation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].NF-κB protein is usually composed of p65 and p50 to form a homologous / heterologous dimer, which binds to the inhibitor protein IkB to form a trimer complex and is in an inactive state [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. When the upstream signal factor is activated, it will lead to the phosphorylation of IκB protein and the release of the complex p50 / p65, which enters the nucleus and rapidly activates the transcription of various cytokines and chemokines [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In the current results, the concentrations of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IFN-γ in the serum of DSS-induced colitis mice increased significantly, and rectal administration of Abaecin significantly reduced the concentration of these pro-inflammatory cytokines. IL-10 is an anti-inflammatory cytokine, and its concentration in mouse serum is reduced under the DSS treatment. However, Abaecin did not significantly improve the reduction of IL-10 concentration. To further investigate the mechanism associated with Abaecin alleviation in mice, we measured the expression of two signaling pathway proteins, NF-κB and MAPK, and found that the phosphorylation levels of P38, JNK, ERK1/2, and p65 increased significantly in the mouse model with DSS-induced colitis, while Abaecin decreased significantly the levels of phosphorylation of these proteins. It has been reported that p38, JNK and ERK1/2 are crucial regulators of inflammation, autoimmune, apoptosis, cell proliferation and differentiation, and are closely related to the occurrence and development of inflammation [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This indicates that Abaecin can alleviate DSS-induced colon inflammation and reduce the secretion of pro-inflammatory cytokines by regulating the expression of NF-κB / MAPK signaling pathway.\u003c/p\u003e \u003cp\u003eThe intestinal immune system maintains a state of tolerance to the diversity and beneficial symbiotic intestinal microorganisms while responding to pathogenic microorganisms, invasive pathogens, and microbial products. These two states achieve balance and maintain intestinal health [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Changing the intestinal microbial community will break this immune balance, leading to disease infection or immune disorders [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The results of 16SrRNA analysis showed that the abundance of \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eBarnesiella\u003c/em\u003e and \u003cem\u003eEscherichia\u003c/em\u003e in the intestine increased abnormally. \u003cem\u003eBacteroides\u003c/em\u003e are usually \u0026ldquo;friendly\u0026rdquo; symbionts in the gut, but they tend to become opportunistic pathogens when they are hosted elsewhere [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It is reported that Bacteroides Bfr is the main promoter and promoter of human colorectal cancer [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. \u003cem\u003eBarnesiella\u003c/em\u003e and \u003cem\u003eEscherichia\u003c/em\u003e are Gram-negative bacteria. The main component of their outer membrane is LPS, which has strong pro-inflammatory activity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Therefore, the excessive increase in the abundance of these two bacteria is closely related to the aggravation of intestinal inflammation. Interestingly, the results of the spearman correlation analysis also showed that \u003cem\u003eBacteroides\u003c/em\u003e, \u003cem\u003eBarnesiella\u003c/em\u003e, and \u003cem\u003eEscherichia\u003c/em\u003e were positively correlated with histological damage, pro-inflammatory cytokines, and indicators related to intestinal permeability, which means that the high abundance of these three genera is related to colon tissue damage, increased inflammation, intestinal barrier damage, and weakening of antioxidant status. Administration of Abaecin inhibited the abnormal increase in the abundance of these three genera. On the contrary, the abundance of \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eDesulfovibrio\u003c/em\u003e in the intestine of colitis mice decreased significantly. \u003cem\u003eLactobacillus\u003c/em\u003e has been reported to exhibit enzymatic activity and, by producing bile salt hydrolases, Lactobacillus plays a crucial role in bile acid metabolism [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Furthermore, studies have shown that Lactobacillus and other probiotic species can effectively treat and alleviate Crohn's disease and ulcerative colitis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The results of the Spearman correlation analysis also showed that Lactobacillus and Desulfovibrio were positively correlated with body weight, colon length and anti-inflammatory cytokines in mice, and Abaecin restored the abundance of these bacteria. Therefore, the results of this study indicate that Abaecin can maintain intestinal health by regulating the composition of intestinal flora.\u003c/p\u003e \u003cp\u003eIn summary, ulcerative colitis can cause infiltration of inflammatory cells, structural intestinal damage, and imbalance of intestinal flora. Unbalance intestinal flora will lead to aggravation of the inflammatory reaction and will form a vicious circle. In this study, Abaecin alleviated these injuries, balanced the intestinal flora, and prevented a vicious cycle.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, Abaecin can alleviate tissue damage caused by colitis, improve intestinal barrier damage, and excessive secretion of pro-inflammatory cytokines by regulating inflammatory signaling pathways and improving intestinal flora composition. Therefore, Abaecin can be used as a potential natural substance for the development of specific drugs for ulcerative colitis.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Hainan Provincial Natural Science Foundation High-Level Talents Project (320RC463), the National Natural Science Foundation of China (32260857), and the Innovation and entrepreneurship training project of college students (202310589071).\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTavakoli P, Vollmer-Conna U, Hadzi-Pavlovic D, Grimm MC. A Review of Inflammatory Bowel Disease: A Model of Microbial, Immune and Neuropsychological Integration. 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Am J Gastroenterol. 2005;100(7):1539-46. https://doi: 10.1111/j.1572-0241.2005.41794.x.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Abaecin, Ulcerative colitis, NF-κB, MAPK, Intestinal microorganisms","lastPublishedDoi":"10.21203/rs.3.rs-3540117/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3540117/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe purpose of this study was to determine the effect of Abaecin on dextran sulfate sodium (DSS) -induced ulcerative colitis in mice and to explore its related mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eTwenty-four mice with similar body weight were randomly divided into 4 groups. 2.5% DSS was added to drinking water to induce colitis in mice. Mice were executed after Abaecin administration treatment, and mouse serum and tissues were collected. We measured the concentration of serum inflammatory cytokines in mice and produced colon tissue sections to observe the damage to the colonic structure. Then, we assessed the integrity of the intestinal barrier by the expression of intestinal tight junction proteins. In addition, we determined the phosphorylation levels of NF-κb/MAPK inflammatory signaling pathway proteins and the microbial composition of the intestinal flora to preliminarily investigate the alleviation mechanism of ulcerative colitis by Abaecin.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results showed that Abaecin significantly alleviated histological damage and intestinal mucosal barrier damage caused by colitis, reduced the concentration of pro-inflammatory cytokines and the phosphorylation of NF-κB / MAPK inflammatory signaling pathway proteins, and improved the composition of intestinal microorganisms.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings suggest that Abaecin may have potential prospects for the treatment of ulcerative colitis.\u003c/p\u003e","manuscriptTitle":"The antimicrobial peptide Abaecin alleviates colitis in mice by regulating inflammatory signaling pathways and intestinal microbial composition","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-11-08 00:16:57","doi":"10.21203/rs.3.rs-3540117/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"23670da0-6e5b-4e99-9a8c-d6839f1e1a5b","owner":[],"postedDate":"November 8th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-10T18:59:16+00:00","versionOfRecord":[],"versionCreatedAt":"2023-11-08 00:16:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3540117","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3540117","identity":"rs-3540117","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}