Skip to main content
Download PDF

Cecropin AD ameliorates pneumonia and intestinal injury in mice with mycoplasma pneumoniae by mediating gut microbiota

BMC Veterinary Research volume 21, Article number: 39 (2025) Cite this article

Abstract

Animals infected with mycoplasma pneumoniae not only develop respiratory diseases, but also cause digestive diseases through the lung-gut axis mediated by the intestinal flora, and vice versa. Antimicrobial peptides are characterized by their bactericidal, anti-inflammatory, and intestinal flora-regulating properties. However, the effect of cecropin AD (CAD) against mycoplasma pneumonia remains unclear. To investigate the anti-inflammatory effect of CAD on mycoplasma pneumonia and the associated mechanism, mice were infected with Mycoplasma capricolum subsp. Capripneumoniae(Mccp) to elicit lung inflammation, followed by oral administration of CAD via gavage. The findings showed that mice receiving twice injections of 2.08 × 108 copies of Mccp suffered significant pathological damage to their lungs and colons. Additionally, there was a notable upsurge in inflammatory factors within the affected tissues. 16 S rDNA sequencing revealed alterations in the colonic microbiota, including a decrease in the abundance of beneficial bacteria such as Corynebacterium_glutamicum and Candidatus_Saccharimonas, and an increase in the abundance of potential pathogens like Lachnospiraceae_NK4A136_group and Escherichia-Shigella. As a result, there were abnormal rises in lipopolysaccharide (LPS) levels in both colonic content and blood. Moreover, CAD treatment reversed the microbial dysbiosis and decreased the LPS levels induced by Mccp, thereby suppressing the activation of the TLR-4/NF-κB pathway and the Fas/FasL-caspase-8/-3 pathway. Consequently, this significantly mitigated the morphological and functional damage to the lungs and colons caused by Mccp. The findings offer novel insights and approaches for the clinical management of Mccp infections.

Peer Review reports

Introduction

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is a prevalent pathogen responsible for respiratory diseases in goats, leading to high morbidity and mortality across all age groups of infected herds [1]. These results in substantial economic losses for goat farmers globally. While drug treatment is the primary response to the disease, the frequent emergence of drug-resistant Mccp strains has significantly diminished the efficacy of antibiotic therapy. Furthermore, the vaccines developed to date are typically specific to a single, local epidemic strain, and their protective effects are less than optimal. Consequently, there is an urgent need to develop innovative treatment strategies to alleviate the array of clinical symptoms caused by Mccp.

Billions of bacteria reside in the intestines of animals, forming the largest microbial ecosystem in the body [2]. Therefore, healthy intestinal flora largely determines the overall health of the host. Recent studies have shown that intestinal flora and its metabolites are closely related to the occurrence of multi-organ diseases [3,4,5,6]. For example, LPS, a metabolite of the intestinal flora, can stimulate the development of multi-organ diseases through the inflammatory response of nuclear factor kb (NF-kb) [7, 8]. LPS participates in the inflammatory response by activating Toll-like receptors (TLRs), initiating the signal transduction process of NF-kB, promoting the secretion of downstream inflammatory factors such as IL-17, IL-6, IL-1β and TNF-α. TNF-α is a key factor in initiating apoptosis. It can activate the Fas/FasL-caspase8/3 pathway, induce apoptosis of epithelial cells, and aggravate the degree of damage [9]. Therefore, therapeutic strategies aimed at improving the inflammatory state by modulating the intestinal flora may be a new approach to treating mycoplasma pneumoniae.

Antimicrobial peptides (Amps) not only possess broad-spectrum antimicrobial activity but also play important roles in immune functions, such as anti-apoptosis function and anti-inflammatory activity [10, 11]. Additionally, some studies have found that Amps can treat diseases by regulating intestinal flora. For example, cecropin A can treat DSS-induced inflammatory bowel disease by regulating the intestinal flora of C57BL/6 mice, reducing the abundance of Bacteroidaceae and Enterobacteriaceae, and increasing the abundance of Lactobacillus, Desulfovibrionaceae and Ruminococcaceae [12]. Defensin 5 alpha can increase the abundance of Butyrivibrio Fibrisolvens and Fermicus in G93A mice, thereby alleviating the clinical symptoms of amyotrophic lateral sclerosis [13]. Lysozyme can regulate the degree of lung injury by reducing the abundance of Staphylococcus and increasing the abundance of Corynebacterium and Erwinia in the intestinal flora [14]. AMPs as a promising new alternative to antibiotics, are set to play a significant role in veterinary medicine and livestock industry. They not only possess powerful microbiome-regulating functions, but also have advantages in inhibiting inflammatory responses and promoting overall animal health [15]. Currently, AMPs are widely used as feed additives in the management of several economically important animals, enhancing livestock growth rates and feed efficiency, thus increasing the economic benefits of the animal husbandry industry [16, 17]. Cecropin AD (CAD) is one of the cecropin Amps and is a hybrid peptide that integrates the important functions of cecropin A and cecropin D. Due to its strong antimicrobial activity, immunomodulatory function, and anti-tumor activity without cytotoxicity [18], it is expected to become a new research trend in the future to replace antibiotics in the treatment of diseases. However, the function of CAD in regulating the gut microbiota to alleviate lung inflammation in animals is still unclear.

Additionally, observations on pastures revealed that animals with mycoplasma pneumonia often developed secondary digestive system disease. And intestinal diseases can also cause lung inflammation [19,20,21]. To explore the mechanism by which CAD alleviates Mccp-induced lung inflammation, we used a mouse pneumonia model. Through this study, we aim to provide important data on the anti-mycoplasma pneumonia effect of CAD and offer new ideas and methods for the clinical treatment of Mccp infections. This research may be crucial in addressing the challenges that Mccp poses to the livestock industry.

Materials and methods

Activation and preparation of Mccp

Mycoplasma capricolum subsp. Capripneumoniae was activated by inoculating 500 µL of the suspension into sterile mycoplasma agar medium. After incubating for 7 days under controlled conditions of 5% CO2 and 37 °C, individual clone colonies were picked and transferred into mycoplasma broth medium. The bacterial suspension was then harvested once the color of medium transitioned from red to light orange.

Grouping and drug administration

Six-week-old female BALB/c mice (SPF, N = 30) were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. and placed in an environmentally controlled room free of specific pathogens. They were reared under a light cycle of 8:00–20:00 and a dark cycle of 20:00–8:00, with free access to water and food. After 14 days of acclimatization, the mice were randomly divided into 6 groups (N = 5 per group): control group (Control), model group (Model), low-dose CAD treatment group (CADL), high-dose CAD treatment group (CADH), bacterial depletion group (Baterial depletion) and tylosin treatment group (Tylosin).

The Control group received twice intranasal injections of 20 µL PBS, 12 h apart, followed by daily gavage of 0.4 mL PBS 24 h later. The Model group received twice intranasal injections of 2.08 × 108 copies of Mccp, 12 h apart, followed by daily gavage of 0.4 mL PBS 24 h later. The CADL group received twice intranasal injections of 2.08 × 108copies of Mccp, 12 h apart, followed by intragastric administration of 0.4 mL CAD solution (gift from Viteling Antibiotic-Free Breeding Technology Co., Ltd.) at 100 mg/kg/day, 24 h later. The CADH group received twice intranasal injections of 2.08 × 108copies of Mccp, 12 h apart, followed by intragastric administration of 0.4 mL CAD solution at 200 mg/kg/day, 24 h later. The Bacterial depletion group was first given drinking water containing broad-spectrum antibiotics (ampicillin 1 g/L; neomycin sulfate 1 g/L; metronidazole 1 g/L and vancomycin 0.5 g/L) for three weeks. Two days after stopping the antibiotic water, the mice received twice intranasal injections of 2.08 × 108copies of Mccp, 12 h apart, followed by intragastric administration of 0.4 mL CAD solution at 200 mg/kg/day, 24 h later. The Tylosin group received twice intranasal injections of 2.08 × 108copies of Mccp, 12 h apart, followed by intragastric administration of 0.4 mL tylosin solution at 100 mg/kg/day, 24 h later. After 72 h, all mice were euthanized. Serum and colon contents were stored at −80 °C, and some lung and colon tissues were placed in tissue fixative and some were stored at −80 °C for subsequent analysis.

Disease Activity Index (DAI)

During Mccp infection and treatment, the mice’s weight, food and water intake, activity level, mental state (including behaviors such as curling up, shivering, piloerection, respiratory abnormalities, etc.), and fecal characteristics were recorded every 8 h. Clinical scores were calculated based on average scores for weight change, mental status, and fecal condition. Table 1 outlines the clinical scoring techniques utilized in this research.

Table 1 Calculated disease activity index score [19]
Full size table

Mycoplasma load quantification

Mycoplasma capricolum subsp. Capripneumoniae DNA was quantified using the absolute quantitative method with primers targeting the conserved gene arcD of Mccp (F: 5’-GAACTGAAGAAGGTATGGCTGAAGA-3’; R: 5’-CCTGTTCCAGCACCAACTAAAAC-3’). First, the concentration of the plasmid containing the arcD gene was determined, and calculate its copy number according to the formula: copies/µL=(plasmid DNA concentration ng/µL×10−9)×(6.02 × 1023)/(DNA length×660). Plasmids with known concentrations and copy numbers were serially diluted using Easy dilution (Takara, Dalian, China) and used as standards for real-time PCR performed on a CFX96 qPCR system (Bio-Rad, Hercules, CA, USA) with SYBR Green Premix Pro Taq HS qPCR Kit (ACCURATE BIOTECHNOLOGY, Hunan, China). A standard curve was constructed with the logarithm of the copy number as the horizontal axis and the Ct value as the vertical axis. Subsequently, the initial weight of the samples was unified, followed by addition of 1 mL of PBS for homogenization. After homogenization, the samples were centrifuged at 2000 rpm for 10 min, and the supernatant was collected. Genomic DNA from the lung tissue homogenate supernatant was extracted using a bacterial genomic DNA extraction kit (TIANGEN, Beijing, China). The initial DNA concentration of each sample was unified for Mccp real-time PCR detection.

H.E. Staining and immunohistochemistry

To evaluate tissue damage in the colons and lungs, H.E. staining was conducted. Colon and lung tissues were harvested after euthanizing the mouse and washed with PBS. Tissues were fixed in 4% par-aformaldehyde for 48 h, embedded in paraffin, and sliced into 5 μm sections. These sections were then dewaxed and rehydrated by immersing them in xylene and gradient ethanol, and subsequently stained with the H.E. Stain Kit (Solarbio, Beijing, China). Finally, the sections were sealed with neutral gum, and the tissue damage was observed under an optical microscope (Nikon, Tokyo, Japan). Histological scores were obtained by evaluating the degree of tissue damage in the specimens. For lung tissues, the main observations were alveolar integrity, alveolar hemorrhage or congestion, alveolar wall thickness, and neutrophil infiltration or accumulation in alveoli or vascular walls, as described previously [22]. For colon tissues, the main observations were goblet cell loss, crypt changes, mucosal structural destruction, and inflammatory cell infiltration, as described previously [23].

Paraffin sections of the colon prepared as described above were baked at 65 °C for 2 h, then deparaffinized in xylene, rehydrated in graded ethanol solutions, and rinsed with running water. Antigen retrieval was performed in sodium citrate buffer (pH = 6.0), followed by UltraSensitiveTM SP (mouse/rabbit) IHC Kit (Maixin-Biotech, Fuzhou, China) to block endogenous peroxidase and non-specific staining. Sections were incubated with primary antibodies (ZO-1, Claudin-1, Occludin) overnight at 4 °C, followed by incubation with biotin-labeled IgG polymer at 37 °C for 10 min, and subsequently with streptavidin-peroxidase for 10 min. Finally, the sections were incubated with 3, 3ʹ-diaminobenzidine (DAB) for 5 min at room temperature, rinsed with running water, and counterstained with Mayer’s Hematoxylin Stain Solution, For IHC (Beijing Solarbio Science & Technology Co., Ltd. Beijing, China). The stained sections were evaluated under a microscope (Nikon, Tokyo, Japan) following chromogenic staining with DAB solution (ZSGB-BIO, Beijing, China).

Quantitative reverse tanscription PCR

Total RNA was extracted using TRIzol®reagent (Accurate Biology, Hunan, China). RNA quality was verified using NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA), and samples with a 260 nm and 280 nm absorbance ratio between 1.8 and 2.0 were considered as pure. Subsequently, 1 µg of RNA was subjected to genomic DNA elimination reaction, then the RNA was reverse transcribed into cDNA using the Evo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biology, Changsha, China). The primer sets used for amplifying mRNA are listed in Table 2. Real-time PCR PCR was performed using SYBR Green Premix Pro Taq HS qPCR Kit (ACCURATE BIOTECHNOLOGY, Hunan, China) on a CFX96 qPCR system (Bio-Rad, Hercules, CA, USA). The \(\:{2}^{-{\Delta\:}{\Delta\:}\text{C}\text{t}}\) method was used to compare gene expression levels.

Table 2 Primer sequence used for mRNA RT-qPCR
Full size table

16 S rDNA high throughput sequencing

In this study, colon content samples were collected and transferred to sterile centrifuge tubes. To investigate microbial diversity, 16 S rDNA sequencing was performed on the colon content samples using a high-throughput sequencing platform. The CTAB method was employed to extract the total DNA from the samples. Following quantification and quality assessment of the extracted DNA, the V3-V4 region of the 16 S rDNA gene was amplified using universal primers (341 F: 5’-CCTACGGGNGGCWGCAG-3’; 805R: 5’-GACTACHVGGGTATCTAATCC-3’). After purification of the amplified product, quantitative sequencing was conducted. The paired-end sequencing data were split, and after removing the adapter and barcode sequences, the data were merged ​​and filtered. Denoising was performed using DADA2, and alpha diversity within groups and beta diversity between groups were analyzed.

Enzyme-linked immunosorbent assay

In order to detect LPS in colon and serum samples. PBS was added to the collected colon contents (colon contents mass: PBS volume = 1:9). The contents were homogenized by ultrasound and centrifuged at 4000 rpm for 15 min. The supernatant was aspirated to detect the LPS content in the colon contents. Blood was collected from the eyes of euthanized mice, kept at 4 °C for 30 min, and centrifuged at 3000 rpm for 10 min. Serum samples were collected to detect LPS content. Mouse LPS ELISA KIT was used for detection according to the manufacturer’s instructions (mlbio, Shanghai, China).

Western blot

Tissue protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) by electroblotting. To block the membrane, place the membrane in skim milk (BD Life Sciences, Bridgeport, NJ, USA) dissolved in TBST (50 mmol/L of Tris, 0.1% Tween20, 150 mmol/L of NaCl, and pH 7.6), after blocking for 2 h at 25 °C. After blocking, the membranes were incubated with the primary antibody at 4 °C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody at 25 °C for 3 h. The primary antibodies used were as follows: Occludin (Proteintech, 27260-1-AP, Wuhan, China), Claudin-1 (Santacruz, 3523011327, CA, USA), ZO-1 (Proteintech, 21773-1-AP, Wuhan, China), TLR-4 ( Proteintech, 66350-1-IG, Wuhan, China), Myd88 ( Proteintech, 23230-1-AP, Wuhan, China), NF-κB p65 (Proteintech, 10745-1-AP, Wuhan, China), Phospho-NF-κB p65 (Proteintech, 82335-1-RR, Wuhan, China), IkB (Proteintech, 10268-1-AP, Wuhan, China), Phospho-IkB (Proteintech, 82349-1-RR, Wuhan, China), Caspase8 (Santacruz,,#5800803, CA, USA), Caspase3 (Affinity, 00125840, Jiangsu, China), FADD (Affinity, #237743, Jiangsu, China), Cyt-c(Affinity, #73C2522, Jiangsu, China), BAX (Proteintech, 50599-2-Ig, Wuhan, China), Bcl-2 (Proteintech, 82469-6-RR, Wuhan, China), β-actin (Santacruz, #E2422, CA, USA). The secondary antibodies used were as follows: HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, SA00013-1, Wuhan, China)、 HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00013-2, Wuhan, China). Finally, the protein bands were visualized using the enhanced chemiluminescence detection system imager (ImageQuant800, Cytiva, UK) and enhanced chemiluminescence solution (DiNing, Beijing, China). The relative intensity of each band was evaluated using ImageJ software.

Ethics approval and consent to participate

The Experimental Animal Care and Ethical Review Executive Committee (IACUC) of Northwest A&F University reviewed and approved all experiments (Project Number: IACUC2024-0505). The animal care and use protocol complies with the European Directive of September 22, 2010 (Directive 2010/63/EU) and the U.S. National Institutes of Health.

Statistical analysis

Statistical analyses were performed using SPSS version 28.0 (IBM, IL, USA) and GraphPad Prism version 9.2.0 (GraphPad Software, Inc, CA, USA). Data are presented as mean ± SD. Each measurement was repeated independently at least three times. The six group experiments included detection of Mccp copy number, lung pathological changes, and expression of lung inflammatory factors, T tests were used to compare the Control group with the Model group, the CADH group with the Bacterial depletion group, and the CADH group with the Tylosin group, respectively. Additionally, the Model group, the CADL group, and the CADH group were compared using one-way ANOVA. The four group experiments included the detection of colon pathological changes, inflammatory factor expression, tight junction-related protein expression, and lung inflammation and apoptosis pathway-related protein expression, T test was used to compare the Control group with the Model group, and one-way ANOVA was used to compare the Model group, CADL group, and CADH group, followed by Bonferroni′s post hoc test to assess the significant differences in mean values. The α-diversity data of the intestinal microbiota were analyzed using the Kruskal-Wallis H test followed by the Wilcoxon rank sum test. For the β-diversity of the intestinal microbiota, principal component analysis and principal coordinate analysis were performed on the OTU level and weighted UniFrac phylogenetic distance using similarity analysis of the R package vegan. P < 0.05 was considered statistically significant.

Result

CAD alleviated the severity of mccp-induced pneumonia in mice

The experimental implementation is illustrated as shown in Fig. 1a. The results demonstrated a significant decrease in the weight of mice in the Model group compared to the Control group. Treatment with CAD alleviated mouse weight loss in a dose-dependent manner. There was no significant difference in the weight loss between the Tylosin group and the CADH group, while weight loss was more severe in the Bacterial depletion group compared to the other groups (Fig. 1b). DAI scores were significantly higher in the Model group than in the Control group (Fig. 1c). But the DAI scores decreased significantly after treatment with CAD. No significant differences in DAI scores were found among the CADH group, the Bacterial depletion group, and the Tylosin group.

Fig. 1
figure 1

CAD alleviated the severity of Mccp-induced pneumonia in mice a Animal experimental design. b Changes in the body weight of the mice. c Disease activity index (DAI) scores of pneumonia in each group. d Copy number of MCCP in each group. Data are presented as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001

Full size image

In the lung tissue, the copy number of Mccp in the Model group was 4.96 × 104, which was significantly higher than that in the Control group, indicating that Mccp had invaded and proliferated within the lung tissue. The copy number in the CADL group was 3.16 × 104, and in the CADH group, it was 1.32 × 104 demonstrating a dose-dependent reduction following CAD treatment. The Bacterial depletion group had a copy number of 2.48 × 104, which was significantly higher than that in the CADH group (Fig. 1d).

CAD attenuated the lung inflammatory response

H.E. staining results showed that compared with the Control group, the Model group exhibited significantly thickened and congested alveolar walls, partial detachment of the bronchial endothelium, unclear alveolar sacs and structures in some areas, numerous red blood cells in the alveolar cavity, and extensive infiltration of inflammatory cells. In contrast, lung structural damage was significantly reduced and inflammatory cell infiltration was significantly improved after CAD treatment (Fig. 2a). Furthermore, the lung histological scores of the CAD treated mice were significantly lower than those of the model group (Fig. 2b).

Fig. 2
figure 2

Levels of inflammatory factors in lung tissues and the lung histopathological Changes a H.E. staining of the tissue samples collected from each group. Scale bars, 100 μm. b Lung histopathological score for each group. c IL-1β, d IL-6, e IL-17, and f TNF-α levels measured in lung tissues. Data are presented as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Full size image

RT-PCR experimental results showed that, compared to the Control group, the expression levels of IL-1β, IL-6, IL-17 and TNF-α in the lung tissue homogenate of the Model group mice were significantly increased. Specifically, IL-17, IL-6, TNF-α and IL-1β increased by 272.98 times, 270.5 times, 12.61 times and 4.8 times, respectively. Following CAD administration, the levels of inflammatory factors decreased in a dose-dependent manner (Fig. 2c). The expression of inflammatory factors in the lung tissue homogenate of the Bacterial depletion group was higher than that of the CADH group.

CAD improved gut barrier integrity

To determine the effect of CAD on intestinal barrier function, we used western blot and immunohistochemistry to detect the expression levels of the intestinal tight junction proteins Occludin, Claudin-1, and ZO-1. Western blot results showed that, compared to the Control group, the expression levels of Occludin, Claudin-1, and ZO-1 proteins in the Model group were significantly reduced, while CAD treatment significantly increased the expression levels of these three proteins (Fig. 3d-g). Immunohistochemistry results confirmed these observations (Fig. 3a-c). These data suggest that CAD can upregulate the expression of intestinal tight junction-related proteins, preserve the integrity of the intestinal barrier, and mitigate intestinal damage induced by Mccp. The protective effect of CAD on the intestine is more pronounced at higher doses

Fig. 3
figure 3

CAD enhanced gut barrier integrity in Mccp-induced pneumonia mice (a) Claudin-1、(b) Occludin and (c) ZO-1 protein levels in colon tissue samples were determined using Immunohistochemistry. Scale bar, 100 μm. d Western blot technique was used to detect Claudin-1、 Occludin and ZO-1 protein levels in colon tissue samples. e Relative Claudin-1 protein abundance. f Relative Occludin protein abundance. g Relative ZO-1 protein abundance. *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Full size image

CAD attenuated the colonic inflammatory response

H.E. staining results indicated that the colon tissue structure of mice in the Control group was normal, with an intact and regular mucosal layer, no necrosis or shedding, and well-arranged glands without inflammatory cell infiltration. In contrast, the Model group displayed clear ulcers in the colon tissue, severe mucosal damage, compromised crypts, and extensive infiltration of inflammatory cells. Compared with the Model group, both CADL and CADH can maintain the integrity of the intestinal mucosa, restore damaged crypts, and reduce the number of infiltrating inflammatory cells (Fig. 4a). Similarly, the histological scores were significantly higher in the Model group compared to the Control group, while scores were significantly lower in the CAD treatment groups compared to the Model group (Fig. 4b).

Fig. 4
figure 4

Levels of inflammatory factors in colonic tissues and the colonic histopathological changes a H.E. staining of the colonic tissue samples collected from each group. Scale bars, 100 μm. b Colonic histopathological score in each group. c IL-1β, d IL-6, e IL-17, and f TNF-α levels measured in colonic tissues of mice. Data are presented as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Full size image

RT-PCR results revealed that Mccp infection significantly elevated the expression levels of inflammatory factors IL-1β, IL-6, IL-17 and TNF-α in the colon tissue of mice. CAD treatment significantly reduced the expression levels of these inflammatory factors in a dose-dependent manner (Fig. 4c). These findings suggest that CAD has significant effects on maintaining intestinal health, in addition to the benefits of fighting pneumonia.

CAD regulated the composition of the intestinal microbiota and reduce the lps of colonic contents and serum

16s rDNA sequencing was performed to explore the effect of Mccp infection on intestinal flora and whether CAD can regulate it. Results revealed that the diversity of flora in intestinal contents significantly increased after Mccp infection, indicating a disturbance in the composition of intestinal microorganisms in mice. Compared to the Control group, the Model group showed a significant increase in intestinal flora diversity. However, CAD treatment significantly decreased this diversity in a dose-dependent manner (Fig. 5a-b). Principal component analysis, principal coordinate analysis and sample clustering results demonstrated distinct clustering of microbial groups between the Model and Control groups, indicating substantial differences in the intestinal microbiota structure of Mccp-infected mice. In contrast, the CADL and CADH groups exhibited a microbiota structure more similar to the control group, suggesting that CAD has a regulatory effect on intestinal flora (Fig. 5c-e).

Fig. 5
figure 5

CAD regulated the structure of intestinal microorganisms in Mccp-induced pneumonia mice a Shannon index and b Simpson index represent Alpha diversity analysis. c Principal Component Analysis and d Principal Coordinates Analysis represent Beta diversity analysis. e Sample cluster analysis. Data are presented as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001

Full size image

Specifically, at the phylum level, Mccp infection decreased the relative abundance of Firmicutes, Patescibacteria, Cyanobacteria, Actinobacteriota, and Verrucomicrobiota in the intestinal flora, while increasing the relative abundance of Proteobacteria, Campylobacterota, Desulfobacterota, and Deferribacterota. CAD treatment reversed the dysbiosis caused by lung disease, restoring intestinal flora homeostasis (Fig. 6a-b). At the genus level, the distribution of Lachnospiraceae, Escherichia, Clostridiales, Bacteroides, Clostridium, Desulfovibrionaceae, Colidextribacter, Oscillibacter, Anaerotignum, Odoribacter and other bacterial communities in the intestinal contents of the Model group mice increased significantly, and new microbial communities such as Rodentibacter, Insolitispirillum, Eubacterium_oxidoreducens, and Oscillospira appeared. Conversely, the relative abundance of Lactobacillus, Bacteroidales, Candidatus_Saccharimonas, Alloprevotella, Paramuribaculum, Monoglobus, Veillonella, Ligilactobacillus, and Prevotella decreased significantly (Fig. 6c-d). CAD treatment reversed this trend, regulate the balance of intestinal flora, and restore the function of intestinal flora. To reveal the changes at the species level after Mccp infection and CAD treatment, we also performed sequencing analysis at the species level. Sequencing analysis revealed significant decreases in beneficial bacteria in the Model group, including Lactobacillus_taiwanensis (60-83.4%), Corynebacterium_glutamicum (90.2–94.7%), Candidatus_Saccharimonas (22.4–45.8%), Paramuribaculum_intestinale (97.06–98.96%), Lactobacillus_crispatus (79.25–91.67%), and Clostridia_UCG-014 (9.52–27.78%). Corynebacterium_glutamicum and Paramuribaculum showed the largest decreases, by 7.68–21.24 times and 33–95 times, respectively. Additionally, the relative abundance of conditional pathogens such as uncultured_Oscillibacter_sp, Lachnospiraceae_NK4A136_group, Escherichia-Shigella, Desulfovibrionaceae, Desulfovibrio_sp._UNSW3caefatS, Helicobacter, Anaerotruncus, Rodentibacter, GCA-900066575, and uncultured_Lachnospira_sp in the Model group was greatly increased. Notably, uncultured_Oscillibacter_sp increased by 5.25 ~ 13.75 times, Escherichia-Shigella increased by 13.96 ~ 26.17 times, Desulfovibrio_sp._UNSW3caefatS increased by 4.13 ~ 6.12 times, Helicobacter increased by 2 ~ 6.33 times, and GCA-900066575 increased by 5.33 ~ 22 times (Fig. 6e). LEfSe analysis identified species with significant abundance differences among groups (Fig. 6f). Overall, Mccp infection reduced the relative abundance of beneficial bacteria and increased the relative abundance of conditional pathogens, significantly altering the intestinal flora composition in mice. CAD restored the imbalanced flora, maintaining intestinal homeostasis and normal function (Fig. 6g-h).

Fig. 6
figure 6

CAD regulated the composition of intestinal microorganisms in Mccp-induced pneumonia mice a and c show the relative abundance of colon contents at the phylum (a) and genus (c) levels. b and d are heatmaps generated using R studio, depicting the taxonomic abundance at the phylum (b) and genus (d) level based on metagenomic sequencing analysis results. e Species-level significant difference analysis. f Relative abundance of bacteria in the gut was further analyzed using LEfSe analysis. g Taxonomic abundance of some probiotics. h Taxonomic abundance of some conditional pathogens. i LPS level in colon contents. j LPS level in serum. Data are presented as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Full size image

Since most of the opportunistic pathogens increased in the colon contents of mice in the Model group were gram-negative bacteria and closely related to the production of LPS. In order to verify whether LPS plays an important role in the disease development, we measured the LPS content in the colon contents and serum of each group of mice. The results showed that compared with the Control group, the LPS content in the colon contents and serum of the Model group mice was significantly increased. After treatment with CAD, the LPS content was significantly downregulated in a dose-dependent manner (Fig. 6i).

CAD alleviates inflammation by inhibiting the activation of the TLR4/NF-κB signaling pathway in the lung

Western blot analysis was used to detect the levels of TLR4, MYD88, P65, P-P65, IKB, and P-IKB along the signaling pathway TLR4/NF-kB in lung tissue homogenate following CAD treatment. The results showed that compared with the Control group, the expression levels of TLR4, MYD88, P-P65, and P-IKB in the Model group were significantly increased, along with the ratios of P-P65/P65 and P-IKB/IKB. After CAD intervention, this trend was reversed (Fig. 7). This suggests that CAD can reduce the release of inflammatory factors by inhibiting the activation of the TLR4/NF-kB signaling pathway, with the effect becoming more pronounced with increasing doses.

Fig. 7
figure 7

Protein levels of the inflammation-related proteins in the lung tissues on the TLR4/NF-κB pathway a Western blot of the TLR4、P65、P-P65、IKB、P-IKB and MYD88 proteins. b Relative levels of TLR4. c Relative levels of P65. d Relative levels of P-P65. e The P-P65/P65 ratios. f Relative levels of P-IKB. g Relative levels of IKB. h The P-IKB/IKB ratios. i Relative levels of MYD88. *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Full size image

CAD inhibits apoptosis by inhibiting the activation of the Fas/FasL-caspase-8/-3 signaling pathway in the lung

Early studies have shown that TNF-α could induce apoptosis of epithelial cells by activating the Fas/Fasl-caspase8/3 pathway, thereby exacerbating organ damage. Our previous studies have found that CAD can reduce the secretion of TNF-α by inhibiting the TLR4/NF-κB pathway. Therefore, in order to verify whether Mccp induces apoptosis and whether CAD mitigates this process, we detected the expression levels of Fas/Fasl-caspase8/3 pathway-related proteins caspase 8, FADD, Cyto-c and Cleaved-caspase 3. The results showed that compared with the Control group, the expression levels of caspase 8, FADD, Cyto-c and Cleaved-caspase 3 in the lungs of the Model group were significantly increased, indicating that infection with Mccp would induce apoptosis (Fig. 8). However, in the CAD-treated lungs, compared to the Model group, the expression levels of caspase-8, FADD, Cytochrome c, and Cleaved-caspase-3 were significantly reduced, demonstrating ability of CAD to alleviate Mccp-induced apoptosis.

Fig. 8
figure 8

Protein levels of the apoptosis-related proteins in the lung tissues on the Fas/FasL-caspase-8/-3pathway a Western blot of the Caspase-8, FADD, Cyto-c and Cleaved-Caspase-3 proteins. b Relative levels of Caspase-8. c Relative levels of FADD. d Relative levels of Cyto-c. e Relative levels of Cleaved-Caspase-3. *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Full size image

Additionally, we also assessed the expression levels of BAX and Bcl-2 proteins associated with apoptosis. The results indicated that, compared with the Control group, the expression of BAX in the lungs of the Model group was significantly elevated, while the expression of Bcl-2 and the ratio of Bcl-2/BAX were markedly reduced. In contrast, the CAD-treated group showed significantly decreased BAX expression in the lungs compared to the model group. Moreover, the expression level of Bcl-2 and the ratio of Bcl-2/BAX were notably increased in a dose-dependent manner in the CAD-treated group (Fig. 9).

Fig. 9
figure 9

Protein levels of the BAX and Bcl-2 and Bcl-2/BAX ratios in the lung tissues. a Western blot of the BAX and Bcl-2 proteins. b Relative levels of BAX. c Relative levels of Bcl-2. d The Bcl-2/BAX ratios. *p < 0.05, **p < 0.01, ***p < 0.001, ns p > 0.05

Full size image

Discussion

In this study, we established a mycoplasma pneumonia mouse model by intranasally inoculating Mccp. We evaluated weight fluctuations, DAI scores, Mccp copy numbers in the lungs, pathological alterations, and the expression of several inflammatory factors in each mouse group. The Model group exhibited significant weight loss, increased DAI scores, elevated Mccp copy numbers, and higher levels of inflammatory cytokines, including IL-1β, IL-6, IL-17, and TNF-α, accompanied by more severe pathological damage. These results indicate that Mccp successfully infiltrated the lungs, proliferated, and caused lung inflammation, thus confirming the successful construction of the mycoplasma pneumonia mouse model. The CADL and CADH groups displayed reduced weight loss, significantly lower DAI scores, lower Mccp copy numbers, diminished expression levels of inflammatory factors, and lessened pathological damage compared to the Model group. The CADH group showed a more pronounced effect than the CADL group, demonstrating that CAD has a dose-dependent mitigating effect on Mccp infection. Moreover, the Bacterial depletion group demonstrated higher weight loss, Mccp copy numbers, and inflammatory factor expression levels than the CADH group, suggesting that the intestinal microbiota plays a critical role in CAD’s protective effects against Mccp. The use of Tylosin as a positive control drug further validated CAD’s efficacy against mycoplasma pneumonia.

Studies have shown that lung diseases can affect the intestinal flora and intestinal immune function, ultimately leading to the occurrence of intestinal diseases. Animals infected with influenza virus experience immune damage to both their respiratory and intestinal mucosa. However, tests found no presence of the virus in the intestine, ruling out the possibility of direct viral-induced immune damage, subsequent research revealed that bacteria and viruses in the lungs can mediate a significant influx of lung-derived CCR9 + CD4 + T cells into the intestine through the CCL25/CCR9 pathway, causing immune damage to the intestine [24]. Pneumonia caused by P. aeruginosa has been shown to block the M phase of the cell cycle, resulting in decreased proliferation of intestinal epithelial cells and compromising the integrity of the intestinal epithelium [25]. In addition, studies have shown that lung infections can affect the composition and metabolic of intestinal microorganisms. For example, in patients with pulmonary Mycobacterium tuberculosis infection, the number of beneficial symbiotic bacteria in their intestinal microorganisms will decrease, while the number of pathogenic bacteria such as Prevotella and Enterococcus significantly increases [26]. Research has also found that methicillin-resistant Staphylococcus aureus pneumonia can lead to sepsis, which induces apoptosis of intestinal epithelial cells, reduces crypt cell proliferation, and shortens intestinal villi through the mitochondrial pathway, thereby compromising intestinal integrity [27]. SARS-CoV-2 can regulate the immune status of the digestive tract after infecting the respiratory tract. CCR9 + CD4 + T cells induced by SARS-CoV-2 infection can be recruited to the small intestine, causing intestinal damage by disrupting the distribution of intestinal microbiota [28]. Similarly, influenza virus induces the production of IFN-I in the lungs, which leads to intestinal dysbiosis, increases the host’s susceptibility to secondary Salmonella infection, suppresses intestinal immune function, and promotes intestinal inflammatory responses [29]. In summary, lung diseases can disrupt the homeostasis of intestinal flora by inducing the production of chemokines, causing flora migration, and damaging the intestinal barrier, leading to intestinal diseases. The disordered intestinal flora and its metabolites can directly affect the local immunity of the lungs by recruiting immune cells, regulating the production of cytokines and antibodies, and thus influence the course and prognosis of lung diseases [30,31,32,33]. In this study, the expression of tight junction proteins in the colon of mice infected with Mccp was significantly reduced, and the expression of inflammatory factors such as IL-1β, IL-6, IL-17, and TNF-α was significantly increased. This also shows that lung disease can lead to the occurrence of intestinal diseases.

In recent decades, as people's understanding of intestinal flora has increased, treating diseases by regulating intestinal flora has become a popular method in alternative therapy. Studies have shown that Amps can treat diseases by regulating intestinal flora [31,32,33,34,35,36,37]. However, the mechanism by which CAD fights Mccp by regulating intestinal flora remains unclear. This study confirmed for the first time that after Mccp infection, the abundance of probiotics such as Lactobacillus_taiwanensis, Corynebacterium_glutamicum,Candidatus_Saccharimonas, Paramuribaculum_intestinale, Lactobacillus_crispatus, and Clostridia_UCG-014 in the colon contents was significantly reduced. Studies have demonstrated that the probiotic properties of Lactobacillus_taiwanensis include the acid and bile salt tolerance, adhesion ability, antibacterial activity and in vitro immunomodulatory effects [38]. Corynebacterium_glutamicum alleviates modulated antibiotic and aromatic compound resistance by regulating the secretion of a MarR-family regulator-CarR [39]. Corylin increases the abundance of probiotics such as Candidatus_Saccharimonas in the intestine, reduces the expression of inflammatory factors in the colon, and promotes the expression of intestinal tight junction proteins, showing a therapeutic effect on DSS-induced colitis [40]. Curcumin exerts anti-inflammatory and anti-lipid peroxidation effects by increasing the abundance of Lactobacillus_crispatus [41]. The abundance of opportunistic pathogens such as uncultured_Oscillibacter_sp, Lachnospiraceae_NK4A136_group, Escherichia-Shigella, Desulfovibrionaceae, Desulfovibrio_sp._UNSW3caefatS, Helicobacter, Anaerotruncus, Rodentibacter, GCA-900066575, anduncultured_Lachnospira_sp. increased significantly. Similarly, studies have shown that Desulfovibrio_sp._UNSW3caefatS, uncultured_Lachnospira_sp and Helicobacter associated with the occurrence of inflammatory bowel disease, colorectal cancer and systemic sclerosis [38, 42]. An increased abundance of Rodentibacter can also lead to the development of fatal pneumonia, sepsis and other diseases [43].

It is well known that LPS plays a crucial role in directly or indirectly regulating intestinal barrier integrity and host immune homeostasis [44]. In this study, we found that most of the opportunistic pathogens significantly upregulated in the intestinal flora after infection with Mccp were gram-negative bacteria, which be related to the increased levels of LPS in the intestine and serum. In addition, some studies have shown that certain probiotics possess anti-inflammatory properties, help maintain the integrity of the intestinal barrier, and reduce the transfer of bacterial-derived LPS from the intestine to the blood [45, 46]. Among the probiotics that were significantly downregulated after infection with Mccp in this study, Lactobacillus_taiwanensis, Candidatus_Saccharimonas, Paramuribaculum_intestinale, Lactobacillus_crispatus, and Clostridia_UCG-014 all had antibacterial, anti-inflammatory, and immunomodulatory activities. Specifically, Lactobacillus_taiwanensis, Lactobacillus_crispatus, and Clostridia_UCG-014 have been shown to maintain the integrity of the intestinal barrier [47, 48]. Therefore, the increase in LPS levels in the serum of mice in the Model group was associated with the decrease in these probiotics and the increase in pathogenic bacteria. AMPs are known to regulate the abundance and diversity of the gut microbiota. In this study, CAD significantly reduced the number of opportunistic pathogens in the gut microbiota of the model group. The underlying mechanism may be that CAD, as an amphipathic cationic peptide, forms barrel- or ring-shaped pores in bacterial membranes, further diluting and solubilizing the lipid bilayer, thereby disrupting membrane integrity and ultimately leading to bacterial death [49]. In addition, CAD may interfere with the synthesis of bacterial proteins, RNA, and DNA, thereby affecting the metabolic processes of opportunistic pathogens and ultimately leading to bacterial inactivation [50]. The increase in probiotic abundance may be related to CAD’s selective targeting of opportunistic pathogens. By reducing the number of pathogenic bacteria, CAD improves the intestinal environment, promotes gut health, and creates more favorable conditions for probiotic growth, ultimately increasing the abundance of probiotics [51].

There is increasing evidence that intestinal flora and its products, such as LPS, are pivotal mediators of animal health and disease, and they play a key role in the lung-gut axis [ 7, 8 ]. This study, through in vivo experiments in mice, revealed that after infection with Mccp, the number of gram-negative bacteria in the intestinal flora and the LPS they produce increased significantly, while the number of probiotics that maintain the integrity of the intestinal barrier decreased [45].Our results also showed that infection with Mccp triggered an intestinal inflammatory response, damaged the intestinal barrier, and increasing intestinal permeability. When intestinal permeability increases, excessive intestinal LPS could enter the bloodstream, where it is recognized by the TLRs complex in the lungs, thereby aggravating lung inflammation and promoting the expression of inflammatory factors such as IL-1β, IL-6, IL-17 and TNF-α. Early studies have found that TNF-α is an activator of the Fas/Fasl-caspase8/3 pathway, which can aggravate the injury by inducing apoptosis [9]. This study also confirmed that CAD can reduce lung inflammation and cell apoptosis by inhibiting the activation of the TLR4-NF-κB signaling pathway and the Fas/FasL-caspase-8/-3 signaling pathway. It can be inferred that CAD reduces the LPS content in serum by regulating intestinal flora, inhibiting the secretion of enteric LPS, maintaining the integrity of the intestinal barrier. Thereby alleviating the clinical symptoms related to Mccp infection.

Conclusion

Cecropin AD has a significant effect on alleviating the Mccp-induced mycoplasma pneumonia mouse model and can markedly reduce lung tissue damage. Mechanistically, CAD can regulate the intestinal flora, reduce the secretion of enteric LPS, restore the damaged intestinal barrier, and decrease serum LPS levels. This action inhibits the activation of the TLR4-NF-κB signaling pathway and the Fas/FasL-caspase-8/3 signaling pathway in the lungs, therefore, the inflammation and apoptosis process in the lungs are alleviated. Our findings provide new insights into the role and mechanism of CAD in anti-mycoplasma pneumonia

Data availability

The datasets generated during the current study are available in the Genome Sequence Archive repository, [CRA018388].

References

  1. Ma WT, Gu K, Yang R, Tang XD, Qi YX, Liu MJ, Chen DK. Interleukin-17 mediates lung injury by promoting neutrophil accumulation during the development of contagious caprine pleuropneumonia. Vet Microbiol. 2020;243:108651.

    Article  CAS  PubMed  Google Scholar 

  2. Adak A, Khan MR. An insight into gut microbiota and its functionalities. Cell Mol Life Sci. 2019;76:473–93.

    Article  CAS  PubMed  Google Scholar 

  3. Wong CC, Yu J. Gut microbiota in colorectal cancer development and therapy. Nat Reviews Clin Oncol. 2023;20:429–52.

    Article  Google Scholar 

  4. Parker A, Romano S, Ansorge R, Aboelnour A, Le GG, Savva GM, Pontifex MG, Telatin A, Baker D, Jones E, Vauzour D, Rudder S, et al. Fecal microbiota transfer between young and aged mice reverses hallmarks of the aging gut, eye, and brain. Microbiome. 2022;10:68.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Thomas RM, Jobin C. Microbiota in pancreatic health and disease: the next frontier in microbiome research. Nat Reviews Gastroenterol Hepatol. 2020;17:53–64.

    Article  Google Scholar 

  6. Zhao Q, Huang JF, Cheng Y, Dai MY, Zhu WF, Yang XW, Gonzalez FJ, Li F. Polyamine metabolism links gut microbiota and testicular dysfunction. Microbiome. 2021;9:224.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Zhao Z, Ning J, Bao X, Shang M, Ma J, Li G, Zhang D. Fecal microbiota transplantation protects rotenone-induced Parkinson’s disease mice via suppressing inflammation mediated by the lipopolysaccharide-TLR4 signaling pathway through the microbiota-gut-brain axis. Microbiome. 2021;9:226.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Guo C, Guo D, Fang L, Sang T, Wu J, Guo C, Wang Y, Wang Y, Chen C, Chen J, Chen R, Wang X. Ganoderma Lucidum polysaccharide modulates gut microbiota and immune cell function to inhibit inflammation and tumorigenesis in colon. Carbohydr Polym. 2021;267:118231.

    Article  CAS  PubMed  Google Scholar 

  9. Rex J, Lutz A, Faletti LE, Albrecht U, Thomas M, Bode JG, Borner C, Sawodny O, Merfort I. IL-1β and TNFα differentially influence NF-κB activity and FasL-Induced apoptosis in primary murine hepatocytes during LPS-Induced inflammation. Front Physiol. 2019;10:117.

  10. Torres SB, Dema T, Schilling NA, Janek D, Bornikoel J, Berscheid A, Elsherbini AMA, Krauss S, Jaag SJ, Lämmerhofer M, Li M, Alqahtani N, et al. Commensal production of a broad-spectrum and short-lived antimicrobial peptide polyene eliminates nasal Staphylococcus aureus. Nat Microbiol. 2024;9:200–13.

    Article  Google Scholar 

  11. Lehrer RI, Lu W. α-Defensins in human innate immunity. Immunol Rev. 2012;245:84–112.

    Article  CAS  PubMed  Google Scholar 

  12. Zhai Z, Zhang F, Cao R, Ni X, Xin Z, Deng J, Wu G, Ren W, Yin Y, Deng B. Cecropin A alleviates inflammation through modulating the gut microbiota of C57BL/6 mice with DSS-Induced IBD. Front Microbiol. 2019;10:1595.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Wu S, Yi J, Zhang Y, Zhou J, Sun J. Leaky intestine and impaired microbiome in an amyotrophic lateral sclerosis mouse model. Physiological Rep. 2015;3:e12356.

    Article  Google Scholar 

  14. Abdelgawad A, Nicola T, Martin I, Halloran BA, Tanaka K, Adegboye CY, Jain P, Ren C, Lal CV, Ambalavanan N, O’Connell AE, Jilling T, et al. Antimicrobial peptides modulate lung injury by altering the intestinal microbiota. Microbiome. 2023;11:226.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Valdez-Miramontes CE, De Haro-Acosta J, Aréchiga-Flores CF, Verdiguel-Fernández L, Rivas-Santiago B. Antimicrobial peptides in domestic animals and their applications in veterinary medicine. Peptides. 2021;142:170576.

    Article  CAS  PubMed  Google Scholar 

  16. Liu Q, Yao S, Chen Y, Gao S, Yang Y, Deng J, Ren Z, Shen L, Cui H, Hu Y, Ma X, Yu S. Use of antimicrobial peptides as a feed additive for juvenile goats. Sci Rep. 2017;7(1):12254.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Wang J, Wilson AE, Su B, Dunham RA. Functionality of dietary antimicrobial peptides in aquatic animal health: multiple meta-analyses. Anim Nutr. 2022;12:200–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhang T, Liu Z, Zhi Y, Zhao X, Du M, Zhang Q, Zhang T, Hu G. Cecropin AD reduces viral load and inflammatory response against H9N2 avian influenza virus in chickens. Front Veterinary Sci. 2024;11:1369863.

  19. Parker BJ, Wearsch PA, Veloo ACM, Rodriguez PA. The genus alistipes: gut bacteria with emerging implications to inflammation, cancer, and mental health. Front Immunol. 2020;11:906.

  20. Milliken S, Allen RM, Lamont RF. The role of antimicrobial treatment during pregnancy on the neonatal gut microbiome and the development of atopy, asthma, allergy and obesity in childhood. Exp Opin Drug Saf. 2019;18:173–85.

    Article  CAS  Google Scholar 

  21. Harrison TM, Churgin SM. Acupuncture and traditional Chinese Veterinary Medicine in zoological and exotic Animal Medicine: a review and introduction of methods. Veterinary. Sci 2022;9(2):74.

  22. Wan L, Meng D, Wang H, Wan S, Jiang S, Huang S, Wei L, Yu P. Preventive and therapeutic effects of Thymol in a Lipopolysaccharide-Induced Acute Lung Injury mice Model. Inflammation. 2018;41:183–92.

    Article  CAS  PubMed  Google Scholar 

  23. Cheng J, Liu D, Huang Y, Chen L, Li Y, Yang Z, Fu S, Hu G. Phlorizin mitigates Dextran Sulfate Sodium-Induced Colitis in mice by modulating gut microbiota and inhibiting ferroptosis. J Agric Food Chem. 2023;71:16043–56.

    Article  CAS  PubMed  Google Scholar 

  24. Khailova L, Baird CH, Rush AA, Barnes C, Wischmeyer PE. Lactobacillus rhamnosus GG treatment improves intestinal permeability and modulates inflammatory response and homeostasis of spleen and colon in experimental model of Pseudomonas aeruginosa pneumonia. Clin Nutr. 2017;36:1549–57.

    Article  CAS  PubMed  Google Scholar 

  25. An Z, Yuanyuan L, Li T, Shiping H, Min Y, Lingyi W, Shiming Y, Bo T. Gut microbiota: the emerging link to Lung Homeostasis and Disease. J Bacteriol. 2021;203. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/jb.00454.

  26. Li W, Zhu Y, Liao Q, Wang Z, Wan C. Characterization of gut microbiota in children with pulmonary tuberculosis. BMC Pediatr. 2019;19:445.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Perrone EE, Jung E, Breed E, Dominguez JA, Liang Z, Clark AT, Dunne WM, Burd EM, Coopersmith CM. Mechanisms of methicillin-resistant staphylococcus aureus pneumonia–induced intestinal epithelial apoptosis. Shock. 2012;38(1):68–75.

  28. Nishio J, Honda K. Immunoregulation by the gut microbiota. Cell Mol Life Sci. 2012;69:3635–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Deriu E, Boxx GM, He X, Pan C, Benavidez SD, Cen L, Rozengurt N, Shi W, Cheng G. Influenza virus affects intestinal microbiota and Secondary Salmonella Infection in the gut through type I interferons. PLoS Pathog. 2016;12:e1005572.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Wang L, Cai Y, Garssen J, Henricks PAJ, Folkerts G, Braber S. The bidirectional gut–lung Axis in Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med. 2023;207:1145–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Budden KF, Gellatly SL, Wood DLA, Cooper MA, Morrison M, Hugenholtz P, Hansbro PM. Emerging pathogenic links between microbiota and the gut–lung axis. Nat Rev Microbiol. 2017;15:55–63.

    Article  CAS  PubMed  Google Scholar 

  32. Wang YH, Yan ZZ, Luo SD, Hu JJ, Wu M, Zhao J, Liu WF, Li C, Liu KX. Gut microbiota-derived succinate aggravates acute lung injury after intestinal ischaemia/reperfusion in mice. Eur Respir J. 2023;61:2200840.

    Article  CAS  PubMed  Google Scholar 

  33. Lai HC, Lin TL, Chen TW, Kuo YL, Chang CJ, Wu TR, Shu CC, Tsai YH, Swift S, Lu CC. Gut microbiota modulates COPD pathogenesis: role of anti-inflammatory < em > Parabacteroides goldsteinii lipopolysaccharide. Gut. 2022;71:309.

    Article  CAS  PubMed  Google Scholar 

  34. Zhai Z, Ni X, Jin C, Ren W, Li J, Deng J, Deng B, Yin Y. Cecropin A modulates tight Junction-Related protein expression and enhances the barrier function of Porcine Intestinal epithelial cells by suppressing the MEK/ERK Pathway. Int J Mol Sci. 2018;19(7):1941.

  35. Dai J, Weihao O, Yu G, Ai Q, Zhang W, Zhang Y. The antimicrobial peptide cecropin AD supplement alleviated soybean Meal-Induced intestinal inflammation, barrier damage, and Microbial Dysbiosis in Juvenile Turbot, Scophthalmus maximus. Front Mar Sci. 2020;7:584482.

  36. Beisner J, Filipe RL, Kaden VV, Stolzer I, Günther C, Bischoff SC. Prebiotic inulin and Sodium Butyrate Attenuate obesity-Induced Intestinal Barrier dysfunction by induction of antimicrobial peptides. Front Immunol. 2021;12:678360.

  37. Liang W, Enée E, Andre-Vallee C, Falcone M, Sun J, Diana J. Intestinal cathelicidin antimicrobial peptide shapes a protective neonatal gut microbiota against pancreatic autoimmunity. Gastroenterology. 2022;162:1288–e130216.

    Article  CAS  PubMed  Google Scholar 

  38. O’Toole PW, Snelling WJ, Canchaya C, Forde BM, Hardie KR, Josenhans C, Graham RL, McMullan G, Parkhill J, Belda E, Bentley SD. Comparative genomics and proteomics of Helicobacter mustelae, an ulcerogenic and carcinogenic gastric pathogen. BMC Genomics. 2010;11:164.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Si M, Chen C, Wei Z, Gong Z, Li G, Yao S. CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance. Biochem J. 2019;476:3141–59.

    Article  CAS  PubMed  Google Scholar 

  40. Chang ZY, Liu HM, Leu YL, Hsu CH, Lee TY. Modulation of gut microbiota combined with upregulation of intestinal tight Junction explains anti-inflammatory effect of Corylin on Colitis-Associated Cancer in mice. Int J Mol Sci. 2022;23(5):2667.

  41. Pivari F, Mingione A, Piazzini G, Ceccarani C, Ottaviano E, Brasacchio C, Dei Cas M, Vischi M, Cozzolino MG, Fogagnolo P, Riva A, Petrangolini G et al. Curcumin Supplementation (Meriva®) Modulates Inflammation, Lipid Peroxidation and Gut Microbiota Composition in Chronic Kidney Disease. Nutrients. 2022;14(1):231.

  42. Yuan X, Chen B, Duan Z, Xia Z, Ding Y, Chen T, Liu H, Wang B, Yang B, Wang X, Liu S, Zhou JY, et al. Depression and anxiety in patients with active ulcerative colitis: crosstalk of gut microbiota, metabolomics and proteomics. Gut Microbes. 2021;13:1987779.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Sasaki H, Ueshiba H, Yanagisawa N, Itoh Y, Ishikawa H, Shigenaga A, Benga L, Ike F. Genomic and pathogenic characterization of RTX toxin producing Rodentibacter sp. that is closely related to Rodentibacter Haemolyticus. Infect Genet Evol. 2022;102:105314.

    Article  CAS  PubMed  Google Scholar 

  44. Stevens BR, Goel R, Seungbum K, Richards EM, Holbert RC, Pepine CJ, Raizada MK. Increased human intestinal barrier permeability plasma biomarkers zonulin and FABP2 correlated with plasma LPS and altered gut microbiome in anxiety or depression. Gut. 2018;67:1555.

    Article  CAS  PubMed  Google Scholar 

  45. Kesika P, Suganthy N, Sivamaruthi BS, Chaiyasut C. Role of gut-brain axis, gut microbial composition, and probiotic intervention in Alzheimer’s disease. Life Sci. 2021;264:118627.

    Article  CAS  PubMed  Google Scholar 

  46. Yuan S, Yang J, Jian Y, Lei Y, Yao S, Hu Z, Liu X, Tang C, Liu W. Treadmill Exercise modulates intestinal microbes and suppresses LPS displacement to Alleviate Neuroinflammation in the brains of APP/PS1 mice. Nutrients. 2022;14(19):4134.

  47. Slattery C, Cotter PD, O’Toole W. P. Analysis of health benefits conferred by Lactobacillus species from Kefir. Nutrients. 2019;11(6):1252.

  48. Leibovitzh H, Lee SH, Xue M, Raygoza Garay JA, Hernandez-Rocha C, Madsen KL, Meddings JB, Guttman DS, Espin-Garcia O, Smith MI, Goethel A, Griffiths AM, et al. Altered gut microbiome composition and function are Associated with Gut Barrier Dysfunction in healthy relatives of patients with Crohn’s Disease. Gastroenterology. 2022;163:1364–e137610.

    Article  CAS  PubMed  Google Scholar 

  49. Chen N, Jiang C. Antimicrobial peptides: structure, mechanism, and modification. Eur J Med Chem. 2023;255:115377.

    Article  CAS  PubMed  Google Scholar 

  50. Kumar P, Kizhakkedathu JN, Straus SK. Antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity and biocompatibility in vivo. Biomolecules. 2018;8(1):4.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Zong X, Fu J, Xu B, Wang Y, Jin M. Interplay between gut microbiota and antimicrobial peptides. Anim Nutr. 2020;6(4):389–96.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Funding

Financial support was received from [K4050423759 Integration and demonstration of high-efficiency breeding technology for reducing resistance in mutton sheep].

Author information

Author notes

  1. Bowen Li and Mingming Liu contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, China

    Bowen Li, Mingming Liu, Wenjing Du, Zekang Xu, Xiaoqian Zhang, Yang Zhang & Song Hua

  2. Mianyang Habio Bioengineering Co., Ltd., Mianyang, Sichuan, China

    Shuaidong Wang & Yang Zhang

Authors
  1. Bowen Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Mingming Liu
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Wenjing Du
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Shuaidong Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Zekang Xu
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Xiaoqian Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Yang Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Song Hua
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

S.H. and B.L. conceived and designed the research; B.L., M.L., W.D., S.W. performed the experiments; B.L., M.L., Z.X., X.Z., Y.Z.analyzed the data and interpreted the results of experiments; B.L., M.L., W.D., S.W. and Y.Z. prepared figures and drafted the manuscript; and all authors approved the final version of the manuscript.

Corresponding author

Correspondence to Song Hua.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, B., Liu, M., Du, W. et al. Cecropin AD ameliorates pneumonia and intestinal injury in mice with mycoplasma pneumoniae by mediating gut microbiota. BMC Vet Res 21, 39 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04500-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04500-w

Keywords

BMC Veterinary Research

ISSN: 1746-6148

Contact us