Expression of Toll-like receptors and host defence peptides in the cecum of chicken challenged with Eimeria tenella.

Chicken coccidiosis, caused by Eimeria protozoa, affects poultry farming. Toll-like receptors (TLRs) and host defence peptides (HDPs) help host innate immune responses to eliminate invading pathogens, but their roles in Eimeria tenella infection remain poorly understood. Herein, 14-day-old chickens were treated orally with 50,000 E. tenella oocysts and the cecum was dissected at different timepoints. mRNA expression of 10 chicken TLRs (chTLRs) and five HDPs was measured by quantitative real-time PCR. chTLR7 and chTLR15 were upregulated significantly at 3 h post-infection while other chTLRs were downregulated (p < .05). chTLR1a, chTLR1b, chTLR2b and chTLR4 peaked at 36 h post-infection, chTLR3, chTLR5 and chTLR15 peaked at 72 h post-infection and chTLR21 expression was highest among chTLRs, peaking at 48 h post-infection (p < 0.05). For HDPs, cathelicidin (CATH) 1 to 3 and B1 peaked at 48 h post-infection, liver-expressed antimicrobial peptide 2 peaked at 96 h post-infection, and CATH 2 expression was highest among HDPs. CATH2 and CATH3 were markedly upregulated at 3 h post-infection (p < .05). The results provide insight into innate immune molecules during E. tenella infection in chicken, and indicate that innate immune responses may mediate resistance to chicken coccidiosis.

NOX2 oxidase inhibitor GSK2795039 possess antiviral activity against H1N1 influenza A virus in vitro and vivo.

The continuous zoonotic circulation and reassortment potential of influenza A viruses (IAV) in nature represents an enormous public health threat to humans. Beside vaccination antivirals are needed to efficiently control spreading of the disease. The previous research has shown that NOX2 involved in IAV replication, but the detailed mechanism has not been reported. In the present study we investigated the roles of NOX2 in host inflammatory response and IAV replication using a novel inhibitor GSK2795039. The drug significantly reduced H1N1 virus induced NOX2 activity and ROS release in human lung epithelial cells. The results of time course experiments suggested that GSK2795039 inhibited an early post-entry step of viral infection. Concomitantly, there was a decreased expression of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interferon (IFN)-ß and interleukin (IL)-6) in NOX2 suppressed cells. In vivo, compared with control groups, suppression of NOX2 improved the survival rate of mice infected with H1N1 virus (42.9% in GSK2795039 treated mice versus >0% of control mice) and viral burden also decreased in the GSK2795039 treated group. Thus, our data demonstrated a critical role for NOX2 in the establishment of H1N1 infection and subsequent inflammatory reactions, which suggest that GSK2795039 may be a potential therapeutic drug for IAV infection.

Coexpression analysis of lncRNAs and mRNAs identifies potential regulatory long noncoding RNAs involved in the inflammatory effects of lipopolysaccharide on bovine mammary epithelial cells.

BACKGROUND: The infection of bovine mammary glands by pathogenic microorganisms not only causes animal distress but also greatly limits the development of the dairy industry and animal husbandry. A deeper understanding of the host's initial response to infection may increase the accuracy of selecting drug-resistant animals or facilitate the development of new preventive or therapeutic intervention strategies. In addition to their functions of milk synthesis and secretion, bovine mammary epithelial cells (BMECs) play an irreplaceable role in the innate immune response. To better understand this process, the current study identified differentially expressed long noncoding lncRNAs (DE lncRNAs) and mRNAs (DE mRNAs) in BMECs exposed to Escherichia coli lipopolysaccharide (LPS) and further explored the functions and interactions of these lncRNAs and mRNAs. RESULTS: In this study, transcriptome analysis was performed by RNA sequencing (RNA-seq), and the functions of the DE mRNAs and DE lncRNAs were predicted by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Next, we constructed a modulation network to gain a deeper understanding of the interactions and roles of these lncRNAs and mRNAs in the context of LPS-induced inflammation. A total of 231 DE lncRNAs and 892 DE mRNAs were identified. Functional enrichment analysis revealed that pathways related to inflammation and the immune response were markedly enriched in the DE genes. In addition, research results have shown that cell death mechanisms, such as necroptosis and pyroptosis, may play key roles in LPS-induced inflammation. CONCLUSIONS: In summary, the current study identified DE lncRNAs and mRNAs and predicted the signaling pathways and biological processes involved in the inflammatory response of BMECs that might become candidate therapeutic and prognostic targets for mastitis. This study also revealed several possible pathogenic mechanisms of mastitis.

Regulated lytic cell death in breast cancer.

Breast cancer (BC) is a very common cancer among women and one of the primary causes of death in women worldwide. Because BC has different molecular subtypes, the challenges associated with targeted therapy have increased significantly, and the identification of new therapeutic targets has become increasingly urgent. Blocking apoptosis and inhibiting cell death are important characteristics of malignant tumours, including BC. Under adverse conditions, including exposure to antitumour therapy, inhibition of cell death programmes can promote cancerous transformation and the survival of cancer cells. Therefore, inducing cell death in cancer cells is fundamentally important and provides new opportunities for potential therapeutic interventions. Lytic forms of cell death, primarily pyroptosis, necroptosis and ferroptosis, are different from apoptosis owing to their characteristic lysis, that is, the production of cellular components, to guide beneficial immune responses, and the application of lytic cell death (LCD) in the field of tumour therapy has attracted considerable interest from researchers. The latest clinical research results confirm that lytic death signalling cascades involve the BC cell immune response and resistance to therapies used in clinical practice. In this review, we discuss the current knowledge regarding the various forms of LCD, placing a special emphasis on signalling pathways and their implications in BC, which may facilitate the development of novel and optimal strategies for the clinical treatment of BC.

Necroptosis and its role in infectious diseases.

Necroptosis is a noncaspase-dependent and precisely regulated mechanism of cell death. Necroptosis is mainly initiated by members of the tumor necrosis factor receptor (TNFR) and Toll-like receptor (TLR) families, interferon, intracellular RNA and DNA sensors and other mediators. Subsequently, the protein kinase RIPK1 (receptor-interacting protein kinase 1) and RIPK3 interact with the receptor protein, which transduces death signals and further recruits and phosphorylates MLKL (mixed lineage kinase domain-like protein). MLKL serves as the initiator of cell death and eventually induces necroptosis. It was found that necroptosis is not only involved in the physiological regulation but also in the occurrence, development and prognosis of some necrotic diseases, especially infectious diseases. Intervention in the necroptosis signaling pathway is helpful for removing pathogens, inhibiting the development of lesions, and promoting the remodeling of tissue. In-depth study of the molecular regulation mechanism of necroptosis and its relationship with the pathogenesis of infectious diseases will help to provide new ideas and directions for research of the pathological mechanisms and clinical prevention of infectious diseases.

What role does pyroptosis play in microbial infection?

Pyroptosis, a type of programmed cell death mediated by gasdermin, is characterized by the swelling and rupture of cells, release of cellular contents and a strong inflammatory response, which is critical for controlling microbial infection. Pattern recognition receptors recognize the intracellular and extracellular pathogenic microbial components and stimulate the organism's inflammatory response by activating the pyroptosis signaling pathway and releasing interleukin-1ß (IL-1ß), IL-18, and other inflammatory factors to promote pathogen clearance and prevent infection. In the process of continuous evolution, pathogens have developed multiple strategies to modulate the occurrence of pyroptosis and thus enhance their ability to induce disease; that is, the competition between host cells and pathogens controls the occurrence of pyroptosis. Competition can directly affect tissue inflammation outbreaks and even alter cell survival. Studies have shown that various bacterial infections, including Shigella flexneri, Salmonella, Listeria monocytogenes, and Legionella pneumophila, can lead to pyroptosis. Pyroptosis is associated with the occurrence and development of various diseases caused by microbial infection, and the identification of molecules related to the pyroptosis signaling pathway may provide new drug targets for the treatment of related diseases. This study reviews the molecular mechanisms of pyroptosis and the role of pyroptosis in microbial infection-related diseases.

miR-31 shuttled by halofuginone-induced exosomes suppresses MFC-7 cell proliferation by modulating the HDAC2/cell cycle signaling axis.

Traditional Chinese medicine (TCM) are both historically important therapeutic agents and important source of new drugs. Halofuginone (HF), a small molecule alkaloid derived from febrifugine, has been shown to exert strong antiproliferative effects that differ markedly among various cell lines. However, whether HF inhibits MCF-7 cell growth in vitro and underlying mechanisms of this process are not yet clear. Here, we offer the strong evidence of the connection between HF treatment, exosome production and proliferation of MCF-7 cells. Our results showed that HF inhibits MCF-7 cell growth in both time- and dose-dependent manner. Further microRNA (miRNA) profiles analysis in HF treated and nontreated MCF-7 cell and exosomes observed that six miRNAs are particularly abundant and sorted in exosomes. miRNAs knockdown experiment in exosomes and the MCF-7 growth inhibition assay showed that exosomal microRNA-31 (miR-31) modulates MCF-7 cells growth by specially targeting the histone deacetylase 2 (HDAC2), which increases the levels of cyclin-dependent kinases 2 (CDK2) and cyclin D1 and suppresses the expression of p21. In conclusion, these data indicate that inhibition of exosome production reduces exosomal miR-31, which targets the HDAC2 and further regulates the level of cell cycle regulatory proteins, contributing to the anticancer functions of HF. Our data suggest a new role for HF and the exosome production in tumorigenesis and may provide novel insights into prevention and treatment of breast cancer.

Highly Sensitive Detection of PCV2 Based on Tyramide Signals and GNPL Amplification.

The frequent emergence of secondary infection and immunosuppression after porcine circovirus type 2 (PCV2) infection highlights the need to develop sensitive detection methods. A dual-signal amplification enzyme-linked immunosorbent assay (ELISA) based on a microplate coated with gold nanoparticle layers (GNPL) and tyramide signal amplification (TSA) was established. Results confirmed that the microplates coated with GNPL have a strong binding ability to the antibody without affecting the biological activity of the antibody. The microplates coated with GNPL have strong binding ability to the antibody, and the amplification of the tyramide signal is combined to further improve the sensitivity of PCV2. The PCV2 antibody does not crossreact with other viruses, demonstrating that the method has good specificity. A dual-signal amplification strategy is developed using microplates modified with GNPL and TSA to sensitively detect PCV2.

Inhibitory Effects of Antimicrobial Peptide JH-3 on Salmonella enterica Serovar Typhimurium Strain CVCC541 Infection-Induced Inflammatory Cytokine Release and Apoptosis in RAW264.7 Cells.

The antibiotic resistance of Salmonella has become increasingly serious due to the increased use of antibiotics, and antimicrobial peptides have been considered as an ideal antibiotic alternative. Salmonella can induce macrophage apoptosis and thus further damage the immune system. The antimicrobial peptide JH-3 has been shown to have a satisfactory anti-Salmonella effect in previous research, but its mechanism of action remains unknown. In this study, the effects of JH-3 on macrophages infected with Salmonella Typhimurium CVCC541 were evaluated at the cellular level. The results showed that JH-3 significantly alleviated the damage to macrophages caused by S. Typhi infection, reduced the release of lactic dehydrogenase (LDH), and killed the bacteria in macrophages. In addition, JH-3 decreased the phosphorylation level of p65 and the expression and secretion of interleukin 2 (IL-2), IL-6, and tumor necrosis factor-α (TNF-α) by inhibiting the activation of the mitogen-activated protein kinase (MAPK) (p38) signaling pathway and alleviating the cellular inflammatory response. From confocal laser scanning microscopy and flow cytometry assays, JH-3 was observed to inhibit the release of cytochrome c in the cytoplasm; the expression of TNF-αR2, caspase-9, and caspase-8; to further weaken caspase-3 activation; and to reduce the S.-Typhi-induced apoptosis of macrophages. In summary, the mechanism by which JH-3 inhibits Salmonella infection was systematically explored at the cellular level, laying the foundation for the development and utilization of JH-3 as a therapeutic alternative to antibiotics.

Halofuginone-induced autophagy suppresses the migration and invasion of MCF-7 cells via regulation of STMN1 and p53.

Traditional Chinese medicines have been recognized as especially promising anticancer agents in modern anticancer research. Halofuginone (HF), an analog of quinazolinone alkaloid extracted from Dichroa febrifuga, is widely used in traditional medicine. However, whether HF inhibits the growth of breast cancer cells and/or reduces the migration and invasion of MCF-7 human breast cancer cells, as well as the underlying mechanisms in vitro, remains unclear. In this study, we report that an HF extract inhibits the growth of MCF-7 cells and reduces their migration and invasion, an important feature of potential anticancer agents. In addition, HF significantly increases the activation of autophagy, which is closely associated with tumor metastasis. As STMN1 and p53 have been closely implicated in breast cancer progression, we analyzed their expression in the context of HF extract treatment. Western blot analysis showed that HF suppresses STMN1 and p53 expression and activity in an autophagy-dependent manner. Collectively, these data indicate that activation of autophagy reduces expression of STMN1 and p53, and the migration and invasion of cancer cells contributes to the anti-cancer effects of the HF. These findings may provide new insight into breast cancer prevention and therapy.

Methods for the detection and characterization of Streptococcus suis: from conventional bacterial culture methods to immunosensors.

One of the most important zoonotic pathogens worldwide, Streptococcus suis is a swine pathogen that is responsible for meningitis, toxic shock and even death in humans. S. suis infection develops rapidly with nonspecific clinical symptoms in the early stages and a high fatality rate. Recently, much attention has been paid to the high prevalence of S. suis as well as the increasing incidence and its epidemic characteristics. As laboratory-acquired infections of S. suis can occur and it is dangerous to public health security, timely and early diagnosis has become key to controlling S. suis prevalence. Here, the techniques that have been used for the detection, typing and characterization of S. suis are reviewed and the prospects for future detection methods for this bacterium are also discussed.

The role of natural antimicrobial peptides during infection and chronic inflammation.

Natural antimicrobial peptides (AMPs), a family of small polypeptides that are produced by constitutive or inducible expression in organisms, are integral components of the host innate immune system. In addition to their broad-spectrum antibacterial activity, natural AMPs also have many biological activities against fungi, viruses and parasites. Natural AMPs exert multiple immunomodulatory roles that may predominate under physiological conditions where they lose their microbicidal properties in serum and tissue environments. Increased drug resistance among microorganisms is occurring far more quickly than the discovery of new antibiotics. Natural AMPs have shown promise as 'next generation antibiotics' due to their broad-spectrum curative effects, low toxicity, the fact that they are not residual in animals, and the low rates of resistance exhibited by many pathogens. Many types of synthetic AMPs are currently being tested in clinical trials for the prevention and treatment of various diseases such as chemotherapy-associated infections, diabetic foot ulcers, catheter-related infections, and other conditions. Here, we provide an overview of the types and functions of natural AMPs and their role in combating microorganisms and different infectious and inflammatory diseases.

HJH-1, a Broad-Spectrum Antimicrobial Activity and Low Cytotoxicity Antimicrobial Peptide.

With the overuse of antibiotics, multidrug-resistant bacteria pose a significant threat to human health. Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. This study examines the antimicrobial and membrane activity of HJH-1, a cationic peptide derived from the hemoglobin α-subunit of bovine erythrocytes P3. HJH-1 shows potent antimicrobial activity against different bacterial species associated with infection and causes weaker hemolysis of erythrocytes, at least five times the minimum inhibitory concentration (MIC). HJH-1 has good stability to tolerance temperature, pH value, and ionic strength. The anionic membrane potential probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] and propidium iodide are used as indicators of membrane integrity. In the presence of HJH-1 (1× MIC), Escherichiacoli membranes rapidly depolarise, whereas red blood cells show gradual hyperpolarisation. Scanning electron microscopy and transmission electron micrographs show that HJH-1 (1× MIC) damaged the membranes of Escherichia coli, Staphylococcus aureus, and Candida albicans. In conclusion, HJH-1 damages the integrity of the bacterial membrane, preventing the growth of bacteria. HJH-1 has broad-spectrum antibacterial activity, and these activities are performed by changing the normal cell transmembrane potential and disrupting the integrity of the bacterial membrane.

M protein is sufficient for assembly and release of Peste des petits ruminants virus-like particles.

Peste des petits ruminants virus (PPRV), belonging to paramyxoviruses, has six structure proteins (such as matrix protein (M), nucleocapsid proteins (N), fusion protein (F) and hemagglutinin protein (H)) and could cause high morbidity and mortality in sheep and goats. Although a vaccine strain of PPRV has been rescued and co-expression of M and N could yield PPRV-like particles, the roles of structure proteins in virion assembly and release have not been investigated in detail. In this study, plasmids carrying PPRV cDNA sequences encoding the N, M, H, and F proteins were expressed in Vero cells. The co-expression of all four proteins resulted in the release of virus-like particles (VLPs) with similar release efficiency to that of authentic virions. Moreover, the co-expression of M together with F also resulted in efficient VLPs release. In the absence of M protein, the expression of no combination of the other proteins resulted in particle release. In summary, a VLPs production system for PPRV has been established and M protein is necessary for promoting the assembly and release of VLPs, of which the predominant protein is M protein. Further study will be focused on the immunogenicity of the VLPs.

Development of an Indirect Dot-PPA-ELISA using glutamate dehydrogenase as a diagnostic antigen for the rapid and specific detection of Streptococcus suis and its application to clinical specimens.

Streptococcus suis is an important zoonotic pathogen causing infections in pigs and humans. Bacterial surface-related proteins are often explored as potential vaccine candidates and diagnostic antigens. In the present study, glutamate dehydrogenase, a highly conserved immunogenic extracellular protein, was used to establish a dot horseradish peroxidase enzyme-linked staphylococcal protein A immunosorbent assay (Dot-PPA-ELISA) for diagnosis of S. suis infection. The antigen-antibody reaction was optimised through checkerboard titration involving serial dilutions, followed by selective blocking tests and evaluations of cross-reaction, repeatability, and stability. Comparative analysis by using a conventional plate ELISA kit showed that the specificity and sensitivity of the Dot-PPA-ELISA were 97.5 and 96.6%, respectively. Furthermore, dynamic changes in the levels of antibody in rabbits immunised with a propolis inactivated vaccine were monitored by Dot-PPA-ELISA. A total seroprevalence of 73.1% in 305 pig serum samples indicated the method's applicability to detect S. suis infection. Cumulatively, the results suggested that Dot-PAA-ELISA is a convenient, rapid, sensitive, and specific diagnostic method suitable for studying large numbers of samples obtained from clinical and epidemiological studies, thereby helping reduce important economic losses.

Potential of novel antimicrobial peptide P3 from bovine erythrocytes and its analogs to disrupt bacterial membranes in vitro and display activity against drug-resistant bacteria in a mouse model.

With the emergence of many antibiotic-resistant strains worldwide, antimicrobial peptides (AMPs) are being evaluated as promising alternatives to conventional antibiotics. P3, a novel hemoglobin peptide derived from bovine erythrocytes, exhibited modest antimicrobial activity in vitro. We evaluated the antimicrobial activities of P3 and an analog, JH-3, both in vitro and in vivo. The MICs of P3 and JH-3 ranged from 3.125 µg/ml to 50 µg/ml when a wide spectrum of bacteria was tested, including multidrug-resistant strains. P3 killed bacteria within 30 min by disrupting the bacterial cytoplasmic membrane and disturbing the intracellular calcium balance. Circular dichroism (CD) spectrometry showed that P3 assumed an α-helical conformation in bacterial lipid membranes, which was indispensable for antimicrobial activity. Importantly, the 50% lethal dose (LD50) of JH-3 was 180 mg/kg of mouse body weight after intraperitoneal (i.p.) injection, and no death was observed at any dose up to 240 mg/kg body weight following subcutaneous (s.c.) injection. Furthermore, JH-3 significantly decreased the bacterial count and rescued infected mice in a model of mouse bacteremia. In conclusion, P3 and an analog exhibited potent antimicrobial activities and relatively low toxicities in a mouse model, indicating that they may be useful for treating infections caused by drug-resistant bacteria.

FADD promotes type I interferon production to suppress porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) is an epidemic animal infectious disease worldwide, causing huge economic losses to the global swine industry. Fas-associated death domain (FADD) was previously reported to be an adaptor protein that functions in transferring the apoptotic signals regulated by the death receptors. In the current study, we unravel its unidentified role in promoting type I interferon (IFN) production during PRRS virus (PRRSV) infection. We identified that FADD inhibited PRRSV infection via promotion of type I IFN transcription. Overexpression of FADD suppressed the replication of PRRSV, while knockout of FADD increased viral titer and nucleocapsid protein expression. Mechanistically, FADD promoted mitochondrial antiviral signaling protein (MAVS)-mediated production of IFN-ß and some IFN-stimulated genes (ISGs). Furthermore, FADD exerted anti-PRRSV effects in a MAVS-dependent manner and increased the type I IFN signaling during PRRSV infection. This study highlights the importance of FADD in PRRSV replication, which may have implications for the future control of PRRS.

Occurrence and genotyping of Enterocytozoon bieneusi in flying squirrels (Trogopterus xanthipes) from China.

Enterocytozoon bieneusi is an obligate intracellular microsporidian parasite with a worldwide distribution. As a zoonotic pathogen, E. bieneusi can infect a wide range of wildlife hosts through the fecal-oral route. Although the feces of flying squirrels (Trogopterus xanthipes) are considered a traditional Chinese medicine (as "faeces trogopterori"), no literature is available on E. bieneusi infection in flying squirrels to date. In this study, a total of 340 fresh flying squirrel fecal specimens from two captive populations were collected in Pingdingshan city, China, to detect the prevalence of E. bieneusi and assess their zoonotic potential. By nested PCR amplification of the ITS gene, six specimens tested positive, with positive samples from each farm, with an overall low infection rate of 1.8%. The ITS sequences revealed three genotypes, including known genotype D and two novel genotypes, HNFS01 and HNFS02. Genotype HNFS01 was the most prevalent (4/6, 66.7%). Phylogenetic analysis showed that all genotypes clustered into zoonotic Group 1, with the novel genotypes clustering into different subgroups. To our knowledge, this is the first report of E. bieneusi infection in flying squirrels, suggesting that flying squirrels could act as a potential reservoir and zoonotic threat for E. bieneusi transmission to humans in China.

Title: Occurrence et génotypage d'Enterocytozoon bieneusi chez les écureuils volants (Trogopterus xanthipes) de Chine. Abstract: Enterocytozoon bieneusi est un parasite microsporidien intracellulaire obligatoire présent dans le monde entier. En tant qu'agent pathogène zoonotique, E. bieneusi peut infecter un large éventail d'hôtes sauvages par la voie fécale-orale. Bien que les excréments d'écureuils volants (Trogopterus xanthipes) soient considérés comme un ingrédient de médecine traditionnelle chinoise (comme « faeces trogopterori ¼), aucune littérature n'est disponible à ce jour sur l'infection par E. bieneusi chez les écureuils volants. Dans cette étude, un total de 340 spécimens fécaux frais d'écureuils volants provenant de deux populations captives ont été collectés dans la ville de Pingdingshan, en Chine, pour détecter la prévalence d'E. bieneusi et évaluer leur potentiel zoonotique. Par amplification PCR nichée du gène ITS, six échantillons se sont révélés positifs, avec des échantillons positifs dans chaque ferme, et un taux d'infection global faible, à 1,8 %. Les séquences ITS ont révélé trois génotypes, dont le génotype D connu et deux nouveaux génotypes, HNFS01 et HNFS02. Le génotype HNFS01 était le plus répandu (4/6, 66,7 %). L'analyse phylogénétique a montré que tous les génotypes se regroupaient dans le groupe zoonotique 1, les nouveaux génotypes se regroupant en différents sous-groupes. À notre connaissance, il s'agit du premier rapport d'infection par E. bieneusi chez des écureuils volants, ce qui suggère que les écureuils volants pourraient agir comme un réservoir potentiel et une menace zoonotique pour la transmission d'E. bieneusi aux humains en Chine.

Rhein kills Actinobacillus pleuropneumoniae, reduces biofilm formation, and effectively treats bacterial lung infections in mice.

Background. Actinobacillus pleuropneumoniae, a member of the Pasteurellaceae family, is known for its highly infectious nature and is the primary causative agent of infectious pleuropneumonia in pigs. This disease poses a considerable threat to the global pig industry and leads to substantial economic losses due to reduced productivity, increased mortality rates, and the need for extensive veterinary care and treatment. Due to the emergence of multi-drug-resistant strains, Chinese herbal medicine is considered one of the best alternatives to antibiotics due to its unique mechanism of action and other properties. As a type of Chinese herbal medicine, Rhein has the advantages of a wide antibacterial spectrum and is less likely to develop drug resistance, which can perfectly solve the limitations of current antibacterial treatments.Methods. The killing effect of Rhein on A. pleuropneumoniae was detected by fluorescence quantification of differential expression changes of key genes, and scanning electron microscopy was used to observe the changes in A. pleuropneumoniae status after Rhein treatment. Establishing a mouse model to observe the treatment of Rhein after A. pleuropneumoniae infection.Results. Here, in this study, we found that Rhein had a good killing effect on A. pleuropneumoniae and that the MIC was 25 µg ml-1. After 3 h of action, Rhein (4×MIC) completely kills A. pleuropneumoniae and Rhein has good stability. In addition, the treatment with Rhein (1×MIC) significantly reduced the formation of bacterial biofilms. Therapeutic evaluation in a murine model showed that Rhein protects mice from A. pleuropneumoniae and relieves lung inflammation. Quantitative RT-PCR (Quantitative reverse transcription polymerase chain reaction is a molecular biology technique that combines both reverse transcription and polymerase chain reaction methods to quantitatively detect the amount of a specific RNA molecule) results showed that Rhein treatment significantly downregulated the expression of the IL-18 (Interleukin refers to a class of cytokines produced by white blood cells), TNF-α, p65 and p38 genes. Along with the downregulation of genes such as IL-18, it means that Rhein has an inhibitory effect on the expression of these genes, thereby reducing the activation of inflammatory cells and the production of inflammatory mediators. This helps reduce inflammation and protects tissue from further damage.Conclusions. This study reports the activity of Rhein against A. pleuropneumoniae and its mechanism, and reveals the ability of Rhein to treat A. pleuropneumoniae infection in mice, laying the foundation for the development of new drugs for bacterial infections.

Recombinant Antimicrobial Peptide OaBac5mini Alleviates Inflammation in Pullorum Disease Chicks by Modulating TLR4/MyD88/NF-κB Pathway.

Pullorum disease (PD), caused by Salmonella Pullorum (S. Pullorum), is a serious threat to the poultry industry worldwide. Antimicrobial peptides (AMPs) have drawn extensive attention as new-generation antibiotics because of their broad antimicrobial spectrum, low resistance, and low cytotoxicity. AMP OaBac5mini exhibits strong antibacterial activity against Gram-negative bacteria, but its efficacy and anti-inflammatory effects on chicks with PD remain unclear. The aim of this study was to generate recombinant OaBac5mini via the Escherichia coli (E. coli) recombinant expression system and evaluate its antibacterial effect against S. Pullorum in vitro and in vivo. Real-time cellular analysis (RTCA) results showed that recombinant OaBac5mini exhibited no cytotoxicity on IPEC-J2 and RAW 264.7 cells and significantly alleviated the drop in the cell index of S. Pullorum-infected cells (p < 0.0001). In the chick model of PD, recombinant OaBac5mini significantly attenuated the increase in organ indexes (heart, liver, spleen, and kidney) and bacterial loads (liver and spleen) induced by S. Pullorum. Histopathology examination showed that recombinant OaBac5mini ameliorated histopathological changes and inflammation in chicks with PD, including impaired epithelium of duodenal villi, infiltration of pseudoacidophilic granulocytes in the cecum and bursa of Fabricius, congested blood clots and increased macrophages in the liver, and increased lymphoid nodule and B lymphocytes in the spleen. Western blot and quantitative real-time PCR (qRT-PCR) results indicated that recombinant OaBac5mini alleviated inflammation by modulating innate immunity through the TLR4/MyD88/NF-κB pathway and by suppressing the expression of pro-inflammatory cytokines. These results suggested that recombinant OaBac5mini has good potential as a clinical substitute for antibiotics in PD intervention.