The inhibition effect of caffeic acid on NOX/ROS-dependent macrophages M1-like polarization contributes to relieve the LPS-induced mice mastitis.

The mammary gland is an adipose tissue containing not only adipocytes but also epithelial, endothelial, and immune cells. Epithelial cells and macrophages, as the integral components of the immune system, are on the front line of defense against infection. Our preliminary work proved that caffeic acid (CA) can effectively inhibit the inflammatory cascade of bovine mammary epithelial cells (BMEC) induced by lipopolysaccharide (LPS) and maintain cellular integrity and viability. Here, we investigated the therapeutic effect of CA on LPS-induced mice mastitis and explored its regulatory mechanism on macrophage inflammatory response induced by LPS in vitro. Firstly, the mice mastitis model was established by intramammary injection with 10 µg LPS, after which different CA doses (5, 10, 15 mg/kg) were administered. Then, the pathological section, myeloperoxidase (MPO) activity, proinflammatory factors and chemokines releasement, and redox state of mammary tissues were assessed, confirming CA's effectiveness on mice mastitis. In vitro, we validated the therapeutic relevance of CA in relieving LPS-induced RAW264.7 inflammatory and oxidative stress responses. Moreover, we further provided evidence that CA significantly reduced LPS-induced reactive oxygen species (ROS) generation via NADPH oxidase (NOX), which improved the imbalance relationship between nuclear factor kappa-B (NF-κB) and NF-E2 p45-related factor 2 (Nrf2) and led to a marked weakening of M1 polarization. The NOX-ROS signal inhibited by CA weakened the oxidative burst and neutrophil chemotaxis of macrophages, thus alleviating the immune cascade in mammary gland tissue and reducing the LPS-induced inflammatory damage. Collectively, CA would be a potential candidate or antibacterial synergist for curbing mastitis.

Polysaccharide from Atractylodes macrocephala Koidz Binding with Zinc Oxide Nanoparticles as a Novel Mucosal Immune Adjuvant for H9N2 Inactivated Vaccine.

H9N2 avian influenza poses a significant public health risk, necessitating effective vaccines for mass immunization. Oral inactivated vaccines offer advantages like the ease of administration, but their efficacy often requires enhancement through mucosal adjuvants. In a previous study, we established a novel complex of polysaccharide from Atractylodes macrocephala Koidz binding with zinc oxide nanoparticles (AMP-ZnONPs) and preliminarily demonstrated its immune-enhancing function. This work aimed to evaluate the efficacy of AMP-ZnONPs as adjuvants in an oral H9N2-inactivated vaccine and the vaccine's impact on intestinal mucosal immunity. In this study, mice were orally vaccinated on days 0 and 14 after adapting to the environment. AMP-ZnONPs significantly improved HI titers, the levels of specific IgG, IgG1 and IgG2a in serum and sIgA in intestinal lavage fluid; increased the number of B-1 and B-2 cells and dendritic cell populations; and enhanced the mRNA expression of intestinal homing factors and immune-related cytokines. Interestingly, AMP-ZnONPs were more likely to affect B-1 cells than B-2 cells. AMP-ZnONPs showed mucosal immune enhancement that was comparable to positive control (cholera toxin, CT), but not to the side effect of weight loss caused by CT. Compared to the whole-inactivated H9N2 virus (WIV) group, the WIV + AMP-ZnONP and WIV + CT groups exhibited opposite shifts in gut microbial abundance. AMP-ZnONPs serve as an effective and safe mucosal adjuvant for oral WIV, improving cellular, humoral and mucosal immunity and microbiota in the gastrointestinal tract, avoiding the related undesired effects of CT.

Laponite/lactoferrin hydrogel loaded with eugenol for methicillin-resistant Staphylococcus aureus-infected chronic skin wound healing.

Severe bacterial infections can give rise to protracted wound healing processes, thereby posing a significant risk to a patient's well-being. Consequently, the development of a versatile hydrogel dressing possessing robust bioactivity becomes imperative, as it holds the potential to expedite wound healing and yield enhanced clinical therapeutic outcomes. In this context, the present study involves the formulation of an injectable multifunctional hydrogel utilizing laponite (LAP) and lactoferrin (LF) as foundational components and loaded with eugenol (EG). This hydrogel is fabricated employing a straightforward one-pot mixing approach that leverages the principle of electrostatic interaction. The resulting LAP/LF/EG2% composite hydrogel can be conveniently injected to address irregular wound geometries effectively. Once administered, the hydrogel continually releases lactoferrin and eugenol, mitigating unwarranted oxidative stress and eradicating bacterial infections. This orchestrated action culminates in the acceleration of wound healing specifically in the context of MRSA-infected wounds. Importantly, the LAP/LF/EG2% hydrogel exhibits commendable qualities including exceptional injectability, potent antioxidant attributes, and proficient hemostatic functionality. Furthermore, the hydrogel composition notably encourages cellular migration while maintaining favorable cytocompatibility. Additionally, the hydrogel manifests noteworthy bactericidal efficacy against the formidable multidrug-resistant MRSA bacterium. Most significantly, this hydrogel formulation distinctly expedites the healing of MRSA-infected wounds by promptly inducing hemostasis, curbing bacterial proliferation, and fostering angiogenesis, collagen deposition, and re-epithelialization processes. As such, the innovative hydrogel material introduced in this investigation emerges as a promising dressing for the facilitation of bacterial-infected wound healing and consequent tissue regeneration.

Eugenol alleviates Salmonella Typhimurium-infected cecal injury by modulating cecal flora and tight junctions accompanied by suppressing inflammation.

BACKGROUND: Salmonella enterica serovar Typhimurium (ST) mainly exists in poultry and poultry related products, which are common sources of human salmonellosis. So, ST is an important zoonotic pathogen that threatens public health and safety. Eugenol has been noted for its antibacterial and anti-inflammatory properties, and it is expected to develop into an antibacterial therapy in vivo. METHODS: Herein, the effects of eugenol against ST infection in terms of intestinal flora, cecal tight junction, and cecal inflammation in broilers was evaluated in this study. RESULTS: The results showed that oral eugenol pretreatment obviously relieved the histopathological changes and ultrastructural injury of cecum caused by ST infection. Further analysis found that eugenol lessened the number of ST in the cecum, restrained Proteobacteria and Ruminococcus, and kept the ratio of Firmicutes to Bacteroidetes (F/B), improved the relative abundance of Alistipes compared to the infection control. Moreover, eugenol sustained the expression of ZO-1, claudin-1, and occluding tight junction proteins, reduced the mRNA levels of myeloid differentiation factor 88 (MyD88), toll-like receptor-4 (TLR4) and inducible nitric oxide synthesis (iNOS), suppressed the phosphorylation of p65 and IκBα in the NF-κB signaling pathway, as well as the mRNA levels of TNF-α, IL-1ß, IL-2, and IL-18 in the cecum tissue. CONCLUSION: Taken together, eugenol could protect broilers against ST infection via maintaining intestinal flora, tight junctions involved in mucosal barrier function, then accordingly limiting the excessive inflammation, finally contributed to improving relative weight gains and survival rates of broilers.

The synergy of tea tree oil nano-emulsion and antibiotics against multidrug-resistant bacteria.

AIMS: We determined the synergistic effects of tea tree essential oil nano-emulsion (nanoTTO) and antibiotics against multidrug-resistant (MDR) bacteria in vitro and in vivo. Then, the underlying mechanism of action of nanoTTO was investigated. METHODS AND RESULTS: Minimum inhibitory concentrations and fractional inhibitory concentration index (FICI) were determined. The transepithelial electrical resistance (TEER) and the expression of tight junction (TJ) protein of IPEC-J2 cells were measured to determine the in vitro efficacy of nanoTTO in combination with antibiotics. A mouse intestinal infection model evaluated the in vivo synergistic efficacy. Proteome, adhesion assays, quantitative real-time PCR, and scanning electron microscopy were used to explore the underlying mechanisms. Results showed that nanoTTO was synergistic (FICI ≤ 0.5) or partial synergistic (0.5 < FICI < 1) with antibiotics against MDR Gram-positive and Gram-negative bacteria strains. Moreover, combinations increased the TEER values and the TJ protein expression of IPEC-J2 cells infected with MDR Escherichia coli. The in vivo study showed that the combination of nanoTTO and amoxicillin improved the relative weight gain and maintained the structural integrity of intestinal barriers. Proteome showed that type 1 fimbriae d-mannose specific adhesin of E. coli was downregulated by nanoTTO. Then, nanoTTO reduced bacterial adhesion and invasion and inhibited the mRNA expression of fimC, fimG, and fliC, and disrupted bacterial membranes.

Programmable Bispecific Nano-immunoengager That Captures T Cells and Reprograms Tumor Microenvironment.

Immune checkpoint blockade (ICB) therapy has revolutionized clinical oncology. However, the efficacy of ICB therapy is limited by the ineffective infiltration of T effector (Teff) cells to tumors and the immunosuppressive tumor microenvironment (TME). Here, we report a programmable tumor cells/Teff cells bispecific nano-immunoengager (NIE) that can circumvent these limitations to improve ICB therapy. The peptidic nanoparticles (NIE-NPs) bind tumor cell surface α3ß1 integrin and undergo in situ transformation into nanofibrillar network nanofibers (NIE-NFs). The prolonged retained nanofibrillar network at the TME captures Teff cells via the activatable α4ß1 integrin ligand and allows sustained release of resiquimod for immunomodulation. This bispecific NIE eliminates syngeneic 4T1 breast cancer and Lewis lung cancer models in mice, when given together with anti-PD-1 antibody. The in vivo structural transformation-based supramolecular bispecific NIE represents an innovative class of programmable receptor-mediated targeted immunotherapeutics to greatly enhance ICB therapy against cancers.

Eugenol exposure in vitro inhibits the expressions of T3SS and TIF virulence genes in Salmonella Typhimurium and reduces its pathogenicity to chickens.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a common food-borne pathogen, which has the ability to infect a wide range of hosts. The increasing emergence of drug-resistant strains urgently requires new alternative therapies. Eugenol has been shown to be very effective against drug-resistant strains of Gram-negative and Gram-positive bacteria. The purpose of this study is to explore the effects of eugenol on the virulence factors and pathogenicity of S. Typhimurium. METHODS: The antibacterial activity of eugenol was investigated via the changes of cell morphology, fimbriae related-genes and virulence factors of S. Typhimurium, then the pathogenicity of S. Typhimurium pretreated by eugenol to chickens was evaluated. RESULTS: Susceptibility testing showed that eugenol possessed significant antimicrobial activity. Scanning electron microscope analysis showed eugenol treatment deformed the morphology with damaged fimbriae structure of S. Typhimurium. Real time PCR assay confirmed eugenol significantly down-regulated the expressions of virulence factors (hilA, hilD, sipA, sipC, spiC, misL) of Type III secretion system (T3SS) and adherence genes (fimA, fimH, fimD, fimY, fimZ, stm0551) of Type I fimbriae (TIF). Animal experiment proved that the pathogenicity of S. Typhimurium exposed by eugenol was reduced, which was evidenced by the higher survival rate, weight gains and organs indexes, the lower bacterial loads in organs. Meanwhile, the duodenal histopathological changes were mitigated, with a significantly decline in the expressions of TNF-α, IL-6 and IL-18. CONCLUSION: In summary, eugenol pretreatment may alleviate the pathogenicity of the S. Typhimurium to chickens via wrecking the fimbriae and inhibiting the mRNA expressions of virulence factors and adhesion molecules. These data dedicated the potential mechanisms of eugenol against S. Typhimurium in vitro.

Bactericidal activity of gallic acid against multi-drug resistance Escherichia coli.

The continuous emergence of multidrug-resistant (MDR) bacteria has posed an increasingly serious public health threat which urges people to develop some alternatives. Gallic acid (GA) is a natural ingredient in many traditional Chinese medicines, which has many biological activities, such as antibacterial, and antiseptic. Here, clinical isolates of MDR Escherichia coli (E. coli) were used to evaluate the antibacterial effect of GA and the underlying mechanism. The results revealed that GA exerted bactericidal activity and inhibited the formation of bacterial biofilm. GA enhanced the activities of ceftiofur sodium or tetracycline against E. coli, and facilitated antibiotic accumulation in bacteria. Further analysis of morphological alterations and efflux pump gene expressions confirmed that GA damaged outer and inner membranes, and suppressed the mRNA expressions of acrA, acrB, tolC, acrD and acrF involved in membrane permeability. In addition, GA showed protective effects against bacterial infection and improved the survival rates of Galleria mellonella and BALB/c mice. These data highlight a better understanding of GA against bacteria and provide an alternative strategy for MDR bacterial infection.

Ferulic acid inhibits LPS-induced apoptosis in bovine mammary epithelial cells by regulating the NF-κB and Nrf2 signalling pathways to restore mitochondrial dynamics and ROS generation.

In bovine mammary epithelial cells (BMECs), a cascade of inflammatory reactions induced by lipopolysaccharide (LPS) has been shown to result in cell injury and apoptosis. The present study aims to reveal the protective effect of ferulic acid (FA) on LPS-induced BMEC apoptosis and explore its potential molecular mechanisms. First, we showed that FA had low cytotoxicity to BMECs and significantly decreased cell apoptosis and the proinflammatory response induced by LPS. Next, FA blocked LPS-induced oxidative stress by restoring the balance of the redox state and inhibiting mitochondrial dysfunction, the main contributor to LPS-induced apoptosis and ROS generation. Furthermore, the relief of inflammation and redox disturbance in the FA preconditioning group were accompanied by weaker NF-κB activation, enhanced Nrf2 activation and maintained cell viability compared to the LPS group. When BMECs were treated with FA alone, we observed that Nrf2 activation was induced before the inhibition of NF-κB activation and that the Keap1-Nrf2 relationship was disturbed. We concluded that FA prevented LPS-induced BMEC apoptosis by reversing the dominant relationship between NF-κB and Nrf2.

Characterization of tea tree oil nanoemulsion and its acute and subchronic toxicity.

Tea tree oil (TTO) is a popular topical use to treat skin infections. However, its poor aqueous solubility and stability have substantially limited its widespread application, including oral administration that might be therapeutic for enteric infections. In this study, mechanical ultrasonic methods were used to prepare TTO nanoemulsion (nanoTTO) with a mean droplet diameter of 161.80 nm ± 3.97, polydispersity index of 0.21 ± 0.01, and zeta potential of -12.33 ± 0.72 mV. The potential toxicity of nanoTTO was assessed by studying the oral median lethal dose (LD50) and repeated 28-day oral toxicity to provide a reference for in vivo application. Results showed that nanoTTO had no phase separation under a centrifugation test and displayed good stability during storage at -20, 4 and 25 °C over 60 days. Repeated-dose 28-day oral toxicity evaluation revealed no significant effects on growth and behavior. Assessments of hematology, clinical biochemistry, and histopathology indicated no obvious adverse effects in mice at 50, 100 and 200 mg/mL. These data suggest that nanoTTO can be considered a potential antimicrobial agent by oral administration due to its inhibitory effect on bacteria and relatively lower toxicity.

Rational Design of PLGA Nanoparticle Vaccine Delivery Systems To Improve Immune Responses.

Nanoparticle-based vaccine delivery systems have been extensively used to promote and induce immune responses to protein antigens. The properties of the nanoparticles, such as size, surface charge, and antigen loading mode, have been proved to significantly influence the adjuvant effect and immunoreactivity of nanoparticle-based vaccine delivery systems. The purpose of the study was to investigate how the surface charge and antigen loading mode of nanoparticles impact the immune responses. In this study, three ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles with different surface charges and antigen loading modes were developed. The three nanoparticles were designed as antigen encapsulated with negatively charged (Angelica sinensis polysaccharide (ASP)-PLGA/OVA), antigen encapsulated with polyethylenimine (PEI)-coated (ASP-PLGA/OVA-PEI), and antigen adsorbed on PEI-coated (ASP-PLGA-PEI-OVA) nanoparticles. The Angelica sinensis polysaccharide (ASP) was used as the immunopotentiator and encapsulated into three nanoparticles. The results demonstrated that both PEI-coated (positively charged) nanoparticles promoted the antigen escape from the endosome, which led to the cytoplasmic antigen delivery to generate cross presentation, compared to negatively charged nanoparticles. In addition, PEI-coated nanoparticles activated the DCs in lymph nodes 5 days after the primary vaccination. In vivo experiments demonstrated that both antigen-encapsulated nanoparticles induced more potent and long-term antigen-specific antibody responses, compared to that of antigen-adsorbed nanoparticles. Thus, the PEI-coated and antigen-encapsulated nanoparticles (ASP-PLGA/OVA-PEI) as a vaccine adjuvant delivery system have the potential to induce strong and long-term humoral and cellular immune responses.

Chitosan-coated artesunate protects against ulcerative colitis via STAT6-mediated macrophage M2 polarization and intestinal barrier protection.

Oral delivery of chitosan-coated artesunate (CPA) has been proven to be effective at preventing ulcerative colitis (UC) in mice. However, the anti-inflammatory mechanism is not fully understood. STAT6 is a key transcription factor that promotes anti-inflammatory effects by inducing M2 and Th2 dominant phenotypes, therefore we hypothesized STAT6 might play a key role in the process. To prove it, a STAT6 gene knockout macrophage cell line (STAT6-/- RAW264.7, by CRISPR/Cas9 method), and its corresponding Caco-2/RAW264.7 co-culture system combined with the STAT6 inhibitor (AS1517499, AS) in a mouse UC model were established and studied. The results showed that CPA remarkably suppressed the activation of TLR-4/NF-κB pathway and the mRNA levels of proinflammatory cytokines, while increased the IL-10 levels in RAW264.7. This effect of CPA contributed to the protection of the ZO-1 in Caco-2 which was disrupted upon the stimulation to macrophages. Simultaneously, CPA reduced the expression of CD86 but increase the expression of CD206 and p-STAT6 in LPS-stimulated RAW264.7 cells. However, above alterations were not obvious as in STAT6-/- RAW264.7 and its co-culture system, suggesting STAT6 plays a key role. Furthermore, CPA treatment significantly inhibited TLR-4/NF-κB activation, intestinal macrophage M1 polarization and mucosal barrier injury induced by DSS while promoted STAT6 phosphorylation in the UC mouse model, but this effect was also prominently counteracted by AS. Therefore, our data indicate that STAT6 is a major regulator in the balance of M1/M2 polarization, intestinal barrier integrity and then anti-colitis effects of CPA. These findings broaden our understanding of how CPA fights against UC and imply an alternative treatment strategy for UC via this pathway.

Mucosal immune responses and protective efficacy elicited by oral administration AMP-ZnONPs-adjuvanted inactivated H9N2 virus in chickens.

The avian influenza virus is infected through the mucosal route, thus mucosal barrier defense is very important. While the inactivated H9N2 vaccine cannot achieve sufficient mucosal immunity, adjuvants are needed to induce mucosal and systemic immunity to prevent poultry from H9N2 influenza virus infection. Our previous study found that polysaccharide from Atractylodes macrocephala Koidz binding with zinc oxide nanoparticles (AMP-ZnONPs) had immune-enhancing effects in vitro. This study aimed to evaluate the mucosal immune responses of oral whole-inactivated H9N2 virus (WIV)+AMP-ZnONPs and its impact on the animal challenge protection, and the corresponding changes of pulmonary metabolomics after the second immunization. The results showed that compared to the WIV, the combined treatment of WIV and AMP-ZnONPs significantly enhanced the HI titer, IgG and specific sIgA levels, the number of goblet cells and intestinal epithelial lymphocytes (iIELs) as well as the expression of J-chain, polymeric immunoglobulin receptor (pIgR), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß). In viral attack experiments, WIV combing with AMP-ZnONPs effectively reduced lung damage and viral titers in throat swabs. Interestingly, significant changes of both the IgA intestinal immune network and PPAR pathway could also be found in the WIV+AMP-ZnONPs group compared to the non-infected group. Taken together, these findings suggest that AMP-ZnONPs can serve as a potential mucosal vaccine adjuvant, thereby avoiding adverse stress and corresponding costs caused by vaccine injection.

Eugenol Possesses Colitis Protective Effects: Impacts on the TLR4/MyD88/NF-[Formula: see text]B Pathway, Intestinal Epithelial Barrier, and Macrophage Polarization.

Eugenol (EU) has been shown to ameliorate experimental colitis due to its anti-oxidant and anti-inflammatory bioactivities. In this study, DSS-induced acute colitis was established and applied to clarify the regulation efficacy of EU on intestinal barrier impairment and macrophage polarization imbalance along with the inflammatory response. Besides, the adjusting effect of EU on macrophages was further investigated in vitro. The results confirmed that EU intervention alleviated DSS-induced colitis through methods such as restraining weight loss and colonic shortening and decreasing DAI scores. Microscopic observation manifested that EU maintained the intestinal barrier integrity in line with the mucus barrier and tight junction protection. Furthermore, EU intervention significantly suppressed the activation of TLR4/MyD88/NF-[Formula: see text]B signaling pathways and pro-inflammatory cytokines gene expressions, while enhancing the expressions of anti-inflammatory cytokines. Simultaneously, WB and FCM analyses of the CD86 and CD206 showed that EU could regulate the DSS-induced macrophage polarization imbalance. Overall, our data further elucidated the mechanism of EU's defensive effect on experimental colitis, which is relevant to the protective efficacy of intestinal barriers, inhibition of oxidative stress and excessive inflammatory response, and reprogramming of macrophage polarization. Hence, this study may facilitate a better understanding of the protective action of the EU against UC.

Tea tree oil nanoliposomes: optimization, characterization, and antibacterial activity against Escherichia coli in vitro and in vivo.

The purpose of this study was to formulate tee tree oil nanoliposomes (TTONL) and evaluate its characterization and antibacterial activity. TTONL was prepared by thin film hydration and sonication technique, and the preparation conditions were optimized by Box-behnken response surface method. The characterization (morphology, size, zeta potential, and stability) and antibacterial activity of TTONL against Escherichia coli (E. coli) in vitro and in vivo were evaluated. The optimal preparation conditions for TTONL: lecithin to cholesterol mass ratio of 3.7:1, TTO concentration of 0.5%, and pH of the hydration medium of 7.4, which resulted in a TTONL encapsulation rate of 80.31 ± 0.56%. TTONL was nearly spherical in shape and uniform in size, and the average particle size was 227.8 ± 25.3 nm with negative charge. The specific disappearance of the TTO peak in the infrared spectrum suggested the successful preparation of TTONL, which showed high stability at 4°C within 35 d. The result of MIC test found that the nanoliposomes improved antibacterial activity of TTO against various E. coli strains. TTONL exposure in vitro caused different degrees of structural damage to the E. coli. TTONL by oral administration alleviated the clinical symptoms and intestinal lesion of chickens induced with E. coli challenge. Furthermore, TTONL treatment remarkably lowered the mRNA expression of NLRP3 and NF-κB (p65) in the duodenum and cecum of E. coli-infected chickens. In conclusion, the prepared TTONL had good stability and slow-release property with dose-dependent inhibition and killing effects on different strains of E. coli, and exerted a preventive role against chicken colibacillosis through inhibition.

A transformable nanoplatform with multiple therapeutic and immunostimulatory properties for treatment of advanced cancers.

Cancer is a complex pathological phenomenon that needs to be treated from different aspects. Herein, we developed a size/charge dually transformable nanoplatform (PDR NP) with multiple therapeutic and immunostimulatory properties to effectively treat advanced cancers. The PDR NPs exhibit three different therapeutic modalities (chemotherapy, phototherapy and immunotherapy) that can be used to effectively treat primary and distant tumors, and reduce recurrent tumors; the immunotherapy is simultaneously activated by three major pathways, including toll-like receptor, stimulator of interferon genes and immunogenic cell death, effectively suppresses the tumor development in combination with an immune checkpoint inhibitor. In addition, PDR NPs show size and charge responsive transformability in the tumor microenvironment, which overcomes various biological barriers and efficiently delivers the payloads into tumor cells. Taking these unique characteristics together, PDR NPs effectively ablate primary tumors, activate strong anti-tumor immunity to suppress distant tumors and reduce tumor recurrence in bladder tumor-bearing mice. Our versatile nanoplatform shows great potential for multimodal treatments against metastatic cancers.

Therapeutic effect of a self-made herbal formula on a multi-drug resistant Eimeria tenella isolate infection in broiler chickens.

In-feed prophylactic chemotherapy is widely considered the mainstay of avian coccidiosis control, while serious drug resistance strictly restricts its application. Confronted with the urgent need for an alternative strategy, a traditional Chinese medicine formula (TCMF) was developed. Meanwhile, its potential to iron out complicated clinical coccidiosis was scrutinized in vivo with a field-isolated multi-drug resistant Eimeria tenella (E. tenella) isolate. Birds were inoculated with 5 × 104 sporulated oocysts and administrated with TCMF supplementation in water from 72 h post-infection to the end of the experiment, diclazuril (DIC) was set as a positive control. As a result, TCMF intervention reduced oocyst shedding, cecal lesion and mortality, and enhanced body weight gain. According to the above, anticoccidial index (ACI) was calculated and TCMF exerted a moderate anticoccidial activity. Besides, macroscopic, histopathological, and ultrastructural observations revealed the safeguarding effects of TCMF on E. tenella-induced cecal injury. Following, TCMF treatment presented an obvious inhibition effect on E. tenella caused oxidative stress and inflammatory response. Moreover, TCMF supplementation restored the cecal flora abundance and diversity, reduced the colonization of harmful bacteria, and increased the probiotics abundance. In conclusion, TCMF exhibited a moderate anticoccidial effect along with alleviating E. tenella-induced cecal injury, redox imbalance, and inflammatory response which may be associated with the microflora modulatory effect.

Caffeic acid, but not ferulic acid, inhibits macrophage pyroptosis by directly blocking gasdermin D activation.

Regulated pyroptosis is critical for pathogen elimination by inducing infected cell rupture and pro-inflammatory cytokines secretion, while overwhelmed pyroptosis contributes to organ dysfunction and pathological inflammatory response. Caffeic acid (CA) and ferulic acid (FA) are both well-known antioxidant and anti-inflammatory phenolic acids, which resemble in chemical structure. Here we found that CA, but not FA, protects macrophages from both Nigericin-induced canonical and cytosolic lipopolysaccharide (LPS)-induced non-canonical pyroptosis and alleviates LPS-induced mice sepsis. It significantly improved the survival of pyroptotic cells and LPS-challenged mice and blocked proinflammatory cytokine secretion. The anti-pyroptotic effect of CA is independent of its regulations in cellular lipid peroxidation, mitochondrial function, or pyroptosis-associated gene transcription. Instead, CA arrests pyroptosis by directly associating with gasdermin D (GSDMD) and blocking its processing, resulting in reduced N-GSDMD pore construction and less cellular content release. In LPS-induced septic mice, CA inhibits GSDMD activation in peritoneal macrophages and reduces the serum levels of interleukin-1ß and tumor necrosis factor-α as the known pyroptosis inhibitors, disulfiram and dimethyl fumarate. Collectively, these findings suggest that CA inhibits pyroptosis by targeting GSDMD and is a potential candidate for curbing the pyroptosis-associated disease.

Comparison of endogenous development, invasion ability and apoptotic features between diclazuril resistant and sensitive strains of Eimeria tenella.

Diclazuril (DIC) is widely used in the poultry industry to control coccidiosis. However, drug resistance makes it less effective, and the underlying mechanism remains unclear. One DIC-resistant E. tenella (RE) isolate and one sensitive E. tenella (SE) isolate were used to compare the differences in their endogenous development, pathogenicity, invasion-related gene expression and apoptotic characteristics. Chickens were allocated into four groups to receive RE or SE strain and their corresponding DIC treatment or not. Caeca tissues were sampled at 96 h, 120 h and 144 h post-infection (PI) for pathological analysis. Meanwhile, second-generation merozoites (Mz2) were separated at 120 h PI to detect alterations in mitochondrial membrane potential (MMP), apoptotic rate and caspase-3 activity and mRNA expression of protein phosphatase 5 (PP5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin depolymerizing factor (ADF) and microneme proteins (MICs). Haematoxylin and eosin staining revealed that DIC treatment strictly blocked the development of the SE strain but slightly affected the RE strain. Meanwhile, the number of SE Mz2 and their MMP decreased at the same time the apoptotic rate increased after DIC treatment. Real-time quantitative PCR and caspase-3 activity studies demonstrated that Mz2 from the RE strain had higher mRNA expression of ADF and MICs along with no significant changes in GAPDH and caspase-3 activity under DIC pressure compared to its control; in contrast, the mRNA expression of ADF, MICs and PP5 was markedly suppressed in Mz2 from SE with upregulated caspase-3 activity and GAPDH transcription. In addition, the mRNA expression of GAPDH and PP5 in Mz2 from RE was remarkably higher than that of SE. Taken together, the higher mRNA expression of invasion-related genes and almost unaffected endogenous development provide a better understanding of coccidian resistance to DIC.

Alhagi honey polysaccharides encapsulated into PLGA nanoparticle-based pickering emulsion as a novel adjuvant to induce strong and long-lasting immune responses.

Alhagi honey polysaccharides, extracted from a perennial plant Alhagi pseudalhagi syn, possessed many biological activities such as immune enhancement, anti-tumor effect, and antioxygenation. In this study, we used Alhagi honey polysaccharide encapsulated (poly lactic-co-glycolic acid) (PLGA) nanoparticles to prepare an assembled particles-oil pickering emulsion: PPAS and PEI-PPAS. We investigated the characterization of two pickering emulsions, and the possible mechanism to enhance immune responses. The results showed that PPAS and PEI-PPAS both could load high adsorption of OVA and had ability to sustained controlled release OVA. In vivo experiment, PEI-PPAS/OVA enhanced the levels of IgG and cytokines. Meanwhile, it could effectively target dendritic cells (DCs), promoted the cellular uptake of OVA then activated DCs in lymph nodes. And this effect of PEI-PPAS might be induced through the MHC II and MHC I pathway in DCs. Thus, these findings demonstrated that PEI-PPAS could induce a strong and long-term cellular and humoral immune response, and have potential to applied to vaccine adjuvant delivery system.