Klebsiella pneumoniae (K. pneumoniae) exhibits the ability to form biofilms as a means of adapting to its adverse surroundings. K. pneumoniae in this biofilm state demonstrates remarkable resistance, evades immune system attacks, and poses challenges for complete eradication, thereby complicating clinical anti-infection efforts. Moreover, the precise mechanisms governing biofilm formation and disruption remain elusive. Recent studies have discovered that fingolimod (FLD) exhibits biofilm properties against Gram-positive bacteria. Therefore, the antibiofilm properties of FLD were evaluated against multidrug-resistant (MDR) K. pneumoniae in this study. The antibiofilm activity of FLD against K. pneumoniae was assessed utilizing the Alamar Blue assay along with confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and crystal violet (CV) staining. The results showed that FLD effectively reduced biofilm formation, exopolysaccharide (EPS), motility, and bacterial abundance within K. pneumoniae biofilms without impeding its growth and metabolic activity. Furthermore, the inhibitory impact of FLD on the production of autoinducer-2 (AI-2) signaling molecules was identified, thereby demonstrating its notable anti-quorum sensing (QS) properties. The results of qRT-PCR analysis demonstrated that FLD significantly decreased the expression of genes associated with the efflux pump gene (AcrB, kexD, ketM, kdeA, and kpnE), outer membrane (OM) porin proteins (OmpK35, OmpK36), the quorum-sensing (QS) system (luxS), lipopolysaccharide (LPS) production (wzm), and EPS production (pgaA). Simultaneously, FLD exhibited evident antibacterial synergism, leading to an increased survival rate of G. mellonella infected with MDR K. pneumoniae. These findings suggested that FLD has substantial antibiofilm properties and synergistic antibacterial potential for colistin in treating K. pneumoniae infections.

Toll-like receptor 7 (TLR7) and Stimulator of Interferon Genes (STING) ligands possess a series of immunomodulatory effects such as anti-infection, anti-tumor, and autoimmune-disease-alleviating effects. In this study, porcine TLR7 (pTLR7) and porcine STING (pSTING) were selected as targets, and molecular docking and virtual screening methods were used for screening of dual-target livestock immunomodulators. Finally, two compounds were screened with molecular docking scores higher than the positive control compounds. They have good binding ability with pTLR7 and pSTING proteins, as well as satisfactory predictive safety and pharmacokinetic properties. Molecular dynamics (MD) simulation results also indicated that the above ligands can form stable complexes with two target proteins. The average binding free energies of compound 2 with pTLR7 and pSTING were −28.65 kcal/mol and −30.12 kcal/mol, respectively, and of compound 7 with pTLR7 and pSTING were −35.93 kcal/mol and −31.70 kcal/mol, respectively, which were comparable to that of positive control ligands. The similarity of target proteins between pigs, humans, and mice, as well as the interactions between ligands and TLR7 and STING in different species, were analyzed. And analysis of predicted structure–activity relationship (SAR) was conducted. Briefly, compound 2 and compound 7 were predicted to form stable complexes with pTLR7 and pSTING, with satisfactory predicted physicochemical properties and pharmacokinetic characteristics, and represented candidates for experimental validation. This study supplies a research basis for the development, design, and structural modification of immune enhancers for animals.

Fatty liver hemorrhagic syndrome (FLHS) is a common metabolic disorder in laying hens, leading to reduced egg production and economic losses. Aspirin eugenol ester (AEE) has lipid-lowering, anti-inflammatory, and antioxidant properties, but its effects on FLHS are unknown. This study evaluated the protective effects of AEE using an in vivo FLHS model induced by a high-energy low-protein diet in laying hens and an in vitro steatosis model established by free fatty acid treatment in LMH cells. AEE alleviated liver histopathological damage, reduced oxidative stress (decreased ROS and MDA; increased SOD, GSH, and CAT), and suppressed inflammatory responses. The hepatoprotective effects of AEE were tentatively associated with altered molecular expression of the Nrf2 antioxidant pathway and MAPK/NF-κB inflammatory signaling; however, this correlation was speculated based on molecular detection and incomplete in vitro pharmacological interventions, lacking rigorous causal validation. These findings suggest that AEE alleviates FLHS-related liver injury in laying hens, possibly in association with altered oxidative and inflammatory status. Collectively, these preliminary findings provide a limited theoretical reference for the potential application of AEE as a preventive agent against FLHS in laying hens.

Objective: Toll-like receptor 7 (TLR7) is an important pattern recognition receptor. It mainly participates in the recognition of single-stranded RNA and is closely related to various disease processes. In humans, the enormous potential of TLR7 antagonists in reducing inflammation and alleviating autoimmune damage has been identified. Similar effects have also been observed in livestock and poultry. Therefore, using porcine TLR7 as the target, we combined virtual screening with a heterologous reporter gene assay to identify potential ligands, aiming to provide a research basis for the development of immune modulators. Methods: The structure of porcine TLR7 was predicted through homology modeling, and molecular docking was performed for virtual screening of potential ligands. ADMET and toxicity properties of small molecules were predicted. Subsequently, a mouse TLR7 reporter cell was employed for in vitro detection of TLR7 activity and cytotoxicity. The interaction modes and molecular dynamics simulations between hit compounds and receptors were further analyzed. Results: The porcine TLR7 structure was predicted by homology modeling. According to the scoring ranking of molecular docking and of ADMET and toxicity prediction, fifty compounds with satisfactory pharmacokinetic properties and safety were screened and their inhibitory effects on R848-induced TLR7 activation were verified. Among them, two hit compounds with novel structures, HY02-35 and HY02-46, exhibited significant mouse TLR7 antagonistic activity. HY02-35, with a parent nucleus structure of 2-indolone, exhibited an inhibitory concentration range between 10.25 and 38.23 µM, with a median estimate of 14.73 µM. The core structure of HY02-46 is 1,4-dihydropyrano [2,3-c] pyrazole, and the IC₅₀ falls between 21.92 and 36.00 µM, with a median estimate of 26.61 µM. The above-mentioned hit compounds had strong interactions with porcine TLR7, formed stable complexes with the receptor, and can significantly reduce the mRNA levels of IFN-β and TNF-α. Conclusions: In short, this study obtained two hit compounds with novel structures and moderate inhibitory activity in vitro. These hits provide a starting point for subsequent structural optimization and in vivo pharmacological studies in porcine subjects.