Polygonum hydropiper Compound Extract Inhibits Clostridium perfringens-Induced Intestinal Inflammatory Response and Injury in Broiler Chickens by Modulating NLRP3 Inflammasome Signaling.

Necrotic enteritis (NE) is a critical disease affecting broiler health, with Clostridium perfringens as its primary pathogen. Polygonum hydropiper compound extract (PHCE), formulated based on traditional Chinese veterinary principles, contains primarily flavonoids with antibacterial, anti-inflammatory, and antioxidant properties. However, PHCE's efficacy against Clostridium perfringens-induced NE and its underlying mechanism remain unclear. This study employed network pharmacology and molecular docking to predict PHCE's potential mechanisms in treating NE, followed by determining its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Clostridium perfringens (C. perf). Subsequently, the effects of various PHCE doses on intestinal damage, antioxidant capacity, and inflammatory factors in C. perf-infected broilers were assessed. Network pharmacology and molecular docking suggested that PHCE's therapeutic mechanism for NE involves the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome signaling pathway, with flavonoids such as quercetin, kaempferol, and isorhamnetin as key active components. PHCE exhibited an MIC of 3.13 mg/mL and an MBC of 12.5 mg/mL against C. perf. High PHCE doses effectively reduced intestinal damage scores in both the jejunum and ileum, accompanied by attenuated intestinal pathological changes. Additionally, the high dose significantly increased superoxide dismutase (SOD) levels while decreasing malondialdehyde (MDA), hydrogen peroxide (H2O2), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6) in the jejunum and ileum (p < 0.01 or p < 0.05). PHCE also modulated the expression of caspase-1, IL-1ß, gasdermin D (GSDMD), and NLRP3 mRNA, key components of the NLRP3 inflammasome signaling pathway, in both intestinal segments. These findings collectively indicate that PHCE protects against C. perf-induced oxidative stress and inflammatory damage in NE. By enhancing antioxidant capacity, PHCE likely reduces oxidative stress and inflammatory responses, subsequently modulating NLRP3 inflammasome signaling pathway key factor expression. Overall, this research provides valuable insights into the protective mechanism of the herbal compound PHCE and its potential benefits for avian health.

Molecular characterizations of subcellular localization signals in the nucleocapsid protein of porcine epidemic diarrhea virus.

The nucleolus is a dynamic subnuclear structure, which is crucial to the normal operation of the eukaryotic cell. The porcine epidemic diarrhea virus (PEDV), coronavirus nucleocapsid (N) protein, plays important roles in the process of virus replication and cellular infection. Virus infection and transfection showed that N protein was predominately localized in the cytoplasm, but also found in the nucleolus in Vero E6 cells. Furthermore, by utilizing fusion proteins with green fluorescent protein (GFP), deletion mutations or site-directed mutagenesis of PEDV N protein, coupled with live cell imaging and confocal microscopy, it was revealed that, a region spanning amino acids (aa), 71-90 in region 1 of the N protein was sufficient for nucleolar localization and R87 and R89 were critical for its function. We also identified two nuclear export signals (NES, aa221-236, and 325-364), however, only the nuclear export signal (aa325-364) was found to be functional in the context of the full-length N protein. Finally, the activity of this nuclear export signal (NES) was inhibited by the antibiotic Lepomycin B, suggesting that N is exported by a chromosome region maintenance 1-related export pathway.

[Co-localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and nucleolar phosphoprotein B23.1].

OBJECTIVE: To elucidate the co-localization characteristic between porcine epidemic diarrhea virus (PEDV) N protein and B23.1 phosphoprotein. METHODS: Two pairs of primers used to amplify N gene and B23.1 gene were designed and synthesized according to CV777 N gene sequence (AF353511) and human nucleolar phosphoprotein B23.1 gene sequence (BC050628.1), respectively. The PEDV N gene and B23.1 gene were amplified by RT-PCR from PEDV strain CV777 and Vero E6 cells, respectively; then cloned into eukaryotic expression vector pAcGFP1-C1 and pDsRed2-N1, to generate the recombinant plasmids pAcGFP1-C1/N and pDsRed2-N1/B23.1, respectively. Vero E6 cells were transfected with plasmids pAcGFP1-C1/N and pDsRed2-N1/B23.1. RESULTS: The fusion proteins successfully expressed in transfected Vero E6 cells by western blot analysis, and the PEDV N protein and the B23.1 phosphoprotein showed co-localization features in co-transfected cells through confocal microscopy analysis. CONCLUSION: The results will help to identify the nucleolar localization signals in PEDV N protein and to elucidate the mechanism of N protein located in nucleus.