Osthole (Ost) and icariin (Ica) are extracted from traditional Chinese medicine Cnidium monnieri and Epimedii Folium, respectively, and both exhibit estrogen-like biological activity. This study aimed to determine the efficacy and safety of combining Ost with Ica on the production performance of laying hens and to explore their possible mechanisms. The production performance, egg quality, residues of Ost and Ica in eggs, serum reproductive hormone levels, expression of ovarian reproductive hormone receptor, proliferation of granulosa cells in small yellow follicles (SYF), and progesterone secretion in large yellow follicles (LYF) related genes and proteins expression were detected. The results showed that adding 2 mg/kg Ost + 2 mg/kg Ica to the feed increased the laying rate, average egg weight, Haugh unit, and protein height of laying hens. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and progesterone (P4) levels increased, and the expression of ovarian estrogen receptor (ER), follicle-stimulating hormone receptor (FSHR), and progesterone receptor (PGR) mRNA was up-regulated. Additionally, the mRNA and protein levels of steroidogenesis acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) increased in LYF. Furthermore, mRNA and protein levels of proliferating cell nuclear antigen (PCNA), cyclin E1, and cyclin A2 were up-regulated in SYF. The residues of Ost and Ica in egg samples were not detected by high-performance liquid chromatography (HPLC). In conclusion, dietary supplementation of Ost and Ica increased granulosa cells proliferation in SYF and increased P4 secretion in granulosa cells of LYF, ultimately improving the production performance of laying hens.

Shuanghuanglian oral liquid (SHL) is widely used in companion animals and poultry, but its molecular mechanism in pneumonia–myocarditis comorbidity and heart–lung inflammatory crosstalk remains largely unclear. This computational study investigated the conserved AKT1/HIF1A-mediated immunoregulatory mechanism of SHL and its cross-species translational potential in veterinary medicine. Network pharmacology was integrated with GO, KEGG, and Reactome enrichment analyses, protein–protein interaction network construction, ADMET evaluation, cross-species sequence homology analysis (human, dog, cattle, and pig), molecular docking, and molecular dynamics simulation. A total of 61 active compounds, 251 putative targets, and 52 common targets associated with pneumonia and myocarditis were identified. These targets were mainly enriched in inflammation- and immune-related pathways, including TNF, IL-17, AGE–RAGE, and PPAR signaling. AKT1 and HIF1A showed high sequence conservation across species (85–98%). Key compounds exhibited favorable binding affinity to AKT1, and molecular dynamics simulation suggested the stability of the Baicalein–AKT1 complex. ADMET analysis suggested favorable pharmacokinetic properties and low predicted toxicity. These findings suggest that SHL may potentially alleviate pneumonia and myocarditis through modulation of the conserved AKT1/HIF1A axis and support its potential as a complementary therapeutic approach for managing heart–lung inflammatory diseases in multiple livestock species. This entirely computational study highlights promising mechanisms that should be further validated in vivo.

Zearalenone is a common mycotoxin that impairs reproductive function, particularly by damaging ovarian granulosa cells. This study investigated the protective effect and underlying mechanism of scutellarin against zearalenone-induced injury in porcine ovarian granulosa cells. Cells were isolated and identified by follicle-stimulating hormone receptor immunofluorescence. Cell viability, cell-cycle distribution, and related molecular changes were evaluated using the MTT assay, flow cytometry, qRT-PCR, and Western blot. Scutellarin showed no obvious cytotoxicity within the tested range and attenuated the zearalenone-induced reduction in cell viability. Flow cytometry analysis demonstrated that scutellarin alleviated zearalenone-induced cell-cycle disturbance. At the molecular level, scutellarin upregulated the expression of cell-cycle-related factors, including CDK1, CDK2, CDK4, and PCNA, and increased the expression of Wnt/β-catenin signaling-related proteins, including WNT5A, β-catenin, c-MYC, and CCND1. WNT5A knockdown further indicated that scutellarin-mediated regulation of PCNA, CDK1, and CDK4 is WNT5A-dependent, whereas its effect on CDK2 may involve a WNT5A-independent mechanism. These findings indicate that scutellarin alleviates zearalenone-induced granulosa cell injury partly through WNT5A-associated modulation of cell-cycle-related proteins, providing mechanistic insights for its protective effects.