Bo Wang, Cunxi Nie, Yanfeng Liu, Rongyan Qin, Wenqi Wang, Yanfen Cheng
Background:Peganum harmala L. stem ethanolic extract (PHL) has been scarcely evaluated as a ruminant feed component. We evaluated its effects on serum inflammatory and antioxidant biomarkers and duodenal and hepatic molecular responses in Hu sheep. Methods: Twelve male Hu sheep, approximately 120 days old, were randomly assigned to a control group or a PHL treatment group (n = 6 per group). After a 7-day adaptation period, the PHL group received 3 g/head/day of PHL (total flavonoid content: 65.7 mg rutin equivalents (RE)/g; approximately 197.1 mg RE/head/day) for 50 days. Serum markers, duodenal untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics, liver RNA sequencing, and alternative splicing were analyzed. Results: Serum interleukin-6 and malondialdehyde were lower, whereas interleukin-4 and glutathione peroxidase were higher, in the PHL group than in the control group (p < 0.05). Using variable importance in projection ≥ 1, fold change ≥ 1.2 or ≤0.83, and unadjusted p < 0.05 as screening criteria, 479 LC-MS features met these nominal screening criteria, of which 81 had database annotations. Following Benjamini-Hochberg correction, only two features remained significant (q < 0.05); however, only one was successfully annotated, rendering the pathway analysis exploratory. Liver RNA sequencing identified 279 differentially expressed genes (absolute log2FC ≥ 1 and q ≤ 0.001) and 104 skipped-exon events (false discovery rate < 0.05). Functional enrichment analysis highlighted annotations related to bile secretion, ATP-binding cassette transport, nutrient metabolism, and glutathione metabolism. Conclusions: PHL supplementation was associated with selective changes in serum inflammatory and antioxidant biomarkers, accompanied by hepatic molecular responses. The duodenal metabolomic findings remain exploratory, and dose-response studies incorporating alkaloid quantification and targeted molecular validation are required before practical application can be considered.