Breed-driven bile acid metabolic heterogeneity highlights the potential of UDCA as an anti-inflammatory food additive

This study investigates the breed-specific metabolite interactions and their effects on the liver and intestines in pigs and mice under LPS-induced conditions through a combination of animal experiments and molecular biological methods. A total of 16 Min piglets and 16 Yorkshire piglets (50 days old) were selected from a commercial farm and strictly standardized in terms of growth and feeding conditions. They were housed individually in stainless steel metabolism crates with ad libitum access to water and the same diet. After a 14-day trial, the piglets were randomly assigned to four experimental groups: M-CON (Min piglets control group), M-LPS (Min piglets injected with LPS), Y-CON (Yorkshire piglets control group), and Y-LPS (Yorkshire piglets injected with LPS), with eight replicates per group. The LPS groups received an intraperitoneal injection of LPS (100 μg/kg) and were sacrificed 6 hours post-injection, while the CON groups received an equal volume of saline. Sampling occurred immediately after sacrifice, with serum separated by centrifugation and tissues from the liver, spleen, and intestine collected and stored at −80 °C.In addition, 8-week-old male ICR mice were obtained from Chongqing Tengxin Biotechnology Co. and maintained under controlled conditions in the animal facility of Southwest University. The mice were randomized into three groups (n = 12 per group): control, LPS (LPS injection), and LPS + UDCA (ursodeoxycholic acid, oral gavage at 50 mg/kg/mouse once daily for 7 consecutive days). On the final day, the LPS group received an intraperitoneal injection of LPS (7.5 mg/kg) and was sacrificed 5 hours post-injection.To evaluate the phenotypic effects, organ indices were calculated using the formula: organ index (%) = (organ weight/body weight) × 100%. Liver and ileum tissues were fixed in 10% PBS buffered formalin, embedded in paraffin, sectioned at approximately 5 μm, and stained with hematoxylin and eosin for histological analysis. For immunofluorescence, ileum tissue samples were treated with a permeabilizing agent (e.g., 0.1-0.5% Triton X-100), blocked with a protein-containing solution, incubated with primary antibodies (Claudin-1, ZO-1, Occludin), and then with secondary antibodies. Nuclei were stained with DAPI, and samples were imaged using a fluorescence microscope. Serum samples were analyzed for biochemical indicators (ALT, AST, TP, ALB, GLO, GLU, ALP, UREA, TG, CHO, LDH) using the UnicelDxC 800 Synchron®. Inflammatory cytokines (IL-1β, IL-6, TNF-α) in liver and intestine homogenates were measured by ELISA.Serum metabolomics were performed using an LC-MS system. Metabolites were extracted with 50% methanol buffer, separated using UPLC, and detected by a TripleTOF5600plus mass spectrometer in both positive and negative ion modes. Metabolites were annotated using the KEGG database and an in-house fragment spectrum library. Student t-tests and supervised PLS-DA were used to identify significant differences in metabolite concentrations.For molecular analysis, total RNA was extracted from liver and ileum samples using TRIzol reagent, reverse transcribed, and analyzed by real-time PCR using the 2-ΔΔCt method. Western blot was performed to detect protein expression levels of TLR4, p-NF-κB p65, and β-actin. Statistical analysis was conducted using Student's t-test and one-way ANOVA, with results expressed as mean ± SEM and P < 0.05 considered statistically significant.