The Combination of Chinese Medicine Monomers Reduces 7S Globulin-Induced Damage to IPEC-J2 Cells In Vitro by Modulating NF-κB/MAPK Pathway-Related Markers

This study aimed to explore the protective effects of different concentration combinations of four traditional Chinese medicine (TCM) monomers (Eleutheroside E, Anemoside B4, Forsythoside A, and Esculin) on porcine intestinal epithelial cell (IPEC-J2) damage induced by soybean 7S globulin, providing preliminary in vitro evidence for future studies on dietary-allergen-induced intestinal injury in piglets. An L9 (34) orthogonal experimental design was implemented to identify the optimized ratio of these monomers. IPEC-J2 cells were co-cultured with 5 mg/mL 7S globulin and the selected combinations for 24 h. Cell viability was determined via Cell Counting Kit-8 (CCK-8) assay, while cytokine secretion, oxidative status, and mechanical barrier markers were assessed using enzyme-linked immunosorbent assay (ELISA) and RT-qPCR. The optimal combination was determined to consist of Eleutheroside E at 50 mg/L, Anemoside B4 at 25 mg/L, Forsythoside A at 80 mg/L, and Esculin at 20 mg/L. This formulation significantly alleviated inflammatory damage, upregulated tight-junction-related mRNA expression, and reversed the decrease in cellular viability caused by 7S globulin. Furthermore, Western blot and transcript analyses suggested that this combination exerted its protective effects by modulating the nuclear factor-kappa B/mitogen-activated protein kinase (NF-κB/MAPK) pathway-related markers, reducing the expression of p38 MAPK, c-Jun N-terminal kinase (JNK), NF-κB p65, and inducible nitric oxide synthase (iNOS). In conclusion, this optimized combination of TCM monomers successfully alleviates both inflammatory injury and oxidative stress in IPEC-J2 cells in vitro and holds potential as a promising dietary additive candidate for mitigating dietary-allergen-induced enteropathy in piglets.