Self-Assembled Carrier-Free Nanostructures of Anemoside B4 and Oleanolic Acid Alleviate Intestinal Injury Induced by Intraperitoneal Escherichia coli Challenge in Mice

Abstract

Escherichia coli-induced enteritis imposes a substantial economic burden on the global livestock industry. In the current context of reducing and restricting antibiotic use, there is an urgent need for novel therapeutic strategies. Based on traditional Chinese medicine compatibility, anemoside B4 (AB4) and oleanolic acid (OA) form nanoscale assemblies (AB4-OA NPs) through hydrogen bonds and van der Waals forces. Physicochemical characterization quantified the NPs as spherical particles with an average hydrodynamic diameter of 164.16 nm, a PDI of 0.280, zeta potential of −22.16 mV, and stable particle size for 28 days; the hemolysis ratio remained below 3.38% at concentrations up to 2 mg/mL, confirming favorable biosafety. In vitro assays confirmed that AB4-OA NPs outperformed AB4/OA physical mixtures in anti-inflammatory and antioxidant activity by restraining pro-inflammatory factors and activating antioxidant enzymes, verifying nanotechnology’s potency in boosting their bioactivity. In mice challenged intraperitoneally with E. coli, AB4-OA NPs elevated SOD, CAT, GSH-Px and lowered MDA; they inhibited the NF-κB pathway, activated the Nrf2 pathway, balanced inflammatory cytokines, alleviated intestinal leakage, upregulated tight junction proteins and reshaped gut microbiota by reducing Proteobacteria and enriching Firmicutes. Spearman’s correlation study indicated a strong positive relationship between Lactobacillus and Faecalibacterium with intestinal barrier function (Occludin and ZO-1) as well as antioxidant capacity, while Enterococcus was significantly and positively correlated with pro-inflammatory cytokines. These findings demonstrate that AB4-OA NPs exert pronounced protective therapeutic effects on E. coli-induced enteritis via inhibition of inflammation, attenuation of oxidative stress, protection of the intestinal barrier, and regulation of gut microbiota, and that these effects were greater than those of the corresponding physical mixture. This study proposes a novel therapeutic strategy for managing E. coli-induced enteritis and highlights the therapeutic potential of phytochemical-based self-assembled nanomedicines.