Liposomal Delivery of Macleaya cordata Extract Alleviates Bacterial Diarrhea Through Intestinal Barrier Restoration, Microbiota Remodeling, and Inhibition of Inflammatory Factor Release

<b>Background/Objectives</b>: To overcome bottlenecks in the application of <i>Macleaya cordata</i> extract (MCE) in veterinary traditional Chinese medicine, such as low bioavailability of its active ingredients, gastrointestinal irritation, and muscular toxicity, this study aimed to develop a liposomal nano-delivery system loaded with MCE (MCE-Lips) to achieve the core objective of "enhancing efficacy and reducing toxicity" and to explore its potential application and mechanism of action in treating bacterial diarrhea. <b>Methods</b>: MCE-Lips were prepared using the thin-film dispersion method, and their physicochemical properties-particle size, encapsulation efficiency, and drug loading capacity-were characterized. In vitro, cytotoxicity against skeletal muscle cells and NCM460 intestinal epithelial cells was evaluated using the CCK-8 assay. The release of lactate dehydrogenase (LDH) from skeletal muscle cells was measured with an LDH assay kit. The expression levels of inflammatory factors (TNF-α, IL-6, and IL-1β) in both cell types were determined through ELISA. A fluorescent probe was employed to assess cell membrane integrity. The effect of MCE-Lips on the expression of tight junction proteins (ZO-1, Occludin, and Claudin-5) was evaluated via immunofluorescence. Acute toxicity was examined through H&E staining. A bacterial diarrhea model was established using Escherichia coli in mice, and comprehensive safety and efficacy were assessed through hematological tests and gastrointestinal motility evaluation. Finally, untargeted metabolomics and 16S rRNA sequencing were utilized to investigate the underlying mechanisms of action. <b>Results</b>: The prepared MCE-Lips had an average particle size of 86.49 nm and a high encapsulation efficiency of 89.07%. In vitro experiments demonstrated that MCE-Lips significantly alleviated skeletal muscle cell damage, reduced LDH release (<i>p</i> < 0.05), and effectively inhibited the expression of inflammatory factors IL-6, TNF-α, and IL-1β (<i>p</i> < 0.05). In NCM460 cells, MCE-Lips exhibited a more pronounced inhibitory effect on LPS-induced release of TNF-α (<i>p</i> < 0.01), IL-6 (<i>p</i> < 0.0001), and IL-1β (<i>p</i> < 0.0001) and enhanced intestinal barrier function by upregulating the expression of tight junction proteins ZO-1 (<i>p</i> < 0.001), Occludin (<i>p</i> < 0.01), and Claudin-5 (<i>p</i> < 0.01). In the bacterial diarrhea model, MCE-Lips showed excellent anti-diarrheal efficacy (<i>p</i> < 0.01). Hematological analysis indicated no systemic toxicity. At the endocrine level, the high-dose group significantly reduced motilin (MTL) levels (<i>p</i> < 0.01), which slowed intestinal motility and prolonged chyme retention, thereby alleviating diarrhea symptoms. Mechanistic studies revealed that it acts by regulating the intestinal metabolic profile and microbial community structure, with <i>Desulfovibrio</i>, <i>Enterococcus</i>, and <i>Streptococcus</i> identified as key characteristic differential genera. <b>Conclusions:</b> For the first time, an MCE liposome nanoparticle system was constructed, and untargeted metabolomics combined with 16S rRNA sequencing were employed to elucidate its anti-diarrheal mechanism. MCE-Lips exerts excellent antibacterial diarrhea effects through multiple mechanisms, including direct cytoprotection and anti-inflammatory action, enhancement of the intestinal barrier, regulation of gut function, and remodeling of the gut microecology. This work provides a novel paradigm for plant-derived nano-anti-diarrheal agents. The systematic evaluation of the pharmacodynamics of MCE-Lips in a piglet bacterial diarrhea model will lay a solid foundation for its eventual market application.