[Assessment of the effectiveness of recombinant human lactoferrin using an intestinal barrier and Tohoku hospital pediatrics-1 co-culture model].
摘要
OBJECTIVE: Developing a microfluidic organ chip-based in vitro gut immune co-culture model to assess the anti-inflammatory properties and barrier protection effects of recombinant human lactoferrin(rHLF) in this highly biomimetic system. METHODS: An inflammatory model was established by co-culturing a human-derived intestinal organoid barrier, constructed using organ chips, with the human monocyte line Tohoku hospital pediatrics-1(THP-1). The model was stimulated with lipopolysaccharide(LPS) to activate intestinal epithelial cells and THP-1-induced macrophages. The experiment included a model control group, an LPS group, 200 and 1000 μg/mL rHLF group. High-content imaging was employed to assess cell apoptosis, while a cell resistance meter measured transmembrane resistance(TEER). Enzyme-linked immunosorbent assay(ELISA) was utilized to evaluate fluorescein apparent permeability(Papp), and RT-qPCR was used to quantify the mRNA expression of inflammatory cytokines tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), IL-6, IL-8, chemokines(CCL2), oxidative stress-related enzymes nitric oxide synthase 2(NOS2), prostaglandin-endoperoxide synthase 2(PTGS2), and barrier-related cell junction proteins tight junction protein 1(TJP1). RESULTS: In the co-culture system of THP-1 and small intestine organoids, compared with the LPS group, the apoptosis signal of the rHLF group was significantly weakened, the barrier TEER value was significantly increased and the Papp value was significantly reduced(P<0.05). The expression of IL-1β and IL-8, chemokine CCL2, and oxidative stress-related enzymes NOS2 and PTGS2 was significantly reduced(P<0.05). CONCLUSION: The rHLF may inhibit the expression of IL-1β, IL-8 and CCL2, directly reducing intestinal mucosal inflammation and protecting the intestinal mucosal barrier function. It may also downregulate the oxidative stress pathway enzyme PTGS2 and other mechanisms, providing protective effects against LPS-induced in vitro damage to