关键词: 苦荞来源类外泌体纳米囊泡；巨噬细胞RAW264.7；转录组学；生物信息学；炎症

Abstract: Objective: To investigate the regulatory effect and underlying mechanism of tartary buckwheat-derived exosome-like nanovesicles (TELN) on lipopolysaccharide (LPS)-induced inflammatory response and polarization in murine macrophage RAW264.7 cells. Methods: TELN were extracted from tartary buckwheat using size-exclusion chromatography and characterized. RAW264.7 macrophages were divided into a control, an LPS-treated group, and an LPS + TELN-treated group. Transcriptomic analysis was performed to identify the differences in gene expression between these groups. The secretion levels of the key inflammatory cytokines interleukin (IL)-6, tumor necrosis factor-α (TNF-α), IL-1β, and IL-10 in cell culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). The mRNA expression of these cytokines was detected by real-time quantitative polymerase chain reaction (qPCR). The impact of TELN on LPS-induced macrophage polarization was analyzed using flow cytometry and Western blot. Results: TELN exhibited a homogeneous size distribution with an average diameter of 145.8 nm and a concentration of (1.305 ± 0.074) × 1010 particles/mL. The transcriptomic analysis revealed significant upregulation of the genes encoding macrophage-derived chemotactic factors, ubiquitinated proteins, phosphorylated proteins, and immune/inflammatory regulators (e.g., S100A8) in the TELN-treated group. The ELISA results demonstrated that compared with the LPS group, TELN significantly reduced the levels of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in supernatants by (75.6 ± 0.9)% (P < 0.000 1), (10.9 ± 0.2)% (P < 0.000 1), and (57.8 ± 6.8)% (P < 0.000 1), respectively, while elevating the anti-inflammatory cytokine IL-10 by (69.7 ± 4.6)% (P < 0.000 1). The qPCR results confirmed that TELN significantly downregulated the mRNA expression of IL-6, TNF-α, and IL-1β and upregulated IL-10 mRNA expression at the transcriptional level. Flow cytometry and Western blot analyses consistently showed that TELN significantly suppressed the LPS-induced expression of the M1 polarization marker CD86. However, no detectable CD206 protein expression was observed, indicating that TELN did not promote M2 polarization. Conclusion: TELN exerts an anti-inflammatory effect by bidirectionally modulating cytokine secretion (effectively inhibiting the release of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β while significantly enhancing the level of anti-inflammatory IL-10) and suppressing M1 macrophage polarization. Particularly, TELN has a pronounced inhibitory effect on IL-6 expression, suggesting that its mechanism of action may be related to targeting signal pathways such as nuclear factor kappa-B (NF-κB). This study provides novel insights into the nutritional and immunomodulatory mechanisms of tartary buckwheat.

Key words: tartary buckwheat-derived exosome-like nanovesicles; RAW264.7 macrophages; transcriptomics; bioinformatics; inflammation