关键词: 羊脐带提取物, 小鼠巨噬细胞RAW264.7, 抗炎, M2极化

Abstract:

In this study, sheep umbilical cord extract （S-UCE） was obtained through a multi-step purification process involving enzymatic hydrolysis, homogenization, ultrasonication and filtration. The protein mass concentration of S-UCE, as determined by the bicinchoninic acid （BCA） assay, was （1.64±0.37） mg/mL. The effect of S-UCE on cell viability of human dermal fibroblasts （HDFs）, human immortalized keratinocytes （HaCaT）, and RAW 264.7 macrophages were evaluated using the MTT colorimetric assay. Results demonstrate that S-UCE at protein mass concentrations ranging from 0 to 400 μg/mL exhibit no cytotoxicity and promote cell proliferation by 50%-100% in the aforementioned cell lines. An inflammatory model was established using lipopolysaccharide （LPS） -stimulated RAW264.7 cells. Morphological changes were observed using microscope, while nitric oxide （NO） release in the culture supernatant was quantified using the Griess reagent method. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the expression levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha （TNF-α）, interleukin （IL） -6, and IL-1β. Furthermore, western blotting and immunofluorescence assays were performed to detect the protein expression of polarization markers CD86 （M1 phenotype） and CD206 （M2 phenotype） in RAW264.7 cells. The results indicate that S-UCE at protein mass concentrations of 50-200 μg/mL significantly attenuate LPS-induced morphological alterations in RAW264.7 cells, reduce NO production, and suppress the expression of IL-6, IL-1β, and TNF-α. Notably, S-UCE at 100 μg/mL exhibits the most potent anti-inflammatory effects, downregulating the relative protein expression of CD86 while upregulating CD206 expression. These findings demonstrate that the prepared S-UCE possesses favorable biosafety and anti-inflammatory properties, providing an experimental foundation for the development of novel animal-derived functional ingredients in cosmetic applications.

Key words: sheep umbilical cord extract, RAW264.7 cells, anti-inflammatory, M2 polarization