目的 研究制霜对千金子Euphorbiae Semen总蛋白结构及肠道毒性的影响。方法 采用凝胶电泳、紫外光谱、红外光谱、圆二光谱、荧光光谱等研究千金子总蛋白经模拟制霜前后蛋白结构的变化；通过MTT、ELISA、Western blotting法研究千金子总蛋白经模拟制霜前后对IEC-6细胞存活率、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）和IL-10含量、闭合蛋白（occludin）和紧密连接蛋白-1（claudin-1）蛋白表达的影响；利用HE染色、Western blotting法探讨千金子总蛋白经模拟制霜前后对小鼠结肠组织病理形态及occludin、claudin-1蛋白表达的影响。结果 千金子总蛋白经模拟制霜后总蛋白变性、溶解度降低（P＜0.01），其相对分子质量分布发生改变；280 nm处的紫外特征吸收峰强度降低；荧光强度降低，最大荧光吸收波长发生红移；α-螺旋结构含量减少，β-折叠、β-转角和无规卷曲结构含量增加（P＜0.01）；红外光谱中酰胺I带向低波数发生位移，且峰形趋于平缓。与千金子总蛋白相应剂量组比较，千金子总蛋白模拟制霜品对TNF-α、IL-1β含量的上调作用、对IL-10含量的下调作用以及对occludin、claudin-1蛋白表达的下调作用均明显减弱（P＜0.05、0.01）。结论 千金子制霜减毒的机制可能与制霜后毒性成分总蛋白发生变性，总蛋白结构发生改变，致使千金子总蛋白的肠黏膜刺激性毒性降低有关。

Objective To study the effect of frosting on the structure and intestinal toxicity of total protein from Qianjinzi (Euphorbiae Semen). Methods The changes in the structure of total protein from Euphorbiae Semen before and after simulated frosting were determined by gel electrophoresis, ultraviolet spectroscopy, infrared spectroscopy, circular dichroism and fluorescence spectroscopy. The effects of total protein from Euphorbiae Semen before and after simulated frosting on the viability, the content of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-10, as well as the protein expressions of occludin and claudin-1 in IEC-6 cells were investigated by MTT, ELISA and Western blotting. The effects of total protein from Euphorbiae Semen before and after simulated frosting on the pathological morphology and the protein expressions of occludin and claudin-1 in mice colon tissues were explored using HE staining and Western blotting. Results The total protein from Euphorbiae Semen was denatured after simulated frosting, with reduced solubility and altered molecular weight distribution (P < 0.01). After the simulation of frosting, the ultraviolet spectroscopy characteristic absorption peak intensity at 280 nm of total protein from Euphorbiae Semen decreased, the fluorescence intensity was decreased, the maximum fluorescence absorption wavelength was red-shifted, the content of α-helix structure of the protein was decreased, the content of β-sheet, β-turn, and random coil structure was increased (P < 0.01), the amide Ⅰ band of the infrared spectrum was shifted to low wavenumber and the peak shape tended to be gentle. Compared with the corresponding dose of total protein from Euphorbiae Semen, the up-regulation of TNF-α and IL-1β content, the down-regulation of IL-10 content, as well as the down-regulation of occludin and claudin-1 protein expressions were significantly attenuated by the total protein from Euphorbiae Semen after simulated frosting (P < 0.05, 0.01). Conclusion The mechanism of the toxicity attenuation of Euphorbiae Semen after frosting may be related to the denaturation of the total protein after frosting, and the structure of the total protein are thus altered, which ultimately leads to a reduction in the intestinal mucosal irritant toxicity of the total protein from Euphorbiae Semen.

R285.5

国家自然科学基金项目（82374040）;北京市科技新星计划（20240484548）;中央高校基本科研业务费专项基金（2024-JYB-JBZD-047）;国家中医药管理局高水平建设学科（zyyzdxk-2023272）