目的 探究千金子Euphorbiae Semen制霜前后提取物通过调控脂筏介导Caco-2细胞中Toll样受体4（Toll-like receptor 4，TLR4）信号通路的作用机制。方法 将Caco-2细胞分为千金子生品低、中、高剂量（50、200、800 μg/mL）组及千金子霜品低、中、高剂量（50、200、800 μg/mL）组和对照组，利用流式细胞术检测Caco-2细胞膜脂筏丰度；采用激光共聚焦实验检测Caco-2细胞中TLR4与脂筏的共定位；利用Western blotting检测Caco-2细胞中TLR4、核因子-κB p65（nuclear factor-κB p65，NF-κB p65）、p-NF-κB p65和NOD样受体热蛋白结构域相关蛋白3（NOD-like receptor family pyrin domain containing 3，NLRP3）的蛋白表达；ELISA检测Caco-2细胞上清液中白细胞介素-1β（interleukin-1β，IL-1β）和肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）的含量；Caco-2细胞转染TLR4后检测IL-1β和TNF-α的含量变化。结果 与对照组比较，千金子生品提取物可以显著增加Caco-2细胞膜脂筏的丰度（P<0.01），促进TLR4向脂筏的募集（P<0.01），提高TLR4、NF-κB p65、p-NF-κB p65和NLRP3的蛋白表达以及诱导IL-1β和TNF-α的释放（P<0.05、0.01、0.001）。相同剂量下，与千金子生品提取物相比，千金子霜品提取物对Caco-2细胞膜脂筏丰度、TLR4向脂筏募集、TLR4介导的信号通路传导和促炎因子分泌的影响均明显减弱（P<0.05、0.01、0.001）。体外siTLR4转染试验结果进一步证实，Caco-2细胞转染TLR4后TNF-α和IL-1β的含量显著降低（P<0.05、0.01、0.001）。结论 千金子制霜减毒的作用机制可能与通过调控脂筏丰度干扰TLR4向脂筏募集，最终影响脂筏中包含的TLR4信号蛋白的传导有关。

Objective To study the mechanism of extracts from Qianjinzi (Euphorbiae Semen) and Qianjinzishuang (Euphorbiae Semen Pulveratum) mediating Toll-like receptor 4 (TLR4) signaling pathway in Caco-2 cells via regulating lipid rafts. Methods Caco-2 cells were divided into Euphorbiae Semen low-, medium-and high-dose (50, 200, 800 μg/mL) groups, Euphorbiae Semen Pulveratum low-, medium-and high-dose (50, 200, 800 μg/mL) groups and control group. Flow cytometry was used to detect the lipid rafts abundance of Caco-2 cells membrane. Confocal laser scanning microscope was used for studying the co-localization of TLR4 and lipid rafts in Caco-2 cells. Western blotting was applied to detect TLR4, nuclear factor-κB p65 (NF-κB p65), p-NF-κB p65 and NOD-like receptor family pyrin domain containing 3 (NLRP3) protein expressions in Caco-2 cells. Furthermore, ELISA was applied to determine the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the supernatant of Caco-2 cells. And the levels of IL-1β and TNF-α were detected in Caco-2 cells after transfection with TLR4. Results Compared with control group, Euphorbiae Semen extracts could increase the content of lipid rafts (P < 0.01), promote the recruitment of TLR4 to lipid rafts (P < 0.01), increase the protein expressions of TLR4, NF-κB p65, p-NF-κB p65 and NLRP3, as well as induce the release of IL-1β and TNF-α in Caco-2 cells (P < 0.05, 0.01, 0.001). However, the effects of Euphorbiae Semen Pulveratum extracts on the abundance of lipid rafts, the recruitment of TLR4 to lipid rafts, the conduction of TLR4-mediated signaling pathways and the secretion of pro-inflammatory factors were significantly weaker than those of Euphorbiae Semen extracts at the same dose (P < 0.05, 0.01, 0.001). In addition, the results of siTLR4 transfection in vitro further confirmed that the content of IL-1β and TNF-α were significantly decreased after transfection of TLR4 in Caco-2 cells (P < 0.05, 0.01, 0.001). Conclusion The attenuation mechanism of processing for Euphorbiae Semen may be closely related to interfering the recruitment of TLR4 to lipid rafts by regulating lipid rafts abundance, and ultimately affecting the cascade of TLR4 signaling protein contained in lipid rafts.

R285.5

国家自然科学基金资助项目（82074021）