Department of Pediatric Surgery, Xin Hua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Shanghai Key Laboratory of Pediatric Gastroenterology and Nutrition, Shanghai Institute for Pediatric Research, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Rd, Shanghai, 200092, China.
Lipids Health Dis. 2018 Jun 23;17(1):148. doi: 10.1186/s12944-018-0786-5.
Excessive cell death of enterocytes has been demonstrated to be partially associated with the intravenously-administrated lipid emulsions (LEs) during parenteral nutrition (PN) support. However, as a new generation of LE, the effect of fish oil-derived lipid emulsion (FOLE) on the death of enterocytes remains elusive.
Intestinal epithelial cells (IEC-6 cell line) were treated with FOLE (0.25-1%) for 24 h. Cell survival was measured by CCK-8 assay, and morphological changes were monitored by time-lapse live cell imaging. The expression of receptor-interacting protein 1/3 (RIP1/3) and caspase 8 was assessed by westernblot, and the formation of necrosome (characterized by the assembly of RIP1/3 complex along with the dissociation of caspase 8) was examined by immunoprecipitation. Additionally, the production of intracellular reactive oxygen species (ROS) was detected by using a ROS detection kit with an oxidation-sensitive probe (DCFH-DA).
FOLE dose-dependently induced non-apoptotic, but programmed necroctic cell death (necroptosis) within 4-8 h after treatment. The assembly of RIP1/3 complex along with the dissociation of caspase 8 from RIP1 was observed in FOLE-treated cells. Moreover, FOLE-induced cell death was significantly alleviated by inhibiting RIP1, and was further aggravated by inhibiting caspase 8. In addition, prior to cell death the accumulation of intracellular ROS was significantly increased in FOLE-treated cells (increased by approximately 5-fold versus control, p < 0.001), which could be attenuated by inhibiting RIP1 (decreased by approximately 35% versus FOLE, p < 0.05).
FOLE induces RIP1-dependent and caspase 8-licensed necroptosis through overproduction of ROS in vitro. Our findings may provide novel insights into the clinical applications of FOLE during PN support.
研究表明，肠上皮细胞的过度死亡与肠外营养支持时静脉给予的脂肪乳剂（LE）部分相关。然而，作为新一代 LE，鱼油衍生的脂肪乳剂（FOLE）对肠上皮细胞死亡的影响仍不清楚。
用 FOLE（0.25-1%）处理肠上皮细胞（IEC-6 细胞系）24 小时。通过 CCK-8 测定法测量细胞存活率，并通过延时活细胞成像监测形态变化。通过 Western blot 测定受体相互作用蛋白 1/3（RIP1/3）和半胱天冬酶 8 的表达，通过免疫沉淀检测坏死小体（由 RIP1/3 复合物的组装和半胱天冬酶 8 与 RIP1 的解离特征）的形成。此外，通过使用氧化敏感探针（DCFH-DA）的 ROS 检测试剂盒检测细胞内活性氧（ROS）的产生。
FOLE 处理后 4-8 小时内，呈剂量依赖性诱导非凋亡但程序化的坏死性细胞死亡（坏死）。在 FOLE 处理的细胞中观察到 RIP1/3 复合物的组装以及半胱天冬酶 8 从 RIP1 的解离。此外，抑制 RIP1 可显著减轻 FOLE 诱导的细胞死亡，而抑制半胱天冬酶 8 则进一步加重细胞死亡。此外，在细胞死亡之前，FOLE 处理的细胞中细胞内 ROS 的积累显著增加（与对照相比增加约 5 倍，p<0.001），通过抑制 RIP1 可减弱该增加（与 FOLE 相比降低约 35%，p<0.05）。
FOLE 通过过度产生 ROS 在体外诱导 RIP1 依赖性和半胱天冬酶 8 许可的坏死。我们的发现可能为 FOLE 在肠外营养支持中的临床应用提供新的见解。
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