BACKGROUND Palmitoylethanolamide (PEA) is a pleiotropic endogenous lipid mediator currently used as a "dietary food for special medical purposes" against neuropathic pain and neuro-inflammatory conditions. Several mechanisms underlie PEA actions, among which the "entourage" effect, consisting of PEA potentiation of endocannabinoid signaling at either cannabinoid receptors or transient receptor potential vanilloid type-1 (TRPV1) channels. Here, we report novel molecular mechanisms through which PEA controls mast cell degranulation and substance P (SP)-induced histamine release in rat basophilic leukemia (RBL-2H3) cells, a mast cell model. METHODS RBL-2H3 cells stimulated with SP were treated with PEA in the presence and absence of a cannabinoid type-2 (CB2) receptor antagonist (AM630), or a diacylglycerol lipase (DAGL) enzyme inhibitor (OMDM188) to inhibit the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The release of histamine was measured by ELISA and β-hexosaminidase release and toluidine blue staining were used as indices of degranulation. 2-AG levels were measured by LC-MS. The mRNA expression of proposed PEA targets (Cnr1, Cnr2, Trpv1, Ppara and Gpr55), and of PEA and endocannabinoid biosynthetic (Napepld, Dagla and Daglb) and catabolic (Faah, Naaa and Mgl) enzymes were also measured. The effects of PEA on the activity of DAGL-α or -β enzymes were assessed in COS-7 cells overexpressing the human recombinant enzyme or in RBL-2H3 cells, respectively. RESULTS SP increased the number of degranulated RBL-2H3 cells and triggered the release of histamine. PEA counteracted these effects in a manner antagonized by AM630. PEA concomitantly increased the levels of 2-AG in SP-stimulated RBL-2H3 cells, and this effect was reversed by OMDM188. PEA significantly stimulated DAGL-α and -β activity and, consequently, 2-AG biosynthesis in cell-free systems. Co-treatment with PEA and 2-AG at per se ineffective concentrations downmodulated SP-induced release of histamine and degranulation, and this effect was reversed by OMDM188. CONCLUSIONS Activation of CB2 underlies the inhibitory effects on SP-induced RBL-2H3 cell degranulation by PEA alone. We demonstrate for the first time that the effects in RBL-2H3 cells of PEA are due to the stimulation of 2-AG biosynthesis by DAGLs.

中文翻译：

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棕榈酰乙醇酰胺通过刺激二酰基甘油脂肪酶活性来抵消体外 P 物质诱导的肥大细胞活化。

背景技术棕榈酰乙醇酰胺(PEA)是一种多效性内源性脂质介质，目前用作对抗神经性疼痛和神经炎症病症的“特殊医学用途的膳食食品”。PEA 作用有多种机制，其中“随从”效应包括 PEA 增强大麻素受体或瞬时受体电位香草酸 1 型 (TRPV1) 通道上的内源性大麻素信号传导。在此，我们报道了 PEA 在肥大细胞模型大鼠嗜碱性白血病 (RBL-2H3) 细胞中控制肥大细胞脱颗粒和 P 物质 (SP) 诱导的组胺释放的新分子机制。方法 在存在和不存在 2 型大麻素 (CB2) 受体拮抗剂 (AM630) 或二酰基甘油脂肪酶 (DAGL) 酶抑制剂 (OMDM188) 的情况下，用 PEA 处理经 SP 刺激的 RBL-2H3 细胞，以抑制内源性大麻素 2-花生四烯酰甘油 (2-AG)。通过ELISA测定组胺的释放，并使用β-己糖胺酶释放和甲苯胺蓝染色作为脱粒指标。通过LC-MS测量2-AG水平。还测量了提议的 PEA 靶标（Cnr1、Cnr2、Trpv1、Ppara 和 Gpr55）以及 PEA 和内源性大麻素生物合成酶（Napepld、Dagla 和 Daglb）以及分解代谢酶（Faah、Naaa 和 Mgl）的 mRNA 表达。分别在过表达人重组酶的 COS-7 细胞或 RBL-2H3 细胞中评估 PEA 对 DAGL-α 或 -β 酶活性的影响。结果 SP 增加了脱粒 RBL-2H3 细胞的数量并引发组胺的释放。PEA 以 AM630 拮抗的方式抵消这些影响。PEA 同时增加了 SP 刺激的 RBL-2H3 细胞中 2-AG 的水平，并且这种效应被 OMDM188 逆转。PEA 显着刺激 DAGL-α 和 -β 活性，从而刺激无细胞系统中的 2-AG 生物合成。用本身无效浓度的 PEA 和 2-AG 共同处理可下调 SP 诱导的组胺释放和脱颗粒，并且这种效应可被 OMDM188 逆转。结论 CB2 的激活是单独 PEA 对 SP 诱导的 RBL-2H3 细胞脱颗粒的抑制作用的基础。我们首次证明 PEA 对 RBL-2H3 细胞的作用是由于 DAGL 刺激 2-AG 生物合成。