N-氯代氨基酸的合成及其在 LPS 刺激的 RAW 264.7 细胞中的生物活性。

Synthesis of N-Chloroamino Acids and Their Biological Activities in LPS Stimulated RAW 264.7 Cells.

机构信息

Laboratory of Leukocyte Signaling Research, Department of Pharmacology, Inha University School of Medicine, Incheon, 22212, South Korea.

Convergent Research Center for Metabolism and Immunoregulation, Inha University, Incheon, 22212, South Korea.

出版信息

Adv Exp Med Biol. 2017;975:675-684. doi: 10.1007/978-94-024-1079-2_53.

DOI:10.1007/978-94-024-1079-2_53

PMID:28849491

Abstract

Amino acids (AAs) are essential for protein synthesis, neurotransmission and macro molecule biosynthesis. Ala, Gln, Gly, Lys, Val and taurine (Tau) are the most abundant free AAs in mammals, and some of these react with hypochlorite (HOCl/OCl) produced by myeloperoxidase in activated phagocytes to form N-chloroamino acids (NCAA). In this study, we reacted 20 AAs and Tau with sodium hypochlorite (NaOCl), then classified the products into five types (I-V) based on the change in their absorbance. Type I AAs (Ala, Arg, Gln, Gly, Ile, Lys, Phe, Ser, Tau, Thr and Val) generated a typical monochloramine peak at 252 nm, while Type II AAs (Asn and Tyr) and Type III AAs (Glu and Leu) produced peaks at 275 nm and 225 nm, respectively. The Type IV AAs (His, Met and Trp) did not show any distinct absorption peak, and Type V AAs (Asp, Cys and Pro) did not appear to react with NaOCl. The ArgCl and TauCl were stable, while GlnCl, GlyCl, IleCl, LysCl, PheCl and ValCl were less stable and AlaCl, SerCl and ThrCl were the least stable. Tau is the most abundant non-proteinogenic free AA in cellular fluid and has many physiological functions in the nervous, cardiovascular, renal and immune systems. Tau reacts with HOCl to form TauCl, which inhibits the production of proinflammatory mediators such as superoxide, nitric oxide (NO) and interleukins, while increasing the antioxidant proteins in macrophages. We determined the effects of Type I NCAA on cell viability, NO and TNF-α production in LPS-activated RAW 264.7 cells. All Type I NCAA showed dose-dependent cytotoxicity and inhibited LPS-induced NO production. However, only GlnCl, GlyCl, IleCl, LysCl, SerCl and TauCl inhibited LPS-induced TNF-α production. In summary, Type I NCAA showed dose-dependent cytotoxicity and inhibited NO production, while their effects on TNF-α varied. Our results suggest that Type I NCAA may serve as biological regulators similar to TauCl during inflammation.

摘要

氨基酸（AAs）是蛋白质合成、神经递质和大分子生物合成所必需的。丙氨酸（Ala）、谷氨酰胺（Gln）、甘氨酸（Gly）、赖氨酸（Lys）、缬氨酸（Val）和牛磺酸（Tau）是哺乳动物中最丰富的游离氨基酸，其中一些与髓过氧化物酶在活化吞噬细胞中产生的次氯酸（HOCl/OCl）反应，形成 N-氯氨基酸（NCAA）。在这项研究中，我们将 20 种氨基酸和 Tau 与次氯酸钠（NaOCl）反应，然后根据它们的吸光度变化将产物分为五类（I-V）。I 型氨基酸（Ala、Arg、Gln、Gly、Ile、Lys、Phe、Ser、Tau、Thr 和 Val）在 252nm 处产生典型的单氯胺峰，而 II 型氨基酸（Asn 和 Tyr）和 III 型氨基酸（Glu 和 Leu）分别在 275nm 和 225nm 处产生峰。IV 型氨基酸（His、Met 和 Trp）没有显示出任何明显的吸收峰，V 型氨基酸（Asp、Cys 和 Pro）似乎没有与 NaOCl 反应。ArgCl 和 TauCl 是稳定的，而 GlnCl、GlyCl、IleCl、LysCl、PheCl 和 ValCl 则不太稳定，AlaCl、SerCl 和 ThrCl 则是最不稳定的。Tau 是细胞液中最丰富的非蛋白氨基酸，在神经系统、心血管系统、肾脏和免疫系统中具有许多生理功能。Tau 与 HOCl 反应形成 TauCl，抑制超氧化物、一氧化氮（NO）和白细胞介素等前炎性介质的产生，同时增加巨噬细胞中的抗氧化蛋白。我们测定了 I 型 NCAA 对 LPS 激活的 RAW 264.7 细胞活力、NO 和 TNF-α 产生的影响。所有 I 型 NCAA 均表现出剂量依赖性细胞毒性，并抑制 LPS 诱导的 NO 产生。然而，只有 GlnCl、GlyCl、IleCl、LysCl、SerCl 和 TauCl 抑制 LPS 诱导的 TNF-α 产生。综上所述，I 型 NCAA 表现出剂量依赖性细胞毒性并抑制 NO 的产生，但其对 TNF-α 的影响不同。我们的结果表明，I 型 NCAA 可能在炎症过程中作为类似于 TauCl 的生物调节剂发挥作用。