Kynurenine   中文名称: 犬尿氨酸

Endogenous metabolite

将色氨酸（Trp）转化为犬尿氨酸（Kyn）的主要酶有两种：色氨酸-2,3-双加氧酶（TDO）和吲哚胺2,3-双加氢酶（IDO）。在某些癌症中，Kyn的积累可以促进免疫抑制。在本研究中，我们研究了IDO和TDO在原代人肝细胞（PHH）和肿瘤HepG2细胞中对Trp向Kyn的降解。为了量化Trp降解和Kyn积累，使用反相高压液相色谱法，测定PHH和HepG2细胞培养基中Trp和Kyn的水平。通过用IFN-γ激活IDO和用1-甲基色氨酸（1-DL-MT）抑制IDO来研究IDO在Trp代谢中的作用。使用两种TDO抑制剂之一680C91或LM10研究TDO的作用。使用实时PCR来测量TDO和IDO的表达。Trp在PHH和HepG2细胞中都被降解，但在PHH细胞中降解更高。然而，Kyn在HepG2细胞的上清液中的积累更高。IFN-γ刺激IDO对Trp降解和Kyn积累没有显著影响，尽管它强烈上调了IDO的表达。1-DL-MT抑制IDO对Trp的降解也没有影响。相反，用680C91或LM10抑制TDO显著降低了Trp的降解。TDO的表达与Kyn在HepG2细胞培养基中的积累呈正相关，而IDO的表达则与Kyn的积累无关。此外，TDO在HepG2细胞中降解L-Trp而不降解D-Trp。Kyn是TDO在HepG2细胞中降解Trp的主要代谢产物。Kyn在HepG2细胞中的积累可能是肿瘤免疫抵抗的关键机制。两种TDO抑制剂680C91和LM10可用于肝癌的免疫治疗[1]。

吲哚胺2,3-二加氧酶-1（IDO1）在许多炎症和癌症疾病中催化色氨酸-甘脲代谢。值得注意的是，心脏损伤后立即发生的急性炎症对新生儿心肌细胞增殖和心脏再生至关重要。然而，IDO1催化的色氨酸在心脏再生过程中的代谢在很大程度上尚未被探索。在这里，我们发现心尖新生小鼠心脏切除手术导致心脏IDO1表达和犬尿氨酸积累的快速和持续增加。Ido1基因的心脏缺失或Ido1的化学抑制会损害心脏再生。从机制上讲，犬尿氨酸升高通过激活细胞质芳基烃受体SRC-YAP/ERK途径触发心肌细胞增殖。此外，心肌细胞衍生的犬尿氨酸转运到内皮细胞，并通过促进芳烃受体核转位和增强血管内皮生长因子A的表达来刺激心脏血管生成。值得注意的是，Ahr缺失可阻止吲哚胺2,3-双加氧酶-犬尿氨酸相关的心脏再生。总之，增加吲哚胺2,3-双加氧酶衍生的犬尿氨酸水平通过作为心肌细胞增殖和心脏血管生成的内源性调节因子来促进心脏再生[2]。

如前所述培养HepG2细胞和PHH细胞。PHH和HepG2细胞在William培养基中培养，培养基中Trp浓度增加（60、120和240µmol/L）。在24小时、48小时和72小时检测培养的PHH和HepG2细胞上清液中的Trp和Kyn水平。在下一步中，我们还通过在含有更高量Trp（480µmol/L、960µmol/L和1920µmol/L）的William培养基中培养这些细胞72小时来研究HepG2细胞中Trp的最大降解。为了确定IDO是否是Trp分解的原因，我们用IFN-γ激活IDO，并用1-甲基色氨酸（1-DL-MT）抑制PHH和HepG2细胞中的IDO。在这些实验中，细胞在以下条件之一下培养72小时：不处理，用IFN-γ刺激，用IFN-γ+1-DL-MT处理，和单独用1-DL-MT处理。72小时后，测定上清液中Trp和Kyn的量。为了确定TDO是否是Trp分解的原因，我们用TDO抑制剂LM10和680C91培养细胞。在这些实验中，HepG2细胞在含有240µM Trp的培养基中用增加量的680C91（10、20、40和80µmol/L）和LM10（25、50和75µmol/L）处理72小时，然后测量上清液中的Trp和Kyn水平。为了研究哪种Trp异构体（L-Trp或D-Trp）被TDO降解，我们在含有60、120或240µM L-Trp的培养基中培养HepG2细胞72小时，并在72小时后测量Trp和Kyn水平[1]。

Mice [2]
All procedures involving animals were approved by the Institutional Animal Care and Use Committee at Georgia State University (GSU). Cardiomyocyte (CM)-specific knockout Ido1 (Ido1 mKO) mice were developed by crossing floxed Ido1 (Ido1 F/F) mice with Troponin T (Tnt)-Cre mice. In addition, deficiency of Ido1 in endothelial cell (EC) or vascular smooth muscle cell (VSMC) was obtained by crossbreeding Ido1 F/F with Cdh5-Cre or Myh11-Cre and generated Cdh5-Cre; Ido1 F/F (Ido1 ecKO) or Myh11-Cre; Ido1 F/F (Ido1 vsmcKO) transgenic mice. Their respective littermate of Ido1 F/F were used as controls. The transgenic mice and wild-type mice were in a C57BL/6 background and purchased from Jackson Laboratory (Bar Harbor, ME, USA). Tail biopsy DNA was extracted for genotyping according to the Jackson protocol. All mice were housed in temperature-controlled cages under a 12-h light–dark cycle and given free access to water and food at Georgia State University.
Neonatal cardiac apex resection surgery [2]
The neonatal mouse heart regeneration model was generated by heart apical resection (AR) surgery for 3–4 weeks as described9. Briefly, postnatal day 1 (P1) or P6 mice were subjected to anesthesia by freezing for 3–5 min. A curved forcep was extended into the chest to pull the heart out. Then, the left ventricular apex was truncated with microsurgical scissors until the ventricular chamber was exposed. The mouse chest was sewn up with 8-0 sutures, and the mice were warmed until recovery. The entire procedure required approximately about 10 min. Sham procedures excluded apex amputation. To detect the role of 1-methyl-D-tryptophan (1MT) and kynurenine (Kyn) in heart regeneration, 1MT (100 mg/kg) and Kyn (100 mg/kg) were intraperitoneally injected into wild-type mice every other day from P1 and were subjected to AR surgery at P2 and P6, respectively. The equal volume of PBS was used as vehicle control.

[1]. J Clin Med. 2022 Aug 16;11(16):4794.
[2]. Nat Commun. 2022 Oct 26;13(1):6371.

Kynurenine is a ketone that is alanine in which one of the methyl hydrogens is substituted by a 2-aminobenzoyl group. It has a role as a human metabolite. It is a substituted aniline, an aromatic ketone and a non-proteinogenic alpha-amino acid. It is a conjugate acid of a kynureninate.
Kynurenine has been reported in Drosophila melanogaster, Psychotria punctata, and other organisms with data available.
Kynurenine is a ketone and an amino acid derivative that is synthesized by either tryptophan 2,3-dioxygenase (TDO)- or indoleamine 2,3-dioxygenase (IDO)-mediated oxidation of tryptophan with diverse biological functions, including vasodilatory, immunoregulatory and neuromodulatory activities. Kynurenine is a precursor for niacin. Additionally, kynurenine can be further metabolized into anthranilic acid, kynurenic acid, and 3-hydroxykynurenine; aberrant production of kynurenine is associated with neurological disease-related cognitive deficits and depressive symptoms. Overexpressed in certain cancer cell types, kynurenine could potentially be used as a biomarker to assess cancer risk.
A metabolite of the essential amino acid tryptophan metabolized via the tryptophan-kynurenine pathway.

C10H12N2O3

208.2139

208.084

C, 57.69; H, 5.81; N, 13.45; O, 23.05

343-65-7

846

Light yellow to yellow solid

1.3±0.1 g/cm3

466.6±45.0 °C at 760 mmHg

~235 °C (dec.)

236.0±28.7 °C

0.0±1.2 mmHg at 25°C

1.626

1.09

106.41

3

5

4

15

255

0

O=C(C1=C([H])C([H])=C([H])C([H])=C1N([H])[H])C([H])([H])C([H])(C(=O)O[H])N([H])[H]

YGPSJZOEDVAXAB-UHFFFAOYSA-N

InChI=1S/C10H12N2O3/c11-7-4-2-1-3-6(7)9(13)5-8(12)10(14)15/h1-4,8H,5,11-12H2,(H,14,15)

alpha-2-Diamino-gamma-oxobenzenebutyric acid

DL-Kynurenine; 2-Amino-4-(2-aminophenyl)-4-oxobutanoic acid

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

1M HCl : 20 mg/mL (~96.05 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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