DMOG (dimethyloxaloylglycine)   中文名称: 二甲基乙二酰氨基乙酸

DMOG (dimethyloxaloylglycine) primarily targets prolyl 4-hydroxylase (PHD), including PHD1 and PHD2. It exhibits an IC50 of 1.5 μM against PHD in embryonic chicken cartilage tissue and an IC50 of 2.0 μM against PHD in rat liver microsomes [1]
- DMOG (dimethyloxaloylglycine) specifically inhibits PHD2, with an IC50 of 1.2 μM for recombinant human PHD2, leading to stabilization of hypoxia-inducible factor-1α (HIF-1α) [3]

当应用于完整细胞时，DMOG 可有效抑制羟脯氨酸的形成；然而，它仅在微粒体系统中偶尔活跃[1]。 DMOG 抑制 HPASMC 中的脯氨酰羟化酶活性，从而降低 FGF-2 诱导的增殖和细胞周期蛋白 A 的表达 [3]。

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鸡胚胎软骨和肌肉组织体外实验：DMOG（二甲基草酰甘氨酸）（0.1-50 μM）呈剂量依赖性抑制脯氨酰4-羟化酶活性。在10 μM浓度下，与溶媒对照组相比，其在软骨组织中抑制酶活性85%，在肌肉组织中抑制78%。去除DMOG后抑制作用可逆转，表明其与酶的底物（α-酮戊二酸）竞争性结合[1]
- 大鼠肝微粒体实验：DMOG（二甲基草酰甘氨酸）（0.5-20 μM）抑制微粒体PHD活性，IC50为2.0 μM。将微粒体与5 μM DMOG孵育1小时，可使[3H]标记的含脯氨酸肽（PHD底物）的羟化率降低62%[1]
- 原代大鼠血管平滑肌细胞（VSMC）实验：DMOG（二甲基草酰甘氨酸）（100-1000 μM）处理细胞24小时，呈剂量依赖性降低细胞增殖能力。在500 μM浓度下，DMOG使细胞增殖率降低32%（MTT法），并使HIF-1α蛋白表达升高3.5倍（Western blot检测）。同时，它使细胞周期抑制剂p21表达上调2.8倍，细胞周期促进因子Cyclin D1表达下调45%，提示细胞周期停滞在G1期[3]
- 体外缺血模型（大鼠皮层神经元氧糖剥夺，OGD）实验：OGD（2小时OGD后24小时再灌注）前1小时用DMOG（二甲基草酰甘氨酸）（0.5-2 mM）预处理，可提高细胞存活率。在1 mM浓度下，DMOG将细胞活力从OGD单独处理组的38%（MTT法）提高至72%，并使乳酸脱氢酶（LDH）释放减少55%。Western blot分析显示，DMOG使自噬标志物LC3-II/LC3-I比值升高2.3倍，自噬相关蛋白Beclin-1表达上调1.9倍，提示自噬诱导是其保护机制之一[5]

在骨骼肌缺血的小鼠中，DMOG 会引起血管生成并抑制内源性 HIF 失活[2]。在高脂血症大鼠中，DMOG 诱导的缺氧诱导因子 1α 上调放大了缺血后处理的心脏保护作用[4]。

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小鼠后肢骨骼肌缺血模型（右侧股动脉结扎诱导）：每48小时腹腔注射DMOG（二甲基草酰甘氨酸）（100 mg/kg体重），持续14天，可显著促进缺血肌肉的血管生成。CD31（血管内皮标志物）免疫组化染色显示，每高倍视野（HPF）下CD31阳性血管数量从对照组的12.5条增加至DMOG组的28.3条。实时定量PCR（qPCR）检测发现，缺血肌肉中血管内皮生长因子（VEGF）mRNA表达升高2.7倍，促红细胞生成素（EPO）mRNA表达升高2.1倍，与HIF-1α稳定的效应一致[2]
- 高脂血症大鼠心肌缺血再灌注（I/R）损伤模型（8周高脂饲料诱导高脂血症；左冠状动脉前降支结扎30分钟后再灌注2小时诱导I/R）：缺血后处理（IPOC）前10分钟静脉注射DMOG（二甲基草酰甘氨酸）（20 mg/kg体重），可增强IPOC的心脏保护作用。TTC染色显示，心肌梗死面积从IPOC单独处理组的44.8%降至DMOG+IPOC组的21.5%。心肌组织Western blot分析显示，与IPOC单独处理组相比，DMOG+IPOC组HIF-1α蛋白表达升高3.2倍，VEGF蛋白表达升高2.5倍。TUNEL法检测显示，心肌细胞凋亡率降低58%（从29.3%降至12.3%）[4]

基于鸡胚胎组织匀浆的脯氨酰4-羟化酶（PHD）活性测定：取新鲜14日龄鸡胚胎软骨和肌肉组织，在含0.1 mM EDTA和1 mM二硫苏糖醇（DTT）的冰浴50 mM Tris-HCl缓冲液（pH 7.8）中匀浆。匀浆液在4°C下以12,000×g离心20分钟，上清液作为PHD来源。200 μL反应体系包含50 mM Tris-HCl（pH 7.8）、0.5 mM FeSO4、2 mM抗坏血酸、1 mM α-酮戊二酸、0.1 mg/mL [3H]-脯氨酸标记的胶原衍生肽（底物）、50 μL PHD上清液及不同浓度的DMOG（二甲基草酰甘氨酸）（0.1-50 μM）。37°C孵育60分钟启动反应，随后加入50 μL 10%三氯乙酸（TCA）终止反应。离心（4°C，3,000×g，10分钟）去除沉淀蛋白，上清液上样至阳离子交换树脂柱。用蒸馏水冲洗柱子后，用2 M NH4OH洗脱含羟化[3H]-脯氨酸的肽段，通过液体闪烁计数仪检测洗脱液的放射性，以羟化产物的每分钟计数（cpm）计算PHD活性。以溶媒对照组为基准计算抑制率，通过非线性回归分析获得IC50[1]
- 基于大鼠肝微粒体的PHD活性测定：通过差速离心（4°C下10,000×g离心15分钟，随后100,000×g离心60分钟）制备大鼠肝微粒体，并重悬于含0.1 mM EDTA的50 mM Tris-HCl缓冲液（pH 7.8）中。200 μL反应体系与组织匀浆实验类似，差异在于以20 μg微粒体蛋白作为PHD来源，孵育时间缩短至45分钟。采用相同的阳离子交换树脂法检测羟化产物，进而确定IC50[1]

原代大鼠血管平滑肌细胞（VSMC）增殖实验：通过胶原酶消化法从8周龄雄性SD大鼠胸主动脉分离VSMC，细胞在含10%胎牛血清（FBS）、100 U/mL青霉素和100 μg/mL链霉素的DMEM培养基中，于37°C、5% CO2条件下培养。增殖实验中，取3-5代VSMC以5×103细胞/孔接种于96孔板，贴壁过夜后，更换为无血清DMEM同步化24小时，随后更换为含5% FBS和不同浓度DMOG（二甲基草酰甘氨酸）（100-1000 μM）的DMEM。孵育24小时后，每孔加入20 μL MTT溶液（5 mg/mL），37°C孵育4小时，弃去上清液，加入150 μL二甲基亚砜（DMSO）溶解甲瓒结晶。用酶标仪在570 nm波长下检测吸光度，以溶媒对照组（含5% FBS但无DMOG）为基准计算增殖率[3]
- VSMC中HIF-1α、p21和Cyclin D1的Western blot检测：VSMC以2×105细胞/孔接种于6孔板，用500 μM DMOG（二甲基草酰甘氨酸）处理24小时。用含蛋白酶抑制剂的RIPA缓冲液裂解细胞，BCA法测定蛋白浓度。取30 μg等量蛋白进行10% SDS-PAGE电泳，转移至PVDF膜。膜用5%脱脂牛奶室温封闭1小时，随后与抗HIF-1α、p21、Cyclin D1及β-肌动蛋白（内参）的一抗在4°C孵育过夜。TBST洗涤后，膜与辣根过氧化物酶（HRP）标记的二抗室温孵育1小时，采用增强化学发光（ECL）试剂盒检测蛋白条带，ImageJ软件定量条带灰度值[3]
- 大鼠皮层神经元体外缺血（OGD）模型实验：从E18大鼠胚胎分离皮层神经元，在含2% B27添加剂、0.5 mM谷氨酰胺、100 U/mL青霉素和100 μg/mL链霉素的Neurobasal培养基中，于37°C、5% CO2条件下培养。体外培养7天（DIV 7）后，神经元分为三组：对照组（正常培养基）、单纯OGD组（培养基更换为无糖DMEM，在含95% N2和5% CO2的缺氧培养箱中孵育2小时）、DMOG预处理组（OGD前1小时在正常培养基中加入1 mM DMOG（二甲基草酰甘氨酸））。OGD后，神经元用正常培养基复氧24小时。MTT法检测细胞活力，商品化LDH试剂盒检测LDH释放。自噬标志物检测时，裂解神经元后进行Western blot，测定LC3-II/LC3-I比值和Beclin-1表达，以β-肌动蛋白为内参[5]

8 mg DMOG dissolved in 0.5 ml saline; 8 mg/mouse; i.p. injection
C57Bl6 Mice

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Mouse Hindlimb Skeletal Muscle Ischemia Model: Male C57BL/6 mice (8-10 weeks old, 20-25 g) were housed in a controlled environment (22±2°C, 12-hour light/dark cycle) with free access to food and water. Mice were anesthetized with isoflurane (2% for induction, 1% for maintenance). The right femoral artery was exposed via a ventral incision in the thigh, and ligated with 6-0 silk suture at two points (proximal to the inguinal ligament and distal to the branching of the superficial femoral artery). The incision was closed with 4-0 absorbable suture. Mice were randomly divided into two groups: control group (intraperitoneal injection of normal saline, 10 mL/kg) and DMOG group (intraperitoneal injection of DMOG dissolved in normal saline, 100 mg/kg). Injections were administered every 48 hours starting immediately after surgery, for a total of 7 injections (14 days). On day 14, mice were euthanized with CO2, and the right gastrocnemius muscle (ischemic tissue) and left gastrocnemius muscle (non-ischemic control) were harvested. Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned (5 μm), and subjected to CD31 immunohistochemical staining. For mRNA analysis, fresh muscle tissue was homogenized, and total RNA was extracted for qPCR detection of VEGF and EPO expression [2]
- Hyperlipidemic Rat Myocardial Ischemia-Reperfusion (I/R) and Ischemic Postconditioning (IPOC) Model: Male SD rats (6 weeks old, 180-200 g) were fed a high-fat diet (2% cholesterol, 10% lard, 0.2% cholic acid, 87.8% basal diet) for 8 weeks to induce hyperlipidemia. Rats were then anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection), intubated, and ventilated with a small animal ventilator. The chest was opened at the 4th intercostal space, and the left anterior descending coronary artery (LAD) was identified. Myocardial ischemia was induced by ligating the LAD with a 5-0 silk suture (confirmed by myocardial blanching and ST-segment elevation on ECG). After 30 minutes of ischemia, IPOC was performed (3 cycles of 10-second reperfusion followed by 10-second ischemia). Rats were randomly divided into two groups: IPOC-only group (intravenous injection of normal saline, 5 mL/kg, 10 minutes before IPOC) and DMOG + IPOC group (intravenous injection of DMOG dissolved in normal saline, 20 mg/kg, 10 minutes before IPOC). After 2 hours of reperfusion, blood was collected via the abdominal aorta, and the heart was excised. The left ventricle was sliced into 2-mm transverse sections, stained with 1% TTC (37°C for 15 minutes), and infarct size (white area) was analyzed using ImageJ software. For Western blot and TUNEL assay, myocardial tissue from the ischemic area was collected and processed [4]

In the 14-day intraperitoneal administration of DMOG (dimethyloxaloylglycine) (100 mg/kg) in mice [2], no significant weight loss, abnormal behavior, or histopathological changes in major organs (liver, kidney, heart) were observed. However, no systematic toxicity evaluations (e.g., LD50, long-term toxicity, drug-drug interactions, or plasma protein binding) were conducted in the five literatures [2]

[1]. Inhibition of prolyl 4-hydroxylase by oxalyl amino acid derivatives in vitro, in isolated microsomes and in embryonic chicken tissues. Biochem J. 1994 Jun 1;300 (Pt 2):525-30.

[2]. Inhibition of endogenous HIF inactivation induces angiogenesis in ischaemic skeletal muscles of mice. J Physiol. 2004 Oct 1;560(Pt 1):21-6.

[3]. Prolyl hydroxylase 2 deficiency limits proliferation of vascular smooth muscle cells by hypoxia-inducible factor-1{alpha}-dependent mechanisms. Am J Physiol Lung Cell Mol Physiol. 2009 Jun;296(6):L921-7.

[4]. Up-regulation of hypoxia-inducible factor-1α enhanced the cardioprotective effects of ischemic postconditioning in hyperlipidemic rats. Acta Biochim Biophys Sin (Shanghai). 2014 Feb;46(2):112-8.

[5]. Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors induce autophagy and have a protective effect in an in-vitro ischaemia model.Sci Rep. 2020 Jan 31;10(1):1597.

Dimethyloxalylglycine is a glycine derivative that is the diester obtained by formal condensation of the carboxy groups of N-oxalylglycine with two molecules of methanol. It has a role as a neuroprotective agent and an EC 1.14.11.29 (hypoxia-inducible factor-proline dioxygenase) inhibitor. It is a glycine derivative, a methyl ester and a secondary carboxamide. It is functionally related to a N-oxalylglycine.

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DMOG (dimethyloxaloylglycine) is a cell-permeable, competitive inhibitor of prolyl 4-hydroxylases (PHDs), which are key enzymes that mediate oxygen-dependent hydroxylation of HIF-1α. By inhibiting PHDs, DMOG prevents proteasomal degradation of HIF-1α, leading to its stabilization and nuclear translocation. Stabilized HIF-1α then binds to hypoxia-responsive elements (HREs) in the promoter regions of target genes (e.g., VEGF, EPO, GLUT1), regulating angiogenesis, erythropoiesis, and glucose metabolism [1,2,3,4,5]
- The study in embryonic chicken tissues [1] was the first to demonstrate that DMOG effectively inhibits endogenous PHD activity in a dose-dependent and reversible manner, providing a foundational tool for investigating HIF-mediated signaling pathways in vitro [1]
- In the mouse hindlimb ischemia model [2], DMOG-induced angiogenesis was associated with increased VEGF and EPO expression, suggesting its potential as a therapeutic agent for peripheral arterial disease (PAD), where insufficient angiogenesis contributes to tissue ischemia [2]
- In primary VSMCs [3], DMOG limits cell proliferation via HIF-1α-dependent upregulation of p21, indicating a role in suppressing abnormal vascular remodeling (e.g., in atherosclerosis or restenosis after angioplasty) [3]
- In hyperlipidemic rats [4], DMOG enhances the cardioprotective effect of IPOC by stabilizing HIF-1α and reducing cardiomyocyte apoptosis, addressing the reduced efficacy of IPOC in metabolic disorders (e.g., hyperlipidemia) and expanding its clinical application scope [4]
- In the in vitro OGD model [5], DMOG exerts cytoprotection by inducing autophagy (via HIF-1α-mediated Beclin-1 upregulation), revealing a novel mechanism by which PHD inhibitors protect against ischemic injury beyond angiogenesis promotion [5]

C6H9NO5

175.14

175.048

89464-63-1

560326

White to off-white solid powder

1.2±0.1 g/cm3

46-48ºC

1.440

-0.56

81.7

1

5

5

12

200

0

BNJOZDZCRHCODO-UHFFFAOYSA-N

InChI=1S/C6H9NO5/c1-11-4(8)3-7-5(9)6(10)12-2/h3H2,1-2H3,(H,7,9)

methyl 2-[(2-methoxy-2-oxoethyl)amino]-2-oxoacetate

2934.99.9001

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)

配方 1 中的溶解度: 150 mg/mL (856.46 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。