P2Y6 (EC50 = 15 nM)

内体运输与G蛋白偶联受体（GPCR）的命运和信号传导有着错综复杂的联系。细胞外尿苷二磷酸（UDP）通过选择性激活GPCR P2Y6充当信号分子。尽管最近人们对这种受体在胃肠道和神经系统疾病等病理学中的作用感兴趣，但关于P2Y6受体对其内源性激动剂UDP和合成选择性激动剂5-碘-UDP（MRS2693）的内体运输的信息很少。共聚焦显微镜和细胞表面ELISA揭示了表达人P2Y6的AD293和HCT116细胞对MRS2693与UDP刺激的延迟内化动力学反应。有趣的是，UDP诱导了网格蛋白依赖的P2Y6内化，而MRS2693内吞作用的受体刺激似乎与小窝蛋白依赖机制有关。内化P2Y6与Rab4、5和7阳性囊泡相关，与激动剂无关。我们测量了受体表达与Rab11囊泡、反高尔基体网络和溶酶体共同出现的更高频率，以响应MRS2693。有趣的是，在MRS2693刺激下，较高的激动剂浓度逆转了延迟的P2Y6内化和再循环动力学，而没有改变其依赖于小窝蛋白的内化。这项工作表明，配体依赖性效应影响P2Y6受体内化和内体运输。这些发现可以指导可能影响P2Y6信号传导的偏置配体的开发[1]

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内源性P2Y6受体激动剂UDP和合成激动剂MRS2693以浓度依赖的方式（0.1-10 nM）保护C2C12骨骼肌细胞免于凋亡，如碘化丙啶染色、使用苏木精和赫斯特33258的组织化学分析以及DNA断裂所示。无法克服的P2Y6受体拮抗剂MRS2578阻断了这种保护作用。TNFα诱导C2C12细胞凋亡与转录因子NF-κB的激活有关。10nM MRS2693减弱了NF-κB的激活，激活了抗凋亡ERK1/2通路[2]。

在体内小鼠后肢模型中，MRS2693可保护骨骼肌免受缺血/再灌注损伤。P2Y6受体是一种新型的细胞保护受体，在改善骨骼肌损伤方面值得进一步探索[2]

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小鼠骨骼肌缺血/再灌注体内模型的细胞保护作用 我们使用小鼠后肢缺血/再灌注模型来测试选择性P2Y6激动剂MRS2693的体内保护能力。正如之前在同一模型中对腺苷受体诱导的保护作用的研究[16]所证明的那样，外部收缩剂（90分钟）诱导的缺血再灌注（24小时）导致PBS载体处理的小鼠骨骼肌严重损伤。损伤程度通过EBD对骨骼肌细胞染色的增加来量化，EBD与白蛋白结合，仅进入受损细胞，血清肌酸激酶水平更高。缺血再灌注前给予MRS2693（1mg/kg，腹腔注射）可显著减轻损伤程度（图5）。该化合物减少了骨骼肌损伤，血清CK水平显著降低（3450 U/L±1660 U/L，n=10，SE，与赋形剂治疗的12600 U/L±3300 U/L相比，n=14，P=0.037）。同样，MRS2693也显著降低了EBD染色面积百分比（10.4%±2.0%，SE，n=10，与载体处理的28.3%±5.6%，n=7，P=0.0038）。图6[2]显示了治疗和未治疗小鼠切片中EBD染色的肌细胞。

凋亡的诱导和检测[2]
TNFα用于诱导C2C12细胞（5、7和12天龄）凋亡。洗涤细胞后，用含有5μg/ml环己酰亚胺的新鲜培养基替换培养基，如前所述，在随后的整个培养过程中，环己酰酰亚胺都存在，以促进细胞凋亡。在P2Y6受体拮抗剂MRS2578联合用药的情况下，这是下一个要添加的试剂。将新制备的MRS2578（1 mM）DMSO溶液加入培养基中，使终浓度达到10μM，可选择初始培养20分钟。加入细胞的下一个试剂是P2Y6受体激动剂（MRS2693或UDP）（如果存在）或TNFα（10 ng/ml）。当使用P2Y6受体激动剂时，在激动剂后10分钟加入TNFα。细胞在TNFα和其他试剂存在下保持4小时。然后更换培养基，将培养物在放线菌酮存在下放置16小时。在首次暴露于TNFα后20小时观察到细胞死亡
使用标准末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）方法检测凋亡细胞的DNA断裂。PI阳性细胞和用5-溴-2′-脱氧尿苷5′-三磷酸（BrdUTP）标记的DNA片段末端的荧光表明细胞死亡或凋亡。通过用细胞渗透染料Hoechst 33258对DNA进行荧光标记以染色核染色质来检测活细胞的存在。对于显微镜应用，细胞被沉积在载玻片上。

Protocol for in vivo administration of P2Y6 receptor agonist [2]
The P2Y6 receptor agonist MRS2693 (300 μM) or vehicle alone (0.1% DMSO in PBS) was administered in a sterile 0.1-ml volume by i.p. injection 2 h before the induction of ischemia. This protocol was used in the previous study of adenosine receptor agonists and antagonists in the same model. EBD (1% wt/vol solution to yield 1 mg of EBD/10 g body weight) was also given via a separate i.p. injection 2.5 h before the induction of ischemia.

[1]. Ligand-dependent intracellular trafficking of the G protein-coupled P2Y6 receptor. Biochim Biophys Acta Mol Cell Res. 2023 Jun;1870(5):119476.

[2]. Attenuation of apoptosis in vitro and ischemia/reperfusion injury in vivo in mouse skeletal muscle by P2Y6 receptor activation. Pharmacol Res. 2008 Sep-Oct;58(3-4):232-9.

For the investigation of the role of the P2Y6 receptor in skeletal muscle cell injury, we have chosen to use a novel, potent synthetic agonist, MRS2693. MRS2693 can be considered more selective for this subtype because: 1) MRS2693 itself has no activity at other P2Y subtypes and 2) the corresponding 5′-triphosphate derivative is a much weaker agonist than UTP at other P2Y receptor subtypes. We have demonstrated that this derivative is cytoprotective toward skeletal muscle cells in the mouse both in vitro and in vivo.
Activation of the endogenous P2Y6 receptor in C2C12 cells significantly attenuated TNFα-induced apoptosis. The protection occurred at very low agonist concentrations and correlated with the activation of ERK1/2. The potent antiapoptotic protection by the novel P2Y6 receptor agonist MRS2693 in the C2C12 cell line was concentration-dependent between 0.1 and 10 nM. Similar results were obtained in astrocytoma cells, in which the protection by the appropriate P2Y agonist was clearly P2Y6 receptor-dependent and was absent in untransfected control cells or cells expressing the P2Y4 receptor.
Members of the protein kinase C (PKC) family of serine/threonine protein kinases are involved in many cellular responses across a wide range of cell types. Each PKC isoenzyme may be involved in specific regulatory processes. Various PKC isoenzymes exhibit differences in tissue distribution, intracellular localization, and cofactor requirements, suggesting that they are freely regulated in response to discrete ligands, and that they may act on distinct protein substrates. Our results showed that activation of the P2Y6 receptor by MRS2693 increased the expression level of PKC θ, which might control stimulation of ERK1/2 activation. ERK1/2 is a contributing pathway in the protection by MRS2693 against TNFα-induced cell death in C2C12 cells. Thus, the protection in skeletal muscle cells mechanistically resembles protection in the astrocytoma cells.
There are no competitive antagonists that can be used as pharmacological probes of the P2Y6 receptor, so we used the diisothiocyanate derivative MRS2578 as P2Y6 receptor antagonist. This antagonist blocked the protection provided by MRS2693 against apoptosis in the C2C12 cell line. The reduced protection at higher concentrations of the agonists might be a result of interaction with other extracellular nucleotide binding sites or enzymes that act on nucleotides.
TNFα-induced apoptosis in C2C12 cells correlated with NF-κB activation. This was consistent with numerous previous reports in which TNFα has been noted to activate NF-κB in various systems. NF-κB elevation was also noted to induce damage accompanied by protein degradation in cultured rat skeletal muscle cells. Curiously, P2Y6 receptor activation was previously reported to induce translocation of NF-κB to the nucleus in certain cell types, including osteoblasts. In our study, P2Y6 receptor activation alone had little effect on NF-κB, yet substantially reduced the dramatic elevation of NF-κB induced by TNFα.
In an in vivo mouse model of hindlimb skeletal muscle ischemia/reperfusion injury, MRS2693 was also able to exert a potent cytoprotective effect. The same model was previously applied to study the protective effect of adenosine receptor agonists. We have not yet elucidated the mechanism of the P2Y6 receptor-induced protection in vivo.
In conclusion, the P2Y6 receptor is a novel cytoprotective receptor that warrants exploration for ameliorating skeletal muscle injury. This effort will be aided with the development of even more potent, selective, and stable agonists of the P2Y6 receptor.[2]

C9H10IN2NA3O12P2

596.003209590912

595.844

C, 18.14; H, 1.69; I, 21.29; N, 4.70; Na, 11.57; O, 32.21; P, 10.39

1448858-83-0

1448858-83-0;MRS 2693 trisodium salt;93906-48-0;MRS2693

90488768

Typically exists as solid at room temperature

221

3

12

5

29

704

4

IC1C(NC(N(C=1)[C@H]1[C@@H]([C@@H]([C@@H](COP(=O)([O-])OP(=O)([O-])[O-])O1)O)O)=O)=O.[Na+].[Na+].[Na+]

QWGVSYFNEAILDQ-FCIXCQMASA-K

InChI=1S/C9H13IN2O12P2.3Na/c10-3-1-12(9(16)11-7(3)15)8-6(14)5(13)4(23-8)2-22-26(20,21)24-25(17,18)19;;;/h1,4-6,8,13-14H,2H2,(H,20,21)(H,11,15,16)(H2,17,18,19);;;/q;3*+1/p-3/t4-,5-,6-,8-;;;/m1.../s1

trisodium;[[(2R,3S,4R,5R)-3,4-dihydroxy-5-(5-iodo-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-oxidophosphoryl] phosphate

MRS 2693 trisodium salt; 1448858-83-0; 5-Iodouridine-5'-O-diphosphate trisodium salt; 5-Iodo-UDP trisodium salt; trisodium;[[(2R,3S,4R,5R)-3,4-dihydroxy-5-(5-iodo-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-oxidophosphoryl] phosphate;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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；
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。