NLRP3

在纳摩尔剂量下，MCC950 可防止传统和非规范的 NLRP3 激活。 MCC950 不会特异性抑制 AIM2、NLRC4 和 NLRP1 的激活，而 NLRP3 则会被抑制。使用小鼠骨髓源性巨噬细胞 (BMDM) 和人单核细胞源性巨噬细胞 (HMDM)，研究了 MCC950 对 NLRP3 炎性体激活的影响。 MCC950 在 BMDM 中表现出约 7.5 nM 的抑制能力，在 HMDM 中表现出约 8.1 nM 的抑制能力。此外，MCC950 以剂量依赖性方式减少 IL-1β 分泌，但不减少 TNF-α 分泌。 MCC950 首先刺激非经典途径，然后选择性抑制 caspase-11 介导的 NLRP3 激活和 IL-1β 释放。即使剂量为 10 µM，MCC950 也无法抑制鼠伤寒沙门氏菌诱导的 NLRC4 刺激的 IL-1β 和 TNF-α 产生。 MCC950 对鼠伤寒沙门氏菌响应的 IL-1β 或 caspase-1 激活过程没有影响。 MCC950 处理不会对细胞裂解物中的 pro-caspase-1 和 pro-IL-1β 产生显着影响 [1]。

MCC950 可减轻实验性自身免疫性脑脊髓炎 (EAE)（一种多发性硬化症疾病模型）的严重程度，并减少白介素-1p (IL-1β) 的产生。 MCC950 预处理可降低 IL-1β 和 IL-6 的血清浓度，但不会显着降低 TNF-α 水平。 MCC950 治疗可减轻小鼠 EAE 的严重程度并推迟其发展。将 MCC950 处理的动物与 PBS 处理的小鼠进行比较时，第 22 天处死小鼠的脑单核细胞的细胞内细胞因子标记和 FACS 分析显示，产生 IL-17 和 IFN-γ 的 CD3+ T 细胞的频率略低。产生 IFN-γ 的 CD3+ T 细胞的 CD4+ 和 γδ+ 亚群数量减少，特别是产生 IL-17 的细胞[1]。

炎症小体活化测定[1]
将BMDM以5×0 105/ml或1×0 6/ml接种，HMDM以5倍10 5/ml接种，PBMC以2×10 6/ml或5×0 6 g/ml接种于96孔板中。第二天，更换过夜培养基，并用来自大肠杆菌血清型EH100（ra）TLRgrade™的10 ng/ml LPS刺激细胞3小时。取出培养基，用含有二甲基亚砜（1:1000）、MCC950（0.001-10µM）、格列本脲（200µM），孤雌内酯（10µM）或拜耳半胱氨酰白三烯受体拮抗剂1-（5-羧基-2{3-[4-（3-环己基丙氧基）苯基]丙氧基}苯甲酰基）哌啶-4-羧酸（40µM）的无血清培养基（SFM）代替。然后用炎症小体激活剂刺激细胞30分钟：5 mM腺苷5’-三磷酸二钠盐水合物（ATP）（1小时）、用Lipofectamine 2000™（Invitrogen）转染的1µg/ml聚脱氧腺苷酸胸苷酸钠盐（Poly dA:dT）（3-4小时）、200µg/ml MSU（过夜）和10µM尼格瑞金（1小时。细胞也用25µg/ml聚腺苷酸-多ridylic acid刺激（4小时）。对于非典型炎症小体激活细胞，用100 ng/ml Pam3CSK4引发4小时，移除培养基并用含有DMSO或MCC950的SFM代替，并使用0.25%FuGENE®转染2µg/ml LPS 16小时。根据制造商的说明，移除上清液并使用ELISA试剂盒分析。使用Cytox96®非放射性细胞毒性测定法测量LDH释放。[1]

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飞行时间炎症小体评估（TOFIE）测定[1]
使用Lipofectamine 2000™在24孔板中用以下质粒转染HEK293T细胞（4×105/ml）：pEF6人ASC-GFP、pEF6人类C-mCherry或空载体对照。转染后1小时，用DMSO或MCC950（0.1–50µM）处理细胞。将转染后15小时的细胞移除并悬浮在含有1%FCS和2mM EDTA的DPBS中。使用Gallios™流式细胞仪和FlowJo软件对细胞进行分析。在GFP和Cherry表达上对活细胞进行门控（当共转染时）。通过分析GFP脉冲区域的高度和宽度（低宽度：区域和高高度：区域）来确定含有ASC斑点的细胞的百分比。Sester et al。

蛋白质印迹[1]
通过在50µl 5中直接裂解制备细胞裂解物ィ 莱姆利样品缓冲液。根据制造商的说明，使用StrataClean™树脂浓缩上清液的蛋白质含量。将蛋白质样品在15%SDS-PAGE凝胶上解析，并使用湿转移系统转移到聚偏二氟乙烯（PVDF）膜上。在室温（RT）下，将膜封闭在TBS-T（50mM Tris/HCL，pH 7.6，150mM NaCl和0.1%（v/v）Tween-20）中的5%（w/v）奶粉中1小时。将膜与稀释在TBS-T中的5%（w/v）奶粉中的一级抗体一起孵育，然后与适当的辣根过氧化物酶（HRP）偶联的二级抗体在TBS-T中稀释在5%（w/v）奶粉中孵育1小时。使用20ィ LumiGLO®化学发光试剂。在重新处理之前，使用Restore™PLUS蛋白质印迹剥离缓冲液剥离膜。[1]
将患有CAPS的个体的PBMC以2×0 106/ml的剂量接种在12孔板中，然后用1µg/ml LPS预处理3小时。用含有MCC950（5–1000 nM）的SFM代替培养基。45分钟后，收集细胞培养上清液和细胞裂解物。使用Novex®Tris-Glycine凝胶系统解析样品。[1]

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荧光成像平板阅读器（FLIPR）Ca2+分析[1]
将BMDM（3×104/孔）在37°C下用不洗涤的钙染料（Molecular Devices）在含有0.1%BSA的生理盐水溶液（PSS；成分NaCl 140 mM，葡萄糖11.5 mM，KCl 5.9 mM，MgCl2 1.4 mM，NaH2PO4 1.2 mM，NaHCO3 5 mM，CaCl2 1.8 mM，HEPES 10 mM）中加载30分钟。然后将细胞转移到FLIPRETTRA荧光板读取器上，并使用冷却的CCD相机测量Ca2+响应，激发为470–495 nM，发射为515–575 nM。调节每个板的相机增益和强度，以产生至少1000个任意荧光单位（AFU）的基线荧光。在添加MCC950之前，采集10个基线荧光读数，然后在添加样品后300秒内每秒读取荧光读数，并在添加PSS或ATP（500µM）后再读取300秒。

In vivo LPS challenge[1]
C57BL/6 mice were injected intraperitoneally (i.p.) with 50 mg/kg MCC950 or vehicle control (DMSO/PBS) 1 h h before i.p. injection of 10 mg/kg LPS Escherichia coli 055:B5 or PBS. After for 2 h mice were sacrificed and serum levels of IL-1β, TNF-α and IL-6 were measured by ELISA.[1]

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Induction and Assessment of EAE[1]
C57BL/6 mice were immunized subcutaneously with 150 µg of MOG peptide 35–55 (GenScript) emulsified in CFA containing 4 mg/ml (0.4.mg/mouse) of heat-killed MTB (Chondrex). Mice were injected i.p. with 500 ng pertussis toxin (PT: kaketsuken) on days 0 and 2. MCC950 was administered i.p. to mice (10 mg/kg) at induction of the disease, day 0, 1 and 2 and every 2 days thereafter. Control mice were administered vehicle (PBS) at the same time points. Mice were observed for clinical signs of disease daily (unblinded). Disease severity was scored as follows: no clinical signs, 0; limp tail, 1; ataxic gait, 2; hind limb weakness, 3; hind limb paralysis, 4; and tetra paralysis, 5., Experiments were performed under license (BI00/2412) from The Irish Medicine Board and with approval from the Trinity College Dublin BioResources Ethics Committee.[1]

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FACS analysis of EAE[1]
On day 22 post immunization mononuclear cells were isolated from whole brains of perfused mice with EAE, following homogenisation and centrifugation on a Percoll gradient. Mononuclear cells (MNC) (2 × 106/ml) were stimulated for 4 h with PMA (10 ng/ml) and ionomycin (1 µg/ml) in the presence of brefeldin A (5 µg/ml). Cells were washed in PBS and re-suspended in 50 µL PBS with 1:1,000 LIVE/DEAD® Fixable Aqua Dead Cell Stain kit for 20 min. Surface stains for CD3 (145-2c11) (0.5 µl/106 cells), CD4 (RM4-5) (0.5 µl/106 cells) and γδ TCR (GL3) (1 µl/106 cells) (eBioscience) were added and cells were incubated for a further 20 mins. Cells were then fixed with 2% paraformaldehyde and washed in PBS twice, before being intracellularly stained for IL-17 or IFN-γ in permeabilization buffer (0.2% saponin in PBS + 1% FBS). Flow cytometric analysis of MNC was performed using a BD LSRFortessa™ and analysed with FlowJo software. MNC were first gated on live CD3+ T cells followed by CD4 expression, γδ TCR expression or cytokine production.[1]

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NLRP3 and NLRP1 activating mutation mice[1]
Mice were backcrossed to C57BL/6 at least ten times. Nlrp3A350VneoR mice were provided by Hal M. Hoffman, The University of California, San Diego, U.S.A. and crossed with LysMCre mice (B6.129P2-Lyz2tm1(cre)Ifo/J. MCC950 was administered i.p. (20 mg/kg) every second day starting at day 4 after birth. Mice with an activating mutation in NLRP1, Nlrp1aQ593P were generated on a C57BL/6 background as described previously and administered MCC950 i.p. (20 mg/kg) every second day for 9 days. Blood was collected at the timepoints indicated for analysis of plasma cytokines by ELISA. IL-18 ELISA was performed as described by Westwell-Roper et al. Experiments were performed under AEC Project 2013.011 and were approved by the Animal Ethics Committee of The Walter and Eliza Hall Institute of Medical Research.

[1]. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med. 2015 Mar;21(3):248-55.

The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its aberrant activation is pathogenic in inherited disorders such as cryopyrin-associated periodic syndrome (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease and atherosclerosis. We describe the development of MCC950, a potent, selective, small-molecule inhibitor of NLRP3. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibited activation of NLRP3 but not the AIM2, NLRC4 or NLRP1 inflammasomes. MCC950 reduced interleukin-1β (IL-1β) production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Furthermore, MCC950 treatment rescued neonatal lethality in a mouse model of CAPS and was active in ex vivo samples from individuals with Muckle-Wells syndrome. MCC950 is thus a potential therapeutic for NLRP3-associated syndromes, including autoinflammatory and autoimmune diseases, and a tool for further study of the NLRP3 inflammasome in human health and disease.[1]

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Diabetes is associated with a high risk of developing cognitive dysfunction and neuropsychiatric disabilities, and these disease symptomsare termed diabetic encephalopathy (DEP). Inflammation is involved in the development of DEP. The cleavage and maturation of the proinflammatory cytokine interleukin (IL)-1β is regulated by the NLRP3 inflammasome. Obese and type 2 diabetic db/db mice show anxiety- and depression-like behaviors and cognitive disorders associated with hippocampal inflammation. The purpose of this study was to explore the role of NLRP3 inflammasome in DEP. Results showed that expression levels of inflammasome components including NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1, as well as IL-1β in the hippocampus of diabetic db/db mice were higher than those of non-diabetic db/m mice. Treatment of db/db mice with NLRP3 inflammasome inhibitor MCC950 ameliorated anxiety- and depression-like behaviors as well as cognitive dysfunction, and reversed increased NLRP3, ASC, and IL-1βexpression levels and caspase-1 activity in hippocampus. Moreover, MCC950 treatment significantly improved insulin sensitivity in db/db mice. These results demonstrate that inhibition of NLRP3 inflammasome activation may prove to be a potential therapeutic approach for DEP treatment.[2]

C20H24N2O5S

404.48

404.14

210826-40-7

MCC950 sodium;256373-96-3

9910393

White to light yellow solid

1.4±0.1 g/cm3

239 ºC

1.637

3.2

106Ų

3

5

4

28

684

0

S(C1=C([H])C(=C([H])O1)C(C([H])([H])[H])(C([H])([H])[H])O[H])(N([H])C(N([H])C1=C2C([H])([H])C([H])([H])C([H])([H])C2=C([H])C2C([H])([H])C([H])([H])C([H])([H])C=21)=O)(=O)=O

LFQQNXFKPNZRFT-UHFFFAOYSA-M

InChI=1S/C20H24N2O5S.Na/c1-20(2,24)14-10-17(27-11-14)28(25,26)22-19(23)21-18-15-7-3-5-12(15)9-13-6-4-8-16(13)18;/h9-11,24H,3-8H2,1-2H3,(H2,21,22,23);/q;+1/p-1

sodium;1,2,3,5,6,7-hexahydro-s-indacen-4-ylcarbamoyl-[4-(2-hydroxypropan-2-yl)furan-2-yl]sulfonylazanide

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: ≥ 2.5 mg/mL (6.18 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.18 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.18 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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。