mitochondria-targeted superoxide dismutase mimetic

Mito-tempo（MT）是一种线粒体靶向的超氧化物歧化酶模拟物，通过抑制过氧亚硝酸盐的形成来保护对乙酰氨基酚（APAP）肝毒性的早期阶段。

在这两个时间点，Mito-TEMPO (MT) 均显着抑制 ALT 活性的上升并减少坏死区域，表明 Mito-TEMPO 的保护作用在 APAP 后持续至少 24 小时。在 APAP 肝毒性的后期，Mito-Tempo 可引起继发性细胞凋亡。通过阻断 RIP3，Mito-Tempo 会导致二次细胞凋亡，以响应过多的 APAP[1]。

半胱氨酸蛋白酶活性测定和蛋白质印迹
如所述测量肝脏半胱天冬酶活性（Lawson等人，1999）。简言之，将冷冻肝组织在含有5mM EDTA、2mM DTT和0.1%CHAPS的25mM HEPES缓冲液中匀浆，然后离心得到匀浆。将荧光底物（Ac-DEVD-AFC）加入到匀浆中，并在存在或不存在泛胱天蛋白酶抑制剂（z-VAD-fmk）的情况下测量荧光。结果表示为每单位时间每毫克蛋白质浓度的RFU。使用兔抗胱天蛋白酶3抗体和兔抗β-肌动蛋白抗体以及抗RIP3抗体，如所述（Bajt等，2000）进行蛋白质印迹。使用山羊抗兔HRP缀合的抗体对蛋白质进行可视化。

组织学[1]
将石蜡包埋的肝组织样本切成5μm切片，用苏木精和伊红（H&E）染色，以评估凋亡与坏死（Gujral等人，2002）。如前所述（Knight等人，2002），使用兔多克隆抗硝基酪氨酸抗体和Dako LSAB过氧化物酶试剂盒进行硝基酪氨酸染色。使用切割的胱天蛋白酶-3（Asp175）抗体进行活性胱天蛋白酶3染色。按照制造商的说明，使用原位细胞死亡检测试剂盒AP对细胞死亡进行末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）染色。

Animals
Male C57BL/6J mice 8-12 weeks of age were kept in an environmentally controlled room with a 12h light/dark cycle. RIP3-deficient mice (C57BL/6N background) and C57BL/6N wild type animals were were acclimated before experiments with free access to diet and water.
Experimental design
Overnight fasted mice (16-18h) were treated i.p. with 300 mg/kg APAP dissolved in warm saline. Some mice were treated with 200mg/kg APAP in experiments evaluating effect of RIP3 deficiency. A dose of 20 mg/kg Mito-Tempo dissolved in saline was administered i.p. 1.5 or 3 h after APAP. Some mice were subsequently treated (i.p.) with 10 mg/kg Z-VD fmk (EP1013) dissolved in Tris-buffered saline or vehicle 2 h after APAP. To mimic the clinical care of APAP-overdose patients, some mice received the antidote NAC (i.p., 500 mg/kg) at 1.5 or 3 h after APAP overdose. Groups of mice were euthanized at 0-24 h post-APAP by exsanguination under isoflurane anesthesia. Additional mice were treated i.p. with 100 μg/kg Salmonella abortus equi endotoxin (ET) and 700 mg/kg galactosamine (Gal) for 6 h. Blood was drawn into a heparinized syringe and centrifuged to obtain plasma. Plasma ALT activities were measured using the ALT assay kit from Pointe Scientific, MI. The liver tissue was cut into pieces and fixed in 10% phosphate-buffered formalin for histology or flash frozen in liquid nitrogen and subsequently stored at −80°C.
In vivo morpholino treatment
The antisense sequence used for RIP3 was 5’-TAGGCCATAACTTGACAGAAGACAT-3’. The standard control in vivo oligo sequence from Gene Tools was used for all control morpholino treatments. Morpholinos were used as provided by the manufacturer and administered ip to mice (12.5 mg/kg body weight) every 24h for 2 days. Treatment with APAP was then done on day 3.

[1]. Mito-tempo protects against acute liver injury but induces limited secondary apoptosis during the late phase of acetaminophen hepatotoxicity. Arch Toxicol. 2019 Jan;93(1):163-178.

Researchers previously reported that delayed treatment with Mito-tempo (MT), a mitochondria-targeted superoxide dismutase mimetic, protects against the early phase of acetaminophen (APAP) hepatotoxicity by inhibiting peroxynitrite formation. However, whether this protection is sustained to the late phase of toxicity is unknown. To investigate the late protection, C57Bl/6J mice were treated with 300 mg/kg APAP followed by 20 mg/kg MT 1.5 h or 3 h later. We found that both MT treatments protected against the late phase of APAP hepatotoxicity at 12 and 24 h. Surprisingly, MT-treated mice demonstrated a significant increase in apoptotic hepatocytes, while the necrotic phenotype was observed almost exclusively in mice treated with APAP alone. In addition, there was a significant increase in caspase-3 activity and cleavage in the livers of MT-treated mice. Immunostaining for active caspase-3 revealed that the positively stained hepatocytes were exclusively in centrilobular areas. Treatment with the pan-caspase inhibitor ZVD-fmk (10 mg/kg) 2 h post-APAP neutralized this caspase activation and provided additional protection against APAP hepatotoxicity. Treatment with N-acetylcysteine, the current standard of care for APAP poisoning, protected but did not induce this apoptotic phenotype. Mechanistically, MT treatment inhibited APAP-induced RIP3 kinase expression, and RIP3-deficient mice showed caspase activation and apoptotic morphology in hepatocytes analogous to MT treatment. These data suggest that while necrosis is the primary cause of cell death after APAP hepatotoxicity, treatment with the antioxidant MT may switch the mode of cell death to secondary apoptosis in some cells. Modulation of mitochondrial oxidative stress and RIP3 kinase expression play critical roles in this switch.[1]

C29H36CLN2O2P

511.03510761261

510.22

C, 68.16; H, 7.10; Cl, 6.94; N, 5.48; O, 6.26; P, 6.06

1334850-99-5

MitoTEMPO hydrate;1569257-94-8

134828258

Light yellow to pink solid powder

52.6Ų

2

4

6

35

612

0

QJEOOHMMSUBNGG-UHFFFAOYSA-N

InChI=1S/C29H35N2O2P.ClH/c1-28(2)20-23(21-29(3,4)31(28)33)30-27(32)22-34(24-14-8-5-9-15-24,25-16-10-6-11-17-25)26-18-12-7-13-19-26;/h5-19,23,33H,20-22H2,1-4H3;1H

[2-[(1-Hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)amino]-2-oxoethyl]-triphenylphosphanium Chloride

Mito-TEMPO; 1334850-99-5; [2-[(1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl)amino]-2-oxoethyl]-triphenylphosphanium;chloride; (2-(2,2,6,6-TETRAMETHYLPIPERIDIN-1-OXYL-4-YLAMINO)-2-OXOETHYL)TRIPHENYLPHOSPHONIUM CHLORIDE; Mito-TEMPO?; DTXSID601044299; MFCD26406410;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

DMSO : ~125 mg/mL (~245.08 mM)
H2O : ~60 mg/mL (~117.64 mM)

配方 1 中的溶解度: ≥ 2.25 mg/mL (4.41 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 22.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 2.25 mg/mL (4.41 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 22.5 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: ≥ 2.25 mg/mL (4.41 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 22.5 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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配方 4 中的溶解度: ≥ 2.2 mg/mL (4.4 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline
≥ 2.2 mg/mL (4.4 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)
≥ 2.2 mg/mL (4.4 mM) in 10% DMSO + 90% Corn oil

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配方 5 中的溶解度: 50 mg/mL (98.03 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。