BOC-D-FMK

Caspase

细胞凋亡是由称为半胱天冬酶的蛋白酶家族精心策划的细胞死亡途径。 TNFα 激活时会产生活性氧 (ROS)，但 Boc-D-fmk 可以阻止这种情况发生。 Boc-D-FMK 的 IC50 为 39 µM[1]，可阻断 TNFα 诱导的细胞凋亡。浓度为 50 µM 的 BocD-fmk 可阻止金雀异黄素诱导的 p815 细胞凋亡。根据共聚焦显微镜观察，线粒体凋亡因子的释放受到 BocD-fmk 的抑制 [2]。

在胆管结扎大鼠中，Boc-D-FMK-fmk 显着减少肝细胞凋亡，并可能提高内毒素攻击后的存活率[3]。接受单次 Boc-D-FMK 注射后，MN 可以在 8 周以上的时间内长期免受根撕脱引起的死亡，并且 Boc-D-FMK 治疗的 MN 能够将轴突再生为 PN 移植物已植入并重新支配目标肌肉[4]。

在大多数细胞类型中，组成型和配体诱导的细胞凋亡是一个依赖caspase的过程。然而，在中性粒细胞中，广谱caspase抑制剂z-VAD-fmk增强肿瘤坏死因子α (TNF α)诱导的细胞死亡，这被解释为caspase依赖性和非依赖性细胞死亡途径的证据。我们的目的是确定z-VAD-fmk在中性粒细胞中作用的特异性，并确定其潜在的作用机制。虽然证实z-VAD-fmk(> 100微米)增强TNF α诱导的中性粒细胞凋亡，但较低浓度(1-30微米)完全阻断TNF α刺激的细胞凋亡。Boc-D-fmk是一种类似的广谱caspase抑制剂，z-IETD-fmk是一种选择性caspase-8抑制剂，它们只对TNF α刺激的细胞凋亡产生浓度依赖性抑制。此外，caspase-9抑制剂Ac-LEHD-cmk对TNF α诱导的细胞凋亡没有影响，z-VAD-fmk和Boc-D-fmk抑制TNF α刺激的活性氧(ROS)的产生。这些数据表明，TNF α诱导的中性粒细胞凋亡完全依赖于caspase，并使用线粒体独立途径，z-VAD-fmk的促凋亡作用是化合物特异性的，与ROS无关。[1]

在初步研究中，我们发现，与boc -天冬氨酸(OMe)-氟甲基酮(BocD-fmk)不同，在常规剂量下，苄基氧羰基-缬氨酸- α -asp (OMe)-氟甲基酮(zVAD- fmk)不能阻止染料木黄酮诱导的p815乳母细胞瘤细胞凋亡。本研究旨在揭示zvd -fmk无法阻止这种类型细胞凋亡的机制。我们观察到染料木素处理的细胞14-3-3蛋白水平降低，BocD-fmk而不是zVAD-fmk阻止14-3-3蛋白水平的降低和Bad的释放。我们还证明，在染料木黄酮处理的细胞中，BocD-fmk可以阻止截断的Bad与Bcl-xL的相互作用，而zVAD-fmk则不能。我们的数据表明，与zVAD-fmk相比，BocD- fmk对14-3-3/Bad信号通路具有一定的抑制偏好。我们还阐明了BocD-fmk和zVAD-fmk的这种差异功效是由于抑制caspase-6的效果不同，并且zvd -fmk和caspase-6特异性抑制剂共同处理实质上阻止了染料木黄酮诱导的细胞凋亡。我们的数据显示，caspase-6在染料木黄酮诱导的p815细胞凋亡的Bad/14-3-3通路中发挥作用，并且与BocD-fmk相比，通常剂量的zVAD-fmk不能阻止caspase-6作用于14-3-3/Bad介导的事件。[2]

Male Sprague-Dawley rats, weighing 280-300 g were randomized to three groups of eight rats each. Group 1 (OBBOC-D) underwent common bile duct ligation and simultaneous treatment with Boc-D-FMK-fmk (dissolved in dimethylsulfoxide [DMSO]). Group 2 (OBZFA) underwent common bile duct ligation and simultaneous treatment with ZFA-fmk (dissolved in DMSO). Group 3 (SHAM) underwent sham operation and simultaneous treatment with the same amount of dimethylsulfoxide (DMSO, n = 4) or the same amount of normal saline (n = 4). After 3 days, liver tissue was harvested for histopathological analysis and measurements of apoptosis. Survival rates were measured in a separate experiment in which animals underwent the same protocol. The animals received endotoxin (15 mg/kg) in the afternoon of the third postoperative day. Animals were observed for 48 h and the survival rates were recorded. Results: When compared with sham operation, common bile duct ligation with ZFA-fmk (placebo) significantly increased hepatocyte apoptosis (P < 0.001). When compared with the OBZFA group, Boc-D-FMK significantly diminished the increased hepatocyte apoptosis in the OBBOC-D group (P < 0.001). There is no difference in hepatocyte apoptosis (P = 0.05) between OBBOC-D and SHAM groups. After endotoxin challenge, the 48 h survival rates were 100%, 87.5% and 62.5% for the SHAM, OBBOC-D and OBZFA groups, respectively. Conclusions: Boc-D-FMK-fmk effectively attenuated the hepatocyte apoptosis in bile duct-ligated rats and may improve the survival rates after endotoxin challenge.[3]

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We examined whether (1) a pan-caspase inhibitor, Boc-D-FMK, exerts long-term neuroprotective effects on spinal motoneurons (MNs) after root avulsion in neonatal rats and (2) whether the rescued spinal MNs regenerate their axons into a peripheral nerve (PN) graft and reinnervate a previously denervated target muscle. Eight weeks after root avulsion, 67% of spinal MNs remained in the Boc-D-FMK-treated group, whereas all MNs died in the sham control group. By 12 weeks postinjury, however, all Boc-D-FMK treated MNs died. In the regeneration experiment, a PN graft was implanted at different times after injury. The animals were allowed to survive for 4 weeks following the operation. Without caspase inhibition, MNs did not regenerate at any time point. In animals treated with Ac-DEVD-CHO, a caspase-3-specific inhibitor, and Boc-D-FMK, 44 and 62% of MNs, respectively, were found to regenerate their axons into a PN graft implanted immediately after root avulsion. When the PN graft was implanted 2 weeks after injury, however, MNs failed to regenerate following Ac-DEVD-CHO treatment, whereas 53% of MNs regenerated their axons into the graft after treatment with Boc-D-FMK. No regeneration was observed when a PN graft was implanted later than 2 weeks after injury. In the reinnervation study, injured MNs and the target biceps muscle were reconnected by a PN bridge implanted 2 weeks after root avulsion with administration of Boc-D-FMK. Eight weeks following the operation, 39% of MNs reinnervated the biceps muscle. Morphologically normal synapses and motor endplates were reformed in the muscle fibers. Collectively, these data provide evidence that injured neonatal motoneurons can survive and reinnervate peripheral muscle targets following inhibition of caspases.[4]

[1]. z-VAD-fmk augmentation of TNF alpha-stimulated neutrophil apoptosis is compound specific and does not involve the generation of reactive oxygen species.

[2]. zVAD-fmk, unlike BocD-fmk, does not inhibit caspase-6 acting on 14-3-3/Bad pathway in apoptosis of p815 mastocytoma cells. Exp Mol Med. 2006 Dec 31;38(6):634-42.

[3]. Effect of Boc-D-Fmk on hepatocyte apoptosis after bile duct ligation in rat and survival rate after endotoxin challenge. J Gastroenterol Hepatol. 2008 Aug;23(8 Pt 1):1276-9.

[4]. Inhibition of caspases promotes long-term survival and reinnervation by axotomized spinal motoneurons of denervated muscle in newborn rats. Exp Neurol. 2003 Jun;181(2):190-203.

Surgical procedures[4]
On the day of birth, newborn female Spraque–Dawley rats were anesthetized under deep hypothermia. Under a surgical microscope, a dorsal laminectomy was carried out and the spinal root of the seventh cervical (C7) segment was identified. The C7 ventral root together with the dorsal root were avulsed by a pair of microhemostatic forceps. To study the long-term neuroprotective effect of Boc-D-FMK, animals were divided into two groups. There were six rats in each group at each time point. The first...

Long-term neuroprotective effect of Boc-D-FMK[4]
Motoneurons were identified and counted as described previously (Clarke and Oppenheim, 1995). In brief, only MNs with a large nucleus containing clearly visible nucleoli and a largely distinct cytoplasm were counted. Because the number of MNs on the contralateral intact side of the experimental animals was not significantly different from normal control animals (data not shown), the contralateral side served as an internal control. We have previously reported that by 7 days postlesion, there...

Discussion[4]
The present results indicate that caspases play a key role in the death of spinal MNs after injury in neonates. Inhibition of caspases led to long-term neuroprotection as well as axonal regeneration of avulsed spinal MNs. With a PN bridge between the spinal cord and the denervated muscle target, the caspase inhibitor-treated MNs were able to reinnervate the neuromuscular junction and muscular atrophy was reduced. These results suggest that the inhibition of caspases may be a potent strategy for ...

Conclusion[4]
The experiments presented here provide evidence that following root avulsion, neonatal spinal MNs can survive and reinnervate target muscle if appropriate treatment is provided. A single injection of Boc-D-FMK results in long-term protection of MNs against root avulsion-induced death for more than 8 weeks and the Boc-D-FMK-treated MNs are able to regenerate their axons into an implanted PN graft and reinnervate the target muscle. Taken together, these data suggest that local administration of...

C11H18FNO5

263.26272726059

263.12

C, 50.19; H, 6.89; F, 7.22; N, 5.32; O, 30.39

634911-80-1

634911-80-1

16760348

Boc-DL-Asp(OMe)-CH2F

Solid if <31.2°C; Liquid if >31.2°C; Light yellow to yellow color

1.150

0.9

81.7Ų

1

6

8

18

324

0

FCC(C(CC(=O)OC)NC(=O)OC(C)(C)C)=O

MXOOUCRHWJYCAL-UHFFFAOYSA-N

InChI=1S/C11H18FNO5/c1-11(2,3)18-10(16)13-7(8(14)6-12)5-9(15)17-4/h7H,5-6H2,1-4H3,(H,13,16)

methyl 5-fluoro-3-[(2-methylpropan-2-yl)oxycarbonylamino]-4-oxopentanoate

BOC-D-FMK; 634911-80-1; Caspase Inhibitor III; 3-[[(tert-Butoxy)carbonyl]amino]-5-fluoro-4-oxopentanoic acid methyl ester; methyl 5-fluoro-3-[(2-methylpropan-2-yl)oxycarbonylamino]-4-oxopentanoate; Caspase3-Inhibitor BOC-D-FMK; BOC-D-FMK?; C11H18FNO5;

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)

DMSO: ~100 mg/mL (~379.9 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (9.50 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 (9.50 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 (9.50 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：
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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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