Necrostatin-1 mainly acts on RIP1 in cells [5], Necrostatin-1 is a RIP1 kinase inhibitor with an IC50 value of 0.32 mM[1]. Necrostatin-1 can effectively inhibit RIP1 autophosphorylation, Necrostatin-1 effectively blocks RIP1-RIP3-MLKL signaling by inhibiting RIP1 phosphorylation [7]. Necrostatin-1 is also an IDO inhibitor[2].
Under oxidative stress conditions, CMPC mainly displayed a necrotic phenotype and by pretreatment with Necrostatin-1, we observed a 37 ± 8% reduction in necrotic cell death in CMPCs compared with vehicle. Not find differences in apoptotic-mediated cell death. Therefore, Necrostatin-1 increased the survival of CMPCs by inhibiting necrotic cell death [4]. The ratios of apoptotic and necrotic C2C12 cells were increased following CoCl2 treatment, typical necroptotic morphological characteristics were able to observe by TEM, whereas Necrostatin-1 exhibited a protective effect against CoCl2 induced oxidative stress. Treatment with Necrostatin-1 significantly decreased the levels of RIP1, p ERK1/2, HIF 1α, BNIP3 and ROS induced by CoCl2, and promoted C2C12 differentiation. Necrostatin-1 reversed the CoCl2 induced decrease in mitochondrial membrane potential [6].
In mice,Necrostatin-1 (Nec-1), a specific inhibitor of the receptor-interacting protein 1 (RIP1) kinase domain, prevented osmotic nephrosis and CIAKI, whereas an inactive Necrostatin-1 derivate (Nec-1i) or the pan-caspase inhibitor zVAD did not. Necrostatin-1 prevented RCM-induced dilation of peritubular capillaries, suggesting a novel role unrelated to cell death for the RIP1 kinase domain in the regulation of microvascular hemodynamics and pathophysiology of CIAKI[3].
参考文献:
[1]: Xie T, Peng W, et,al. Structural basis of RIP1 inhibition by necrostatins. Structure. 2013 Mar 5;21(3):493-9. doi: 10.1016/j.str.2013.01.016. PMID: 23473668.
[2]: Degterev A, Huang Z, et,al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005 Jul;1(2):112-9. doi: 10.1038/nchembio711. Epub 2005 May 29. Erratum in: Nat Chem Biol. 2005 Sep;1(4):234. PMID: 16408008.
[3]: Linkermann A, Heller JO, et,al. The RIP1-kinase inhibitor necrostatin-1 prevents osmotic nephrosis and contrast-induced AKI in mice. J Am Soc Nephrol. 2013 Oct;24(10):1545-57. doi: 10.1681/ASN.2012121169. Epub 2013 Jul 5. Erratum in: J Am Soc Nephrol. 2014 Dec;25(12):2942. PMID: 23833261; PMCID: PMC3785275.
[4]: Feyen D, Gaetani R, et,al. Increasing short-term cardiomyocyte progenitor cell (CMPC) survival by necrostatin-1 did not further preserve cardiac function. Cardiovasc Res. 2013 Jul 1;99(1):83-91. doi: 10.1093/cvr/cvt078. Epub 2013 Apr 3. PMID: 23554461.
[5]: Degterev A, Hitomi J, et,al.Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008 May;4(5):313-21. doi: 10.1038/nchembio.83. PMID: 18408713; PMCID: PMC5434866.
[6]: Chen R, Xu J, et,al.Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia. Int J Mol Med. 2018 May;41(5):2565-2572. doi: 10.3892/ijmm.2018.3466. Epub 2018 Feb 6. PMID: 29436688; PMCID: PMC5846651.
[7]: Degterev A, Huang Z, et,al. Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 2005 Jul;1(2):112-9. doi: 10.1038/nchembio711. Epub 2005 May 29. Erratum in: Nat Chem Biol. 2005 Sep;1(4):234. PMID: 16408008.
Necrostatin-1主要作用于细胞[5]中的RIP1，Necrostatin-1是一种RIP1激酶抑制剂，IC50值为0.32 mM[1]。 Necrostatin-1可有效抑制RIP1自身磷酸化，Necrostatin-1通过抑制RIP1磷酸化有效阻断RIP1-RIP3-MLKL信号通路[7]。 Necrostatin-1 也是一种 IDO 抑制剂[2]。
在氧化应激条件下，CMPC 主要表现出坏死表型，通过使用 Necrostatin-1 进行预处理，我们观察到与载体相比，CMPC 中的坏死细胞死亡减少了 37 ± 8%。未发现凋亡介导的细胞死亡差异。因此，Necrostatin-1 通过抑制坏死细胞死亡来增加 CMPCs 的存活率[4]。 CoCl2 处理后 C2C12 细胞凋亡和坏死的比例增加，通过 TEM 能够观察到典型的坏死形态特征，而 Necrostatin-1 对 CoCl2 诱导的氧化应激具有保护作用。用 Necrostatin-1 处理可显着降低 CoCl2 诱导的 RIP1、p ERK1/2、HIF 1α、BNIP3 和 ROS 的水平，并促进 C2C12 分化。 Necrostatin-1逆转了CoCl2诱导的线粒体膜电位降低[6]。
在小鼠中，Necrostatin-1 (Nec-1) 是一种受体相互作用蛋白 1 (RIP1) 激酶结构域的特异性抑制剂，可预防渗透性肾病和 CIAKI，而无活性的 Necrostatin-1 衍生物 (Nec-1i) 或泛半胱天冬酶抑制剂 zVAD 则没有。 Necrostatin-1 阻止 RCM 诱导的管周毛细血管扩张，表明 RIP1 激酶结构域在调节 CIAKI 的微血管血流动力学和病理生理学方面具有与细胞死亡无关的新作用[3]。
| Kinase experiment [1]: | |
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Preparation Method |
Cells were lysed, Immunoprecipitation was carried out for 16 h at 4 °C using anti-FLAG M2 agarose beads, Beads were incubated in 15 ul of the reaction buffer for 15 min at 23-25 °C in the presence of different concentrations of necrostatins (including Necrostatin-1). Kinase reaction was initiated by addition of 10 μM cold ATP and 1 μCi of [y-32P]ATP, and reactions were carried out for 30 min at 30°C. |
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Reaction Conditions |
RIP1 kinase assay（ Necrostatin-1） was performed at 30°C for 30 min |
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Applications |
Necrostatin-1 is a RIP1 kinase inhibitor, RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. |
| Cell experiment [2]: | |
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Cell lines |
Cardiomyocyte progenitor cells (CMPCs) |
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Preparation Method |
CMPCs were pretreated with vehicle or Necrostatin-1 for 30 min prior to the addition of tert-Butyl hydroperoxide in serum-free M199 medium. |
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Reaction Conditions |
30 μM Necrostatin-1 for 30 min |
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Applications |
Under oxidative stress conditions, CMPC mainly displayed a necrotic phenotype and by pretreatment with Necrostatin-1, we observed a 37 ± 8% reduction in necrotic cell death in CMPCs compared with vehicle. Not find differences in apoptotic-mediated cell death. Therefore, Necrostatin-1 increased the survival of CMPCs by inhibiting necrotic cell death. |
| Animal experiment [3]: | |
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Animal models |
Eight-week-old male C57Bl/6 mice |
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Preparation Method |
Mice undergo sham surgery or bilateral renal pedicle clamping 24 hours before intraperitoneal injection of either PBS, the highly specific RIP1 kinase inhibitor Necrostatin-1, or the inactive derivate of necrostatin-1 (Necrostatin-1i) in the presence or absence of RCM. |
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Dosage form |
Necrostatin-1 (1.65 mg/kg body weight) was applied intraperitoneally 15 min before RCM-injection. |
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Applications |
CIAKI Is Attenuated by Necrostatin-1, an Inhibitor of the Kinase Domain of Receptor-Interacting Protein Kinase-1. |
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参考文献: [1]. Degterev A, Hitomi J,et,al.Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 2008 May;4(5):313-21. doi: 10.1038/nchembio.83. PMID: 18408713; PMCID: PMC5434866. [2]. Feyen D, Gaetani R,et,al. Increasing short-term cardiomyocyte progenitor cell (CMPC) survival by necrostatin-1 did not further preserve cardiac function. Cardiovasc Res. 2013 Jul 1;99(1):83-91. doi: 10.1093/cvr/cvt078. Epub 2013 Apr 3. PMID: 23554461. [3]. Linkermann A, Heller JO,et,al. Nec-1The RIP1-kinase inhibitor necrostatin-1 prevents osmotic nephrosis and contrast-induced AKI in mice. J Am Soc Nephrol. 2013 Oct;24(10):1545-57. doi: 10.1681/ASN.2012121169. Epub 2013 Jul 5. Erratum in: J Am Soc Nephrol. 2014 Dec;25(12):2942. PMID: 23833261; PMCID: PMC3785275. |
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