| 名稱 | Necrostatin-1 |
| 描述 | Necrostatin-1 (Nec-1) is a necrotic apoptosis inhibitor and RIP1 inhibitor with specificity. Necrostatin-1 inhibits TNF-α-induced necrotic apoptosis. Necrostatin-1 also inhibits IDO. |
| 細胞實驗 | Determination of EC50 was performed in FADD-deficient Jurkat cells treated with human TNFα as previously described. Briefly, cells were seeded into 96-well plates and treated with a range of necrostatin concentrations (30 nM to 100 μM, 11 dose points) in the presence and absence of 10 ng ml–1 human TNFα for 24 h. For these and all other cellular assays, compound stocks (in DMSO) were diluted to appropriate concentrations in DMSO before addition to the cells to maintain final concentration of DMSO for all samples at 0.5%. Cell viability was determined using CellTiter-Glo luminescent cell viability assay. Ratio of luminescence in compound and TNF-treated wells to compound-treated, TNF-untreated wells was calculated (viability, %) [1]. |
| 激酶實驗 | The assay was performed essentially as described. 293T cells were transfected with pcDNA3-FLAG-RIP1 vector, vectors encoding RIP1 mutant proteins or pcDNA3-RIP2-Myc and pcDNA3-FLAG-RIP3 vectors using standard Ca3(PO4)2 precipitation procedure. Culture medium was replaced 6 h after the transfection and cells were lysed 48 h later in the TL buffer consisting of 1% Triton X-100, 150 mM NaCI, 20 mM HEPES, pH 7.3, 5 mM EDTA, 5 mM NaF, 0.2 mM NaVO3 and complete protease inhibitor cocktail. Immunoprecipitation was carried out for 16 h at 4 °C using anti-FLAG M2 agarose beads, followed by three washes with TL buffer and two washes with 20 mM HEPES, pH 7.3. Beads were incubated in 15 μl of the reaction buffer containing 20 mM HEPES, pH 7.3, 10 mM MnCl2 and 10 mM MgCl2 for 15 min at 23–25 °C in the presence of different concentrations of necrostatins. For these assays, compound stocks (in DMSO) were diluted to appropriate concentrations in DMSO before the addition to the reactions to maintain final concentration of DMSO for all samples at 3%. Kinase reaction was initiated by addition of 10 μM cold ATP and 1 mCi of [γ-32P] ATP, and reactions were carried out for 30 min at 30 °C. Reactions were stopped by boiling in SDS-PAGE sample buffer and subjected to 8% SDS-PAGE. RIP1 band was visualized by analysis in a Storm 8200 Phosphorimager. Similar protocol was used for endogenous RIP1 kinase reactions, except mouse monoclonal RIP1 antibody and protein magnetic beads or rabbit RIP1 antibody-coupled agarose beads were used. For recombinant baculovirally expressed RIP1, protein was expressed in Sf9 cells according to manufacturer's instructions and purified using glutathione-sepharose beads. Protein was eluted in 50 mM Tris-HCl, pH 8.0 supplemented with 10 mM reduced glutathione, and eluted protein was used in the kinase reactions, supplemented with 5 × kinase reaction buffer (100 mM HEPES, pH 7.3, 50 mM MnCl2, 50 mM MgCl2, 50 μM cold ATP and 5 μCi of [γ-32P]ATP) [1]. |
| 動物實驗 | 24 hours after reperfusion, mice received intravenous application of 200 μl PBS or RCM via the tail vein. A single dose of zVAD (10 mg/kg body weight) or Nec-1 (1.65 mg/kg body weight) was applied intraperitoneally 15 min. before RCM-injection. To test the activity of zVAD, we applied zVAD from the same byculture to anti-Fas-treated Jurkat cells to assure its quality before mice were treated with this compound. Mice were harvested another 24 hours after RCM-application (48 hours after reperfusion). Blood samples were obtained from retroorbital bleeding and serum levels of urea and creatinine 5 were determined according to clinical standards in the central laboratory of the University Hospital Schleswig-Holstein, Campus Kiel, Germany, employing an enzymatic ultraviolettest for urea and an enzymatic peroxidase-dependent test for creatinine according to the manufacturer's instructions. Kidneys were conserved for histology. In addition to the demonstrated experiments, we compared the PBS group to mice that only received IRI without 200 μl of PBS and detected no changes in serum concentrations of urea and creatinine or histologically [3]. |
| 體外活性 | 方法：人肝癌細胞 Huh7 和 SK-HEP-1 用 Necrostatin-1 (10-20 μM) 預處理 1 h，再用 sulfasalazine、 erastin 或 RSL3 處理24 h，使用 CellTiter Glo? assay 檢測細胞活力。
結果：Necrostatin-1 顯著阻斷了 sulfasalazine 和 erastin 在兩種細胞系中誘導的細胞活力下降，部分逆轉了 SK-HEP-1 細胞中 RSL3 引起的細胞活力下降。[1]
方法：人組織細胞淋巴瘤細胞 U937 用 Necrostatin-1 (1-20 μM)、zVAD.fmk (100 μM) 和 TNFα (10 ng/mL)處理 72 h，使用 ATP-based viability assay 檢測細胞活力。
結果：Necrostatin-1 以濃度依賴的方式有效阻斷 U937 細胞的壞死性死亡。[2]
方法：H/R 損傷誘導的人腎乳頭瘤狀細胞 HK-2 用 Necrostatin-1 (30 mmol/L) 處理 2-12 h，使用 Flow Cytometry 方法分析細胞死亡情況。
結果：Necrostatin-1 部分保護 HK-2 細胞免受 H/R 誘導的壞死。[3] |
| 體內活性 | 方法：為研究造影劑誘導的 AKI (CIAKI) 的病理生理學，將 Necrostatin-1 (1.65 mg/kg) 單次腹腔注射給 C57BL/6 小鼠，15 min 后使用放射性造影劑 (RCM) 誘導 CIAKI。
結果：Necrostatin-1 可以預防滲透性腎病和 CIAKI。Necrostatin-1 阻止了 RCM 誘導的管周毛細血管擴張，這表明 RIP1 激酶結構域在調節(jié) CIAKI 的微血管血液動力學和病理生理學中具有與細胞死亡無關的新作用。[4]
方法：為研究對小鼠肝炎的保護作用及其機制，將 Necrostatin-1 (1.8 mg/kg) 單次腹腔注射給C57BL/6 小鼠，1 h 后使用 concanavalin A 誘導 肝炎。
結果：注射 Necrostatin-1 的小鼠中觀察到肝功能和組織病理學變化的改善以及炎癥細胞因子產生的抑制。注射 Necrostatin-1 的小鼠中 TNF-α、IFN-γ、IL2、IL6 和 RIP1 的表達顯著降低。Necrostatin-1 處理顯著減少了自噬體的形成。結果表明，Necrostatin-1 通過 RIP1 相關和自噬相關途徑預防 concanavalin A 誘導的肝損傷。[5] |
| 存儲條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice. |
| 溶解度 | DMSO : 55 mg/mL (212.08 mM)
10% DMSO+40% PEG300+5% Tween 80+45% Saline : 4 mg/mL (15.42 mM), Please add co-solvents sequentially, clarifying the solution as much as possible before adding the next one. Dissolve by heating and/or sonication if necessary. Working solution is recommended to be prepared and used immediately.
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| 關鍵字 | Receptor-interacting protein kinases | Inhibitor | Necrostatin-1 | RIP kinase | RIPK | Nec 1 | Ferroptosis | Necrostatin1 | Nec1 | inhibit | Autophagy | Indoleamine 2,3-Dioxygenase (IDO) |
| 相關產品 | L-Cystine | Guanidine hydrochloride | Naringin | Valproic Acid | L-Glutamic acid | Gefitinib | Aceglutamide | Hydroxychloroquine | Stavudine | Acetylcysteine | Paeonol | Sodium 4-phenylbutyrate |
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