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.

RIP1:490 nM(EC50, Jurkat cells)

方法：人肝癌细胞 Huh7 和 SK-HEP-1 用 Necrostatin-1 (10-20 µM) 预处理 1 h，再用 sulfasalazine、 erastin 或 RSL3 处理24 h，使用 CellTiter Glo® assay 检测细胞活力。
结果：Necrostatin-1 显著阻断了 sulfasalazine 和 erastin 在两种细胞系中诱导的细胞活力下降，部分逆转了 SK-HEP-1 细胞中 RSL3 引起的细胞活力下降。[1]
方法：人组织细胞淋巴瘤细胞 U937 用 Necrostatin-1 (1-20 µM)、zVAD.fmk (100 μM) 和 TNFα (10 ng/mL)处理 72 h，使用 ATP-based viability assay 检测细胞活力。
结果：Necrostatin-1 以浓度依赖的方式有效阻断 U937 细胞的坏死性死亡。[2]
方法：H/R 损伤诱导的人肾乳头瘤状细胞 HK-2 用 Necrostatin-1 (30 mmol/L) 处理 2-12 h，使用 Flow Cytometry 方法分析细胞死亡情况。
结果：Necrostatin-1 部分保护 HK-2 细胞免受 H/R 诱导的坏死。[3]

方法：为研究造影剂诱导的 AKI (CIAKI) 的病理生理学，将 Necrostatin-1 (1.65 mg/kg) 单次腹腔注射给 C57BL/6 小鼠，15 min 后使用放射性造影剂 (RCM) 诱导 CIAKI。
结果：Necrostatin-1 可以预防渗透性肾病和 CIAKI。Necrostatin-1 阻止了 RCM 诱导的管周毛细血管扩张，这表明 RIP1 激酶结构域在调节 CIAKI 的微血管血液动力学和病理生理学中具有与细胞死亡无关的新作用。[4]
方法：为研究对小鼠肝炎的保护作用及其机制，将 Necrostatin-1 (1.8 mg/kg) 单次腹腔注射给C57BL/6 小鼠，1 h 后使用 concanavalin A 诱导 肝炎。
结果：注射 Necrostatin-1 的小鼠中观察到肝功能和组织病理学变化的改善以及炎症细胞因子产生的抑制。注射 Necrostatin-1 的小鼠中 TNF-α、IFN-γ、IL2、IL6 和 RIP1 的表达显著降低。Necrostatin-1 处理显著减少了自噬体的形成。结果表明，Necrostatin-1 通过 RIP1 相关和自噬相关途径预防 concanavalin A 诱导的肝损伤。[5]

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].

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].

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].