Myosin II (IC50: 0.5 to 5 μM)

Blebbistatin 的 IC50 值范围为 0.5 至 5 μM，可有效抑制脊椎动物非肌肉肌球蛋白 IIA 和 IIB 以及多种横纹肌肌球蛋白。对平滑肌肌球蛋白仅有轻微抑制作用 (IC50=80 μM)[1]。肌球蛋白亚片段 1 与肌球蛋白亚片段 1 的核苷酸结合不具有竞争性。该抑制剂通过优先与 ATP 酶中间体、ADP 和活性位点的磷酸盐结合来抑制磷酸盐的释放。它抑制与肌动蛋白亲和力较低的复合物中的肌球蛋白头部基团 [2]。布雷他汀被证明可以在体外改变活化肝星状细胞的外观和功能。星细胞经历树突形态、收缩并失去含有纽蛋白和肌球蛋白 IIA 的粘着斑和应力纤维。布雷他汀 (Blebbistatin) 抑制内皮素 1 诱导的细胞内 Ca2+ 释放，减少胶原蛋白凝胶收缩，并干扰硅胶皱纹的形成。它促进伤口诱导的细胞迁移[3]。

blebbistatin 以剂量依赖的方式完全放松由 KCl 和卡巴胆碱引发的大鼠逼尿肌以及由内皮素-1 引起的人类膀胱收缩。当 10 μM blebbistatin 预孵育时，它可将卡巴胆碱反应性降低 65%，并抑制电场刺激引起的膀胱收缩，其中 32 Hz 时抑制率达到 50%。

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与模型组相比，Blebbistatin（1mg/kg）抑制颈动脉AT的发展，减少炎性细胞的浸润，并防止血管组织损伤。此外，Blebbistati还降低了TF的促凝活性。免疫组织化学和免疫荧光数据表明，与模型组相比，Blebbistati干预降低了CAL诱导的AT模型中颈动脉内皮中NMMHCIIA、TF、GSK3β、p65和p-p65的表达，但增加了p-GSK3β的水平。Blebbistatin可以抑制CAL模型中NMMHCIIA mRNA的表达[3]。

[Ca2+]i[2]的测量
使用多模式台式酶标仪分析了blebbistatin对凝血酶和ATP诱导的Ca2+瞬变的影响。将细胞（每孔8000个）接种到96孔板上，并在2至3天内达到融合。然后在室温下用Fura-2AM（终浓度为1.25μg/mL）装载细胞30分钟。通过分别在510 nm处获得发射，在340 nm和380 nm处获得激发，从而获得比率[Ca2+]i测量值。使用R编程的DRC包（版本1.2.0），使用Michaelis-Menten模型拟合ATP剂量-反应曲线。

全器官移植培养[4]
从出生后第3天（P）的野生型FVB小鼠中解剖耳蜗感觉上皮，并在添加了2%B27、1%N-2和50μg/ml氨苄青霉素的DMEM/F12中培养。在实验组中，耳蜗用0.5 mM新霉素和1μMblebbistatin（溶解在DMSO中）处理12小时，并允许再恢复12小时。将等量的DMS添加到对照组和仅新霉素组中。组织在37°C和5%CO2下培养。

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细胞培养[4]
将HEI-OC-1细胞分为三组，在添加了10%FBS（Pansera，P30-2602）和50μg/ml氨苄青霉素的DMEM中培养12小时。在此初始培养后，实验组在6孔板中用2 mM新霉素和0.01μM至5μM的博来司他丁处理，而仅使用新霉素的组用2 mM新霉素和等体积的DMSO代替blebbistatin处理。再培养24小时后，用PBS彻底洗涤细胞，并在含有氨苄青霉素的DMEM中培养12小时。用等体积的DMSO处理不含新霉素或blebbistatin的对照细胞，并在相同条件下孵育。最后，用倒置相差显微镜对细胞进行成像。

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CCK-8含量测定[4]
使用细胞计数CCK-8试剂盒（蛋白质生物技术，CC201-01）测量细胞死亡。简而言之，将HEI-OC-1细胞暴露于96孔板中的2 mM新霉素中12小时。去除新霉素后，让组织再恢复12小时。在实验组的整个过程中加入blebbistatin，在仅使用新霉素的组中加入等体积的DMSO。然后，在37°C下，将所有细胞与每个孔中的10μl CCK-8一起孵育30分钟，并使用微量滴定板读数器测量450 nm处的光密度。

Model of carotid-artery ligation (CAL)[3]
A mouse model of CAL was generated using a modified method based on previous reports. Briefly, C57BL/6 J mice were anesthetized, as determined by assessment of the righting reflex. Neck hair was removed and a 1-cm midline incision made in the neck to expose the right side of the carotid artery. Two knots were tied in the upper end of the isolated carotid artery (external carotid artery) and internal carotid artery using 6.0 non-absorbable sutures. During carotid surgery, a length of ∼1 cm, between the upper-artery bifurcation and the carotid artery from the first lower ligation point, was tied using two 6.0 non-absorbable silk knots. The wound was rinsed with physiologic (0.9 %) saline, after closing muscle and skin (model group). Sham-operated mice underwent carotid-artery surgery after anesthesia without silk ligation (sham group). The blebbistatin was dissolved in absolute ethanol to the concentration of 1 × 10−2 M, and suspended at 0.5 % CMC-Na before use. In the blebbistatin group, mice were injected with blebbistatin (1 mg/kg, i.v.) to inhibit thrombosis. The blebbistatin was injected to the mice at the 0,4.7 day from the ligation. Six mice were included in each group. After 7 days, blood vessels from all groups were collected.[3]

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5-25 μM

Zebrafish embryos model

[1]. Absolute Stereochemical Assignment and Fluorescence Tuning of the Small Molecule Tool, (–)‐Blebbistatin. Eur J org Chem. 2005, 2005 (9), 1736-1740. doi.org/10.1002/ejoc.200500103
[2]. The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellularcalcium wave propagation in corneal endothelial cells. Invest Ophthalmol Vis Sci. 2008 Nov;49(11):4816-27.
[3]. An inhibitor of myosin II, blebbistatin, suppresses development of arterial thrombosis. Bomed Pharmacother . 2020 Feb:122:109775.
[4]. Blebbistatin Inhibits Neomycin-Induced Apoptosis in Hair Cell-Like HEI-OC-1 Cells and in Cochlear Hair Cells. Front Cell Neurosci. 2020 Feb 5;13:590.

(S)-blebbistatin is the (S)-enantiomer of blebbistatin. It is a blebbistatin and a tertiary alpha-hydroxy ketone.

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(–)-Blebbistatin (1), a recently discovered small molecule inhibitor of the ATPase activity of non-muscle myosin II has been prepared from methyl 5-methylanthranilate (6) in three steps. This flexible synthetic route has also been used to prepare a nitro group-containing analogue 12 that has modified fluorescence properties and improved stability under microscope illumination. The key step in the synthesis of 1 and its analogues was the asymmetric hydroxylation of the quinolone intermediate 3 using the Davis oxaziridine methodology. The absolute stereochemistry of (–)-blebbistatin (1) was shown to be S by X-ray crystal structure analysis of a heavy atom (bromine) containing analogue 11, which was subsequently reduced and shown to be identical to 1.[1]

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Purpose: Thrombin inhibits intercellular Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs) through a mechanism dependent on myosin light chain (MLC) phosphorylation. In this study, blebbistatin, a selective myosin II ATPase inhibitor, was used to investigate whether the effect of thrombin is mediated by enhanced actomyosin contractility. Methods: BCECs were exposed to thrombin (2 U/mL) for 5 minutes. MLC phosphorylation was assayed by immunocytochemistry. Ca(2+) waves were visualized by confocal microscopy with Fluo-4AM. Fluorescence recovery after photobleaching (FRAP) was used to investigate intercellular communication (IC) via gap junctions. ATP release was measured by luciferin-luciferase assay. Lucifer yellow (LY) uptake was used to investigate hemichannel activity, and Fura-2 was used to assay thrombin- and ATP-mediated Ca(2+) responses. Results: Pretreatment with blebbistatin (5 microM for 20 minutes) or its nitro derivative prevented the thrombin-induced inhibition of the Ca(2+) wave. Neither photo-inactivated blebbistatin nor the inactive enantiomers prevented the thrombin effect. Blebbistatin also prevented thrombin-induced inhibition of LY uptake, ATP release and FRAP, indicating that it prevented the thrombin effect on paracrine and gap junctional IC. In the absence of thrombin, blebbistatin had no significant effect on paracrine or gap junctional IC. The drug had no influence on MLC phosphorylation or on [Ca(2+)](i) transients in response to thrombin or ATP. Conclusions: Blebbistatin prevents the inhibitory effects of thrombin on intercellular Ca(2+) wave propagation. The findings demonstrate that myosin II-mediated actomyosin contractility plays a central role in thrombin-induced inhibition of gap junctional IC and of hemichannel-mediated paracrine IC.[2]

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Arterial thrombosis (AT) causes various ischemia-related diseases, which impose a serious medical burden worldwide. As an inhibitor of myosin II, blebbistatin has an important role in thrombosis development. We investigated the effect of blebbistatin on carotid artery ligation (CAL)-induced carotid AT and its potential underlying mechanism. A model of carotid AT in mice was generated by CAL. Mice were divided into three groups: CAL model, blebbistatin-treated, and sham-operation. After 7 days, blood vessels were harvested from mice in each group. The procoagulant activity of tissue factor (TF) was tested by a chromogenic assay, and thrombus severity assessed by histopathology scores. Expression of non-muscle myosin heavy chain II A (NMMHCIIA), TF, glycogen synthase kinase 3β (GSK3β), and nuclear factor-kappa B (NF-κB) was detected by immunohistochemical and immunofluorescence staining. mRNA expression was measured by quantitative polymerase chain reaction. Blebbistatin (1 mg/kg) inhibited development of carotid AT, reduced infiltration of inflammatory cells, and prevented vascular-tissue damage, relative to the model group. Furthermore, blebbistatin also reduced the procoagulant activity of TF. Immunohistochemical and immunofluorescence data demonstrated that, compared with the model group, blebbistatin intervention reduced expression of NMMHCIIA, TF, GSK3β, p65, and p-p65 in carotid-artery endothelia in the CAL-induced AT model, but it increased levels of p-GSK3β. Blebbistatin could inhibit expression of NMMHCIIA mRNA in the CAL model. Overall, our data demonstrated that blebbistatin could inhibit TF expression and AT development in arterial endothelia (at least in part) via GSK3β/NF-κB signaling.[3]

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Aging, noise, and ototoxic drug-induced hair cell (HC) loss are the major causes of sensorineural hearing loss. Aminoglycoside antibiotics are commonly used in the clinic, but these often have ototoxic side effects due to the accumulation of oxygen-free radicals and the subsequent induction of HC apoptosis. Blebbistatin is a myosin II inhibitor that regulates microtubule assembly and myosin-actin interactions, and most research has focused on its ability to modulate cardiac or urinary bladder contractility. By regulating the cytoskeletal structure and reducing the accumulation of reactive oxygen species (ROS), blebbistatin can prevent apoptosis in many different types of cells. However, there are no reports on the effect of blebbistatin in HC apoptosis. In this study, we found that the presence of blebbistatin significantly inhibited neomycin-induced apoptosis in HC-like HEI-OC-1 cells. We also found that blebbistatin treatment significantly increased the mitochondrial membrane potential (MMP), decreased ROS accumulation, and inhibited pro-apoptotic gene expression in both HC-like HEI-OC-1 cells and explant-cultured cochlear HCs after neomycin exposure. Meanwhile, blebbistatin can protect the synaptic connections between HCs and cochlear spiral ganglion neurons. This study showed that blebbistatin could maintain mitochondrial function and reduce the ROS level and thus could maintain the viability of HCs after neomycin exposure and the neural function in the inner ear, suggesting that blebbistatin has potential clinic application in protecting against ototoxic drug-induced HC loss.[4]

C18H16N2O2

292.33

292.121

C, 73.95; H, 5.52; N, 9.58; O, 10.95

856925-71-8

Blebbistatin;674289-55-5 (racemic); 856925-71-8 (S-isomer); 1177356-70-5 (R-isomer)

5287792

Light yellow to yellow solid powder

1.3±0.1 g/cm3

486.7±55.0 °C at 760 mmHg

210-212ºC

248.1±31.5 °C

0.0±1.3 mmHg at 25°C

1.681

0.93

52.9

1

3

1

22

497

1

CC1=CC2=C(C=C1)N=C3[C@](C2=O)(CCN3C4=CC=CC=C4)O

LZAXPYOBKSJSEX-GOSISDBHSA-N

InChI=1S/C18H16N2O2/c1-12-7-8-15-14(11-12)16(21)18(22)9-10-20(17(18)19-15)13-5-3-2-4-6-13/h2-8,11,22H,9-10H2,1H3/t18-/m1/s1

1,2,3,3a-tetrahydro-3aS-hydroxy-6-methyl-1-phenyl-4H-Pyrrolo[2,3-b]quinolin-4-one

(S)-Blebbistatin; (-)-Blebbistatin; 856925-71-8; (S)-(-)-Blebbistatin; (S)-blebbistatin; Blebbistatin, (-)-; (-)Blebbistatin; Blebbistatin (S)-form [MI]; CHEBI:75388; Blebbistatin.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： (1). 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。  (2). 该产品在溶液状态不稳定，请现配现用。

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

配方 1 中的溶解度: ≥ 1 mg/mL (3.42 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 10.0 mg/mL澄清DMSO储备液加入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: 1 mg/mL (3.42 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 10.0 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: ≥ 1 mg/mL (3.42 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 10.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，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：
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。