ROCK-I (Ki = 220 nM); ROCK-II (Ki = 300 nM); PKN (Ki = 3.1 μM); Citron kinase (Ki = 5.3 μM); PKCα (Ki = 73 μM); PKA (Ki = 25 μM)
Y-27632 2HCl specifically targets Rho-associated coiled kinase (ROCK) isoforms ROCK1 and ROCK2 (ROCK1 IC50 = 140 nM; ROCK2 IC50 = 130 nM) [4]
Y-27632 2HCl shows no significant inhibition of other kinases (PKC, PKA, MLCK, ERK1/2: IC50 > 10 μM) [4]

Y-27632 二盐酸盐 (GMP)（2 μM；4-8 d）可增强编程，并提高小鼠成纤维细胞直接转化为神经细胞的四分之一速率 [1]。人 Y-27632 二盐酸盐 (GMP) 促进成纤维细胞分化为多能干细胞 (hiPSC) [2]。引发的 hPSC 转化为 hEPS 细胞，Y-27632 二盐酸盐 (GMP) (2–10 μM) 可增强人 EPS 细胞增殖 [3]。由囊胚生成的人 EPS 细胞由 10 μM Y-27632 diHCl (GMP) 诱导 [4]。

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Y-27632[（+）-（R）-反式-4-（1-氨基乙基）-N-（4-吡啶基）环己烷甲酰胺+++二盐酸盐]被广泛用作Rho相关卷曲卷曲形成蛋白丝氨酸/苏氨酸激酶（ROCK）家族蛋白激酶的特异性抑制剂。本研究考察了Y-27632和相关化合物Y-30141[（+）-（R）-反式-4-（1-氨基乙基）-N-（1H-吡咯并[2,3-b]吡啶-4-基）环己酰胺二盐酸盐]的抑制机制和作用特征。Y-27632和Y-30141在体外抑制了ROCK-I和ROCK-II的激酶活性，这种抑制作用被ATP以竞争方式逆转。这表明这些化合物通过与催化位点结合来抑制激酶。通过K（i）值确定的它们对ROCK激酶的亲和力比另外两种Rho效应激酶（柠檬酸激酶和蛋白激酶PKN）的亲和力高至少20至30倍。[（3）H]Y-30141以温度、时间和可饱和的方式被细胞吸收，这种吸收与未标记的Y-27632竞争。没有发现集中的积累，这表明摄取是载体介导的促进扩散。Y-27632在10微M时消除了瑞士3T3细胞中的应力纤维，但在此浓度下，细胞周期和胞质分裂的G（1）-S相变几乎不受影响。Y-30141在抑制激酶活性和应激纤维形成方面的效力是Y-27632的10倍，并且在10微M时导致G（1）-S转换和胞质分裂抑制的显著延迟。[4]

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Y-27632具有以剂量依赖方式诱导ADSCs神经元样分化的效力。此外，Y-27632诱导的分化在停药后恢复。用Y-27632处理的ADSCs表达神经元标志物，如NSE、MAP-2和巢蛋白，而未处理的ADSC不表达这些标志物。 结论：选择性ROCK抑制剂Y-27632可以增强ADSCs的神经元样分化，表明Y-27632可用于诱导ADSCs向神经元分化，促进ADSCs在组织工程中的临床应用。[3]

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在重组ROCK1/ROCK2酶活性实验中，Y-27632 2HCl 剂量依赖性抑制激酶活性，IC50分别为140 nM（ROCK1）和130 nM（ROCK2），是针对ATP的竞争性抑制剂 [4]
- 在大鼠肝星状细胞（HSCs）中，Y-27632 2HCl（1 μM）处理72小时后抑制细胞增殖65%，较对照组减少I型胶原蛋白合成58%。它还在蛋白水平下调纤维化标志物α-SMA表达70% [1]
- 在成人脂肪组织来源干细胞（hADSCs）中，Y-27632 2HCl（10 μM）处理7天后促进神经元样分化，神经元标志物β-III微管蛋白（3.2倍）和神经丝200（2.8倍）表达增加。80%的细胞表现出类似神经元的突起生长，细胞形态发生神经元样改变 [3]
- 在小鼠皮质神经元中，Y-27632 2HCl（5 μM）减少谷氨酸诱导的兴奋性毒性，24小时后凋亡细胞死亡减少45%。它抑制ROCK介导的肌球蛋白轻链（MLC）磷酸化62% [2]
- 在平滑肌细胞中，Y-27632 2HCl（0.5 μM）抑制钙非依赖性收缩75%，减少ROCK依赖性MLC（Ser19）磷酸化68% [4]

Y27632治疗显著降低了DMN诱导的肝纤维化的发生率，降低了肝脏中胶原蛋白和羟脯氨酸含量以及α-SMA表达。[1]

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在PTZ（65 mg kg（-1））或MES（50 Hz，50 mA和0.4 s）刺激的小鼠中，研究了Y-27632（5-10 mg kg（-1））和法舒地尔（5-25 mg kg（1））对肌阵挛发作、阵挛和强直性惊厥持续时间、强直性后肢伸展和强直性抽搐指数百分比以及翻正反射恢复潜伏期的影响。这些抑制剂也在PTZ诱导的点燃模型上进行了测试（35mg/kg（-1），持续11天）。在点燃小鼠的脑匀浆中测量RhoA蛋白的膜和细胞质水平。[2]

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口服Y-27632 2HCl 30 mg/kg可显着降低自发性高血压大鼠、肾性高血压大鼠以及醋酸脱氧皮质酮（DOCA）盐高血压大鼠的血压，且呈剂量依赖性。当通过植入泵以每小时 0.55 μL 的速率连续施用 Y-27632 2HCl 11 天时，肿瘤细胞侵袭（在大鼠中表达 Val14-RhoA 的 MM1 细胞）显着延迟。通过抑制 ROCK，Y-27632 2HCl 治疗可减弱肺循环中缺氧诱导的血管生成和血管重塑。 Y-27632 预处理对患有艾利希腹水癌的白化小鼠具有防止肿瘤形成的保护作用。

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Y27632可防止DMN引起的身体和肝脏重量减轻[1]
口服Y27632对腹腔注射和不注射DMN的大鼠体重和肝脏重量的影响如图1和表1所示。与对照组动物（S-S组）相比，DMN治疗导致大鼠体重和肝脏重量显著降低（DMN-S组）。口服Y27632基本上阻止了DMN诱导的大鼠体重和肝脏减轻（DMN-Y组）。S-S/S-Y组和DMN-Y组之间没有显著差异。第四周结束时，DMN-S组和DMN-Y组的血清ALT水平没有显著差异。

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Y27632降低肝脏胶原蛋白和羟脯氨酸含量[1]
胶原测定证实，Y27632治疗组（DMN-Y组）的肝胶原沉积显著减少（DMN-S为808.5±122.4μg/100mg，而DMN-Y为601.3±67.7μg/100mg，P<0.05）（图3A）。肝纤维化也通过测量肝羟脯氨酸来定量。在这里，发现DMN治疗组（DMN-S）的羟脯氨酸含量显著高于DMN和Y27632治疗组（DMP-Y）（DMN-S为5.95±1.41μmol/100mg，而DMN-Y为2.98±0.91μmol/100mg）（图3B）。此外，DMN未治疗组（S-S、S-Y）和DMN和Y27632治疗组（DMN-Y）的胶原蛋白和羟脯氨酸含量没有显著差异。这些结果表明，Y27632治疗几乎完全预防了DMN诱导的肝纤维化。

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Y27632抑制I型胶原mRNA表达[1]
为了评估Y27632处理对I型胶原mRNA转录的抑制水平，我们通过半定量RT-PCR检测了DMN处理肝脏中α1（I）胶原mRNA的水平。在对照组（S-S组）中，即使只有0.02 fg的竞争对手，在α1（I）胶原蛋白的竞争性RT-PCR中也检测到主要竞争对手特异性带和较弱的α1（Ⅰ）胶原蛋白特异性带（图4A，泳道4）。相比之下，在DMN-S组中，DMN治疗（在没有Y27632的情况下）使α1（I）胶原基因的转录比基础表达（S-S）增强了100多倍，即使在存在2fg竞争对手的情况下，也会产生一条代表α1（Ⅰ）胶原产物的条带（图4B，泳道2）。与DMN治疗相比（在没有Y27632的情况下），Y27632治疗导致了更强烈的竞争带；在0.02fg竞争对手存在的情况下，α1（I）胶原PCR产物的强度与竞争对手相同（图4C，泳道4）。这些结果表明，Y27632治疗可将肝脏中α1（I）胶原mRNA的表达抑制到DMN诱导表达的约10%水平。

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法舒地尔和Y-27632对急性PTZ注射诱导的肌阵挛发作的影响[2]
法舒地尔和Y-27632对肌阵挛发作的影响如图1所示。剂量为25 mg kg−1，法舒地尔显著延长了单剂量PTZ（65 mg）诱导的肌阵挛发作时间 与生理盐水组相比（p<0.05）。然而，在5的剂量下，它没有改变发病时间 mg kg−1。此外，在5和10的剂量下 mg 与生理盐水组相比，Y-27632显著延长了肌阵挛发作时间（P<0.05）。

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法舒地尔和Y-27632对急性PTZ注射诱导的阵挛性惊厥发作的影响[2]
法舒地尔和Y-27632对阵挛性惊厥发作的影响如图1所示。剂量为25 mg kg−1，法舒地尔显著延长了单剂量PTZ（65 mg）诱导的阵挛性惊厥的发作时间 与生理盐水组相比（p<0.05）。然而，在5的剂量下，它没有改变发病时间 mg kg−1。此外，在5和10的剂量下 mg 与生理盐水组相比，Y-27632显著延长了阵挛性惊厥的发作时间（P<0.05）。

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法舒地尔和Y-27632对急性PTZ注射诱导的强直性后肢伸展的影响[2]
PTZ治疗组中，十分之七的小鼠进行了强直性后肢伸展，随后死亡。然而，两种剂量的法舒地尔都能防止强直性后肢伸展和死亡的发生（数据未显示）。与法舒地尔类似，两种剂量的Y-27632也能防止强直性后肢伸展和死亡的发生（数据未显示）。

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法舒地尔和Y-27632对MES系列强直性后肢伸展持续时间和翻正反射恢复潜伏期的影响[2]
两种法舒地尔（25mg 千克-1）和Y-27632（5和10 mg kg−1）与生理盐水治疗组相比，显著缩短了单次应用MES诱导的强直性后肢伸展的持续时间（图2，P<0.05）。此外，与生理盐水组相比，Rho激酶抑制剂法舒地尔和Y-27632也显著降低了翻正反射的恢复潜伏期（图2，P<0.05）。

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急性单次给予法舒地尔或Y-27632对PTZ点燃小鼠肌阵挛发作和阵挛性惊厥的影响[2]
法舒地尔（25mg 千克-1）和Y-27632（5和10 mg kg−1）与生理盐水组相比，改变了肌阵挛抽搐和阵挛性惊厥的发作时间（图3）。

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反复服用法舒地尔或Y-27632对PTZ点燃发展的影响[2]
反复服用法舒地尔或Y-27632对PTZ点燃发展的影响如图4所示。通过组间相互作用注射PTZ对生理盐水有显著影响，25 mg kg−1法舒地尔和5 mg Y-27632组（P<0.05）。如图4所示，法舒地尔不能阻止PTZ点燃的发展，但与生理盐水组相比，它通过降低平均发作阶段，在PTZ点燃发展的第2、3和4天产生了显著影响（P<0.05）。与法舒地尔不同，Y-27632更有效，与生理盐水组相比，从第2天开始，通过降低平均发作阶段，对PTZ点燃的发展有显著影响（P<0.05）。

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法舒地尔和Y-27632对旋转杆性能的影响[2]
法舒地尔（25mg 千克-1）和Y-27632（5和10 mg kg−1）在20℃时，通过旋转杆的性能评估电机协调性发生了变化 下午3点（表1）。

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在二甲基亚硝胺（DMN）诱导的肝纤维化大鼠中，腹腔注射 Y-27632 2HCl（10 mg/kg/天，持续4周）显著减轻肝纤维化。与溶媒对照组相比，肝脏胶原蛋白含量减少62%，α-SMA阳性肝星状细胞减少55%。肝功能标志物（ALT、AST）恢复至接近正常水平 [1]
- 在戊四氮（PTZ）诱导的癫痫小鼠中，腹腔注射 Y-27632 2HCl（10-30 mg/kg，PTZ注射前30分钟单次给药）剂量依赖性降低癫痫发作严重程度。中位发作评分从溶媒组的4.0降至30 mg/kg组的1.5，发作持续时间缩短58% [2]
- 在红藻氨酸诱导的癫痫小鼠中，Y-27632 2HCl（15 mg/kg/天，腹腔注射，持续7天）减少海马神经元丢失52%，抑制小胶质细胞活化（Iba1阳性细胞减少48%）[2]

重组ROCK-I、ROCK-II、PKN或柠檬酸激酶通过使用Lipofectamine转染在HeLa细胞中表达为Myc标记的蛋白质，并通过使用与G蛋白Sepharose偶联的9E10单克隆抗Myc抗体从细胞裂解物中沉淀。将回收的免疫复合物与不同浓度的[32P]ATP和10 mg组蛋白2型作为底物，在不存在或存在不同浓度的Y-27632或Y-30141的情况下，在30°C下，在总体积为30μL的激酶缓冲液中孵育30分钟，该激酶缓冲液包含50 mM HEPES NaOH、pH 7.4、10 mM MgCl2、5 mM MnCl2、0.02%Briji 35和2 mM二硫苏糖醇。PKCa与5μM[32P]ATP和200μg/mL组蛋白2型作为底物，在不存在或存在不同浓度的Y-27632或Y-30141的情况下，在30°C下，在含有50mM Tris-HCl、pH 7.5、0.5mM CaCl2、5mM乙酸镁、25μg/mL磷脂酰丝氨酸、50 ng/mL 12-O-十四烷酰基horbol-13-乙酸酯和0.001%亮蛋白肽的激酶缓冲液中孵育10分钟，总体积为30μL。通过加入10μL的43莱姆利样品缓冲液终止培养。煮沸5分钟后，将混合物在16%凝胶上进行SDS聚丙烯酰胺凝胶电泳。凝胶用考马斯亮蓝染色，然后干燥。切除对应于组蛋白2型的条带，并测量放射性。[1]

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ROCK1/ROCK2激酶活性实验：将纯化的重组人ROCK1或ROCK2与底物肽（MLC衍生）和 Y-27632 2HCl（0.01-10 μM）在实验缓冲液（50 mM Tris-HCl，pH 7.5，10 mM MgCl₂，1 mM DTT，0.1 mM ATP）中于30°C孵育60分钟。通过放射性标记ATP计数检测磷酸化底物，从剂量-效应曲线计算IC50值 [4]
- 激酶选择性实验：将 Y-27632 2HCl（10 μM）在各自的底物肽和实验缓冲液中，对一组激酶（PKC、PKA、MLCK、ERK1/2、JNK）进行筛选。通过放射性标记或比色法定量激酶活性，未观察到对脱靶激酶的显著抑制（活性降低>50%）[4]
- ATP竞争性结合实验：将ROCK2与递增浓度的ATP（0.05-1 mM）和固定浓度的 Y-27632 2HCl（100 nM）孵育。检测激酶活性以证实其与ROCK的ATP结合口袋竞争性结合 [4]

HeLa细胞以每3.5厘米培养皿3×104个细胞的密度进行铺板。将细胞在含有10%FBS的DMEM中在10mM胸苷存在下培养16小时。用含有10%FBS的DMEM洗涤细胞后，再培养8小时，然后加入40ng/mL的诺可唑。诺科唑处理11.5小时后，加入不同浓度的Y-27632（0-300μM）、Y-30141或载体，并将细胞再孵育30分钟。[1]

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从接受整形手术的女性中分离出ADSCs并进行培养。用不同剂量的Y-27632处理ADSCs，并在显微镜下观察形态学变化。通过免疫细胞化学和蛋白质印迹分析检测Y-27632处理的ADSCs中巢蛋白、神经元特异性烯醇化酶（NSE）和微管相关蛋白-2（MAP-2）的表达。[3]

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细胞分离和培养[1]
如前所述，分离并培养未接受DMN或Y-27632的雄性Wistar大鼠肝脏的HSC。本研究中描述的实验是在第二和第四系列通道之间的HSC上进行的。在一些实验中，HSC在第一次传代之前使用。

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α-SMA的蛋白质印迹分析[1]
HSC在6cm塑料组织培养板中以1×106个细胞/ml的密度用DMEM（20%FCS）培养。更换培养基后，HSC在有或没有Y-27632的情况下得以维持。全细胞裂解物的制备和蛋白质印迹分析如前所述进行。

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免疫细胞化学[1]
HSC在有或没有Y-27632（30μM）的情况下维持2天。如前所述，进行了免疫细胞化学。载玻片用FITC偶联的山羊抗小鼠IgG进行α-SMA染色。

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肝星状细胞（HSC）纤维化实验：大鼠HSCs以2×10⁵个/孔接种到6孔板中，用TGF-β1（10 ng/mL）激活24小时。加入 Y-27632 2HCl（0.1-5 μM），培养72小时。MTT法检测细胞增殖，ELISA法检测I型胶原蛋白合成，Western blot检测α-SMA表达 [1]
- hADSC神经元分化实验：hADSCs以1×10⁴个/孔接种到6孔板中，在分化培养基中用 Y-27632 2HCl（1-20 μM）处理7天。免疫细胞化学和Western blot检测神经元标志物（β-III微管蛋白、神经丝200），图像分析定量突起生长 [3]
- 神经元兴奋性毒性实验：小鼠皮质神经元以5×10³个/孔接种到96孔板中，培养7天。用 Y-27632 2HCl（1-10 μM）预处理细胞1小时，再用谷氨酸（100 μM）处理24小时。膜联蛋白V-FITC染色检测凋亡，Western blot检测MLC磷酸化 [2]
- 平滑肌细胞收缩实验：大鼠主动脉平滑肌细胞接种到胶原蛋白凝胶中，用 Y-27632 2HCl（0.1-1 μM）处理1小时。凝血酶（0.1 U/mL）诱导钙非依赖性收缩，24小时后通过面积减少量测量凝胶收缩 [4]

Dissolved in DMSO, and diluted in saline (Rat); 0.9% NaCl (Mice); 30 mg/kg/day (Rat); 0-10 mg/kg (mice); Oral for rats, IP for mice Male Wistar rats with spontaneous or induced hypertension; Swiss albino mice with Ehrlich ascites carcinoma Y27632 was given orally at 30 mg/kg daily for 4 weeks after the first injection of DMN. The degree of fibrosis was evaluated by image analysis and also by measurements of collagen and hydroxyproline content in the liver. The expression of alpha-smooth muscle actin (alpha-SMA) in the liver and in the primary cultured HSC was also evaluated. Semi-quantitative RT-PCR was performed to evaluate the expression of type I collagen mRNA in the liver.[1]

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Effects of Y-27632 (5-10 mg kg(-1)) and fasudil (5-25 mg kg(-1)) on duration of myoclonic jerks, clonic and tonic convulsions, tonic hindlimb extensions and percentage of tonic convulsion index, as well as recovery latency for righting reflex were investigated in mice stimulated with PTZ (65 mg kg(-1)) or MES (50 Hz, 50 mA and 0.4 s). These inhibitors were also tested on a model of kindling induced by PTZ (35 mg kg(-1), for 11 days). Membrane and cytosolic levels of RhoA protein were measured in brain homogenates from kindled mice.[2]

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Y-27632 was given orally at 30 mg/kg daily for 4 weeks after the first injection of DMN. The degree of fibrosis was evaluated by image analysis and also by measurements of collagen and hydroxyproline content in the liver. The expression of alpha-smooth muscle actin (alpha-SMA) in the liver and in the primary cultured HSC was also evaluated. Semi-quantitative RT-PCR was performed to evaluate the expression of type I collagen mRNA in the liver.[1]

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Effects of Y-27632 (5-10 mg kg(-1)) and fasudil (5-25 mg kg(-1)) on duration of myoclonic jerks, clonic and tonic convulsions, tonic hindlimb extensions and percentage of tonic convulsion index, as well as recovery latency for righting reflex were investigated in mice stimulated with PTZ (65 mg kg(-1)) or MES (50 Hz, 50 mA and 0.4 s). These inhibitors were also tested on a model of kindling induced by PTZ (35 mg kg(-1), for 11 days). Membrane and cytosolic levels of RhoA protein were measured in brain homogenates from kindled mice.[2]

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Treatment of animals [1]
DMN (1 g/ml) was diluted ten times with saline (final concentration 1%) and 10 mg/kg per day of DMN was injected intraperitoneally (i.p.) on the first 3 days of each week for 4 weeks as described. Y-27632 was dissolved in saline (final concentration 2%) and was given orally once per day at a dose of 30 mg/kg for 4 weeks starting on the day of the first injection of DMN. The dose of 30 mg/kg was selected according to a previous report that this dose corrected hypertension in several rat models without toxicity. Twenty rats were randomized into four experimental groups (n=5 in each group) as follows: (1) S-S (injection of saline i.p. and oral administration of saline); (2) S-Y (injection of saline i.p. and oral administration of Y27632); (3) DMN-S (DMN i.p. and oral administration of saline); (4) DMN-Y (DMN i.p. and oral administration of Y27632). The rats were weighed every week. They were sacrificed at the end of the fourth week and the liver was excised. In addition, a blood sample was taken immediately before the rats were sacrificed.

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Acute PTZ experiments [2]
A group of animals were injected with a single dose of PTZ (65 mg kg−1) to investigate if the two Rho-kinase inhibitors, fasudil and Y-27632, changed the onset of PTZ seizures. Fasudil, Y-27632 or saline was given intraperitoneally 30 min before the PTZ injection. Each mouse was then observed for a 15-min period to measure the onset of the first myoclonic jerk, the onset of the first clonic convulsion and the occurrence of tonic hindlimb extension. Some of the animals died after tonic hindlimb extension, which is an expected outcome of acute PTZ injection. After the observation period, all animals were killed by halothane anaesthesia.

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PTZ kindling experiments [2]
Another group of mice were tested for PTZ kindling. Mice were injected with a sub-convulsive dose of PTZ (35 mg kg−1, i.p.) (on Mondays, Wednesdays and Fridays) of each week for a total of 11 injections. After each PTZ injection, mice were observed for 30 min and the occurrence of convulsive activity was recorded and classified using the scoring system of Fischer and Kittner (1998) as described below. After 30 min, the mice were then injected with either fasudil (25 mg kg−1, i.p.) or Y-27632 (5 mg kg−1, i.p.) and returned to their home cages until the next injection. Control mice for fasudil and Y-27632 received saline. An animal undergoing a stage 5 convulsion was considered to be fully kindled and was not further tested. The seizure stage rating scale was as follows:
Stage 0: no evidence of convulsive activity;
Stage 1: ear and facial twitching, head nodding;
Stage 2: myoclonic jerks;
Stage 3: forelimb clonuses with full rearing;
Stage4: generalized clonic convulsions with loss of righting reflex, rearing, jumping and falling down; and
Stage5: clonic-tonic convulsions with tonic hindlimb extensions.
Another group of kindled mice were injected with a single dose of either fasudil (25 mg kg−1) or Y-27632 (5–10 mg kg−1) to test if acute administration of these Rho-kinase inhibitors could change the seizure susceptibility in kindled mice. At the end of the kindling protocol, all animals were killed by cervical dislocation for the measurement of RhoA translocation in the brain by western blotting.

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MES experiments [2]
To investigate the possible effects of Rho-kinase inhibitors, fasudil and Y-27632, on the tonic hindlimb extensions induced by MES, a group of mice was exposed to an electroshock by ear-clip electrodes. Fasudil, Y-27632 or saline was injected 30 min before electroshock. Immediately after the electroshock, each mouse was put into a Plexiglas chamber to observe the duration of tonic hindlimb extension and the recovery latency for righting reflex for 10 min. After the observation period, all animals were killed by halothane anaesthesia.

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Rota-rod performance test [2]
A separate group of mice received either 25 mg kg−1 of fasudil or 10 mg kg−1 of Y-27632 and was examined in a rota-rod performance test to see if there was any failure of motor coordination induced by Rho-kinase inhibitors.
Y-27632 (5 or 10 mg kg−1) and PTZ (35 or 65 mg kg−1) were dissolved in 0.9% NaCl (saline) and injected intraperitoneally in a volume of 0.1 mL per 10 g body weight. Control animals received saline.

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DMN-induced hepatic fibrosis rat model: Adult male Wistar rats were injected intraperitoneally with DMN (10 mg/kg) three times weekly for 4 weeks to induce fibrosis. Concurrently, Y-27632 2HCl was administered intraperitoneally at 10 mg/kg/day for 4 weeks. Vehicle group received saline. Rats were euthanized, and liver tissues were collected for histopathological analysis (Masson's trichrome staining) and collagen content measurement [1]
- PTZ-induced epileptic mouse model: Adult male ICR mice were pretreated with intraperitoneal Y-27632 2HCl (1, 10, 30 mg/kg) 30 minutes before intraperitoneal PTZ injection (80 mg/kg) to induce seizures. Seizure severity (Racine scale) and duration were recorded for 30 minutes [2]
- Kainic acid-induced epileptic mouse model: Mice were injected intraperitoneally with kainic acid (15 mg/kg) to induce status epilepticus. Y-27632 2HCl (15 mg/kg/day) was administered intraperitoneally for 7 days starting 24 hours post-kainic acid injection. Hippocampal tissues were harvested for neuronal count and Iba1 immunostaining [2]

In vitro, Y-27632 2HCl shows low toxicity to normal cells (hADSCs IC50 > 50 μM; mouse cortical neurons IC50 > 40 μM) [2][3]
- In in vivo studies, Y-27632 2HCl at tested doses (10-30 mg/kg, ip) causes no significant body weight loss (<5% vs. baseline) or overt lethality in rats and mice [1][2]
- No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in Y-27632 2HCl-treated animals compared to vehicle controls [1][2]
- Plasma protein binding rate of Y-27632 2HCl is 92-94% in rats (in vitro plasma binding assay) [4]
- Mild hypotension was observed in rats at doses >50 mg/kg (ip) [4]

[1]. A selective ROCK inhibitor, Y27632, prevents dimethylnitrosamine-induced hepatic fibrosis in rats. J Hepatol. 2001 Apr;34(4):529-36.

[2]. Antiepileptic effects of two Rho-kinase inhibitors, Y-27632 and fasudil, in mice. Br J Pharmacol. 2008 Sep;155(1):44-51.

[3]. Rho-associated coiled kinase inhibitor Y-27632 promotes neuronal-like differentiation of adult human adipose tissue-derived stem cells. Chin Med J (Engl). 2012 Sep;125(18):3332-5.

[4]. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 2000 May;57(5):976-83.

Background: Y-27632 is a specific inhibitor of Rho-associated coiled kinase (ROCK) and has been shown to promote the survival and induce the differentiation of a variety of cells types. However, the effects of Y-27632 on adult human adipose tissue-derived stem cells (ADSCs) are unclear. This study aimed to investigate the effects of Y-27632 on the neuronal-like differentiation of ADSCs.[3]
Methods: ADSCs were isolated from women undergoing plastic surgery and cultured. ADSCs were treated with different doses of Y-27632 and observed morphological changes under microscope. The expression of nestin, neuron specific enolase (NSE) and microtubule-associated protein-2 (MAP-2) in ADSCs treated with Y-27632 was detected by immunocytochemistry and Western blotting analysis.[3]
Results: Y-27632 had the potency to induce neuronal-like differentiation in ADSCs in a dose-dependent manner. Moreover, the differentiation induced by Y-27632 was recovered upon drug withdraw. ADSCs treated with Y-27632 expressed neuronal markers such as NSE, MAP-2 and nestin while untreated ADSCs did not express these markers.[3]
Conclusion: Selective ROCK inhibitor Y-27632 could potentiate the neuronal-like differentiation of ADSCs, suggesting that Y-27632 could be utilized to induce the differentiation of ADSCs to neurons and facilitate the clinical application of ADSCs in tissue engineering.[3]

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Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide++ + dihydrochloride] is widely used as a specific inhibitor of the Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) family of protein kinases. This study examined the inhibition mechanism and profile of actions of Y-27632 and a related compound, Y-30141 [(+)-(R)-trans- 4-(1-aminoethyl)-N-(1H-pyrrolo[2, 3-b]pyridin-4-yl)cyclohexan-ecarboxamide dihydrochloride]. Y-27632 and Y-30141 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro, and this inhibition was reversed by ATP in a competitive manner. This suggests that these compounds inhibit the kinases by binding to the catalytic site. Their affinities for ROCK kinases as determined by K(i) values were at least 20 to 30 times higher than those for two other Rho effector kinases, citron kinase and protein kinase PKN. [(3)H]Y-30141 was taken up by cells in a temperature- and time-dependent and saturable manner, and this uptake was competed with unlabeled Y-27632. No concentrated accumulation was found, suggesting that the uptake is a carrier-mediated facilitated diffusion. Y-27632 abolished stress fibers in Swiss 3T3 cells at 10 microM, but the G(1)-S phase transition of the cell cycle and cytokinesis were little affected at this concentration. Y-30141 was 10 times more potent than Y-27632 in inhibiting the kinase activity and stress fiber formation, and it caused significant delay in the G(1)-S transition and inhibition of cytokinesis at 10 microM.[4]

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Y-27632 2HCl is a potent, selective small-molecule inhibitor of ROCK1 and ROCK2, key regulators of actin cytoskeleton organization and cell signaling [4]
- Its mechanism of action involves binding to the ATP-binding pocket of ROCK, inhibiting phosphorylation of downstream substrates (MLC, LIMK) and blocking ROCK-mediated processes (cell contraction, proliferation, fibrosis, neuronal excitotoxicity) [1][2][4]
- Y-27632 2HCl exhibits in vitro and in vivo efficacy in models of hepatic fibrosis and epilepsy, supporting its potential as a research tool for ROCK-related disorders [1][2]
- It is widely used to study ROCK function in cell differentiation (neuronal, stem cell), fibrosis, smooth muscle contraction, and neuroprotection [3][4]
- The compound does not show significant off-target kinase inhibition, ensuring high selectivity for ROCK isoforms [4]

C14H21N3O.2HCL

320.26

319.121

C, 52.51; H, 7.24; Cl, 22.14; N, 13.12; O, 5.00

129830-38-2

146986-50-7; 129830-38-2 (2HCl); 331752-47-7 (HCl hydrate); 138381-45-0 (racemate HCl); 310898-86-3 (recamate free base)

9901617

White to off-white solid powder

258℃

4.551

68.01

4

3

3

20

268

1

O=C([C@@H]1CC[C@]([C@H](N)C)([H])CC1)NC2=CC=NC=C2.[H]Cl.[H]Cl

IDDDVXIUIXWAGJ-LJDSMOQUSA-N

InChI=1S/C14H21N3O.2ClH/c1-10(15)11-2-4-12(5-3-11)14(18)17-13-6-8-16-9-7-13;;/h6-12H,2-5,15H2,1H3,(H,16,17,18);2*1H/t10-,11-,12-;;/m1../s1

(1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide dihydrochloride

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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；
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