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Blockade of Rho-associated kinase prevents inhibition of axon regeneration of peripheral nerves induced by anti-ganglioside antibodies

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Neural Regeneration Research 2024年第4期19卷 895-899页

作者： Andrés Berardo Cristian R.Bacaglio Bárbara B.Báez Rubén Sambuelli Kazim A.Sheikh Pablo H.H.Lopez Laboratorio de Neurobiología Instituto de Investigación Médica Mercedes y Martin Ferreyra(INIMEC)-Consejo Nacional de Investigaciones Cientificas y Técnicas(CONICET)Universidad Nacional de Córdoba Departamento de Química Biológica-Centro de Investigaciones en Química Biológica de Córdoba(CIQUIBIC)-Consejo Nacional de Investigaciones Cientificas y Técnicas(CONICET) Facultad de Cs.QuímicasUniversidad Nacional de Córdoba Servicio de Anatomía Patológica Clínica Universitaria Reina FabiolaUniversidad Católica de Córdoba Department of Neurology University of Texas Medical School at Houston

Anti-ganglioside antibodies are associated with delayed/poor clinical recovery in Guillain-Barrèsyndrome,mostly related to halted axon regeneration.Cross-linking of cell surface gangliosides by anti-ganglioside antibodies triggers inhibition of nerve repair in in vitro and in vivo paradigms of axon regeneration.These effects involve the activation of the small GTPase Rho A/ROCK signaling pathways,which negatively modulate growth cone cytoskeleton,similarly to well stablished inhibitors of axon regeneration described so far.The aim of this work was to perform a proof of concept study to demonstrate the effectiveness of y-27632,a selective pharmacological inhibitor of ROCK,in a mouse model of axon regeneration of peripheral nerves,where the passive immunization with a monoclonal antibody targeting gangliosides GD1a and GT1b was previously reported to exert a potent inhibitory effect on regeneration of both myelinated and unmyelinated fibers.Our results demonstrate a differential sensitivity of myelinated and unmyelinated axons to the pro-regenerative effect of y-27632.Treatment with a total dosage of 9 mg/kg of y-27632 resulted in a complete prevention of anti-GD1a/GT1b monoclonal antibody-mediated inhibition of axon regeneration of unmyelinated fibers to skin and the functional recovery of mechanical cutaneous sensitivity.In contrast,the same dose showed toxic effects on the regeneration of myelinated fibers.Interestingly,scale down of the dosage of y-27632 to 5 mg/kg resulted in a significant although not complete recovery of regenerated myelinated axons exposed to anti-GD1a/GT1b monoclonal antibody in the absence of toxicity in animals exposed to only y-27632.Overall,these findings confirm the in vivo participation of Rho A/ROCK signaling pathways in the molecular mechanisms associated with the inhibition of axon regeneration induced by anti-GD1a/GT1b monoclonal antibody.Our findings open the possibility of therapeutic pharmacological intervention targeting Rho A/Rock pathway in immune neuropathies associated wi

关键词： anti-ganglioside antibodies anti-glycan antibodies axon regeneration ganglioside Guillain-Barrésyndrome nerve repair ROCK y-27632

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Effect of y-27632 on the cultured retinal neurocytes of rats

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International Journal of Ophthalmology(English edition) 2013年第1期6卷 15-18页

作者： Pei-Li Feng Jing Wang Zi-Jian yang Xiao-Hong Liu yi-Sheng Zhong Department of Ophthalmology Ruijin Hospital Affiliated Medical SchoolShanghai Jiaotong University

AIM:To investigate the effect of y-27632 on the survival and neurite outgrowth of the cultured retinal neurocytes. METHODS:After the postnatal day 2-3, Sprague-Dawley retinal neurocytes were cultured for 48 hours, the culture media was replaced with serum-free media (control group) and serum-free media contained 30μmol/L y-27632 (y-27632 group), and the cells were continually cultured another 48 hours. The cultured retinal neurocytes were identified with anti-neuron specific enolase (NSE) immunocytochemistry. The survival state of those cells was estimated by MTT assay, and the neurite outgrowth of those cells was evaluated by the computerized image-analysis system. RESULTS:Compared with the control group, the absorbance values of cells survival in y -27632 group increased 12.90% and 33.33% respectively after 72 and 96 hours culture. y-27632 had no significant effect on the diameter of cultured retinal neurocytes. Compared with the control group, y-27632 induced a stable improvement of neurite outgrowth of retinal neurocytes after 72 and 96 hours culture (P =0.001). CONCLUSION:y-27632 could promote the survival and neurite outgrowth of the early postnatal cultured retinal neurocytes.

关键词： y-27632 retinal neurocytes cell culture neurites

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Rho-associated coiled kinase inhibitor y-27632 promotes neuronal-like differentiation of adult human adipose tissue-derived stem cells

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Chinese Medical Journal 2012年第18期125卷 3332-3335页

作者： XUE Zhong-wen SHANG Xiao-ming XU Hong, LU Song-hui, DONG Tian-wei LIANG Chao-hui yUAN yuan Department of Internal Medicine Hebei Medical UniversityShijiazhuang China Hebei 050017 Medical Research Center Hebei United University Tangshan China Hebei 063000 Department of Cardiology Tangshan WorkerTs Hospital langslaan China Hebei 063000 Department of Obstetrics Beijing Fangshan District Maternal andChild Health Hospital China Beijing 100000 Department of Cardiology Affiliated Hospital of JiamusiUniversity Jiamusi China Heilongjiang 154000

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. 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. 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. 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.

关键词： Rho-associated coiled kinase y-27632 adipose tissue-derived stem cells, cell differentiation

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Interference of y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts

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International Journal of Ophthalmology(English edition) 2012年第5期5卷 576-581页

作者： Xiao-Hui Zhang, Jian-Ming Wang Department of Ophthalmology the Second Affiliated Hospital College of Medicine Xi'an Jiaotong University Xi'an 710004 Shaanxi Province China Department of Pharmacology Xijing Hospital Fourth Military Medical University Xi'an 710032 Shaanxi Province China

AIM: To investigate the interfering effect of y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and y-27632 had no remarkable difference from that in control group. y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. y-27632 suppressed the expression of collagen I mRNA without inducti

关键词： y-27632 ocular Tenon's capsule fibroblasts transforming growth factor beta type 1 α-smooth muscular actin connective tissue growth factor collagen I

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Therapeutic potential of Rho-associated kinase inhibitor y27632 in corneal endothelial dysfunction:an in vitro and in vivo study

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International Journal of Ophthalmology(English edition) 2021年第1期14卷 19-25页

作者： yao-Wen Song Jun-yu Chen Xu Li Li Wang Zhi-Qiang Pan Shanxi Eye Hospital Taiyuan 030002Shanxi ProvinceChina Beijing Tongren Eye Center Beijing Tongren HospitalCapital Medical UniversityBeijing Ophthalmology and Visual Science Key LaboratoryBeijing 100730China

AIM:To investigate the effects of a selective inhibitor of Rho-associated kinase(ROCK),y-27632,on inbred Wuzhishan porcine corneal endothelial cells(PCECs)in vitro and in vivo studies.METHODS:Primary PCECs were trypsinized from Wuzhishan miniature porcine corneal tissues.The optimal concentration of y-27632 on PCECs was determined through MTT and 5-ethynyl-2'-deoxyuridine(EdU)-labeling assays.Seven New Zealand rabbits were used as a corneal endothelial dysfunction model,and a PCECs suspension supplemented with y-27632 was injected into the anterior chamber of the rabbits.The progression of rabbit corneal opacity and edema were observed by slit lamp examination.The rabbits were sacrificed,and rabbit globes were enucleated for trypan blue-alizarin red staining,hematoxylineosin staining,and immunofluorescence analysis.RESULTS:Administration of 100μmol/L y-27632 facilitated PCECs'proliferation obviously.The rabbit corneas injected with PCECs suspension and 100μmol/L y-27632 were restored to transparency significantly after 14d.CONCLUSION:The 100μmol/L y-27632 treatment improves PCECs'proliferation significantly.And our results suggest that y-27632 and PCECs can be used to treat corneal endothelial dysfunction.

关键词： corneal endothelial dysfunction Rho kinase inhibitor y-27632 porcine corneal endothelial cells cell proliferation

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Rho signaling inhibitor,y-27632,inhibits invasiveness of metastastic hepatocellular carcinoma in a mouse model

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Chinese Medical Journal 2007年第24期120卷 2304-2307页

作者： XUE Feng ZHANG Jian-jun QIU Feng ZHANG Ming CHEN Xiao-song LI Qi-gen HAN Long-zhi XI Zhi-feng XIA Qiang Organ Transplantation Center Renji Hospital Shanghai Jiao TongUniversity School of Medicine Shanghai 200127 China

Hepatocellular carcinoma(HCC)is among the mostcommon tumors worldwide.Despite extensiveexploration of novel therapies,the prognosis of HCCpatients with intrahepatic metastasis is still poor,and notreatment is effective against metastases.Therefore,more effective therapeutic strategies for treatment ofmetastasis are urgently needed.

关键词：肝细胞癌肿瘤转移细胞凋亡信号抑制剂 y-27632

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y-27632及人脐带间充质干细胞条件培养液对角质形成细胞增殖和衰老的影响

Y-27632及人脐带间充质干细胞条件培养液对角质形成细胞增殖和衰...

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作者：黄俊群南方医科大学

学位级别：硕士

研究背景目前大面积烧伤患者治疗上遇到的最大问题在于供皮区有限,无法快速修复创面。近年来,自体种子细胞组织工程皮肤的出现为解决这一问题又提供了新思路,但其最重要的种子细胞——角质形成细胞,无法在体外快速增殖,导致组织工程皮... 详细信息

研究背景目前大面积烧伤患者治疗上遇到的最大问题在于供皮区有限,无法快速修复创面。近年来,自体种子细胞组织工程皮肤的出现为解决这一问题又提供了新思路,但其最重要的种子细胞——角质形成细胞,无法在体外快速增殖,导致组织工程皮肤的发展及临床应用受到限制。因此,如何促进角质形成细胞体外快速增殖成为其关键环节。既往研究表明ROCK抑制剂能抑制人胚胎干细胞在分离过程中诱导的凋亡,后续不少学者发现,ROCK抑制剂y-27632对骨髓间充质干细胞、牙周膜上皮细胞、角膜内皮细胞等均有促进增殖、延缓衰老、抑制分化的作用;还能影响细胞的旁分泌作用,增强其分泌多种细胞因子的能力。本课题组既往研究表明,脐带间充质干细胞与角质形成细胞直接共培养和间接共培养时均可促进角质形成细胞的增殖y-27632可促进脐带间充质干细胞增殖,延缓其衰老。而间充质干细胞条件培养液也被证实具有促进靶细胞增殖、影响细胞分化潜能的作用。在体试验中,y-27632及间充质干细胞条件培养液对创面修复也有积极影响。但目前尚未见y-27632对人头部刃厚皮片来源角质形成细胞增殖和衰老的影响,也无y-27632刺激后人脐带间充质干细胞条件培养液对角质形成细胞增殖和衰老影响的报道。研究目的通过研究y-27632对无血清无异种细胞培养条件下人角质形成细胞增殖、衰老及迁移的影响,以及y-27632刺激人脐带间充质干细胞后制备的条件培养液对角质形成细胞增殖、衰老及迁移能力的影响,探讨体外快速扩增人角质形成细胞的方法。研究方法1.从头部刃厚皮片中分离获得人角质形成细胞(KC),将细胞随机分为对照组和y-27632组,分别在DKSFM和添加了 5μM y-27632的DKSFM中进行传代培养。观察细胞的形态、生长情况并进行细胞生物学鉴定。对各组细胞进行形态学观察;免疫荧光染色法检测细胞表面标志物CK14及β1-整合素的表达。MTT法测OD值以检测细胞增殖能力及活性;β-半乳糖苷酶染色法检测细胞衰老情况;划痕试验检测各组细胞迁移能力。2.从人脐带中分离获取人脐带间充质干细胞(hUC-MSCs),将hUC-MSCs培养至60%-70%融合后,随机分为y-27632条件培养液组和单纯干细胞条件培养液组,将获得的hUC-MSCs在含和不含5μM y-27632的DMEM培养基中培养,48h后收集干细胞培养上清液制备干细胞条件培养液,ELISA法测定两组干细胞条件培养液中EGF及VEGF含量;用DMEM培养基(对照组)及上述两组条件培养液处理角质形成细胞,MTT法测OD值检测细胞增殖能力及活性;划痕试验检测各组细胞迁移能力;β-半乳糖苷酶染色法检测细胞衰老情况。结果1.从人头部刃厚皮片中分离出具有增殖活性和干性的人角质形成细胞,表达CK14及β1-整合素。而且在实验中发现原代KC在培养至融合度为30%～40%时,每天更换1次培养液较每2-3天换1次培养液,能延缓KC在体外培养过程中的老化,获得更多活力旺盛的KC;若2-3天换液一次,在培养至第3代后KC就会迅速膨大衰老,不利于KC的大量扩增。y-27632可显著促进原代KC分离培养过程中的KC贴壁,而对KC细胞形态无明显影响。(1)y-27632可促进角质形成细胞增殖。MTT试验结果显示:y-27632组OD值明显高于对照组(Py-27632组细胞增殖能力及细胞活性高于对照组。(2)y-27632可延缓角质形成细胞衰老。β-半乳糖苷酶染色结果显示:对照组衰老细胞明显较多,β-半乳糖苷酶阳性细胞占比达(20.719±4.405)%,而y-27632组阳性细胞占比仅为(12.424±1.043)%(Py-27632可促进角质形成细胞迁移。划痕试验结果显示:不同时间点y-27632组划痕愈合百分比均高于对照组(Py-27632刺激后脐带间充质干细胞培养上清液中EGF和VEGF含量较单纯干细胞培养上清液中明显增多(Py-27632增强干细胞条件培养液对角质形成细胞的增殖作用。MTT结果显示:单纯干细胞条件培养液组及y-27632刺激后干细胞条件培养液组OD值均高于未加条件培养液的对照组;且y-27632刺激后干细胞条件培养液组OD值高于单纯干细胞条件培养液组(P0.05)。(4)y-27632增强干细胞条件培养促进角质形成细胞迁移。单纯干细胞条件培养液组及y-27632刺激后干细胞条件培养液组不同时间点划痕愈

关键词：人角质形成细胞人脐带间充质干细胞 y-27632 条件培养液

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Rho激酶抑制剂y-27632对MRL/lpr狼疮小鼠的保护作用

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细胞与分子免疫学杂志 2017年第7期33卷 875-880页

作者：王元元贾孝云王其一叶明陆杨张佳佳陈升李杨磊吴俊英谢长好蚌埠医学院组织学与胚胎学教研室安徽蚌埠233030 蚌埠医学院安徽省组织移植重点实验室安徽蚌埠233030 蚌埠医学院第一附属医院风湿免疫科安徽蚌埠233030 蚌埠医学院生理学教研室安徽蚌埠233030 蚌埠医学院临床医学系安徽蚌埠233030 蚌埠医学院免疫学教研室安徽蚌埠233030

目的探讨Rho激酶抑制剂y-27632对MRL/lpr狼疮小鼠的保护作用机制。方法 MRL/lpr狼疮小鼠20只随机分为MRL/lpr对照组、5 mg/kg y-27632处理组,每组10只;野生型对照组C57BL/6小鼠10只。采用ELISA检测各组小鼠血清、脾脏组织中超氧化物歧化... 详细信息

目的探讨Rho激酶抑制剂y-27632对MRL/lpr狼疮小鼠的保护作用机制。方法 MRL/lpr狼疮小鼠20只随机分为MRL/lpr对照组、5 mg/kg y-27632处理组,每组10只;野生型对照组C57BL/6小鼠10只。采用ELISA检测各组小鼠血清、脾脏组织中超氧化物歧化酶(SOD)、丙二醛(MDA)水平,ELISA检测血清核因子κB(NF-κB)相关炎症因子白细胞介素6(IL-6)、IL-1β、肿瘤坏死因子α(TNF-α)含量;采用Western blot法检测各组小鼠脾脏组织中硫氧还蛋白结合蛋白(Txnip)/硫氧还蛋白(Trx)、丝裂原激活蛋白激酶(MAPK)相关蛋白胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)和p38MAPK以及NF-κB的水平;Western blot法检测各组小鼠脾脏T淋巴细胞Txnip、p38MAPK、NF-κB蛋白水平;ELISA检测T淋巴细胞上清液IL-6、IL-1β、TNF-α水平。结果 y-27632提高MRL/lpr狼疮小鼠血清及脾脏组织SOD水平;降低血清及脾脏组织MDA水平;降低血清、脾脏组织和脾脏T淋巴细胞上清液IL-6、IL-1β、TNF-α水平;抑制脾脏和脾脏T淋巴细胞Txnip、MAPK相关蛋白ERK、JNK和p38MAPK以及NF-κB表达,增加Trx含量。结论 Rho激酶抑制剂y-27632对MRL/lpr狼疮小鼠有保护作用。

关键词：系统性红斑狼疮 y-27632 硫氧还蛋白结合蛋白/硫氧还蛋白(Txnip/Trx) 丝裂原激活蛋白激酶(MAPK) 核因子κB(NF-κB)

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农业生物技术学报 2012年第7期20卷 726-734页

作者：成德高星李珍珍高祎刘亚军鲍建昌王华岩西北农林科技大学动物医学院陕西省干细胞工程技术研究中心

猪作为重要的疾病模型动物,目前猪诱导多能干细胞(iPS 细胞)已有建系,但是细胞的冻存和传代的效率较低,影响后续的实验研究。Rho 相关蛋白激酶(ROCK)抑制剂 y-27632 可提高人胚胎干细胞的解冻存活率,促进其克隆形成。本实验以猪(Sus scr... 详细信息

猪作为重要的疾病模型动物,目前猪诱导多能干细胞(iPS 细胞)已有建系,但是细胞的冻存和传代的效率较低,影响后续的实验研究。Rho 相关蛋白激酶(ROCK)抑制剂 y-27632 可提高人胚胎干细胞的解冻存活率,促进其克隆形成。本实验以猪(Sus scrofa)iPS 细胞为研究对象,通过在猪 iPS 细胞冻存和解冻过程中添加y-27632,发现其可以减少猪 iPS 细胞在冻存和解冻复苏过程中的凋亡。在细胞的传代过程中,使用 y-27632 可以促进猪 iPS 细胞的贴壁和克隆形成。虽然高浓度 y-27632 会对猪 iPS 克隆的形态产生一定的影响,但并未影响细胞的碱性磷酸酶(AP)活性和多能性基因 Oct4、Sox2 和 Nanog 的表达水平。最后将转座子报告质粒 PB[Act-RFP]DS 导入猪 iPS 细胞中,经流式筛选后得到的带有红色荧光的细胞,并将其注射于猪孤雌胚胎中进行发育能力检测,发现 y-27632 的处理能减少细胞在流式筛选和胚胎注射中的凋亡,促进其在胚胎内的嵌合发育。研究结果说明,ROCK 抑制剂 y-27632 可以提高猪 iPS 细胞冻存和传代的效率,促进其在胚胎中的嵌合。该研究有助于猪 iPS 细胞及其他多能干细胞的保存,传代和筛选等相关研究。

关键词： y-27632 猪诱导多能干细胞(iPS 细胞) Rho相关蛋白激酶(ROCK) 冻存凋亡

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Rho激酶抑制剂y-27632对急性高眼压大鼠视网膜损伤的保护作用

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中国医科大学学报 2011年第8期40卷 712-714,728页

作者：宋海珊刘昕高殿文解放军第二二医院沈阳110003 中国医科大学附属盛京医院眼科沈阳110004

目的探讨Rho激酶抑制剂y-27632对急性高眼压大鼠视网膜损伤的保护作用。方法 50只SD大鼠随机分为正常对照组(n=10)、生理盐水对照组(n=20,玻璃体腔内注射生理盐水)和y-27632治疗组(n=20,玻璃体腔内注射100 nmoly-27632),选取右眼做为实... 详细信息

目的探讨Rho激酶抑制剂y-27632对急性高眼压大鼠视网膜损伤的保护作用。方法 50只SD大鼠随机分为正常对照组(n=10)、生理盐水对照组(n=20,玻璃体腔内注射生理盐水)和y-27632治疗组(n=20,玻璃体腔内注射100 nmoly-27632),选取右眼做为实验眼。正常对照组直接处死取材。生理盐水对照组及y-27632治疗组分别用前房加压灌注法建立大鼠急性高眼压损伤模型,并于损伤后24和168 h取标本。HE染色观察视网膜组织病理学的改变,测量视网膜厚度;TUNEL染色检测细胞凋亡情况,计算凋亡指数;Western blot检测Caspase-3蛋白表达的变化。结果急性高眼压损伤24 h后,y-27632治疗组视网膜凋亡细胞数量和Caspase-3蛋白表达水平均低于生理盐水对照组(Py-27632治疗组视网膜厚度大于生理盐水对照组(Py-27632可以减轻大鼠急性高眼压引起的视网膜损伤,具有神经保护作用。

关键词： y-27632 视网膜高眼压凋亡

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