Rho激酶(ROCK)在糖尿病并发症中的研究

ROCK(Rho-associated protein kinase),即Rho相关蛋白激酶,是Rho/ROCK通路的重要蛋白。ROCK与GTP(guanosine triphosphate)结合蛋白Rho相互作用,通过磷酸化激活多种下游蛋白或核因子,在机体的各项调节功能中起到重要的作用。研究表明,糖尿病患者体内ROCK异常上调,可能是导致糖尿病并发症的重要原因,最终危及患者心血管系统、泌尿系统乃至生殖系统。而对ROCK的深入研究表明,使用ROCK的拮抗剂,如法舒地尔等,可有效抑制ROCK/Rho通路,最终缓解糖尿病并发症。     

Rho/ROCK信号通路与神经可塑性

在神经网络中,神经可塑性是大脑响应内在和外在刺激的重要特征。越来越多的研究已经阐明了神经可塑性与神经损伤性疾病之间的相关性。Rho/Rho相关卷曲螺旋形成蛋白激酶(Rho/Rho associ-ated coiledcoil forming protein kinase, Rho/ROCK)通路是生物体广泛存在的经典信号通路,参与细胞迁移、树突发育和轴突延伸,并且与帕金森、精神发育迟滞和阿尔茨海默症等多种神经退行性或损伤性疾病有关。本文对Rho/ROCK信号通路与神经可塑性的研究进展予以综述,讨论了ROCK抑制剂对各种神经疾病的潜在治疗前景。     

脊髓损伤中RhoA/ROCK通路的作用机制研究

脊髓损伤是一种严重的中枢神经系统损伤,常导致患者瘫痪或死亡,预后差。脊髓损伤主要包括机械损伤和继发性损伤两个过程。在继发性损伤过程中,多种信号通路被激活,在脊髓损伤的发病机制中起重要作用,其中,RhoA/Rho信号通路在脊髓变性和再生中起着特殊的作用。本文讨论RhoA/Rho激酶信号介导的脊髓发病机制,以及针对RhoA/ROCK通路靶向药物的治疗进展。     

ERM蛋白在微血管内皮细胞通透性调控中的研究进展

埃兹蛋白(Ezrin)/根蛋白(Radixin)/膜突蛋白(Moesin)(ERM)是细胞膜与胞内骨架的连接蛋白,具有高度同源性。细胞外刺激因子可通过多种信号通路磷酸化ERM蛋白,使细胞骨架重构,从而调控微血管内皮细胞通透性,在感染、炎症、代谢异常等病理过程中发挥作用。ERM功能调节的一个重要环节就是其羧基末端苏氨酸残基磷酸化后引起ERM构象的改变,暴露的羧基末端尾部的肌动蛋白(actin)-细胞骨架结合位点;故通过ERM的桥接作用,可将肌动蛋白微丝与细胞膜相连,使血管内皮细胞屏障功能发生变化。目前已知能使ERM磷酸化的激酶有蛋白激酶C(PKC)、促分裂原活化蛋白激酶(MAPK)、Rho相关激酶(ROCK),分别通过p38-MAPK、Rho/ROCK、PKC信号通路参与微血管内皮屏障功能的调控。本文旨在阐述ERM及其相关信号通路在微血管内皮细胞通透性调控中发挥的作用。     

利多卡因调节RhoA/ROCK轴对结直肠癌细胞生物学行为的影响

该文主要探讨利多卡因(lidocaine, Lido)通过调节Ras同源基因家族成员A(Rho A)/Rho相关的卷曲螺旋激酶(ROCK)轴对结直肠癌(CRC)细胞生物学行为的影响。该研究使用0～1 250μmol/L的利多卡因处理人结直肠癌细胞LS513, CCK-8法检测细胞活力筛选适宜药物浓度。将细胞分为对照组(Control组)、利多卡因低浓度组(Lido-L组, 500μmol/L Lido)、利多卡因中浓度组(Lido-M组, 750μmol/L Lido)、利多卡因高浓度组(Lido-H组, 1 000μmol/L Lido)和利多卡因高浓度+ROCK信号通路激活剂LPA组(Lido-H+LPA组, 1 000μmol/L Lido+10μmol/L LPA)。Edu检测细胞增殖;划痕愈合实验和Transwell小室实验分别检测细胞迁移和侵袭能力;流式细胞仪检测细胞凋亡情况;Westernblot检测PCNA、Bax、Bcl-2、RhoA、ROCK1、E-cadherin和N-cadherin蛋白表达情况。该研究得出与0μmol/L利多卡因相比, 500μmol/L、75...     

乳腺癌相关透明质酸变化对淋巴内皮细胞紧密连接分子ZO-1分布及细胞增殖的影响

该研究旨在探讨乳腺癌来源的透明质酸(hyaluronic acid,HA)分子大小变化对淋巴内皮细胞紧密连接分子ZO-1(zonula occludens-1)分布情况以及细胞增殖的影响。采用免疫组化实验检测8对良性乳腺疾病和乳腺癌患者组织HA的表达水平,利用酶联免疫吸附实验检测20对正常人与乳腺癌患者血清中透明质酸分解酶(hyaluronidase,Hyase)含量;通过免疫荧光法、荧光素钠渗透实验、MTT增殖实验和Western blot分别观察HA和寡分子透明质酸(oligosaccharides of Hyaluronic acid,o HA)作用淋巴内皮细胞后ZO-1分布、单层细胞渗透性、细胞增殖以及下游ROCK1/Rho A信号通路变化。结果表明,与良性乳腺疾病对照组比较,乳腺癌患者HA表达量明显增加(P0.01);与正常人相比,乳腺癌患者血清Hyase水平显著升高(P0.01),提示乳腺癌发生时,HA升高伴随其降解酶增多,HA代谢活性增加。HA增强细胞间ZO-1的连接,对ROCK1/Rho A蛋白表达水平无明显影响;相反,o HA促使淋巴内皮细胞ZO-1由胞膜向胞浆分布,导致细胞连接呈褶皱状,形成细胞间隙,淋巴管渗透性增加,上调ROCK1/Rho A蛋白表达水平,促进淋巴内皮细胞增殖(P0.01)。研究提示,o HA可能在乳腺癌淋巴管形态及增殖中发挥重要作用。     

草鱼ROCK1基因全长克隆、生物信息学分析及组织差异表达

ROCK1是ROCK(即Rho激酶)家族的成员之一,ROCK家族是小G蛋白Rho的下游效应因子,被Rho蛋白激活后ROCK1作用于下游效应分子,对其介导的信号通路进行调节,参与细胞增殖、迁移、形态改变等。ROCK1与组织纤维化、癌症、心血管等疾病的发生密切相关。本实验采用c DNA末端快速扩增法克隆草鱼ROCK1基因全长序列,预测并分析其编码的蛋白,采用实时荧光定量PCR法检测草鱼ROCK1基因的组织差异表达。实验首次克隆获得了草鱼ROCK1基因全序列,长4 604 bp,编码1 361个氨基酸;草鱼ROCK1氨基酸序列与金线鲃、斑马鱼、鲤鱼ROCK1的氨基酸序列同源性分别达95%、94%、94%,同源性较高;草鱼ROCK1蛋白分子量158 035.73,理论等电点5.69,为亲水性非分泌蛋白,无信号肽;其二级结构包括α-螺旋(α-helix)、β-重叠(β-strand)和环(loop)。组织差异表达显示草鱼ROCK1基因在各组织中均有表达,其中在脑和血液中表达量最高(p0.05),其次为鳃、心脏、肾脏、脾脏和前肠组织,肌肉和肝脏组织表达量较低,皮肤组织表达量最低,所得结果将为后续研究草鱼组织纤维化奠定基础。     

RhoB/ROCK 蛋白分子在重度子痫前期的表达

目的:探讨RhoB/ROCK蛋白分子在重度子痫前期(severe preeclampsia,sPE)患者胎盘组织中表达水平以及意义。方法:取sPE产妇和正常产妇胎盘组织各20例,通过Western blot和免疫组化检测胎盘组织中Rho亚家族蛋白(RhoB、RhoC)及其Rho激酶即ROCK(ROCKI、ROCKII)蛋白的表达水平,应用Spearman等级相关分析法检验RhoB、RhoC与ROCKI、ROCKII蛋白表达水平的相关性。结果:RhoB、ROCKI、ROCKII蛋白在sPE表达水平增加(P<0.05);sPE患者胎盘组织中RhoB与ROCKI、ROCKII蛋白表达水平均呈正相关(r=0.793,r=0.901,P<0.05)。结论:RhoB及其与下游分子ROCKI、ROCKII构成的信号通路,在sPE的发病中可能发挥着重要的作用。     

Rho/Rho激酶信号通路与脉管收缩

Rho/Rho激酶信号通路在正常血管、淋巴管等脉管收缩过程中发挥重要的调节作用,并参与休克后血管反应性和钙敏感性的双相调节以及休克淋巴管低反应性的发生.以Rho/Rho激酶为靶点,对于干预休克脉管系统低反应性的发生具有重要意义.本文综述Rho/Rho激酶信号通路在脉管收缩中的调节机制.     

mTOR信号通路在糖尿病肾病发病机制中作用的研究进展

糖尿病肾病是目前终末期肾脏疾病的主要原因,给患者家庭及社会带来沉重负担,如何预防及治疗糖尿病肾病成为亟需解决的问题。然而,糖尿病肾病的发病机制极其复杂,其中mTOR信号通路在其中扮演着重要角色。该综述主要总结了mTOR信号通路对糖尿病肾病的影响并阐述其可能存在的机制,希望能够给予同行些许借鉴。     

Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway

Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood–brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.     

RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement

RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and an enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs.     

Impact of engagement of FcepsilonRI and CC chemokine receptor 1 on mast cell activation and motility

CC chemokines participate in the recruitment and activation of immune cells through CC chemokine receptors (CCRs). Here, we report that cross-talk between CCR1-mediated signaling pathway and FcepsilonRI-mediated signaling pathway affects degranulation positively but affects chemotaxis of mast cells adversely. Costimulation via FcepsilonRI engagement with IgE/antigen and CCR1 engagement with recombinant human CCL3 synergistically enhanced degranulation in rat basophilic leukemia-2H3 cells expressing human CCR1 (RBL-CCR1). Interestingly, FcepsilonRI engagement inhibited CCL3-mediated chemotaxis and membrane ruffling of RBL-CCR1 cells. Small GTP-binding proteins of the Rho family, Rac, Cdc42, and Rho control chemotaxis by mediating the reorganization of the actin cytoskeleton. Both a Rho inhibitor C3 exoenzyme and a Rho kinase (ROCK) inhibitor Y-27632 inhibited chemotaxis of RBL-CCR1 cells toward CCL3, indicating that activation of the Rho/ROCK signaling pathway is required for the CCL3-mediated chemotaxis of the cells. Costimulation with IgE/antigen and CCL3 enhanced Rac and Cdc42 activation but decreased ROCK activation in RBL-CCR1 cells compared with that in the cells stimulated with CCL3 alone. These results suggest that costimulation via FcepsilonRI and CCR1 engagements induced 1) inhibition of membrane ruffling, 2) decreased ROCK activation, and 3) reciprocal imbalance between Small GTP-binding proteins of the Rho family, which result in the inhibition of chemotaxis of RBL-CCR1 cells. The cross-talk between FcepsilonRI-mediated signaling pathway and CCR-mediated signaling pathway would induce optimal activation and arrested chemotaxis of mast cells, thus contributing to allergic inflammation.     

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.     

Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation

Retinal pigment epithelium (RPE) cells is the outermost layer of the retina and RPE dysfunction is a key factor in the disease pathogenesis of age-related macular degeneration (AMD). Transplantation therapy using induced pluripotent stem cell (iPSC)-derived RPEs has recently received much attention as a treatment for AMD. Preserving these cells under the best possible conditions is important, and preservation methods using Y-27632 have been reported. Rho-associated coiled-coil containing kinase (ROCK) inhibitors are known to inhibit cell death, emerging as important drug candidates for stem cell differentiation and regenerative medicine. However, it has recently been shown that ROCK inhibitors may have a vasodilatory effect on human retinal arterioles, a side effect that should ideally be avoided in RPE transplantation. Although ROCK inhibitors hold great potential, optimizing efficacy while minimizing adverse reactions is critical for translation into a clinical treatment. We examined the effect of transient exposure of RPE cells to ROCK inhibitor Y-27632 to determine whether the extracellular presence of the drug is necessary for ongoing Rho/ROCK downregulation. Human RPE cells were subcultured as a suspension for 4 h in drug-free medium following exposure to Y-27632 for 2 h. A Y-27632 concentration of >10 μM improved cell survival beyond 4 h and cell proliferation in recovery culture medium. ROCK2 expression levels were specifically downregulated by Y-27632 in the Rho/ROCK signaling pathway. In conclusion, we demonstrated that the effect of Y-27632 is not dependent on its extracellular availability and can last beyond the 2 h of exposure. The lasting Rho/ROCK signaling pathway downregulation by Y-27632 suggests that RPE cell transplantation with ROCK inhibitor-free media is possible, which can minimize side effects to host tissue and have wider implications for transplantation methods requiring ROCK inhibition.     

Mechanisms of NOS2 regulation by Rho GTPase signaling in airway epithelial cells

The aberrant dysregulation of the inducible form of nitric oxide synthase (NOS2) is thought to play a role in many inflammatory disorders including cystic fibrosis (CF). The complex regulation of NOS2 expression is the subject of intense investigation, and one intriguing regulatory pathway known to influence NOS2 expression is the Rho GTPase cascade. We examined NOS2 regulation in response to inflammatory cytokines in a human alveolar epithelial cell line treated with inhibitors of different upstream and downstream components of the Rho GTPase pathway to better define potential signaling mechanisms. Statin-mediated 3-hydroxy-3-methylglutaryl-CoA reductase inhibition increased cytokine-dependent activation of the NOS2 promoter, reversible by the addition of geranylgeranyl pyrphosphate. However, inhibition of Rho-associated kinase (ROCK) with Y-27632 resulted in a decrease in NOS2 promoter activity, yet an increase in NOS2 mRNA and protein levels. Our results suggest that prenylation events influence NOS2 promoter activity independently of the Rho GTPase pathway and that Rho GTPase signaling mediated through ROCK suppresses NOS2 production downstream of promoter function at the message and protein level.     

Molecular mechanisms of low intensity pulsed ultrasound in human skin fibroblasts

Soluble factors such as polypeptide growth factors, mitogenic lipids, inflammatory cytokines, and hormones are known regulators of cell proliferation. However, the effect of mechanical stimuli on cell proliferation is less well understood. Here we examined the effect of low intensity pulsed ultrasound (US), which is used to promote wound healing, on the proliferation of primary human foreskin fibroblasts and the underlying signaling mechanisms. We show that a single 6-11-min US stimulation increases bromodeoxyuridine incorporation. In addition, an increase in the total cell number is observed after sequential US stimulation. US induced stress fiber and focal adhesion formation via activation of Rho. We further observed that US selectively induced activation of extracellular signal-regulated kinase (ERK) 1/2. Inhibition of Rho-associated coiled-coil-containing protein kinase (ROCK) prevented US-induced ERK1/2 activation, demonstrating that the Rho/ROCK pathway is an upstream regulator of ERK activation in response to US. Consequently, activation of ROCK and MEK-1 was required for US-induced DNA synthesis. Finally, an integrin beta(1) blocking antibody as well as a RGD peptide prevented US-induced DNA synthesis. In addition, US slightly increased phosphorylation of Src at Tyr(416), and Src activity was found to be required for ERK1/2 activation in response to US. In conclusion, our data demonstrate for the first time that US promotes cell proliferation via activation of integrin receptors and a Rho/ROCK/Src/ERK signaling pathway.     

Wnt-signaling mediates the anti-adipogenic action of lysophosphatidic acid through cross talking with the Rho/Rho associated kinase (ROCK) pathway

Lysophosphatidic acid (LPA) is a bioactive phospholipid with a diverse range of biological activities including the modulation of adipogenesis. Treatment of 3T3-L1 cells and 3T3F44A cells with LPA inhibits adipogenesis and reduces expression of PPARγ through activation of RhoGTPase and its downstream Rho associated kinase (ROCK). The mechanism of suppression of PPARγ expression by Rho/ROCK is poorly understood. By treating the differentiating 3T3-L1 cells with various combinations of LPA and ROCK inhibitors, Y-27632 and fasudil, we observed that LPA treatment resulted in attenuation of adipogenesis and a significant reduction in PPARγ mRNA as early as 3?d post-induction. LPA treatment also resulted in significant but delayed upregulation of components of the canonical Wnt signaling, namely Wnt10b mRNA, β-catenin protein, and mRNA expression of β-catenin target genes, detectable at day 7, but not day 3. Treatment of the 3T3-L1 cells with ROCK inhibitors Y-27632 and fasudil revealed a tonic activation of β-catenin/target genes by ROCK. This study identified the existence of a novel cross talk between the Rho/ROCK pathway and the Wnt-signaling pathway. The LPA/Rho/ROCK pathway inhibits expression of PPARγ and adipogenesis in part through a delayed activation of the canonical Wnt-signaling pathway based on increased Wnt10b expression and β-catenin induction.     

ROCK抑制剂Y27632对小胶质细胞炎性分泌的影响

目的观察ROCK抑制剂Y27632对小胶质细胞活化及其炎性分泌的作用。方法采用小胶质细胞系BV2细胞复苏后传代培养,分为对照组和Y27632干预组;在0、3h、6h、18h和24h收取细胞和细胞培养基;采用免疫荧光细胞染色测定各组BV2细胞上Ibal的表达,ELISA法测定各组培养基中IL-1B和TNF-α的浓度变化。结果Y27632可明显改变BV2细胞形态,干预组细胞比对照组细胞的胞体更大,细胞突起增多增长,细胞形态改变在干预6h时最为显著。与对照组相比,Y27632干预后BV2细胞分泌的IL-1β和TNF-α降低,在18h时抑制效果最为显著（P〈0．01）。结论ROCK抑制剂Y27632可诱导BV2细胞活化及炎性分泌降低,提示Rho／ROCK信号通路在小胶质细胞活化及炎性因子分泌中发挥了重要作用。