Rho激酶及其抑制剂的研究进展

Rho激酶是近十年来发现参与细胞运动的主要激酶之一，对细胞的分裂、收缩、粘附、迁移、分泌等活动具有重要调节作用。Rho激酶的高表达或过度激活与许多心脑血管疾病的发生发展密切相关，Rho激酶现在已经成为新药研发的重要靶点，而Rho激酶抑制剂的不断发现为心血管、神经系统等疾病的治疗提供了新的希望。为此，本文就Rho激酶及其抑制剂的研究做一简要综述。     

哮喘治疗新靶点——RhoA/Rho激酶及其抑制剂开发

RhoA/Rho激酶参与细胞有丝分裂、黏附、细胞骨架调整、肌细胞收缩及肿瘤细胞浸润等过程.RhoA/Rho激酶信号通路在气道炎症细胞迁移、趋化和浸润过程中起关键性的调控作用,是哮喘治疗药物的潜在靶点.本文综述RhoA/Rho激酶信号通路在哮喘气道炎症、气道重塑和气道高反应性(AHR)的作用及其机制以及Rho激酶抑制剂的开发近况.     

Rho激酶信号通路在两肾一夹肾性高血压大鼠心肌肥大中的作用及Fasudil干预

目的：了解Rho激酶介导的信号转导通路在两肾一夹（two-kidneys-one-clip,2K1C）肾性高血压大鼠心肌肥大发病机制中的作用以及特异性Rho激酶抑制剂Fasudil干预效应。方法：制备两肾一夹肾性高血压大鼠模型,实验设假手术组、模型组、实验组（Fasudil 10 mg·kg^-1.d^-1腹腔注射）。电子天平称量左心室质量并计算左心室质量与体质量比值;光学显微镜观察心肌组织形态学变化;Western blotting检测肌球蛋白结合亚基磷酸化（phosphorylation of myosin-binding submet,MBS-P）表达作为Rho激酶功能活化标志;RT-PCR检测Rho激酶mRNA表达。结果：术后12周,模型组大鼠出现较显著的心肌肥大（P〈0.01）,心肌组织Rho激酶活性及mRNA表达显著增加（P〈0.01）且Rho激酶活性及mRNA表达与心肌肥大程度间呈显著正相关（P〈0.05）。Rho激酶特异性抑制剂Fasudil在抑制大鼠心肌组织Rho激酶活化和mRNA表达的同时可有效抑制心肌肥大的发展（P〈0.01）。结论：Rho激酶介导的信号转导通路在2K1C肾性高血压大鼠心肌肥大发病机制中可能具有重要作用,Rho激酶特异性抑制剂Fasudil可有效抑制心肌肥大的发展。     

Rho激酶与疼痛及其抑制剂的临床应用

小分子GTP结合蛋白Rho及其下游Rho激酶对细胞的收缩、迁移、增殖、黏附和凋亡等活动具有重要的调控作用。该文通过阐述Rho/Rho激酶信号通路在疼痛的产生和持续过程中的作用及其抑制剂在临床研究中的使用案例,探讨Rho/Rho激酶作为镇痛药物的作用靶点的可能性。大量的实验和临床研究表明,Rho/Rho激酶通过其介导的细胞信号传导系统参与神经性疼痛、炎症性疼痛、糖尿病性周围神经疼痛、脑血管痉挛、血管痉挛性心绞痛及骨癌痛等,因此,Rho/Rho激酶可作为防治疼痛的潜在药物作用靶点,为临床上预防和治疗疼痛提供新的思路和策略。     

依立卢对大鼠心脏舒张功能的保护作用研究

目的探讨升主动脉缩窄压力超负荷心力衰竭大鼠左室舒张功能的变化及依立卢的干预机制。方法将升主动脉缩窄术后20周心力衰竭大鼠随机分为实验组（B组）和治疗组（C组），同时制备对照组（A组），4周后观察各项指标变化。结果B组与A组对比，LVDEP明显增高，而LVSP和±dp／dkmax明显减低；心肌组织RhoA、Rho激酶mRNA表达明显上调，RhoGTPases活性增高。C组与B组相比，LVDEP明显减低，LVSP和±dp／dtmax明显增高；心肌组织RhoA、Rho激酶mRNA表达明显降低，RhoGTPases活性降低。结论治疗组（C组）心肌舒张功能明显改善，提示Rho激酶抑制剂依立卢有心脏保护作用，可能与其干预Rho激酶的表达和合成有关。     

法舒地尔治疗肺动脉高压研究

法舒地尔是目前唯一在临床上使用的Rho激酶抑制药,Rho激酶现已成为心血管疾病治疗的重要靶点之一,在肺动脉高压的发病机制和发展过程中起重要作用。法舒地尔能够安全有效地治疗肺动脉高压且不良反应小,有望成为治疗肺动脉高压的新型药物。     

Rho激酶抑制剂Y-27632对人肝癌HepG2细胞增殖的抑制作用

目的 研究Rho激酶特异性抑制剂Y-27632对人肝癌HepG2细胞增殖的抑制作用.方法 体外培养人肝癌HepG2细胞,分为正常对照组(C组)以及不同浓度Y-27632 2.5、5、10、25、50μmol/L处理组(分别为A1组、A2组、A3组、A4组、A5组).镜下观察HepG2细胞的生长变化,MTT法检测各组HepG2细胞增殖情况.结果 在Y-27632作用下,HepG2细胞失去原有“铺路石”样排列,细胞间连接减少,细胞间隙增大,细胞胞体皱缩、变圆,细胞黏附性下降,有倾向凋亡趋势.与C组相比,不同浓度Y-27632作用后的HepG2细胞光密度值减少,细胞活力相对下降,且呈浓度依赖性(P＜0.05).结论 Rho激酶抑制剂Y-27632能有效抑制人肝癌HepG2细胞增殖.     

Rho/Rho激酶信号转导通路与他汀类药物

他汀类药物的多效性与Rho/Rho激酶信号转导通路密切相关。他汀类药物通过抑制类异戊二烯的生成而影响Rho的膜定位功能及其对下游效应分子Rho激酶的激活作用,继而引起一系列细胞生物学效应的改变。Rho/Rho激酶信号转导通路与他汀类药物关系的阐明,至少可部分解释他汀类药物调脂作用以外的更广泛的药理活性,以指导其在临床的应用。     

新型蛋白酪氨酸激酶抑制剂类抗肿瘤药物的研究进展

目的探讨蛋白酪氨酸激酶抑制剂抗肿瘤作用机理及其研究进展。方法综述了最新发现的小分子酪氨酸激酶抑制剂的化学结构、抗肿瘤作用及其作用机制及其发展方向等内容。结果结论蛋白酪氨酸激酶与细胞的增殖、分化、迁移和凋亡有着密切的关系,在细胞生命活动的信号转导途径中扮演着十分关键的角色,筛选酪氨酸激酶抑制剂已经成为开发抗肿瘤药物的新途径。     

新型脑、心血管活性药——法舒地尔

法舒地尔是目前唯一临床可用的Rho激酶抑制剂暨新型细胞内Ca2+拮抗剂,能强效扩血管、保护缺血脑组织,临床主要用于蛛网膜下腔出血手术后脑血管痉挛、脑缺血等的防治。鉴于Rho蛋白／Rho激酶系统在细胞分子水平生命活动中的重要意义,近年来,对法舒地尔的研究不断深入,其脑、心血管系统保护作用尤其明了。该药更多的适应证已处于临床试验后期中,有望成为颇具前景的新型脑、心血管活性药物。     

波依定对心力衰竭大鼠Rho/Rho激酶表达的影响

目的探讨波依定对升主动脉缩窄压力超负荷心力衰竭大鼠心肌细胞内Rho/Rho激酶表达的影响。方法制备升主动脉缩窄心力衰竭大鼠及假手术组模型。将升主动脉缩窄术后Wistar雌性大鼠随机分为3组,一组为假手术组,给予氧化钠溶液0.1ml,1次/d灌胃;一组为心力衰竭组,给予氯化钠溶液0.1ml,1次/d灌胃;一组为波依定组,给予波依定5mg/kg,1次/d灌胃;治疗4周。每组各10只大鼠。观察各组大鼠各项指标变化。结果心力衰竭组鼠心肌细胞RhoA、Rho激酶mRNA表达显著增高,波依定组心肌细胞RhoAmRNA、Rho激酶mRNA表达下降。结论心肌细胞RhoA、Rho激酶表达与充血性心力衰竭密切相关,波依定可有效降低心肌细胞内RhoAmRNA、Rho激酶mRNA表达,不恶化心力衰竭症状,可能为其可用于心力衰竭治疗的机制。     

Small guanine nucleotide-binding protein Rho and myocardial function

RhoA and Rho-kinase (ROCK) participate in a wide variety of cell signal functions such as cell growth, smooth and cardiac muscle contraction, cytoskeleton rearrangement, cell migration and proliferation. In vascular smooth muscle cells, RhoA and ROCK play an important role in Ca^2 sensitization and regulate vascular smooth muscle tone. In the heart, RhoA and ROCK mediate hypertrophic response leading to cardiac hypertrophy. Recent cellular and molecular biology studies using ROCK inhibitors such as Y-27632 and fasudil have indicated a pivotal role of the RhoA-ROCK cascade in many aspects of cardiovascular function such as cardiac hypertrophy and ventricular remodeling following myocardial infarction. Inhibition of the RhoA-ROCK signaling pathway may be a suitable target for a number of cardiovascular diseases including hypertension, atherosclerosis, diabetes and hypertrophic heart failure. This review focuses on the current understanding of the RhoA-ROCK signal pathway in heart diseases and discusses the use of ROCK inhibitors as therapeutic agents for heart diseases ranging from hypertensive cardiomyopathy to heart failure.     

Stepwise high-throughput virtual screening of Rho kinase inhibitors from natural product library and potential therapeutics for pulmonary hypertension

Abstract

Context: Pulmonary hypertension (PH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling. The activation of RhoA/Rho-kinase (ROCK) pathway plays a central role in the pathologic progression of PH and thus the Rho kinase, an essential effector of the ROCK pathway, is considered as a potential therapeutic target to attenuate PH.

Objective: In the current study, a synthetic pipeline is used to discover new potent Rho inhibitors from various natural products.

Materials and methods: In the pipeline, the stepwise high-throughput virtual screening, quantitative structure–activity relationship (QSAR)-based rescoring, and kinase assay were integrated. The screening was performed against a structurally diverse, drug-like natural product library, from which six identified compounds were tested to determine their inhibitory potencies agonist Rho by using a standard kinase assay protocol.

Results: With this scheme, we successfully identified two potent Rho inhibitors, namely phloretin and baicalein, with activity values of IC₅₀?=?0.22 and 0.95?μM, respectively.

Discussion and conclusion: Structural examination suggested that complicated networks of non-bonded interactions such as hydrogen bonding, hydrophobic forces, and van der Waals contacts across the complex interfaces of Rho kinase are formed with the screened compounds.     

VEGFR－2酪氨酸激酶抑制剂研究进展

血管内皮生长因子受体是一种可诱导血管生成的受体型酪氨酸激酶,临床研究发现该激酶在肿瘤组织中过度表达。以抑制血管内皮生长因子受体为基础的抗血管生成疗法已成为癌症治疗的新策略,小分子血管内皮生长因子受体-2抑制剂的设计和合成获得了广泛关注。对近5年内血管内皮生长因子受体一2抑制剂的研究进展进行了综述。     

Beneficial effect of arginine vasopressin on hemorrhagic shock through improving the vascular reactivity

The vascular reactivity and calcium sensitivity were decreased following hemorrhagic shock. Arginine vasopressin (AVP) was beneficial to endotoxic, infectious/spetic and hemorrhagic shock. Our previous studies found that Rho kinase played an important role in the occurrence of calcium desensitization following shock. It was reported that AVP was with stimulation effect of Rho kinase. So we hypothesized that AVP might have beneficial effect on shock via activation of Rho kinase to regulate the calcium sensitivity and vascular reactivity. Hemorrhagic shock (40 mmHg for 2 h) Wistar rats in vivo were adopted to observe the effects of small dose of AVP on hemodynamics, 24-h survival rate, the pressor effect of norepinephrine (NE) and the contractility of superior mesenteric artery (SMA). Isolated SMAs from hemorrhagic shock rats were adopted to observe the effects of AVP on vascular reactivity and calcium sensitivity and its relationship to Rho kinase with an isolated organ perfusion system. The results show that AVP at the concentration of 0.1 U/kg and 0.4 U/kg significantly improved the hemodynamic parameters and the 24-h survival rate of hemorrhagic shock rats. Meanwhile, these dosages of AVP significantly increased the pressor effect of NE and the contractile response of SMA to NE. Y-27632 (3 μg/kg), a Rho kinase specific inhibitor, abolished the beneficial effects of AVP. In vitro, the calcium sensitivity and vascular reactivity of SMA to calcium and NE were significantly decreased following hemorrhagic shock. AVP at the concentration of 0.5 nmol/L and 5 nmol/L significantly increased the calcium sensitivity and vascular reactivity. These effects of AVP were abolished by Y-27632 (10 μmol/L). Taken together, the results suggest that AVP at 0.1 U/kg and 0.4 U/kg is beneficial to hemorrhagic shock by improving the vascular reactivity, which involves activation of Rho kinase.     

Rho kinase as potential therapeutic target for cardiovascular diseases: opportunities and challenges

Rho kinase (ROCK) belongs to a family of Ser/Thr protein kinases that are activated via interaction with the small GTP-binding protein RhoA. Growing evidence suggests that RhoA and ROCK participate in a variety of important physiological functions in vasculature including smooth muscle contraction, cell proliferation, cell adhesion and migration, and many aspects of inflammatory responses. As these processes mediate the onset and progression of cardiovascular disease, modulation of the Rho/ROCK signalling pathway is a potential strategy for targeting an array of cardiovascular indications. Two widely employed ROCK inhibitors, fasudil and Y-27632, have provided preliminary but compelling evidence supporting the potential cardiovascular benefits of ROCK inhibition in preclinical animal disease models and in the clinic. This review summarises the molecular biology of ROCK and its biological functions in smooth muscle, endothelium and other vascular tissues. In addition, there will be a focus on recent progress demonstrating the benefits of ROCK inhibition in several animal models of cardiovascular diseases. Finally, recent progress in the identification of novel ROCK inhibitors and challenges associated with their development for clinical use will be discussed.     

Rho kinase as potential therapeutic target for cardiovascular diseases: opportunities and challenges

Rho kinase (ROCK) belongs to a family of Ser/Thr protein kinases that are activated via interaction with the small GTP-binding protein RhoA. Growing evidence suggests that RhoA and ROCK participate in a variety of important physiological functions in vasculature including smooth muscle contraction, cell proliferation, cell adhesion and migration, and many aspects of inflammatory responses. As these processes mediate the onset and progression of cardiovascular disease, modulation of the Rho/ROCK signalling pathway is a potential strategy for targeting an array of cardiovascular indications. Two widely employed ROCK inhibitors, fasudil and Y-27632, have provided preliminary but compelling evidence supporting the potential cardiovascular benefits of ROCK inhibition in preclinical animal disease models and in the clinic. This review summarises the molecular biology of ROCK and its biological functions in smooth muscle, endothelium and other vascular tissues. In addition, there will be a focus on recent progress demonstrating the benefits of ROCK inhibition in several animal models of cardiovascular diseases. Finally, recent progress in the identification of novel ROCK inhibitors and challenges associated with their development for clinical use will be discussed.     

Calcium Sensitization Induced by Sodium Fluoride in Permeabilized Rat Mesenteric Arteries

It was hypothesized that NaF induces calcium sensitization in Ca²⁺-controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric arteries were permeabilized with β-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to Ca²⁺ (decreased EC₅₀ and increased E_max). Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in Ca²⁺-controlled solution at pCa 7.0 or pCa 6.5, but not at pCa 8.0. NaF-induced vasocontraction and GTPγS-induced vasocontraction were not additive. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase inhibitors H1152 or Y27632 but not with a MLCK inhibitor ML-7 or a PKC inhibitor Ro31-8220. NaF induces calcium sensitization in a Ca²⁺-dependent manner in β-escin-permeabilized rat mesenteric arteries. These results suggest that NaF is an activator of the Rho kinase signaling pathway during vascular contraction.     

Vascular rho kinases and their potential therapeutic applications

Over the last decade, numerous advances have been made in characterizing the various roles of rho kinase in vascular function. In light of extensive evidence for the involvement of rho kinase in the development of several cardiovascular disorders, including hypertension and arterial spasm, this protein is now considered to be an important therapeutic target. Several rho kinase inhibitors are already in experimental use, and further novel and structurally diverse inhibitors have been described. This review focuses on recent developments in our understanding of how rho kinase contributes to vascular dysfunction, and thus the potential of rho kinase inhibitors as therapeutic agents for vascular diseases.