Rho-associated protein kinase modulates neurite extension by regulating microtubule remodeling and vinculin distribution

Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulat- ing Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite out- growth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased mem- brane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vin- culin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.

Downregulation of rho-associated protein kinase 1 by mi R-124 in colorectal cancer

AIM: To investigate the roles and interactions of rhoassociatedprotein kinase (ROCK)1 and miR-124 inhuman colorectal cancer (CRC).METHODS: Expression of ROCK1 protein wasexamined by Western blotting, and quantitativereverse transcriptase PCR was performed to measureexpression of ROCK1 mRNA and miR-124. Two cancercell lines were transfected with pre-miR-124 (mimic)and anti-miR-124 (inhibitor) and the effects onROCK1 protein and mRNA expression were observed.In addition, cell proliferation was assessed via a5-ethynyl-2′ deoxyuridine assay. Soft agar formationassay, and cell migration and invasion assays wereused to determine the effect of survivin on thetransformation and invasion activity of CRC cells.RESULTS: miR-124 was significantly downregulated inCRC compared to normal specimens (0.603 ± 0.092 vs1.147 ± 0.286, P = 0.016) and in metastatic comparedto nonmetastatic CRC specimens (0.416 ± 0.047 vs0.696 ± 0.089, P = 0.020). Expression of miR-124 wassignificantly associated with CRC metastasis, tumor Tand N stages, and tumor grade (all P < 0.05). ROCK1protein was significantly increased in CRC comparedto normal tissues (1.896 ± 0.258 vs 0.866 ± 0.136,P = 0.026), whereas ROCK1 mRNA expression wasunaltered (2.613 ± 0.251 vs 2.325 ± 0.246). miR-124and ROCK1 were inversely expressed in CRC tissuesand cell lines. ROCK1 mRNA was unaltered in cellstransfected with miR-124 mimic and miR-124 inhibitor,compared to normal controls. There was a significantreduction in ROCK1 protein in cells transfected withmiR-124 mimic and a significant increase in cells transfected with miR-124 inhibitor (P s < 0.05).Transformation and invasion of cells transfectedwith miR-124 inhibitor were significantly increasedcompared to those in normal controls (P < 0.05). Cellstransfected with miR-124 inhibitor showed increasedcell proliferation.CONCLUSION: miR-124 promotes hyperplasia andcontributes to invasion of CRC cells, but downregulatesROCK1. ROCK1 and miR-124 may play important rolesin CRC.

Inhibition of RhoA/Rho-kinase pathway suppresses the expression of extracellular matrix induced by CTGF or TGF-β in ARPE-19

AIM:To investigate the role of Rho-associated protein kinase (ROCK) inhibitor, Y27632, in mediating the production of extracellular matrix (ECM) components including fibronectin, matrix metallo-proteinase-2 (MMP-2) and type I collagen as induced by connective tissue growth factor(CTGF) or transforming growth factor-β (TGF-β) in a human retinal pigment epithelial cell line, ARPE-19. METHODS:The effect of Y27632 on the CTGF or TGF-β induced phenotype in ARPE-19 cells was measured with immunocytochemistry as the change in F-actin. ARPE-19 cells were treated with CTGF (1, 10, 100ng/mL)and TGF-β (10ng/mL) in serum free media, and analyzed for fibronectin, laminin, and MMP-2 and type I collagen by RT-qPCR and immunocytochemistry. Cells were also pretreated with an ROCK inhibitor, Y27632, to analyze the signaling contributing to ECM production. ·RESULTS:Treatment of ARPE-19 cells in culture with TGF-β or CTGF induced an ECM change from a cobblestone morphology to a more elongated swirl pattern indicating a mesenchymal phenotype. RT-qPCR analysis and different gene expression analysis demonstrated an upregulation in expression of genes associated with cytoskeletal structure and motility. CTGFor TGF-β significantly increased expression of fibronectin mRNA (P =0.006, P =0.003 respectively), laminin mRNA (P =0.006, P =0.005), MMP-2 mRNA (P =0.006, P =0.001), COL1A1 mRNA (P =0.001, P =0.001), COL1A2 mRNA (P = 0.001, P =0.001). Preincubation of ARPE-19 with Y27632 (10mmol/L) significantly prevented CTGF or TGF-β induced fibronectin (P=0.005, P=0.003 respectively), MMP-2 (P = 0.003, P =0.002), COL1A1 (P =0.006, P =0.003), and COL1A2 (P =0.006, P =0.004) gene expression, but not laminin (P =0.375, P =0.516). CONCLUSION:Our study demonstrated that both TGF-β and CTGF upregulate the expression of ECM components including fibronectin, laminin, MMP-2 and type I collagen by activating the RhoA/ROCK signaling pathway. During this process, ARPE-19 cells were shown to change from an epithelial to a mesenchymal phenotype in vitro. Y27632, a ROCK inhibitor, inhibited the transcription of fibronectin, MMP-2 and type I collagen, but not laminin. The data from our work suggest a role for CTGF as a profibrotic mediator. Inhibiting the RhoA/ROCK pathway represents a potential target to prevent the fibrosis of retinal pigment epithelial (RPE) cells. This might lead to a novel therapeutic approach to preventing the onset of early proliferative vitreoretinopathy(PVR).

Rho kinase:A new target for treatment of cerebral ischemia/reperfusion injury

Rho kinase inhibitor fasudil hydrochloride has been shown to reduce cerebral vasospasm, to inhibit inflammation and apoptosis and to promote the recovery of neurological function. However, the effect of fasudil hydrochloride on claudin-5 protein expression has not been reported after cerebral ischemia/reperfusion. Therefore, this study sought to explore the effects of fasudil hydrochloride on blood-brain barrier permeability, growth-associated protein-43 and claudin-5 protein expression, and to further understand the neuroprotective effect of fasudil hydrochloride. A focal cerebral ischemia/reperfusion model was established using the intraluminal suture technique. Fasudil hydrochloride （15 mg/kg） was intraperitoneally injected once a day. Neurological deficit was evaluated using Longa＇s method. Changes in permeability of blood-brain barrier were measured using Evans blue. Changes in RhoA, growth-associated protein-43 and claudin-5 protein expression were detected using immunohistochemistry and western blotting. Results revealed that fasudil hydrochloride noticeably contributed to the recovery of neurological function, improved the function of blood-brain barrier, inhibited RhoA protein expression, and upregulated growth-associated protein-43 and claudin-5 protein expression following cerebral ischemia/reperfusion. Results indicated that Rho kinase exhibits a certain effect on neurovascular damage following cerebral ischemia/reperfusion. Intervention targeted Rho kinase might be a new therapeutic target in the treatment of cerebral ischemia/reperfusion.

Desensitization of G-protein-coupled receptors induces vascular hypocontractility in response to norepinephrine in the mesenteric arteries of cirrhotic patients and rats

BACKGROUND: The increased β-arrestin-2 and its combination with G-protein-coupled receptors (GPCRs) lead to GPCRs desensitization. The latter may be responsible for decreased contractile reactivity in the mesenteric arteries of cirrhotic patients and rats. The present study is to investigate the machinery changes of α-adrenergic receptors and G proteins and their roles in the contractility of mesenteric arteries of cirrhotic patients and animal models. METHODS: Patients with cirrhosis due to hepatitis B and cirrhotic rats induced by CCl 4 were studied. Mesenteric artery contractility in response to norepinephrine was determined by a vessel perfusion system. The contractile effect of G protein-coupled receptor kinase-2 (GRK-2) inhibitor on the mesenteric artery was evaluated. The protein expression of the α 1 adrenergic receptor, G proteins, β-arrestin-2, GRK-2 as well as the activity of Rho associated coiled-coil forming protein kinase-1 (ROCK-1) were measured by Western blot. In addition, the interaction of α 1 adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation. RESULTS: The portal vein pressure of cirrhotic patients and rats was significantly higher than that of controls. The doseresponse curve to norepinephrine in mesenteric arteriole was shifted to the right, and EC 50 was significantly increased in cirrhotic patients and rats. There were no significant differences in the expressions of the α 1 adrenergic receptor and G proteins in the cirrhotic group compared with the controls. However, the protein expressions of GRK-2 and β-arrestin-2 were significantly elevated in cirrhotic patients and rats compared with those of the controls. The interaction of the α 1 adrenergic receptor and β-arrestin-2 was significantly aggravated. This interaction was significantly reversed by GRK-2 inhibitor. Both the protein expression and activity of ROCK-1 were significantly decreased in the mesenteric artery in patients with cirrhosis compared with those of the controls, and this phenomenon was not shown in the cirrhotic rats. Norepinephrine significantly increased the activity of ROCK-1 in normal rats but not in cirrhotic ones. Norepinephrine significantly increased ROCK-1 activity in cirrhotic rats when GRK-2 inhibitor was used. CONCLUSIONS: β-arrestin-2 expression and its interaction with GPCRs are significantly upregulated in the mesenteric arteries in patients and rats with cirrhosis. These upregulations result in GPCR desensitization, G-protein dysfunction and ROCK inhibition. These may explain the decreased contractility of the mesenteric artery in response to vasoconstrictors.

杜仲叶总黄酮通过RhoA/ROCK信号通路参与脑出血大鼠神经功能修复

目的从Ras同源基因家族成员A(RhoA)/Rho相关卷曲螺旋蛋白激酶(ROCK)通路探讨杜仲叶总黄酮促进脑出血大鼠神经功能修复的机制。方法取SD雄性大鼠,用改良二次注血法建立脑出血模型,并按随机数字表法分为假手术组、模型组、杜仲叶总黄酮组、RhoA抑制剂组、RhoA激动剂组、杜仲叶总黄酮+RhoA激动剂组,每组20只。杜仲叶总黄酮组灌胃200 mg/kg杜仲叶总黄酮,RhoA抑制剂组腹腔注射1 mg/kg的RhoA抑制剂Y27632,RhoA激动剂组腹腔注射30μg/kg的RhoA激动剂U-46619,杜仲叶总黄酮+RhoA激动剂组灌胃200 mg/kg杜仲叶总黄酮的同时腹腔注射30μg/kg U-46619,模型组及假手术组灌胃10 mL/kg生理盐水,1次/d,连续7 d。给药结束后,观察大鼠行为变化,采用改良神经功能缺损评分(mNSS)对神经功能缺损症状进行评估;取脑组织后称质量,计算脑组织含水量,然后制备脑组织切片并计算脑血肿体积百分比;透射电镜观察血肿周围组织神经突触结构变化;TUNEL染色法观察血肿周围组织神经元凋亡;鬼笔环肽染色观察血肿周围组织神经元骨架变化;蛋白免疫印迹法检测血肿周围组织RhoA、ROCK、细胞骨架蛋白(F-actin)、丝切蛋白(cofilin)、磷酸化cofilin(p-cofilin)、促神经元及突触生长相关蛋白[神经生长因子(NGF)、神经营养因子3(NT3)、突触后致密蛋白-95(PSD-95)、突触素(SYP)]的表达。结果与假手术组相比,模型组大鼠神经功能缺损评分、脑组织含水量、脑血肿体积百分比升高,血肿周围组织神经元凋亡及骨架结构损伤,神经突触结构改变严重,RhoA/ROCK通路激活,促神经元及突触生长相关蛋白表达降低(P<0.05)。杜仲叶总黄酮或RhoA抑制剂干预治疗均可抑制RhoA/ROCK通路激活介导的神经元骨架结构改变,提高促神经元及突触生长相关蛋白表达,缓解脑出血后血肿周围组织神经元损伤及凋亡,促进神经功能修复(P<0.05)。RhoA激动剂可促进RhoA/ROCK通路激活,加重脑出血后神经功能损伤,并削弱杜仲叶总黄酮的促神经功能修复作用(P<0.05)。结论杜仲叶总黄酮可通过抑制RhoA/ROCK通路活化来改善脑出血大鼠出血症状,促进神经功能修复。

Rho相关卷曲螺旋蛋白激酶1在动脉粥样硬化血管壁中的表达及其与基质金属蛋白酶2及转化生长因子1的相关性

目的研究Rho相关卷曲螺旋蛋白激酶1(ROCK1)在动脉粥样硬化血管壁中的表达及其与基质金属蛋白酶2(MMP2)、转化生长因子1(TGF-β1)的相关性。方法选择30只载脂蛋白E基因敲除小鼠为实验组,高脂饲料喂养,另选30只C57BL/6小鼠为对照组,普通饲料喂养。在喂养的第10、16、22、28及34周,取眼球血监测小鼠血脂水平;取小鼠腹主动脉作为标本,包埋切片并进行苏木精-伊红染色观察血管壁形态;应用免疫组化染色观察血管壁中ROCK1、MMP2、TGF-β1的表达;使用Image Pro Plus 6.0软件测量切片中血管壁厚度、斑块面积、血管壁中ROCK1、MMP2及TGF-β1的表达量。采用SPSS 27.0统计软件进行数据分析。采用单因素方差分析进行组间比较,两两比较采用Tukey检验。采用Pearson相关与线性回归分析ROCK1与血管壁厚度及斑块面积、MMP2、TGF-β1的关系。结果成功建立动脉粥样硬化小鼠模型。在喂养第10、16、22、28及34周,实验组血脂水平明显高于对照组,差异有统计学意义(P<0.05)。自喂养第16周起,实验组小鼠血管内均有斑块形成,随着喂养时间的延长,斑块面积和血管壁厚度不断增加,差异有统计学意义(P<0.05);血管壁内ROCK1的表达逐步增高,其表达与斑块面积及血管壁厚度呈正相关(r=0.821,0.730;P<0.05)。线性相关分析及回归分析显示,ROCK1与MMP2、TGF-β1表达均呈正相关(r=0.801,0.906;P<0.05)。结论在动脉粥样硬化血管壁中存在ROCK1蛋白的表达,且表达量随着血管壁厚度增加而增高,ROCK1的表达与MMP2、TGF-β1呈显著正相关性。鉴于ROCK1蛋白的致血管痉挛作用,提示动脉粥样硬化血管壁可能易于痉挛,具体机制需进一步研究。

Rho/ROCK信号通路介导肾脏纤维化的机制研究进展

肾脏纤维化是几乎所有肾脏疾病进展至终末期肾病的共同病理改变,严重威胁人类健康。Rho/Rho相关卷曲螺旋蛋白激酶(ROCK)信号通路是生物体内广泛存在的经典信号通路,可通过调节骨架蛋白的聚合状态影响细胞收缩、黏附、迁移等生物学行为。Rho/ROCK信号通路的异常激活参与肾脏纤维化的发生发展,有望成为临床治疗肾脏纤维化的有效靶点。因此,深入研究Rho/ROCK信号通路介导肾脏纤维化发生发展的机制,可以为慢性肾脏病的临床诊治和新药研发提供思路。

绿茶提取物介导Rho/ROCK通路缓解脑梗死大鼠脑组织损伤的实验研究

目的:探讨绿茶提取物对Ras同源基因(Rho)/Rho相关螺旋卷曲蛋白激酶(ROCK)通路的调控作用及对脑梗死大鼠脑损伤的影响,初步探讨其作用机制。方法:用线栓法复制大鼠脑梗死模型,并随机分为假手术组、模型组、绿茶提取物组(200 mg/kg)、Rho抑制剂组(给予盐酸法舒地尔15 mg/kg)、Rho激动剂组(给予溶血性磷脂酸50 mmol/kg)、联合组(绿茶提取物+Rho激动剂),每组18只。给药结束后,观察大鼠行为变化;氯化三苯基四氮唑(TTC)染色观察脑梗死状况;尼氏及脱氧核糖核苷酸末端转移酶介导的黏性末端标记(TUNEL)染色法观察神经元损伤及凋亡情况;鬼笔环肽染色观察神经元细胞骨架变化;透射电镜下观察神经突触结构变化;免疫组化法观察Rho阳性表达水平;蛋白免疫印迹法(Western Blot)检测Rho/ROCK通路蛋白、促神经生长因子[神经生长因子(NGF)、脑源性神经营养因子(BDNF)、神经营养因子3(NT3)]、神经生长抑制因子[轴突生长抑制因子(NogoA)、髓鞘相关蛋白(MAG)、切丝蛋白(cofilin)]、突触再生重塑相关蛋白[突触后致密物质(PSD-95)、突触素(SYP)等]蛋白表达。结果:与假手术组比较,模型组大鼠神经功能缺损评分、脑梗死体积均增加,脑皮层神经元损伤和凋亡、突触小泡破裂融合等结构损伤及细胞骨架破坏严重,Rho/ROCK通路活化,及其介导的促神经生长因子表达降低,而神经抑制因子表达升高(P<0.05)。绿茶提取物及Rho抑制剂均可减轻脑皮层神经元损伤和凋亡、突触小泡破裂融合等结构损伤及细胞骨架破坏等病理损伤,降低神经功能缺损评分及脑梗死体积,抑制Rho/ROCK通路活化和神经抑制因子表达,促进促神经生长因子表达(P<0.05)。Rho激动剂可逆转绿茶提取物的上述作用(P<0.05)。结论:绿茶提取物可抑制Rho/ROCK活化,促进脑梗死后神经元及突触的再生及重塑,改善神经功能损伤,发挥脑保护作用。

JTE-013介导RhoA/ROCK1/Drp1信号轴调控线粒体损伤和凋亡缓解过敏性鼻炎

目的探究JTE-013通过RhoA/ROCK1/Drp1通路调控线粒体损伤和凋亡对过敏性鼻炎(allergic rhinitis,AR)保护作用及机制。方法用卵清蛋白(OVA)建立小鼠AR模型。造模结束后,记录揉鼻、打喷嚏的次数。然后鼻组织切片进行HE、TUNEL和DHE染色。在体外,通过人重组白细胞介素-13(IL-13)刺激人鼻黏膜上皮细胞(HNEpCs),Western blot法检测JTE-013和Y27632对RhoA、ROCK1、Drp1及其相关磷酸化的蛋白和细胞凋亡相关蛋白表达影响。免疫荧光观察JTE-013和Y27632对细胞总ROS、线粒体膜电位和线粒体ROS生成,Drp1易位情况以及Cyt-c的表达情况。结果JTE-013减少了AR小鼠的揉鼻和打喷嚏频率,减轻鼻黏膜增厚并降低嗜酸细胞浸润。TUNEL和DHE染色结果提示,JTE-013可以抑制小鼠鼻上皮细胞凋亡并减少ROS表达。Western blot结果表明,JTE-013和Y27632都能明显降低RhoA、ROCK1、Drp1及p-Drp1(616),抑制凋亡蛋白Bax、cleaved-caspase-3、Cyt-c、cleaved-caspase-9表达并上调了p-Drp1(637)、Bcl-2表达。免疫荧光显示JTE-013或ROCK1的抑制剂几乎阻断了IL-13介导鼻黏膜上皮细胞ROS和mtROS生成增加、抑制了线粒体膜电位降低,阻滞了Cyt-c表达和Drp1易位。结论JTE-013能够通过抑制RhoA/ROCK1/Drp1信号轴调节线粒体的形态和功能,从而缓解AR小鼠鼻黏膜上皮细胞炎症。

2种Rho相关卷曲螺旋形成蛋白激酶对经皮冠状动脉介入治疗无复流的预测价值

目的探讨Rho相关卷曲螺旋形成蛋白激酶(ROCK)1和ROCK2对急性ST段抬高型心肌梗死(STEMI)患者经皮冠状动脉介入治疗(PCI)无复流的预测价值。方法选取168例接受PCI的STEMI患者作为研究对象,根据是否发生无复流分为无复流组和血流正常组,并分别根据ROCK1、ROCK2表达情况分为高表达组和低表达组。使用酶联免疫吸附试剂盒检测患者血清ROCK1、ROCK2水平,分析无复流组与血流正常组的血清ROCK1、ROCK2水平差异,分析STEMI患者PCI无复流的危险因素,并分析ROCK1、ROCK2对STEMI患者PCI无复流的预测价值。结果168例STEMI患者中,46例发生无复流,发生率为27.38%。无复流组的Killip分级、发病至入院时间、未使用预防性无复流药物者占比、血清ROCK1水平、血清ROCK2水平高于或长于血流正常组,差异有统计学意义(P<0.05)。ROCK1高表达组的无复流发生率高于ROCK1低表达组,ROCK2高表达组的无复流发生率高于ROCK2低表达组,差异有统计学意义(P<0.05)。多因素Logsitic回归分析显示,Killip分级为Ⅲ~Ⅳ级、发病至入院时间较长、未使用预防性无复流药物、血清ROCK1水平较高、血清ROCK2水平较高均为STEMI患者PCI无复流的独立危险因素(P<0.05)。受试者工作特征曲线显示,ROCK1、ROCK2对STEMI患者PCI无复流的预测价值较高,且两者联用后预测价值提升,曲线下面积为0.789(95%CI:0.711~0.867)。结论血清ROCK1、ROCK2水平较高是STEMI患者PCI无复流的危险因素,两者联合检测对PCI无复流具有较高的预测价值。

IgA肾病患者血清Rho相关卷曲螺旋形成蛋白激酶2、视黄醇结合蛋白4表达水平及在评估病情和预测预后中的价值研究

目的检测IgA肾病(immunoglobulin A nephropathy,IgAN)患者血清Rho相关卷曲螺旋形成蛋白激酶2(rho associated coiled coil containing protein kinase 2,ROCK2)、视黄醇结合蛋白4(retinol-binding protein 4,RBP4)表达水平,分析二者在评估患者病情及预测预后中的价值。方法选取2016年6月至2019年6月98例中国贵航集团302医院已确诊收治的IgAN患者为IgAN组,其中轻度IgAN 36例、中度IgAN 32例、重度IgAN 30例;同期选择在中国贵航集团302医院体检的健康者100名为对照组。酶联免疫吸附法检测血清中ROCK2、RBP4水平;采用Kaplan-Meier生存曲线分析ROCK2、RBP4表达水平与IgAN患者预后的关系;Pearson相关性分析ROCK2与RBP4表达相关性;受试者工作特征曲线(receiver operating characteristic curve,ROC)分析ROCK2、RBP4对IgAN的诊断价值;比例风险回归模型法分析IgAN患者预后的影响因素。结果IgAN组血清中ROCK2、RBP4表达水平分别为(24.25±5.31)μg/L、(59.70±9.43)μg/L,显著高于对照组的(15.57±4.16)μg/L、(32.14±7.82)μg/L,差异具有统计学意义(t=12.818、22.404,均P<0.001);轻度、中度、重度IgAN患者血清中ROCK2水平分别为(18.69±4.36)μg/L、(23.75±5.31)μg/L、(31.46±5.62)μg/L,RBP4表达水平分别为(45.37±7.86)μg/L、(62.48±8.12)μg/L、(73.94±8.65)μg/L。随着病情的加重,IgAN患者血清ROCK2、RBP4表达水平逐渐升高,差异具有统计学意义(F=51.854、102.234,均P<0.05);IgAN患者血清中ROCK2、RBP4表达水平与Lee氏分级、IgA/C3比值、病情程度有关(P<0.05);ROCK2、RBP4高表达组患者预后生存率分别为60.00%、74.19%,均显著低于低表达组的89.71%、91.67%,差异具有统计学意义(log rankχ^(2)=4.674,P=0.031);ROC曲线结果显示,血清ROCK2诊断IgAN的曲线下面积(area under curve,AUC)为0.889,敏感度为80.61%,特异性为80.00%,截断值为18.79μg/L;RBP4诊断IgAN的AUC为0.986,敏感度为96.94%,特异性为95.00%,截断值为41.31μg/L;二者联合检测的AUC为0.997,敏感度为98.98%,特异性为94.86%。Pearson相关性分析显示,IgAN患者血清ROCK2与RBP4表达水平呈显著正相关(r=0.701,P<0.001);多因素比例风险回归模型法分析表明,IgA/C3(OR=2.683,95%CI:1.060~6.794)、ROCK2(OR=1.831,95%CI:1.027~3.264)、RBP4(OR=1.517,95%CI:1.104~2.084)均是IgAN患者预后生存的影响因素(均P<0.05)。结论ROCK2、RBP4在IgAN患者血清中高表达,二者与IgAN患者的临床病理特征、病情严重程度及预后关系密切,ROCK2、RBP4表达水平对IgAN的诊断具有一定的价值。

TGF-β_1介导的RhoA/ROCK通路在大鼠肺肌成纤维细胞分化中的调节作用

目的:探讨转化生长因子β1(TGF-β1)介导的RhoA/Rho相关卷曲螺旋形成蛋白激酶(ROCK)通路在调控肺成纤维细胞向肌成纤维细胞分化过程中的作用。方法:胰酶消化法获得原代培养的肺成纤维细胞,进行以下实验:(1)观察TGF-β1诱导肺成纤维细胞不同时间后p-RhoA、ROCK、磷酸化肌球蛋白磷酸酶靶亚基(pMBS)、血清应答因子(SRF)、α-平滑肌肌动蛋白(α-SMA)、I型和III型胶原蛋白表达变化;(2)将细胞分为对照组、TGF-β1诱导分化组和Y-27632(ROCK通路阻滞剂)干预组,免疫细胞化学染色与Western blotting法分别观察与检测ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白在细胞内的定位分布与表达。结果:(1)经TGF-β1诱导24 h后,大鼠肺成纤维细胞胞体内出现大量平行或交叉排列的α-SMA抗体标记的肌丝。随着TGF-β1诱导刺激时间的延长,p-RhoA、ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白表达逐渐增高,其中RhoA/ROCK信号(p-RhoA、ROCK、p-MBS)、SRF和α-SMA蛋白分别在诱导后6 h、12 h和24 h达到高峰。I型胶原和III型胶原蛋白表达均在24 h达高峰,分别为诱导前的2.19和3.04倍(P<0.05)。(2)与TGF-β1诱导分化组比较,当给予Y-27632干预后,在相应时点ROCK、p-MBS、SRF、α-SMA、I型和III型胶原蛋白表达均明显降低,差异有统计学意义(P<0.05)。结论:TGF-β1通过ROCK信号转导通路的活化,刺激了大鼠肺成纤维细胞向肌成纤维细胞分化,进而促进了胶原蛋白的合成,可能在(矽)肺纤维化形成过程中发挥着重要作用。

川陈皮素对食管癌KYSE150细胞存活、侵袭和凋亡的影响及机制实验研究

目的:探究川陈皮素对食管癌KYSE150细胞存活、侵袭及凋亡的影响及可能的机制。方法:将培养至对数生长期的KYSE150细胞分为对照组(常规培养)、顺铂组(0.5μg/ml顺铂)、川陈皮素低(10μg/ml)、中(20μg/ml)、高(40μg/ml)浓度组。采用MTT法检测KYSE150细胞存活率。采用Transwell实验检测KYSE150细胞侵袭能力。采用流式细胞术检测KYSE150细胞凋亡率。采用荧光定量PCR法检测KYSE150细胞Ras同源基因家族成员A(RhoA)、Rho相关卷曲螺旋蛋白激酶1(ROCK1)、ROCK2 mRNA表达。采用Western bolt法检测KYSE150细胞RhoA、ROCK1、ROCK2蛋白表达。结果:与对照组比较,顺铂组及川陈皮素低、中、高浓度组KYSE150细胞存活率、侵袭数目以及RhoA、ROCK1、ROCK2 mRNA和蛋白表达水平降低,凋亡率升高(均P<0.05)。与顺铂组比较,川陈皮素低、中浓度组KYSE150细胞存活率、侵袭数目以及RhoA、ROCK1、ROCK2 mRNA和蛋白表达水平升高,凋亡率降低(均P<0.05)。川陈皮素高浓度组和顺铂组上述指标比较差异无统计学意义(均P>0.05)。川陈皮素低、中、高浓度组KYSE150细胞存活率、侵袭数目以及RhoA、ROCK1、ROCK2 mRNA和蛋白表达水平依次降低,凋亡率依次升高(均P<0.05)。结论:川陈皮素能够抑制KYSE150细胞的存活和侵袭,促进凋亡,其机制可能与抑制RhoA/ROCK信号通路有关。

RhoA/ROCK信号通路在动脉粥样硬化发生中的作用

Rho A/ROCK通路参与细胞肌动蛋白骨架的调节、细胞的收缩、黏附、增生、分化及基因的表达等。越来越多的证据表明,其在动脉粥样硬化的发生发展中起着重要作用。对Rho A/ROCK通路的研究将拓展对有关心血管疾病的认识,有望为心血管疾病提供新的治疗靶点。

miR-335靶向Rho相关卷曲螺旋形成蛋白激酶1抑制人骨肉瘤细胞MG-63侵袭转移的实验研究

背景与目的:微小RNA(micro RNA,mi RNA)是一类小分子内源性RNA,主要在转录后水平调节靶基因的表达。micro RNA-335(mi R-335)作为一种肿瘤抑制因子,参与了多种人类肿瘤的发生、发展过程。本研究旨在探讨mi R-335是否靶向抑制Rho相关卷曲螺旋形成蛋白激酶1(Rho associated coiled-coil forming protein kinase,ROCK1)基因的表达,并以此调控人骨肉瘤细胞MG-63侵袭及转移。方法:理论预测并通过荧光素酶基因报告验证mi R-335与ROCK1基因的3'-非翻译区(untranslated region,UTR)的特异性结合作用;real-time PCR和蛋白质印迹法(Western blot)分别从基因和蛋白水平检测mi R-335对ROCK1表达的负性调控作用;Transwell小室法检测mi R-335过表达及下调ROCK1表达后MG-63侵袭及转移能力的变化。结果:Targetscan预测显示,mi R-335与ROCK1 3'-UTR存在结合位点。荧光素酶基因报告实验结果显示,mi R-335 mimic和ROCK1 3'-UTR能够靶向结合;mi R-335在MG-63细胞中低表达,ROCK1则呈高表达。Western blot检测结果显示,转染mi R-335 mimic或转染ROCK1 si RNA后ROCK1的蛋白表达减少。Transwell小室法检测结果显示,过表达mi R-335或下调ROCK1后穿过基膜的细胞数目明显下降。结论:mi R-335能特异性结合于ROCK1基因的3'-UTR并下调ROCK1的表达,抑制人骨肉瘤细胞MG-63侵袭转移。

地塞米松联合垂体后叶素预防产后出血的效果及对RhoA、ROCK蛋白影响

目的:分析地塞米松联合垂体后叶素预防产后出血效果及对RhoA、Rho相关卷曲螺旋形成蛋白激酶(ROCK)的影响。方法:选择2017年6月-2018年12月本院分娩的88例高危妊娠产妇为研究对象,根据简单随机法分为对照组(n=46)和观察组(n=42)。对照组采用地塞米松,观察组在对照组基础上联合垂体后叶素,比较两组止血效果,干预前后RhoA、ROCKⅠ、ROCKⅡ蛋白、血压、心率和不良反应。结果:干预后观察组总有效率(97.6%)高于对照组(84.8%),产后2h(172.0±23.2ml)、产后24h出血量(255.1±31.2ml)、产后出血率(2.4%)均低于对照组(287.3±38.7ml、340.2±45.3ml、15.2%),产后24h血红蛋白(121.1±16.0 g/L)高于对照组(113.1±13.3 g/L),RhoA(0.59±0.07 ng/ml)、ROCKⅠ(0.40±0.06 ng/ml)、ROCKⅡ(0.38±0.05 ng/ml)蛋白高于对照组(P<0.05);两组舒张压、收缩压、心率和不良反应发生无差异(P>0.05)。结论:地塞米松联合垂体后叶素可加强子宫收缩,调节RhoA、ROCK蛋白表达,预防宫缩乏力所致的产后出血,且用药安全。

子宫平滑肌RhoA/Rho激酶表达与宫缩乏力性产后出血的关系

目的:探讨产妇子宫平滑肌组织RhoA/Rho激酶表达与宫缩乏力性产后出血的关系。方法:选择30例宫缩乏力性产后出血产妇为病例组,同期无产后出血产妇30例为对照组。采用肌条等长张力测定法检测子宫平滑肌自发收缩活动力、加入Rho激酶抑制剂Y-27632后子宫平滑肌收缩活动力;采用实时荧光定量反转录聚合酶链反应(RT-PCR)法及蛋白质印迹法检测子宫平滑肌组织RhoA,ROCKⅠ和ROCKⅡmRNA及蛋白表达水平。结果:①病例组子宫平滑肌自发收缩活动力水平低于对照组,差异有统计学意义(P<0.01);病例组和对照组加入Y-27632后收缩活动力水平均低于加入前,差异有统计学意义(P<0.01)。②病例组子宫平滑肌组织RhoA,ROCKⅠ和ROCKⅡmRNA表达水平均低于对照组,差异均有统计学意义(P<0.05或P<0.01)。③病例组子宫平滑肌组织RhoA,ROCKⅠ和ROCKⅡ蛋白表达水平均低于对照组,差异有统计学意义(P<0.05或P<0.01)。④2组子宫平滑肌组织RhoA mRNA及蛋白与ROCKⅠ和ROCKⅡmRNA及蛋白表达水平均呈正相关。⑤2组子宫平滑肌组织RhoA,ROCKⅠ和ROCKⅡmRNA及蛋白水平均与子宫平滑肌基础收缩活动力水平呈正相关。结论:宫缩乏力性产后出血产妇子宫平滑肌组织RhoA,ROCKⅠ和ROCKⅡ水平均降低,且均与子宫平滑肌收缩活动力呈正相关,提示子宫平滑肌组织RhoA/Rho激酶信号通路表达降低可能是发生宫缩乏力性产后出血的分子机制之一。

Rho相关激酶Rock在心血管疾病中的研究进展

Rho相关的卷曲蛋白激酶(Rho-associated coiled-coil protein kinase,ROCK)是新近发现的与细胞凋亡相关的激酶,在多种细胞中均有表达,ROCK参与调节细胞的多种行为与功能,包括收缩、游走黏附、增殖及调亡。研究表明ROCK与血管再狭窄、心肌损伤、心肌细胞凋亡及心力衰竭等有关联。

特发性膜性肾病患者血清ROCK2、renalase水平及其诊断价值

目的探讨Rho相关卷曲螺旋形成蛋白激酶2(ROCK2)、肾胺酶(renalase)在特发性膜性肾病(IMN)患者血清中的表达变化及其对IMN的诊断价值。方法选取2019年1月—2020年11月河北以岭医院肾病科收治IMN患者120例为IMN组,另选取同期医院进行健康体检的志愿者120例为健康对照组。酶联免疫吸附法(ELISA)检测受试者血清ROCK2、renalase水平,Pearson法分析ROCK2、renalase水平与部分临床指标的相关性;采用受试者工作特征(ROC)曲线分析血清ROCK2、renalase及二者联合诊断IMN的价值。结果IMN组ROCK2、renalase水平均显著高于健康对照组(t/P=12.837/<0.001、11.066/<0.001)。Ⅰ期、Ⅱ期、Ⅲ期、Ⅳ期IMN患者血清ROCK2逐次升高(F/P=77.154/<0.001),而Ⅰ期、Ⅱ期、Ⅲ期血清renalase水平逐次升高,但Ⅳ期血清renalase水平低于Ⅲ期(F/P=163.042/<0.001)。Alb与血清ROCK2、renalase水平呈负相关(r/P=-0.302/0.009、-0.402/<0.001),PLA2R抗体、BUN、SCr、24 h尿蛋白与血清ROCK2、renalase水平均呈正相关(r/P=0.336/<0.001、0.264/0.011、0.315/0.007、0.320/<0.001,0.359/<0.001、0.284/0.010、0.420/<0.001、0.412/<0.001);ROC曲线显示:血清ROCK2、renalase及二者联合诊断IMN的AUC分别为0.908、0.907、0.965,二者联合优于各自单独预测效能(Z=3.597,3.755,P均<0.001)。结论ROCK2与renalase在IMN患者血清中均高表达,二者联合诊断IMN的价值较高。