Deleted in liver cancer 1 suppresses the growth of prostate cancer cells through inhibiting Rho-associated protein kinase pathway

Objective:Deleted in liver cancer 1(DLC1)is a GTPase-activating protein that is reported as a suppressor in certain human cancers.However,the detailed biological function of DLC1 is still unclear in human prostate cancer(PCa).In the present study,we aimed to explore the function of DLC1 in PCa cells.Methods:Silencing and overexpression of DLC1 were induced in an androgen-sensitive PCa cell line(LNCaP)using RNA interference and lentiviral vector transduction.The Cell Counting Kit-8 assay was performed to determine cell proliferation.The cell cycle was examined by performing a propidium iodide staining assay.Results:Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of LNCaP cells.Moreover,DLC1 expression was negatively correlated with Rho-associated protein kinase(ROCK)expression in LNCaP cells.Importantly,this study showed that the ROCK inhibitor Y27632 restored the function of DLC1 in LNCaP cells and reduced the tumorigenicity of LNCaP cells in vivo.Conclusion:Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of PCa cells and negatively correlated with ROCK expression in PCa cells and tissue.

Advantages of Rho-associated kinases and their inhibitor fasudil for the treatment of neurodegenerative diseases

Ras homolog(Rho)-associated kinases(ROCKs)belong to the serine-threonine kinase family,which plays a pivotal role in regulating the damage,survival,axon guidance,and regeneration of neurons.ROCKs are also involved in the biological effects of immune cells and glial cells,as well as the development of neurodegenerative disorders such as Alzheimer’s disease,Parkinson’s disease,and multiple sclerosis.Previous studies by us and others confirmed that ROCKs inhibitors attenuated the symptoms and progression of experimental models of the abovementioned neurodegenerative diseases by inhibiting neuroinflammation,regulating immune imbalance,repairing the blood-brain barrier,and promoting nerve repair and myelin regeneration.Fasudil,the first ROCKs inhibitor to be used clinically,has a good therapeutic effect on neurodegenerative diseases.Fasudil increases the activity of neural stem cells and mesenchymal stem cells,thus optimizing cell therapy.This review will systematically describe,for the first time,the effects of abnormal activation of ROCKs on T cells,B cells,microglia,astrocytes,oligodendrocytes,and pericytes in neurodegenerative diseases of the central nervous system,summarize the therapeutic potential of fasudil in several experimental models of neurodegenerative diseases,and clarify the possible cellular and molecular mechanisms of ROCKs inhibition.This review also proposes that fasudil is a novel potential treatment,especially in combination with cell-based therapy.Findings from this review add support for further investigation of ROCKs and its inhibitor fasudil for the treatment of neurodegenerative diseases.

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.

Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase Ⅱ in spinal cord injury rats

Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase（ROCK） signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan（GV3）, Dazhui（GV14）, Zusanli（ST36） and Ciliao（BL32） and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the m RNA and protein expression of Rho-A and Rho-associated kinase Ⅱ（ROCKⅡ） of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKⅡ. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKⅡ. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of Rho A and ROCKⅡ. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.

应用组织芯片技术检测KAI1、MRP-1、FAK蛋白在肺癌组织中的表达

背景与目的:肿瘤的发生发展、浸润转移与多基因改变密切相关。目前Kang-ai-1(KAI1)、移动相关蛋白(motility-relatedprotein-1,MRP-1)和局部粘着斑激酶(focaladhesionkinase,FAK)3种基因在肺癌中的共同作用研究较少。本研究旨在探讨这3种基因的蛋白产物在肺癌发生发展中的作用及其在肿瘤诊断和预后判断中的价值。方法:应用免疫组化SP方法检测包含240个点的肺癌组织芯片中KAI1、MRP-1和FAK蛋白的表达。结果:KAI1在肺癌原发灶中阳性率为25.9%,MRP-1为42.6%,与正常肺组织(100%)相比均显著下调;FAK蛋白在肺癌组织中阳性率为44.4%,与正常肺组织相比显著增高;KAI1、FAK两种蛋白在肺癌组织中的表达与患者的年龄、性别、肿瘤的大体类型及组织类型无关,而与肿瘤的分化程度、临床分期及是否伴有淋巴结转移密切相关,两种蛋白的表达呈显著负相关。MRP-1蛋白在肺癌组织中的表达与组织类型有关,小细胞肺癌与非小细胞肺癌相比差异显著,MRP-1与KAI1呈显著正相关,与FAK呈显著负相关。结论:KAI1、MRP-1和FAK的异常表达与肺癌的浸润转移有关,联合检测这3项指标对肺癌的发生发展有重要的预测作用。

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).

Tolerance of neurite outgrowth to Rho kinase inhibitors decreased by cyclooxygenase-2 inhibitor

In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors （Y27632 and Fasudil）, a cyclooxygenase-1 selective inhibitor （SC560）, and a cyclooxygenase-2 inhibitor （NS398）. We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.

Regulation of enolase activation to promote neural protection and regeneration in spinal cord injury

Spinal cord injury(SCI)is a debilitating condition characterized by damage to the spinal cord resulting in loss of function,mobility,and sensation with no U.S.Food and Drug Administration-approved cure.Enolase,a multifunctional glycolytic enzyme upregulated after SCI,promotes pro-and anti-inflammatory events and regulates functional recovery in SCI.Enolase is normally expressed in the cytosol,but the expression is upregulated at the cell surface following cellular injury,promoting glial cell activation and signal transduction pathway activation.SCI-induced microglia activation triggers pro-inflammatory mediators at the injury site,activating other immune cells and metabolic events,i.e.,Rho-associated kinase,contributing to the neuroinflammation found in SCI.Enolase surface expression also activates cathepsin X,resulting in cleavage of the C-terminal end of neuron-specific enolase(NSE)and non-neuronal enolase(NNE).Fully functional enolase is necessary as NSE/NNE C-terminal proteins activate many neurotrophic processes,i.e.,the plasminogen activation system,phosphatidylinositol-4,5-bisphosphate 3-kinase/protein kinase B,and mitogen-activated protein kinase/extracellular signal-regulated kinase.Studies here suggest an enolase inhibitor,ENOblock,attenuates the activation of Rho-associated kinase,which may decrease glial cell activation and promote functional recovery following SCI.Also,ENOblock inhibits cathepsin X,which may help prevent the cleavage of the neurotrophic C-terminal protein allowing full plasminogen activation and phosphatidylinositol-4,5-bisphosphate 3-kinase/mitogen-activated protein kinase activity.The combined NSE/cathepsin X inhibition may serve as a potential therapeutic strategy for preventing neuroinflammation/degeneration and promoting neural cell regeneration and recovery following SCI.The role of cell membrane-expressed enolase and associated metabolic events should be investigated to determine if the same strategies can be applied to other neurodegenerative diseases.Hence,this review discusses the importance of enolase activation and inhibition as a potential therapeutic target following SCI to promote neuronal survival and regeneration.

Axonal growth inhibitors and their receptors in spinal cord injury:from biology to clinical translation

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.

Effect of manipulation on cartilage in rats with knee osteoarthritis based on the Rho-associated protein kinase/LIM kinase 1/Cofilin signaling pathways

OBJECTIVE:To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase(ROCK)/LIM-kinase1(LIMK1)/Cofilin signaling pathway.METHOD:Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups(n=8 each):blank group,model group,manipulation group,celecoxib group,and manipulation combined with celecoxib group(MC group).The osteoarthritis model was established by injecting 0.2 m L 4%papain into the articular disc of the rats.After successfully establishing the model,we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan(EX-LE4),Waixiyan(EX-LE5),Xuehai(SP10),Liangqiu(ST34),and Zusanli(ST36)acupoints for 10 min each time.Also,the celecoxib group was gavaged with 24 mg·kg^(-1)·d^(-1 )celecoxib,while the MC group was treated using both of these two methods.After four weeks,the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue.The expressions of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum were observed using the enzyme-linked immunosorbent assay.Besides,we detected the expressions of ROCK,LIMK1,Phospho-LIM-kinase1(Phospho-LIMK1),Cofilin,and Phospho-Cofilin by Western blot.RESULTS:Compared to the model group,the manipulation group,celecoxib group,and MC group all exhibited superior results concerning pathological morphologic changes of cartilage,as observed by hematoxylin-eosin staining and calculated using the Mankin score.Besides,in contrast to the blank group,the model group exhibited elevated serum levels of IL-1βand TNF-α(P<0.01),while the expression of ROCK,LIMK1,Phospho-LIMK1,Cofilin,and Phospho-Cofilin in cartilage were all higher(P<0.01).Also,the serum levels of IL-1βand TNF-αin each treatment group were lower(P<0.01)than in the model group.Moreover,there were lower expressions of ROCK,LIMK1,Phospho-LIMK1,Cofilin,and Phospho-Cofilin in cartilage in the manipulation group and the MC group(P<0.01).Compared with the model group,the expression of ROCK,LIMK1,PhosphoLIMK1,Cofilin,and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different(P>0.05).CONCLUSION:In this study,we established that manipulation has a better curative effect than celecoxib.Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.

Rho-Associated Kinase Inhibitors Promote Microglial Uptake Via the ERK Signaling Pathway

Microglia are immunocompetent cells in the cen- tral nervous system that take up tissue debris and pathogens. Rho-associated kinase （ROCK） has been identified as an important regulator of uptake, proliferation, secretion, and differentiation in a number of cell types. Although ROCK plays critical roles in the microglial secretion of inflammatory factors, naigration, and morphology, its effects on microglial uptake activity have not been well characterized. In the present study, we found that treatment of BV2 microglia and primary microglia with the ROCK inhibitors Y27632 and fasudil increased uptake activity and was associated with morpholog- ical changes. Furthermore, western blots showed that this increase in uptake activity was mediated through the extracel- lular-signal-regulated kinase （ERK） signaling cascade, indi- cating the importance of ROCK in regulating microglial uptake activity.

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

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.

The sphingosine-1-phosphate/RhoA/Rho associated kinases/myosin light chain pathway in detrusor of female rats is down-regulated in response to ovariectomy

Background:Dysuria is one of the main symptoms of genitourinary syndrome of menopause,which causes serious disruption to the normal life of peri-menopausal women.Studies have shown that it is related to decrease of detrusor contractile function,but the exact mechanism is still poorly understood.Previous results have suggested that the sphingosine-1-phosphate(S1P)pathway can regulate detrusor contraction,and this pathway is affected by estrogen in various tissues.However,how estrogen affects this pathway in the detrusor has not been investigated.In this study,we detected changes of the S1P/RhoA/Rho associated kinases(ROCK)/myosin light chain(MLC)pathway in the detrusor of ovariectomized rats in order to explore the underlying mechanism of dysuria during peri-menopause.Methods::Thirty-six female Sprague-Dawley rats were randomly divided into SHAM(sham operation),OVX(ovariectomy),and E groups(ovariectomy+estrogen),with 12 rats in each group.We obtained bladder detrusor tissues from each group and examined the mRNA and protein levels of the major components of the S1P/RhoA/ROCK/MLC pathway using quantitative real-time polymerase chain reaction and Western blotting,respectively.We also quantified the content of S1P in the detrusor using an enzyme linked immunosorbent assay.Finally,we compared results between the groups with one-way analysis of variance.Results::The components of the S1P pathway and the RhoA/ROCK/MLC pathway of the OVX group were significantly decreased,as compared with SHAM group.The percent decreases of the components in the S1P pathway were as follows:sphingosine kinase 1(mRNA:39%,protein:45%)(both P<0.05),S1P(21.73±1.09 nmol/g vs.18.86±0.69 nmol/g)(P<0.05),and S1P receptor 2/3(S1PR2/3)(mRNA:25%,27%,respectively)(P<0.05).However,the protein expression levels of S1PR2/3 and the protein and mRNA levels of SphK2 and S1PR1 did not show significant differences between groups(P>0.05).The percent decreases of the components in the RhoA/ROCK/MLC pathway were as follows:ROCK2(protein:41%,mRNA:36%)(both P<0.05),p-MYPT1(protein:54%)(P<0.05),and p-MLC20(protein:47%)(P<0.05),but there were no significant differences in the mRNA and protein levels of RhoA,ROCK1,MYPT1,and MLC20(all P>0.05).In addition,all of the above-mentioned decreases could be reversed after estrogen supplementation(E group vs.SHAM group)(all P>0.05).Conclusion::In this study,we confirmed that ovariectomy is closely associated with the down-regulation of the S1P/RhoA/ROCK/MLC pathway in the rat detrusor,which may be one mechanism of dysuria caused by decreased contractile function of the female detrusor during peri-menopause.

RhoA／ROKα反义寡核苷酸抑制TGF-β1诱导的血管外膜成纤维细胞α-SM-actin表达

目的α-平滑肌-肌动蛋白(a-SM-actin)的表达是血管外膜成纤维细胞/肌成纤维细胞表型转化的分子标记。本研究观察阻断RhoA-ROKα信号转导通路对于转化生长因子β1(transfor- ming growth factorβ1,TGF-β1)诱导的血管外膜成纤维细胞α-SM-肌动蛋白(actin)表达的影响,以揭示RhoA-ROKα信号通路在血管外膜成纤维细胞表型转化为肌成纤维细胞过程中的作用。方法使用贴壁法体外培养大鼠胸主动脉外膜成纤维细胞；用Western blot技术测定RhoA和ROKα在血管外膜成纤维细胞表型转化为肌成纤维细胞过程中的表达。结果RhoA-ROKα在血管外膜成纤维细胞表达,TGF-β1可以诱导RhoA表达上调。用反义寡核苷酸技术抑制RhoA或ROKα的表达后能够抑制α-SM-actin的表达。结论RhoA-ROKα信号转导通路参与了TGF-β1诱导的血管外膜成纤维细胞表型转化为肌成纤维细胞的过程。

烟酸对冠心病患者外周血单核细胞环一磷酸腺苷与三磷酸腺苷结合盒转运体A1表达的影响

目的以冠心病患者的外周血单核细胞为研究对象,观察烟酸对体外培养的单核细胞中环一磷酸腺苷及ATP结合盒转运体A1表达的影响,分析烟酸调节ATP结合盒转运体A1的表达与环一磷酸腺苷—蛋白激酶A途径的相关性。方法运用逆转录聚合酶链反应和Westernblot蛋白印迹分别检测ATP结合盒转运体A1mRNA与ATP结合盒转运体A1蛋白的表达,采用低pH值EIA法测定细胞内环磷酸腺苷水平。结果(1)各组外周血单核细胞中ATP结合盒转运体A1的表达及环一磷酸腺苷的水平在3h和48h的变化无明显差异(P>0.05);(2)冠心病高血脂患者单核细胞中ATP结合盒转运体A1的表达较正常人明显降低(P<0.05),而正常血脂组单核细胞中ATP结合盒转运体A1的表达较正常人无明显差异(P>0.05);(3)烟酸单独作用能明显增加冠心病人单核细胞中ATP结合盒转运体A1的表达(P<0.05),同时使单核细胞中环一磷酸腺苷的含量增加(P<0.05);(4)蛋白激酶A抑制剂Ro31-8220单独作用能明显降低冠心病人单核细胞中ATP结合盒转运体A1的表达(P<0.05),同时使单核细胞中环—磷酸腺苷的含量降低(P<0.05);(5)先用Ro31-8220作用30min后,再加入烟酸,则烟酸使单核细胞中ATP结合盒转运体A1表达上调及环一磷酸腺苷的含量增加的作用被完全抑制(P<0.05)。结论烟酸对单核细胞中ATP结合盒转运体A1表达的影响,是通过环一磷酸腺苷-蛋白激酶A途径发挥作用的,烟酸上调外周血单核细胞中ATP结合盒转运体A1表达的作用,与环一磷酸腺苷—蛋白激酶A途径存在明确的相关性。

AG490对重症急性胰腺炎大鼠 IL-6、IL-18表达的影响

目的探讨AG490对重症急性胰腺炎（severe acute pancreatitis,SAP）大鼠血清白介素-6（IL-6）、白介素-18（IL-18）的表达影响。方法以4%牛磺胆酸钠胰胆管逆行注射诱导大鼠 SAP 模型。56只 SD大鼠随机分为正常对照组（NC组）、SAP组和 AG490组（JAK2抑制剂组）。检测并比较三组血清淀粉酶（AMY）、IL-6、IL-18含量,光镜下观察胰腺组织病理变化。结果与 NC 组比较,SAP 组各时间点血清 AMY 水平均明显升高,在SAP发展的6~18 h 内,血清 IL-6、IL-18的表达明显增加,但与 AG490组同时间点血清 IL-6、IL-18的表达比较明显降低。结论 SAP时 IL-6、IL-18表达明显升高,升高程度与病情严重程度呈正相关；抑制JAK/STAT通路的活化可下调血清 IL-6、IL-18的表达,可能减轻 SAP时急性炎症反应。

Rho/ROCK signaling in motility and metastasis of gastric cancer

Gastric cancer is one of the most frequent and lethal malignancies worldwide because of high frequency of metastasis. Tumor cell motility and invasion play fundamental roles in cancer metastasis. Recent studies have revealed that the Rho/Rho-associated protein kinases(ROCK) pathway plays a critical role in the regulation of cancer cell motility and invasion. In addition,the Rho/ROCK pathway plays important roles in invasion and metastasis on the basis of its predominant function of cell cytoskeletal regulation in gastric cancer. According to the current understanding of tumor motility,there are two modes of tumor cell movement:mesenchymal and amoeboid. In addition,cancer cell movement can be interchangeable between the mesenchymal and amoeboid movements under certain conditions. Control of cell motility through the actin cytoskeleton creates the potential for regulating tumor cell metastasis. In this review we discuss Rho GTPases and ROCK signaling and describe the mechanisms of Rho/ROCK activity with regard to motility and metastasis in gastric cancer.In addition,we provide an insight of the therapeutic potential of targeting the Rho/ROCK pathway.

The role of the Rho/ROCK signaling pathway in inhibiting axonal regeneration in the central nervous system

The Rho/Rho-associated coiled-coil containing protein kinase（Rho/ROCK） pathway is a major signaling pathway in the central nervous system, transducing inhibitory signals to block regeneration. After central nervous system damage, the main cause of impaired regeneration is the presence of factors that strongly inhibit regeneration in the surrounding microenvironment. These factors signal through the Rho/ROCK signaling pathway to inhibit regeneration. Therefore, a thorough understanding of the Rho/ROCK signaling pathway is crucial for advancing studies on regeneration and repair of the injured central nervous system.

Stem cells in neuroinjury and neurodegenerative disorders: challenges and future neurotherapeutic prospects

The prevalence of neurodegenerative diseases and neural injury disorders is increasing worldwide. Research is now focusing on improving current neurogenesis techniques including neural stem cell therapy and other biochemical drug-based approaches to ameliorate these disorders. Unfortunately, we are still facing many obstacles that are rendering current neurotherapies ineffective in clinical trials for reasons that are yet to be discovered. That is why we should start by fully understanding the complex mechanisms of neurogenesis and the factors that affect it, or else, all our suggested therapies would fail since they would not be targeting the essence of the neurological disorder but rather the symptoms. One possible paradigm shift is to switch from neuroprotectant therapies towards neurodegeneration/neurorestorative approaches. In addition, other and our laboratories are increasingly focusing on combining the use of pharmacological agents（such as Rho-associated kinase（ROCK） inhibitors or other growth factors（such as brain-derived neurotrophic factor（BDNF）） and stem cell treatment to enhance the survivability and/or differentiation capacity of transplanted stem cells in neurotrauma or other neurodegeneration animal models. Ongoing stem cell research is surely on the verge of a breakthrough of multiple effective therapeutic options for neurodegenerative disorders. Once, we fully comprehend the process of neurogenesis and its components, we will fully be capable of manipulating and utilizing it. In this work, we discuss the current knowledge of neuroregenerative therapies and their associated challenges.