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.

Mitochondrial oxidative damage and apoptosis induced by high glucose through Rho kinase signal pathway in renal tubular epithelial cells

Objective:To investigate the role of oxidative stress in human renal tubular epithelial cells(HK-2)induced by high glucose and the underlying signal pathway in vitro.Methods:MYPT1,pro-caspase-3,PGC-1α,and Drpl protein expressions were measured by Western blot.MnSOD2,Drp1 and PGC-1αmRNA expressions were detected by real time PCR.Results:Results showed that high glucose significantly up-regulated the protein expressions of MYPT1,pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells;while Rho kinase inhibitor fasudil and ROCK1 siRNA inhibited protein expressions of pro-caspase-3 and the mRNA expression of MnSOD2 in HK-2 cells induced by high glucose.Importantly,fasudil and ROCK1 siRNA markedly inhibited the expressions of mitochondrial motor proteins Drp1 and mitochondrial gene PGC-la in HK-2 cell=s induced by high glucose.Conclusions:Our findings suggest that Rho kinase signal pathway is involved in mitochondrial oxidative damage and apoptosis in high glucose-induced renal tubular epithelial cells by regulating mitochondrial motor proteins Drp1 and mitochondrial gene PGC-1α.Targeting Rho kinase signal pathway might be a potential strategy for the treatment of diabetic nephropathy.

Rosuvastatin inhibits the smooth muscle cell proliferation by targeting TNFα mediated Rho kinase pathway

Objective To investigate whether Tumor Necrosis Factor-alpha （TNFα） is capable of activating Rho kinase pathway which leads to smooth muscle cell proliferation and the intervention function of Rosuvastatin, and clarify the mechanism and intervention manner of anti-atherosclerosis by Rosuvastatin. Methods Wistar neonate rat smooth muscle cells were cultured, and the activity of cell proliferation was determined by methyl thiazolyl tetrazolium （MTT）. The expression of Rho kinase genes after the stimulation of TNFα was evaluated by RT-PCR. Western blot method was used to measure the protein expression of proliferating cell nuclear antigen （PCNA） after TNFα stimulation and Rosuvastatin intervention in smooth muscle cell. Results The TNFα stimulation significantly enhanced the expression of Rho kinase and increased the expression of PCNA protein in smooth muscle cells （P 〈 0.05）. These effects were positively correlated with prolonged treatment whereas additional Rosuvastatin administration inhibited the above-mentioned effects （P 〈 0.05）. Conclusions The activation of TNFα mediated Rho kinase signaling pathway can significantly promote smooth muscle cell proliferation, and Rosuvastatin can not only inhibit this pathway but also the induced proliferation.

Rho A/Rho kinase in spinal cord injury

A spinal cord injury refers to an injury to the spinal cord that is caused by a trauma instead of diseases. Spinal cord injury includes a primary mechanical injury and a much more complex secondary injury process involving inflammation, oxidation, excitotoxicity, and cell death. During the secondary injury, many signal pathways are activated and play important roles in mediating the pathogenesis of spinal cord injury. Among them, the Rho A/Rho kinase pathway plays a particular role in mediating spinal degeneration and regeneration. In this review, we will discuss the role and mechanism of Rho A/Rho kinase-mediated spinal cord pathogenesis, as well as the potential of targeting Rho A/Rho kinase as a strategy for promoting both neuroprotection and axonal regeneration.

Long-term culture-induced phenotypic difference and efficient cryopreservation of small intestinal organoids by treatment timing of Rho kinase inhibitor

To investigate a suitable long-term culture system and optimal cryopreservation of intestinal organoid to improve organoid-based therapy by acquiring large numbers of cells.METHODSCrypts were isolated from jejunum of C57BL/6 mouse. Two hundred crypts were cultured in organoid medium with either epidermal growth factor/Noggin/R-spondin1 (ENR) or ENR/CHIR99021/VPA (ENR-CV). For subculture, organoids cultured on day 7 were passaged using enzyme-free cell dissociation buffer (STEMCELL Technologies). The passage was performed once per week until indicated passage. For cryopreservation, undissociated and dissociated organoids were resuspended in freezing medium with or without Rho kinase inhibitor subjected to different treatment times. The characteristics of intestinal organoids upon extended passage and freeze-thaw were analyzed using EdU staining, methyl thiazolyl tetrazolium assay, qPCR and time-lapse live cell imaging.RESULTSWe established a three-dimensional culture system for murine small intestinal organoids using ENR and ENR-CV media. Both conditions yielded organoids with a crypt-villus architecture exhibiting Lgr5+ cells and differentiated intestinal epithelial cells as shown by morphological and biochemical analysis. However, during extended passage (more than 3 mo), a comparative analysis revealed that continuous passaging under ENR-CV conditions, but not ENR conditions induced phenotypic changes as observed by morphological transition, reduced numbers of Lgr5+ cells and inconsistent expression of markers for differentiated intestinal epithelial cell types. We also found that recovery of long-term cryopreserved organoids was significantly affected by the organoid state, i.e., whether dissociation was applied, and the timing of treatment with the Rho-kinase inhibitor Y-27632. Furthermore, the retention of typical morphological characteristics of intestinal organoids such as the crypt-villus structure from freeze-thawed cells was observed by live cell imaging.CONCLUSIONThe maintenance of the characteristics of intestinal organoids upon extended passage is mediated by ENR condition, but not ENR-CV condition. Identified long-term cryopreservation may contribute to the establishment of standardized cryopreservation protocols for intestinal organoids for use in clinical applications.

TGF-β1-induced LPP Expression Dependant on Rho Kinase during Differentiation and Migration of Bone Marrow-derived Smooth Muscle Progenitor Cells

Lipoma preferred partner(LPP) has been identified as a protein which is highly selective for smooth muscle progenitor cells(SMPCs) and regulates differentiation and migration of SMPCs,but mechanisms of LPP expression are not elucidated clearly.The aim of the present study was to discuss the mechanisms by which LPP expression is regulated in the differentiation and migration of SMPCs induced by TGF-β1.It was found that TGF-β1 could significantly increase the expression of LPP,smooth muscle α-actin,smooth muscle myosin heavy chain(SM-MHC),and smoothelin in SMPCs.Moreover,inactivation of Rho kinase(ROK) with ROK inhibitors significantly inhibited LPP mRNA expression in TGF-β1-treated SMPCs and mouse aortic smooth muscle cells(MAoSMCs).At the same time,LPP silencing with short interfering RNA significantly decreased SMPCs migration.In conclusion,LPP appears to be a ROK-dependant SMPCs differentiation marker that plays a role in regulating SMPCs migration.

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.

RhoA/Rho kinase： a novel therapeutic target in diabetic complications

Objective To reveal the roles of Rho kinase （ROCK） in the mechanisms of complications in diabetes by reviewing the correlations between ROCK and related complications in diabetes.Data sources The data used in the present article were mainly from PubMed with relevant English articles published from 1998 to 2010. The search terms were ＂ROCK＂ and ＂diabetes＂.Study selection Original articles including the roles of ROCK or its inhibitors in diabetic complications and review articles about the biological character of ROCK were selected.Results The activity and expression of ROCK were up-regulated in the models of type 1 or type 2 diabetes animals and the cultured cells with concentrations of high glucose, ROCK activation was associated with the development or progression of complications in diabetes. Inhibition of RhoA/ROCK pathway prevented or ameliorated the pathologic changes of diabetic complications, and ROCK has been regarded as a key target for treatment of these complications.Conclusion RhoA/ROCK signaling plays important roles in the pathogenesis of long-term complications in diabetes and ROCK inhibitors are becoming a promising solution to treatments of complications in diabetes.

Tanshinone ⅡA Protects against Lipopolysaccharides-lnduced Endothelial Cell Injury via Rho/Rho Kinase Pathway

Objective： To test whether tanshinone ⅡA （Tan ⅡA）, a highly valued herb derivative to treat vascular diseases in Chinese medicine, could protect endothelial cells from bacterial endotoxin （lipopolysaccharides, LPS）-induced endothelial injury. Methods： Endothelial cell injury was induced by treating human umbilical vein endothelial cells （HUVECs） with 0.2 μg/mL LPS for 24 h. Y27632 and valsartan were used as positive controls. The effects of tanshinone Ⅱ A on the LPS-induced cell viability and apoptosis rate of HUVECs were tested by flow cytometry, cell migration by transwell, adhesion by a 96-well plate pre-coated with vitronectin and cytoskeleton reorganization by immunofluorescence assay. Rho/Rho kinase （ROCK） pathway- associated gene and protein expression were examined by microarray assay; quantitative real-time polymerase chain reaction and Western blotting were used to confirm the changes observed by microarray. Results： Tan ][ A improved cell viability, suppressed apoptosis and protected cells from LPS-induced reductions in cell migration and adhesion at a comparable magnitude to that of Y27632 and valsartan. Tan II A, Y27632 and valsartan also normalized LPS-induced actomyosin contraction and vinculin protein aggregation. A microarray assay revealed increased levels of fibronectin, integrin A5 （ITG A5）, Ras homolog gene family member A （RheA）, myosin light chain phosphatase, phosphatidylinositol-4,5-bisphosphate 3-kinase （PI3K, or PIP2 in Western blotting）, focal adhesion kinase, vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in the damaged HUVECs, which were attenuated to different degrees by Tan ⅡA, Y27632 and valsartan. Conclusion： Tan ⅡA exerted a strong protective effect on HUVECs, and the mechanism was caused, at least in part, by a blockade in the Rho/ROCK pathway, presumably through the down-regulation of ITG A5.

抑制Rho-kinase在柔红霉素诱导的肾小球硬化中的作用及机制

目的:研究抑制Rho-kinase对柔红霉素诱导的肾小球硬化的调控作用,并探讨Rho-kinase抑制剂法舒地尔改善肾小球硬化的作用机制。方法:36只雄性SD大鼠,随机分为三组:假手术(Sham)组,单侧肾脏切除+柔红霉素(模型)组,单侧肾脏切除+柔红霉素+法舒地尔(干预)组,每组各12只。模型组和干预组大鼠在切除左肾后第7、14天,从尾静脉各注射柔红霉素5 mg/kg 1次,同时Sham组大鼠以等剂量的生理盐水尾静脉注射。干预组在第2次注射柔红霉素后从腹腔每天注射法舒地尔3 mg/kg,完成上述处理后的第2、4周,随机取各组大鼠6只处死留取肾标本,处死前收集24 h尿液检测尿蛋白排泄,用HE,PAS染色,免疫组化,透射电镜进行肾组织病理学分析,应用RT-PCR检测Rho-kinase,P27的核酸表达水平。结果:(1)模型组较Sham组24 h尿蛋白明显升高(P<0.01),而法舒地尔干预后,干预组24 h尿蛋白较模型组显著降低(P<0.05)。(2)模型组大鼠足细胞第4周时出现了弥漫性足突融合,系膜基质增殖明显,出现典型的肾小球节段性硬化,免疫组化PCNA表达升高,P27表达下降;而干预组可以改善柔红霉素诱导的肾小球硬化大鼠足细胞的病理改变,系膜细胞增生受抑制,系膜基质蓄积减少,较模型组PCNA表达下降,P27升高。(3)模型组较Sham组Rho-kinase的mRNA表达升高,细胞周期抑制蛋白P27 mRNA的表达下降,而干预组较模型组Rho-kinase的mRNA表达显著降低,P27 mRNA的表达明显升高。结论:(1)抑制Rho-kinase的表达能明显改善柔红霉素诱导的大鼠的肾小球硬化。(2)法舒地尔的作用机制可能是通过改善足细胞的病理改变,减少蛋白尿,升高细胞周期抑制蛋白P27的表达,从而抑制系膜基质增殖,延缓肾小球硬化的进展。

Therapeutic potential of Rho-associated kinase inhibitor Y27632 in corneal endothelial dysfunction:an in vitro and in vivo study

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.

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.

Rho/Rho激酶信号通路与轴突导向和再生的研究进展

Rho是小分子量GTPases超家族Rho亚家族成员．是Ras超家族的哺乳动物基因同系物。Rho可以通过其下游效应因子Rho激酶（Rock或Rho—kinase）调节细胞肌动蛋白骨架的重组，从而广泛参与细胞迁移、运动、凋亡、基因转录、神经再生等生物学过程。过去的研究证实。Rho蛋白及其相关的信号分子参与并介导了轴突的再生、延伸、纤维的投射等生物学过程。本文就Rho蛋白及其下游调节因子Rho激酶在轴突导向和再生中的作用做一综述。

DL0805 derivatives protect the pulmonary arterial cells via the RhoA/ROCK pathway

OBJECTIVE Pulmonary artery hypertension(PAH)is a severe disease characterized by the mean pulmonary artery pressure exceeding 25 mm Hg at rest.PAH could induce right heart failure and has a very high mortality rate.At present,several kinds of drugs have been used in the treatment of PAH.However,most of these drugs aim to relax pulmonary arteries and do not inhibit the injury of vessels.In other words,the drugs available for PAH treatment do not improve the survival rate of PAH patients and cannot satisfy the needs in clinic.To discover and develop novel candidate compounds effective on the treatment of pulmonary artery injury and remodeling will be very important.Based on these background,the present study aimed to study the protective effect of two novel Rho-kinases(Rho-associated coiledcoil forming protein serine/threonine kinase,ROCK)inhibitors,DL0805 derivatives(DL0805-1and DL0805-2),on pulmonary arterial cells and further evaluate the underlying mechanisms and the possibility of DL0805 derivatives become therapeutic drugs for PAH.METHODS The primary cultured pulmonary arterial cells including human pulmonary artery endothelium cells(HPAECs)and human pulmonary artery smooth muscle cells(HPASMCs)were used in this study.HPAECs were injured under hypoxia environment(1%O2)and treated with or without DL0805 derivatives.After 48 h,the proliferation and oxidative stress were observed.CCK8 was used to detect cell viability.DCFH-DA was used as probe for reactive oxygen species(ROS)under fluorescence imaging system.HPASMCs was stimulated by growth factors including platelet-derived growth factor-BB(PDGF-BB)and Fetal Bovine Serum(FBS).The proliferation was observed in the cells treated with or without DL0805 derivatives.HPASMCs treated with or without DL0805 derivatives were further incubated with endothelin(ET-1),the proliferation and cytoskeleton remodeling of cells were detected by immunofluorescence assay.At last,Western blotting(WB)and immunofluorescence assay were employed to analysis the underlying mechanisms in the above experiments.RESULTS 10μmol·L-1DL0805-2 could inhibit the proliferation of HPAECs induced by hypoxia.Each concentration of DL0805-1 and DL0805-2attenuated the production of ROS in HPAECs.Results from WB indicated that DL0805 derivatives decreased the injury of HPAECs induced by hypoxia through the inhibition of the expression of Rho A and the activity of ROCK.On HPASMCs,DL0805 derivatives reduced the proliferation induced by PDGF-BB and FBS and inhibited cytoskeleton remodeling induced by ET-1.Immunofluorescence assay showed that DL0805 derivatives inhibited ROCK activity and down regulated the phosphorylation levels of ROCK substrates.CONCLUSION DL0805derivatives exhibited protective effect on pulmonary arterial cells including endothelium cells and smooth muscle cells.Among the above experiments,DL0805-2 showed stronger potency than DL0805-1.These two compounds might protect the cells through the inhibition of Rho A/ROCK pathway and they probably have the potential in the treatment of PAH and deserve further evaluation.

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.

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.

Fasudil-modified macrophages reduce inflammation and regulate the immune response in experimental autoimmune encephalomyelitis

Multiple sclerosis is characterized by demyelination and neuronal loss caused by inflammatory cell activation and infiltration into the central nervous system.Macrophage polarization plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis,a traditional experimental model of multiple sclerosis.This study investigated the effect of Fasudil on macrophages and examined the therapeutic potential of Fasudil-modified macrophages in experimental autoimmune encephalomyelitis.We found that Fasudil induced the conversion of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type,as shown by reduced expression of inducible nitric oxide synthase/nitric oxide,interleukin-12,and CD16/32 and increased expression of arginase-1,interleukin-10,CD14,and CD206,which was linked to inhibition of Rho kinase activity,decreased expression of toll-like receptors,nuclear factor-κB,and components of the mitogen-activated protein kinase signaling pathway,and generation of the pro-inflammatory cytokines tumor necrosis factor-α,interleukin-1β,and interleukin-6.Crucially,Fasudil-modified macrophages effectively decreased the impact of experimental autoimmune encephalomyelitis,resulting in later onset of disease,lower symptom scores,less weight loss,and reduced demyelination compared with unmodified macrophages.In addition,Fasudil-modified macrophages decreased interleukin-17 expression on CD4^(+)T cells and CD16/32,inducible nitric oxide synthase,and interleukin-12 expression on F4/80^(+)macrophages,as well as increasing interleukin-10 expression on CD4^(+)T cells and arginase-1,CD206,and interleukin-10 expression on F4/80^(+)macrophages,which improved immune regulation and reduced inflammation.These findings suggest that Fasudil-modified macrophages may help treat experimental autoimmune encephalomyelitis by inducing M2 macrophage polarization and inhibiting the inflammatory response,thereby providing new insight into cell immunotherapy for multiple sclerosis.

Fasudil hydrochloride hydrate, a Rho-kinase inhibitor, ameliorates hepatic fibrosis in rats with type 2 diabetes

Background Hyperglycemia may accelerate liver fibrosis.Currently,there is no effective treatment for liver fibrosis induced by type 2 diabetes.The study aim was to investigate whether RhoA/Rho kinase （ROCK） pathway is involved in liver fibrosis in the rats with type 2 diabetes and define the protective effects of fasudil on livers.Methods A rat model of type 2 diabetes was established by high fat diet combined with streptozotocin （30 mg/kg,intraperitoneal injection）.Animals were randomly assigned to 3 groups：control rats,untreated diabetic rats that received vehicle and fasudil-treated diabetic rats that received ROCK inhibitor fasudil hydrochloride hydrate （10 mg/kg per day,intraperitoneal injection,for 14 weeks）.The morphological features of liver were observed by HE staining.Accumulation of collagen in livers was determined by Masson staining and the measurement of hydroxyproline.The mRNA expression of transforming growth factor-β1 （TGFβ1）,connective tissue growth factor （CTGF）,type-Ⅰ,and type-Ⅲ procollagen was assessed with real-time polymerase chain reaction.The phosphorylation of myosin phosphatase target subunit-1 （MYPT1）and the protein levels of TGFβ1 and α-smooth muscle actin （α-SMA） were evaluated by Western blotting.Results Compared with control rats,untreated diabetic rats showed higher values of collagen and hydroxyproline in livers （P ＜0.01）,the phosphorylation of MYPT1 and the protein levels of TGFβ1 and α-SMA were increased （P ＜0.01）,and the mRNA expression of TGFβ1,CTGF,type-Ⅰ,and type-Ⅲ procollagen was upregulated （P ＜0.01）; compared with untreated diabetic rats,treatment with fasudil signifcantly reduced values of collagen and hydroxyproline （P ＜0.01）,and decreased the phosphorylation of MYPT1 and the levels of TGFβ1 and α-SMA （P ＜0.01）,concomitant with the downregulation of TGFβ1/CTGF,type-Ⅰ,and type-Ⅲ procollagen mRNA expression （P ＜0.01）.Conclusions Fasudil ameliorates liver fibrosis in rats with type 2 diabetes at least partly by inhibiting TGFβ1/CTGF pathway and α-SMA expression.Inhibition of RhoA/ROCK may be a novel therapeutic target for liver fibrosis in diabetic non-alcoholic steatohepatitis.

Upregulation of Nogo receptor expression induces apoptosis of retinal ganglion cells in diabetic rats

The Nogo receptor is an essential factor for neuronal apoptosis, but the changes in Nogo receptor expression in the retina and the effects of the Nogo receptor on retinal ganglion cell apoptosis in diabetes mellitus remain unclear. We found that Nogo receptor expression was mainly visible in retinal ganglion cells of a rat model of diabetes mellitus induced by streptozotocin. At 12 weeks after onset of diabetes mellitus, Nogo receptor and Rho kinase expression signiifcantly increased in the retina, and retinal ganglion cell apoptosis was apparent. When RNA interference was used to suppress Nogo receptor expression in rat retina, Rho kinase expression was obviously inhibit-ed, and retinal ganglion cell apoptosis was evidently reduced in rats with diabetes mellitus. These results indicate that upregulation of Nogo receptor expression is an important mechanism of retinal ganglion cell apoptosis in rats with diabetes mellitus.

Endothelin-1 stimulates contraction and migration of rat pancreatic stellate cells

AIM： To examine the ability of FT-1 to affect the cell functions of PSCs and the underlying molecular mechanisms. METHODS： PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase, and cells between passages two and five were used. Expression of ET-1 and FT receptors was assessed by reverse transcription-PCR and immunostaining. Phosphorylation of myosin regulatory light chain （MLC）, extracellular-signal regulated kinase （FRK）, and Akt was examined by Western blotting. Contraction of PSCs was assessed on hydrated collagen lattices. Cell migration was examined using modified Boyden chambers. Ceil proliferation was assessed by measuring the incorporation of 5-bromo-2＇- deoxyuridine. RESULTS： Culture-activated PSCs expressed ETA and ETB receptors, and ET-1. ET-1 induced phosphorylation of NLC and FRK, but not Akt. ET-1 induced contraction and migration, but did not alter proliferation of PSCs. FT-1-induced contraction was inhibited by an ETA receptor antagonist BQ-123 and an ETB receptor antagonist BQ-788, whereas migration was inhibited by BQ-788 but not by BQ-123. A Rho kinase inhibitor Y-27632 abolished both contraction and migration. CONCLUSION： ET-1 induced contraction and migration of PSCs through El receptors and activation of Rho-Rho kinase. ETA and FTB receptors play different roles in the regulation of these cellular functions in response to ET-1.