Effects Of Fasudil Research Articles

Antipsychotic-like effects of fasudil, a Rho-kinase inhibitor, in a pharmacologic animal model of schizophrenia

Current antipsychotics used to treat schizophrenia have associated problems, including serious side effects and treatment resistance. We recently identified a significant association of schizophrenia with exonic copy number variations in the Rho GTPase activating protein 10 (ARHGAP10) gene using genome-wide analysis. ARHGAP10 encodes a RhoGAP superfamily member that is involved in small GTPase signaling. In mice, Arhgap10 gene variations result in RhoA/Rho-kinase pathway activation. We evaluated the pharmacokinetics of fasudil and hydroxyfasudil using liquid chromatography-tandem mass spectrometry in mice. The antipsychotic effects of fasudil on hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits were also investigated in a MK-801-treated pharmacological mouse schizophrenia model. Fasudil and its major metabolite, hydroxyfasudil, were detected in the brain at concentrations above their respective Ki values for Rho-kinase after intraperitoneal injection of 10 mg kg−1 fasudil. Fasudil improved the hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits in MK-801-treated mice in a dose-dependent manner. Following oral administration of fasudil, brain hydroxyfasudil was detected at concentration above the Ki value for Rho-kinase whilst fasudil was undetectable. MK-801-induced hyperlocomotion was also improved by oral fasudil administration. These results suggest that fasudil has antipsychotic-like effects on the MK-801-treated pharmacological mouse schizophrenia model. There are two isoforms in Rho-kinase, and further investigation is needed to clarify the isoforms involved in the antipsychotic-like effects of fasudil in the MK-801-treated mouse schizophrenia model.

High Glucose Aggravates Retinal Endothelial Cell Dysfunction by Activating the RhoA/ROCK1/pMLC/Connexin43 Signaling Pathway.

PurposeThis research aims to explore the mechanism underlying the relationship between RhoA/ROCK signaling and Connexin43 (Cx43) in retinal endothelial cell dysfunction and to evaluate the protective effect of ROCK inhibitors against retinal endothelial cell dysfunction in diabetic retinopathy (DR) models.MethodsTUNEL staining, hematoxylin and eosin staining, a retinal digestion assay, and Evans blue assay were conducted to explore the effect of fasudil in alleviating retinal dysfunction induced by DR. ELISA, the CCK-8 assay, and flow cytometry were conducted to study inflammation, viability, and apoptosis of mouse retinal microvascular endothelial cells treated with high glucose and ROCK inhibitors. The qRT–PCR and Western blotting were used to evaluate the expression of RhoA, ROCK1, ROCK2, MLC, pMLC, and Cx43. Co-immunoprecipitation was used to verify the interaction between pMLC and Cx43. Immunofluorescence and scrape-loading and dye transfer were used to evaluate the expression and function of Cx43.ResultsMarked endothelial cell dysfunction resulting from the activation of RhoA/ROCK1 signaling was found in in vivo and in vitro models of DR. Via interaction with pMLC, which is downstream of RhoA/ROCK1, a significant downregulation of Cx43 was observed in retinal endothelial cells. Treatment with ROCK inhibitors ameliorated retinal endothelial dysfunction in vitro. The ROCK inhibitor, fasudil, significantly alleviated retinal dysfunction as shown by a decrease of retinal acellular capillaries, an improvement of vascular permeability, and a reduction of cell apoptosis in vivo.ConclusionsOur study highlights a novel mechanism that high glucose could activate RhoA/ROCK1/pMLC signaling, which targets the expression and localization of Cx43 and is responsible for cell viability, apoptosis, and inflammation, resulting in retinal endothelial cell injury. ROCK inhibitors markedly ameliorate endothelial cell dysfunction, suggesting their therapeutic potential for diabetic retinopathy.

Fasudil Attenuated 6-OHDA Cytotoxicity in PC12 Cells through Inhibition of JAK/STAT and Apoptosis Pathways

Background: 6-Hydroxydopamine (6-OHDA) is widely used to induce neurotoxicity and investigate the mechanisms of Parkinson disease. 6-OHDA causes cell injury through various mechanisms including oxidative stress, inflammation and apoptosis. The selective Rho-kinase inhibitor, fasudil displays neuroprotective effects in several neurodegenerative disorders. The aim of this study was to assess the protective effect of fasudil in PC12 cytotoxicity induced by 6-OHDA. Methods: PC12 cells were exposed to 5, 10, 25, and 50 µM of fasudil concentrations. After 24 h, the IC50 value of 6-OHDA (150 µM) was added. Twenty-four hours later, the viability of cells was evaluated via MTT assay and the formation of reactive oxygen species (ROS) was measured by the fluorimetric method. At the 50 µM concentration of fasudil, with or without 6-OHDA, the changes of protein levels including STAT3, P-STAT3, JAK2, P-JAK2, and caspase-3 were determined via western blotting. Results: Our results showed that 6-OHDA increased the intracellular level of ROS, reduced cell viability, upregulated p-STAT3/STAT3 and p-JAK2/JAK2 ratios and significantly raised cleaved caspase-3 in comparison to control group. Furthermore, pretreatment of cells with fasudil (50 µM) for 24 h could reverse all changes induced by 6-OHDA. Conclusion: 6-OHDA caused cytotoxicity in PC12 cells through inducing oxidative stress and activating JAK/STAT and apoptosis pathways, while pretreatment with fasudil exhibited protective effect on 6-OHDA-induced neurotoxicity via the inhibition of oxidative stress and prevention of these pathways.

A ROCK1 Inhibitior Fasudil Alleviates Cardiomyocyte Apoptosis in Diabetic Cardiomyopathy by Inhibiting Mitochondrial Fission in a Type 2 Diabetes Mouse Model.

Diabetes mellitus (DM) often involves cardiovascular complications; however, treatment regimens are limited. ROCK1 (rho-associated coiled-coil containing protein kinase 1) serves as a pathological factor in several diabetic complications. Herein, we aimed to explore the effect of Fasudil (a ROCK1 inhibitor) on the progress of cardiac dysfunction in type 2 DM (T2DM), and to explore the possible mechanisms. Type II diabetic mice models were established by inducing insulin resistance through a high-fat diet combined with low-dose streptozotocin (STZ) injection. NMCMs (neonatal mouse ventricular cardiac myocytes) in the control group were treated with 5.5 mM glucose, while those in the High Glucose (HG) group were treated with 33 mM glucose and 10 nmol/L insulin. In vivo, we found that type 2 diabetes enhanced the expression and activation of ROCK1 (p < 0.05). The ROCK1 inhibitor, Fasudil, prevented cardiac dysfunction, fibrosis, oxidative stress and myocardial ultrastructural disorders (p < 0.05) in the diabetic mice. In vitro, ROCK1 was upregulated in HG-induced cardiomyocytes, and ROCK1 inhibition using Fasudil reversed the increased apoptosis, consistent with in vivo results. Mechanistically, ROCK1 inhibition abrogated apoptosis, relieved mitochondrial fission, and efficiently attenuated the escalated production of reactive oxygen species in vitro and in vivo. The content of Ser616-phosphorylated dynamin-related protein 1 (Drp1) increased while ROCK1 led to apoptosis in HG-treated cardiomyocytes, which could be partly neutralized by ROCK1 inhibition with Fasudil, consistent with the in vivo results. Fasudil attenuated the cardiac dysfunction in diabetes by decreasing excessive mitochondrial fission via inhibiting Drp1 phosphorylation at serine 616.

Effects of fasudil on blood–brain barrier integrity

BackgroundCerebral infarction accounts for 85% of all stroke cases. Even in an era of rapid and effective recanalization using an intravascular approach, the majority of patients have poor functional outcomes. Thus, there is an urgent need for the development of therapeutic agents to treat acute ischemic stroke. We evaluated the effect of fasudil, a Rho kinase inhibitor, on blood brain barrier (BBB) functions under normoxia or oxygen–glucose deprivation (OGD) conditions using a primary cell-based in vitro BBB model.MethodsBBB models from rat primary cultures (brain capillary endothelial cells, astrocytes, and pericytes) were subjected to either normoxia or 6 h OGD/24 h reoxygenation. To assess the effects of fasudil on BBB functions, we evaluated real time impedance, transendothelial electrical resistance (TEER), sodium fluorescein permeability, and tight junction protein expression using western blotting. Lastly, to understand the observed protective mechanism on BBB functions by fasudil we examined the role of cyclooxygenase-2 and thromboxane A2 receptor agonist U-46619 in BBB-forming cells.ResultsWe found that treatment with 0.3–30 µM of fasudil increased cellular impedance. Fasudil enhanced barrier properties in a concentration-dependent manner, as measured by an increased (TEER) and decreased permeability. Fasudil also increased the expression of tight junction protein claudin-5. Reductions in TEER and increased permeability were observed after OGD/reoxygenation exposure in mono- and co-culture models. The improvement in BBB integrity by fasudil was confirmed in both of the models, but was significantly higher in the co-culture than in the monoculture model. Treatment with U-46619 did not show significant changes in TEER in the monoculture model, whereas it showed a significant reduction in TEER in the co-culture model. Fasudil significantly improved the U-46619-induced TEER reduction in the co-culture models. Pericytes and astrocytes have opposite effects on endothelial cells and may contribute to endothelial injury in hyperacute ischemic stroke. Overall, fasudil protects the integrity of BBB both by a direct protective effect on endothelial cells and by a pathway mediated via pericytes and astrocytes.ConclusionsOur findings suggest that fasudil is a BBB-protective agent against acute ischemic stroke.

ROCK inhibitor fasudil reduces the expression of inflammatory factors in LPS-induced rat pulmonary microvascular endothelial cells via ROS/NF-\u03baB pathway

BackgroundInflammation plays a major role in the pulmonary artery hypertension (PAH) and the acute lung injury (ALI) diseases. The common feature of these complications is the dysfunction of pulmonary microvascular endothelial cells (PMVECs). Fasudil, the only Rho kinase (ROCK) inhibitor used in clinic, has been proved to be the most promising new drug for the treatment of PAH, with some anti-inflammatory activity. Therefore, in the present study, the effect of fasudil on lipopolysaccharide (LPS)-induced inflammatory injury in rat PMVECs was investigated.MethodsLPS was used to make inflammatory injury model of rat PMVECs. Thereafter, the mRNA and protein expression of pro-inflammatory factors was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) assay respectively. Intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) were determined by using commercial kits according to the manufacturer’s instructions. Western blot assay was used to detect the protein expression of nuclear factor kappa B (NF-κB) p65.ResultsFasudil effectively prevented inflammatory injury induced by LPS, which is manifested by the decrease of pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chenotactic protein-1 (MCP-1). Meanwhile, fasudil dramatically reduced the levels of ROS and MDA, and also elevated the activities of SOD and GSH-Px. Furthermore, the nuclear translocation of NF-κB p65 induced by LPS was also suppressed by fasudil. Additionally, the ROS scavengers N-Acetylcysteine (N-Ace) was also found to inhibit the nuclear translocation of NF-κB and the mRNA expression of IL-6 and MCP-1 induced by LPS, which suggested that ROS was essential for the nuclear translocation of NF-κB.ConclusionsThe present study revealed that fasudil reduced the expression of inflammatory factors, alleviated the inflammatory and oxidative damage induced by LPS in rat PMVECs via ROS-NF-κB signaling pathway.

Rho-Kinase inhibition decreases focal cerebral ischemia-induced glial activation in rats.

BackgroundRho-kinase inhibition in a rat middle cerebral artery occlusion (MCAO) model is reported to improve neurological functions and decrease infarction size.ObjectiveThe objective of this study is to investigate the underlying mechanisms of such improvement by evaluating the effects of Rho-kinase inhibition on astrocytes and microglial accumulation and activation in this condition.MethodsAdult male Sprague-Dawley (SD) rats were used to generate the MCAO model, which received an I.P injection of a chemical Rho-kinase inhibitor (Fasudil- 5 mg/kg/day) or vehicle (PBS) for 2 and 4 days.ResultsFasudil treatment significantly decreased the stroke volumes and water content in the lesion areas, as revealed by MRI. Immunostaining and Western blotting results demonstrated that Fasudil significantly decreased the levels of Aquaporin-4, a water channel protein. The number of GFAP+ astrocytes and Iba-1+ macrophage/microglia was decreased in the lesion areas. Proinflammatory transcription factor NF-κB protein levels were decreased in the Fasudil group 2 days after MCAO. Also, proinflammatory mediators including TNF-α, IL-1β, and iNOS levels were decreased. In vitro migration study using a human microglial cell line (HMO6) confirmed the inhibitory effects of Fasudil on the process. Fasudil also decreased combined IL-1β and IFNγ-induced NF-κB nuclear translocation in HMO6. Moreover, Fasudil transiently decreased combined IL-1β and IFNγ-induced iNOS, TNFα, and IL-1β mRNA levels in HMO6.ConclusionOur study demonstrates the inhibitory effects of Rho-kinase on NF-κB-mediated glial activation and cerebral edema, which might be a promising therapeutic target in acute cerebral ischemia conditions.

Can We Make the Gold Standard Better?

Antispastic Effect of Fasudil and Cocktail of Fasudil and Nitroglycerin in Internal Thoracic ArteryThe Annals of Thoracic SurgeryPreviewSpasm of arterial grafts in coronary artery bypass grafting is a clinical problem and can occasionally be lethal. Perioperative spasm in the internal thoracic artery (ITA) and coronary arteries occurs in 0.43% of patients. This study aimed to investigate the antispastic effect of a RhoA/Rho-kinase (Rho-associated coiled-coil-containing protein kinase [ROCK]) inhibitor (fasudil) with and without nitroglycerin in combination in the ITA. Full-Text PDF

Antispastic Effect of Fasudil and Cocktail of Fasudil and Nitroglycerin in Internal Thoracic Artery

BackgroundSpasm of arterial grafts in coronary artery bypass grafting is a clinical problem and can occasionally be lethal. Perioperative spasm in the internal thoracic artery (ITA) and coronary arteries occurs in 0.43% of patients. This study aimed to investigate the antispastic effect of a RhoA/Rho-kinase (Rho-associated coiled-coil-containing protein kinase [ROCK]) inhibitor (fasudil) with and without nitroglycerin in combination in the ITA. MethodsIsolated human ITA rings taken from 68 patients who were undergoing coronary bypass were studied in a myograph. Cumulative concentration-relaxation curves for fasudil (−9 to −3.5 log M) were established in the ITA, which was precontracted with potassium chloride or U46619. The inhibitory effect of fasudil (−6.3 or −5.3 log M) or fasudil in combination with nitroglycerin were also tested. The ROCK2 protein was measured by Western blot. ResultsFasudil caused similar relaxation in ITA rings contracted by potassium chloride or U46619. Pretreatment with −5.3 log M fasudil significantly depressed contraction induced by potassium chloride (P = .004 vs control; P = .017 vs −6.3 log M) and U46619 (P = .010 vs control; P = .041 vs. −6.3 log M). Fasudil in combination with nitroglycerin had more effect and more rapid and sustained relaxation than either vasodilator alone. Fasudil caused a decrease of ROCK2 protein content (P = .014). ConclusionsFasudil fully relaxes some vasoconstrictor-induced contraction and decreases ROCK2 protein content in the ITA. The combination of fasudil and nitroglycerin has a superior effect than either vasodilator alone. The new cocktail solution composed of fasudil and nitroglycerin (pH 7.4) has effective antispastic action and may prove to be a new antispastic method for arterial conduits during coronary bypass surgery.

Protective Effect of Fasudil on Hydrogen Peroxide-Induced Oxidative Stress Injury of H9C2 Cardiomyocytes.

Objective Oxidative damage is a pathological factor that causes cardiovascular damage in the clinic and is increasingly serious. This study focused on the effect of fasudil on H2O2-induced oxidative damage in cardiomyocytes. Materials and Methods H9C2 cardiomyocytes were cultured in vitro and divided into three groups: control group (Con group), H2O2 treatment (H2O2 group), and fasudil and H2O2 cotreatment (H2O2+fasudil group). The content levels of LDH and MDA in the supernatant were detected, and the morphology of H9C2 cardiomyocytes was observed by light microscopy. 8-OHdG staining was observed by a fluorescence inversion microscope. Cell Counting Kit (CCK-8), western blotting, real-time polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA) were used to investigate the effect of fasudil on the Rho/ROCK signaling pathway. Results Our results showed that after H2O2 treatment, the H9C2 cardiomyocytes were irregular in shape and elliptical. But the morphology of the H2O2+fasudil group was similar to that of the Con group. The green fluorescence of the H2O2 group was significantly enhancer than that of the Con group, while the green fluorescence of the H2O2+fasudil group was weaker than those of the H2O2 group. By detecting the supernatant, it was found that the contents of LDH were significantly increased, and the contents of SOD and CAT in the H2O2 group were significantly decreased. And the expression of antioxidant indicators in the H2O2 group was significantly decreased by western blotting. The results of RT-PCR showed that SOD1 and SOD2 mRNA in the H2O2 group was significantly reduced, and the contents of GPX1 and GPX3 in the H2O2 group were significantly decreased by enzyme-linked immunosorbent assay (ELISA). The expression of ROCK1, ROCK2, and downstream phosphorylation of myosin phosphatase target subunit-1 (p-MYPT-1) was significantly increased in the H2O2 group, while fasudil inhibited the increase of ROCK1, ROCK2, and p-MYPT-1. Conclusions Fasudil can inhibit the Rho/ROCK signaling pathway induced by H2O2 and reduce oxidative stress response, inhibit apoptosis, and improve antioxidant enzyme activity in H9C2 cardiomyocytes thereby delaying cell senescence.

Inhibition of Rho Kinase by Fasudil Ameliorates Cognition Impairment in APP/PS1 Transgenic Mice via Modulation of Gut Microbiota and Metabolites.

Background: Fasudil, a Rho kinase inhibitor, exerts therapeutic effects in a mouse model of Alzheimer's disease (AD), a chronic neurodegenerative disease with progressive loss of memory. However, the mechanisms remain unclear. In addition, the gut microbiota and its metabolites have been implicated in AD.Methods: We examined the effect of fasudil on learning and memory using the Morris water-maze (MWM) test in APPswe/PSEN1dE9 transgenic (APP/PS1) mice (8 months old) treated (i.p.) with fasudil (25 mg/kg/day; ADF) or saline (ADNS) and in age- and gender-matched wild-type (WT) mice. Fecal metagenomics and metabolites were performed to identify novel biomarkers of AD and elucidate the mechanisms of fasudil induced beneficial effects in AD mice.Results: The MWM test showed significant improvement of spatial memory in APP/PS1 mice treated with fasudil as compared to ADNS. The metagenomic analysis revealed the abundance of the dominant phyla in all the three groups, including Bacteroidetes (23.7–44%) and Firmicutes (6.4–26.6%), and the increased relative abundance ratio of Firmicutes/Bacteroidetes in ADNS (59.1%) compared to WT (31.7%). In contrast, the Firmicutes/Bacteroidetes ratio was decreased to the WT level in ADF (32.8%). Lefse analysis of metagenomics identified s_Prevotella_sp_CAG873 as an ADF potential biomarker, while s_Helicobacter_typhlonius and s_Helicobacter_sp_MIT_03-1616 as ADNS potential biomarkers. Metabolite analysis revealed the increment of various metabolites, including glutamate, hypoxanthine, thymine, hexanoyl-CoA, and leukotriene, which were relative to ADNS or ADF microbiota potential biomarkers and mainly involved in the metabolism of nucleotide, lipids and sugars, and the inflammatory pathway.Conclusions: Memory deficit in APP/PS1 mice was correlated with the gut microbiome and metabolite status. Fasudil reversed the abnormal gut microbiota and subsequently regulated the related metabolisms to normal in the AD mice. It is believed that fasudil can be a novel strategy for the treatment of AD via remodeling of the gut microbiota and metabolites. The novel results also provide valuable references for the use of gut microbiota and metabolites as diagnostic biomarkers and/or therapeutic targets in clinical studies of AD.

Renal ROCK Activation and Its Pharmacological Inhibition in Patients With Diabetes

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine/threonine kinase with essential roles in cytoskeletal functions. Substantial evidence implicates ROCK as a critical regulator in the inception and progression of diabetic nephropathy through a mechanism involving mesangial fibrosis, podocyte apoptosis, and endothelial inflammation. Despite these experimental observations, human data is lacking. Here we show that the phosphorylated form of myosin phosphatase targeting subunit 1 (MYPT1), a ROCK substrate, was increased in both the glomerular and tubulointerstitial areas in patients with histologically confirmed diabetic nephropathy. We also conducted a retrospective pilot analysis of data from patients with diabetes to assess the renoprotective effects of fasudil, an ATP-competitive ROCK inhibitor licensed in Japan for the prevention of vasospasm following subarachnoid hemorrhage. Fifteen subjects (male, n = 8; female, n = 7; age 65.7 ± 14.7 years; body height, 161.1 ± 12.6 cm; body weight, 57.6 ± 13.7 kg; body mass index, 22.4 ± 3.7 kg/m2) were enrolled to evaluate blood pressure and the renal outcome after fasudil treatment. Of note, proteinuria was significantly reduced at the end of the fasudil treatment without affecting the blood pressure or estimated glomerular filtration rate. Taken together, these findings suggest that the administration of fasudil could be associated with a better renal outcome by inhibiting the ROCK activity in patients with diabetes.

Fasudil prevents liver fibrosis via activating natural killer cells and suppressing hepatic stellate cells.

BACKGROUNDFasudil, as a Ras homology family member A (RhoA) kinase inhibitor, is used to improve brain microcirculation and promote nerve regeneration clinically. Increasing evidence shows that Rho-kinase inhibition could improve liver fibrosis.AIMTo evaluate the anti-fibrotic effects of Fasudil in a mouse model of liver fibrosis induced by thioacetamide (TAA). METHODSC57BL/6 mice were administered TAA once every 3 d for 12 times. At 1 wk after induction with TAA, Fasudil was intraperitoneally injected once a day for 3 wk, followed by hematoxylin and eosin staining, sirius red staining, western blotting, and quantitative polymerase chain reaction (qPCR), and immune cell activation was assayed by fluorescence-activated cell sorting. Furthermore, the effects of Fasudil on hepatic stellate cells and natural killer (NK) cells were assayed in vitro.RESULTSFirst, we found that TAA-induced liver injury was protected, and the positive area of sirius red staining and type I collagen deposition were significantly decreased by Fasudil treatment. Furthermore, western blot and qPCR assays showed that the levels of alpha smooth muscle actin (α-SMA), matrix metalloproteinase 2 (MMP-2), MMP-9, and transforming growth factor beta 1 (TGF-β1) were inhibited by Fasudil. Moreover, flow cytometry analysis revealed that NK cells were activated by Fasudil treatment in vivo and in vitro. Furthermore, Fasudil directly promoted the apoptosis and inhibited the proliferation of hepatic stellate cells by decreasing α-SMA and TGF-β1. CONCLUSIONFasudil inhibits liver fibrosis by activating NK cells and blocking hepatic stellate cell activation, thereby providing a feasible solution for the clinical treatment of liver fibrosis.

Inhibition of RhoA/Rho kinase signaling pathway by fasudil protects against kainic acid-induced neurite injury.

AimRhoA/Rho kinase pathway is essential for regulating cytoskeletal structure. Although its effect on normal neurite outgrowth has been demonstrated, the role of this pathway in seizure‐induced neurite injury has not been revealed. The research examined the phosphorylation level of RhoA/Rho kinase signaling pathway and to clarify the effect of fasudil on RhoA/Rho kinase signaling pathway and neurite outgrowth in kainic acid (KA)‐treated Neuro‐2A cells and hippocampal neurons.MethodWestern blotting analysis was used to investigate the expression of key proteins of RhoA/Rho kinase signaling pathway and the depolymerization of actin. After incubated without serum to induce neurite outgrowth, Neuro‐2A cells were fixed, and immunofluorescent assay of rhodamine‐phalloidin was applied to detect the cellular morphology and neurite length. The influence of KA on neurons was detected in primary hippocampal neurons. Whole‐cell patch clamp was conducted in cultured neurons or hippocampal slices to record action potentials.ResultKA at the dose of 100–200 μmol/L induced the increase in phosphorylation of Rho‐associated coiled‐coil‐containing protein kinase and decrease in phosphorylation of Lin11, Isl‐1 and Mec‐3 kinase and cofilin. The effect of 200 μmol/L KA was peaked at 1–2 hours, and then gradually returned to baseline after 8 hours. Pretreatment with Rho kinase inhibitor fasudil reversed KA‐induced activation of RhoA/Rho kinase pathway and increase in phosphorylation of slingshot and 14‐3‐3, which consequently reduced the ratio of G/F‐actin. KA treatment induced inhibition of neurite outgrowth and decrease in spines both in Neuro‐2a cells and in cultured hippocampal neurons, and pretreatment with fasudil alleviated KA‐induced neurite outgrowth inhibition and spine loss.ConclusionThese data indicate that inhibiting RhoA/Rho kinase pathway might be a potential treatment for seizure‐induced injury.

Fasudil enhances the phagocytosis of myelin debris and the expression of neurotrophic factors in cuprizone-induced demyelinating mice

Multiple sclerosis (MS) is mainly associated with the neuroinflammation and demyelination in the central nervous system (CNS), in which the failure of remyelination results in persistent neurological dysfunction. Fasudil, a typical Rho kinase inhibitor, has been exhibited beneficial effects on several models of neurodegenerative disorders. In this study, we showed that Fasudil promoted the uptake of myelin debris by microglia via cell experiments and through a cuprizone (CPZ)-induced demyelinating model. In vitro, microglia with phagocytic debris exhibited enhanced expression of brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor (GDNF), and the conditioned medium promoted the maturation of oligodendrocyte precursor cells (OPCs). Meanwhile, Fasudil upregulated TREM2/DAP12 pathway, which positively regulated the phagocytosis of myelin debris by microglia. Similarly, in vivo, Fasudil intervention enhanced the clearance of myelin debris, upregulated the expression of BDNF and GDNF on microglia, and promoted the formation of Oligo2+/PDGFRα+ OPCs and the maturation of MBP + oligodendrocytes in the brain. Our results showed that Fasudil targeted the phagocytic function of microglia, effectively clearing myelin debris produced during pathological process possibly by upregulating TREM2/DAP12 pathway, accompanied by increased expression of BDNF and GDNF. However, the precise mechanism underlying the effects of Fasudil in promoting phagocytic effects and neurotrophic factors remains to be elucidated.

Fasudil Dichloroacetate Alleviates SU5416/Hypoxia-Induced Pulmonary Arterial Hypertension by Ameliorating Dysfunction of Pulmonary Arterial Smooth Muscle Cells.

BackgroundPulmonary arterial hypertension (PAH) is an incurable disease that urgently needs therapeutic approaches. Based on the therapeutic effects of fasudil and dichloroacetate (DCA) on PAH, we aimed to explore the effects and potential mechanism of a new salt, fasudil dichloroacetate (FDCA), in a SU5416 plus hypoxia (SuHx)-induced rat model of PAH.MethodsThe rat model of PAH was established by a single subcutaneous injection of SU5416 (20 mg/kg) followed by hypoxia (10% O2) exposure for 3 weeks. FDCA (15, 45, or 135 mg/kg i.g. daily) or the positive control, bosentan (100 mg/kg i.g. daily), were administered from the first day after SU5416 injection. After 3-week hypoxia, hemodynamic parameters, and histological changes of the pulmonary arterial vessels and right ventricle (RV) were assessed. Additionally, in vitro, the effects of FDCA (50 μM), compared with equimolar doses of fasudil, DCA, or fasudil+DCA, on the proliferation, migration, and contraction of human pulmonary arterial smooth muscle cell (PASMC) under hypoxia (1% O2) were evaluated.ResultsFDCA dose-dependently attenuated SuHx-induced PAH, with significant reductions in RV systolic pressure, pulmonary artery wall thickness, pulmonary vessel muscularization, perivascular fibrosis, as well as RV hypertrophy and fibrosis. In vitro, FDCA inhibited hypoxia-induced PASMC proliferation, migration, and contraction to a greater degree than fasudil or DCA alone by restoring mitochondrial function, reducing intracellular Ca2+, and inhibiting calcium/calmodulin-dependent kinase (Ca2+/CaMK) activity as well as Rho-kinase activity.ConclusionFDCA ameliorates hypoxia-induced PASMC dysfunction by inhibiting both Ca2+/CaMK and Rho-kinase signaling pathways, as well as maintaining mitochondrial homeostasis, thus alleviating SuHx-induced PAH.

Fasudil alleviated insulin resistance through promotion of proliferation, attenuation of cell apoptosis and inflammation and regulation of RhoA/Rho kinase/insulin/nuclear factor-κB signalling pathway in HTR-8/SVneo cells.

The aim of this study was to evaluate the effects of fasudil on insulin resistance (IR) in HTR-8/SVneo cells. HTR-8/SVneo cells were treated with insulin or/and fasudil. Cell proliferation, apoptosis, inflammation and related signalling pathways were assessed. Insulin treatment significantly enhanced the protein expressions of RhoA and Rho kinase (ROCK1 and ROCK2), but decreased glucose consumption. Administration of fasudil effectively promoted glucose uptake. Moreover, fasudil enhanced cell viability and the level of proliferating cell nuclear antigen (PCNA). Insulin-mediated cell apoptosis was inhibited by fasudil via the down-regulation of bax and cleaved-caspase-3, and the up-regulation of bcl-2. At the same time, fasudil led to the reduction of IL-1β, TNF-α, IL-6 and IL-8 mRNA levels in insulin-treated cells. In addition, RhoA, ROCK2 and phosphorylated myosin phosphatase target subunit-1 (p-MYPT-1) expressions were down-regulated by fasudil. Importantly, fasudil activated insulin receptor substrate-1 (IRS-1) through increasing p-IRS-1 (Tyr612) and p-Akt expressions. The nuclear NF-κB p65 and p-IκB-α levels were reduced via the administration of fasudil in insulin-treated cells. Fasudil mitigated IR by the promotion of cell proliferation, inhibition of apoptosis and inflammation and regulation of RhoA/ROCK/insulin/NF-κB signalling pathway through in vitro studies.

Fasudil alleviates acetaminophen-induced liver injury via targeting Rhoa/ROCK signal pathway.

Fasudil is an inhibitor of Rhoa/ROCK signaling, which is involved in anti-inflammatory and anti-injury effects. The purpose of this study was to explore the effects of Fasudil on acetaminophen (APAP)-induced liver injury and reveal its potential molecular mechanism. In this study, C57BL/6 J mice were divided into different groups and treated with APAP and specified dose of Fasudil. HE staining was used to detect the changes of liver pathological tissues induced by APAP. ELISA assay was performed to detected the level of related factors. Western blot was used to detect the expressions of Rhoa, ROCK1, ROCK2. CD86 and CD6 were determined by RT-PCR and immunohistochemical staining detected the difference in CD86 expression. Rhoa/ROCK expression was increased in APAP-induced liver injury, and Fasudil targeted the expression of Rhoa/ROCK. Fasudil inhibits APAP-induced hepatic pathological changes and liver function injury. Fasudil inhibits the release of APAP-induced systemic inflammatory factors in liver tissue. Fasudil inhibits the activity of antioxidant enzymes, lipid peroxidation and macrophage infiltration induced by APAP in liver tissues. Fasudil alleviates APAP-induced liver injury via targeting Rhoa/ROCK signal pathway, indicating the possibility for clinical use of Fasudil in APAP-induced liver injury.

Fasudil ameliorates cognitive deficits, oxidative stress and neuronal apoptosis via inhibiting ROCK/MAPK and activating Nrf2 signalling pathways in APP/PS1 mice.

Alzheimer's disease (AD) is asevere neurodegenerative disorder of the central nervous system (CNS) characterized by neuron loss and dementia. Previous abundant evidence demonstrates that the first critical step in the course of AD is the state of oxidative stress and the neuronal loss is closely related to the interaction of several signalling pathways. The neuroprotective efficacy of Rho-associated protein kinase (ROCK) inhibitor in the treatment of AD has been reported, but its exact mechanism has not been well elucidated. The purpose of this study is to investigate the therapeutic effects of Fasudil on amyloid precursor protein/presenilin-1 (APP/PS1) mice and to discover the potential underlying mechanism. Sixteen 8-month-old APP/PS1 mice were divided into model and Fasudil treatment groups and 8 wild-type mice were used as anormal control group. After the behavioural test, all mice were sacrificed for immunofluorescence and other biochemical tests. The results showed that the administration of Fasudil improved learning and memory ability, elevated the concentration of antioxidative substances and decreased lipid peroxides, as well as inhibited neuronal apoptosis by increasing the expression of B-cell lymphoma-2 (Bcl-2) (p < 0.05), reducing Bcl-2 Associated X (Bax) (p < 0.05) and cleaved caspase-3 (p < 0.05) of APP/PS1 mice. Moreover, Fasudil treatment also ameliorated the phosphorylation of p38 (p < 0.01), c-Jun N-terminal kinase (JNK) (p < 0.001) and extracellular regulated protein kinases (ERK) (p < 0.001), and accelerated the nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) (p < 0.01) expression and its antioxidative downstream molecules (p < 0.05, p < 0.05, and p < 0.05, respectively). Data from the present study demonstrate that Fasudil significantly restored cognitive function, restrained oxidative stress and reduced neuronal apoptosis in the hippocampus, probably by inhibiting ROCK/MAPK and activating Nrf2 signalling pathways in APP/PS1 mice.

A Rho kinase inhibitor (Fasudil) suppresses TGF-β mediated autophagy in urethra fibroblasts to attenuate traumatic urethral stricture (TUS) through re-activating Akt/mTOR pathway: An in vitro study

AimsTransforming growth factor-β (TGF-β) mediated super-activation of urethra fibroblasts contributes to the progression of traumatic urethral stricture (TUS), and the Rho-associated kinase inhibitors, Fasudil, might be a novel therapeutic agent for TUS, but the underlying mechanisms had not been studied. Materials and methodsThe primary urethral fibroblasts (PUFs) were isolated from rabbit urethral scar tissues and cultured in vitro, and the PUFs were subsequently treated with TGF-β (10 μg/L) to simulate the realistic conditions of TUS pathogenesis. Next, the PUFs were exposed to Fasudil (50 μM) and autophagy inhibitor 3-methyladenine (3-MA) treatment. Genes expression was examined by Western Blot and immunofluorescence staining, and cellular functions were determined by MTT assay and Transwell assay. Key findingsTGF-β promoted cell proliferation, migration, autophagy, and secretion of extracellular matrix (ECM), including collagen I and collagen III, which were reversed by co-treating cells with both Fasudil and 3-MA. In addition, TGF-β treatment decreased the expression levels of phosphorylated Akt (p-Akt) and mTOR (p-mTOR) to inactivate the Akt/mTOR pathway in the PUFs, which could be re-activated by Fasudil. Then, the fibroblasts were treated with the Pan-Akt inhibitor (GDC-0068), and we surprisingly found that GDC-0068 abrogated the inhibiting effects of Fasudil on cell autophagy and proliferation in the PUFs treated with TGF-β. SignificanceFasudil regulated Akt/mTOR pathway mediated autophagy to hamper TGF-β-mediated super-activation in PUFs, which supported that Fasudil might be an ideal candidate therapeutic agent for TUS treatment for clinical utilization.