Rho kinase inhibitor Y-27632 downregulates IL-1ß expression in mice with experimental autoimmune myocarditis.

Autoimmune myocarditis is the limited or diffuse inflammation of the myocardium due to dysfunctional cellular and humoral immunity mechanisms. We constructed mouse models of experimental autoimmune myocarditis (EAM) using peptide MyHC-α614-629. On the day after secondary immunization, the mice were intraperitoneally injected with Rho kinase (ROCK) inhibitor Y-27632. On day 21, the cardiac tissues were harvested and weighed. The hearts of EAM mice were significantly enlarged and whitened. Furthermore, body weight (BW) slowly increased during the treatment period, the heart weight (HW) and the ratio of HW/eventual BW were increased, and inflammatory infiltration and fibrosis were aggravated in the myocardial tissue. Y-27632 treatment improved the aforementioned phenotypic and pathological features of EAM mice. Mechanistic analysis revealed a significant increase in Notch1, Hes1, Jag2, Dil1, Toll-like receptor (Tlr) 2, and interleukin (IL)-1ß expression in the myocardial tissue of EAM mice. Notably, IL-1ß expression was correlated with that of Notch1 and Tlr2. Following Y-27632 treatment, the expression of key target genes of the Notch signaling pathway (Notch1, Hes1, Dil1, and Jag2) and Tlr2 were obviously decreased. Y-27632 treatment also decreased the number of monocytes in the spleen of EAM mice. Thus, ROCK inhibitor Y-27632 exerted a protective effect in EAM mice by downregulating IL-1ß expression. This study aimed to provide a reference point for the future treatment of myocarditis in clinical settings.

Rho-kinase inhibitor alleviates CD4+T cell mediated corneal graft rejection by modulating its STAT3 and STAT5 activation.

Penetrating keratoplasty remains the most common treatment to restore vision for corneal diseases. Immune rejection after corneal transplantation is one of the major causes of graft failure. In recent years, Rho-associated protein kinase (ROCK) inhibitors have been found to be associated with the activation of the STATs pathway and are widely studied in autoimmune diseases. Therefore, it may be possible that the ROCK inhibitors also participate in the local and systemic immune regulation in corneal transplantation through activation of the STATs pathway and affect the CD4+ T cell differentiation. This study aimed to explore the role of ROCK-STATs pathway in the occurrence of immune rejection in corneal transplantation by applying Y27632, a ROCK inhibitor, to the recipient mice and peripheral CD4+ T cells. We found that Y27632 significantly up-regulated the phosphorylation level of STAT5 in both spleen and lymph nodes, down-regulated the phosphorylation level of STAT3 in the CD4+ T cells in the spleen. It also increased the proportion of CD4+CD25+Foxp3+Helios+ Tregs while decreased CD4+IL17A+ -Th17 cells. Moreover, Y27632 also reduced the proportion of dendritic cells in both spleen and lymph nodes, as well as the expression level of CD86 on their surfaces in the spleen, while the proportion of macrophages was not affected. The expression levels of ROCK1, ROCK2, CD11c and IL-17A mRNA were also found to be low in the graft tissue while the expression of Helios was upregulated. Rho-kinase inhibitor can modulate the balance of Tregs/Th17 by regulating the phosphorylation levels of both STAT3 and STAT5, thereby inhibiting the occurrence of immune rejection in allogeneic corneal transplantation.

"Keep on ROCKIn": Repurposed ROCK inhibitors to boost corneal endothelial regeneration.

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.

Rho Kinase Inhibitors: Strategies in Glaucoma Treatment in Older Adults.

Glaucoma is a leading cause of irreversible blindness which preferentially affects older individuals. No medications or therapies which are currently in our arsenal actually treat glaucoma itself. We know that intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma. The primary treatments for glaucoma include medications, laser therapies, and surgical therapies. The Rho kinase inhibitors are the newest class of medications currently on the market and in development for topical IOP-lowering therapy. Studies have shown their ability to lower eye pressure individually and in combination with other medications. Their ability to potentially provide neuroprotective effects for disease modification also gives this class exciting potential for glaucoma treatment.

Inhibition of RhoA/ROCK signalling pathway activity improves neural damage and cognitive deficits in the fluorosis model.

Excessive fluoride intake poses health risks to humans and animals. Many studies have indicated that fluoride exposure can damage the cytoskeleton and synapses, which has negative effects on the intellectual development of humans and animals. Our previous study suggested that the RhoA/ROCK signalling pathway is activated by NaF exposure in HT-22 cells and plays a vital role in cytoskeletal assembly and synaptogenesis. However, the mechanism underlying RhoA/ROCK-mediated cytoskeletal injury induced by fluoride remains unclear. In this study, Neuro-2A cells and ICR mice were used to investigate the effects of RhoA/ROCK activation inhibition on NaF-induced synaptic dysfunction and cognitive impairment. We detected the expression of GAP, RhoA, ROCK1/2, and (p)-MLC in vivo and in vitro model. The results showed that NaF exposure activated the RhoA/ROCK/MLC signalling pathway. We measured the effects of RhoA/ROCK inhibition on synaptic injury and intellectual impairment induced by NaF exposure. In vitro, Y-27632 suppressed activated RhoA/ROCK, attenuated morphological and ultrastructural damage, and decreased the survival rate and synapse-functional protein expression caused by NaF. In vivo, the results showed that the RhoA/ROCK/MLC pathway was inhibited by fasudil and improved pathological damage in the hippocampus, cognitive impairment, and decreased expression of neurofunctional proteins induced by NaF. Overall, these results suggest that fasudil and Y-27632 can reverse neurotoxicity caused by fluoride exposure. Furthermore, inhibition of RhoA/ROCK may be a future treatment for CNS injury, and more detailed studies on other neurodegenerative disease models are required to confirm its effectiveness.

Rho-associated protein kinase 1 inhibition in hepatocytes attenuates nonalcoholic steatohepatitis.

BACKGROUND: NASH is the progressive form of NAFLD characterized by lipotoxicity, hepatocyte injury, tissue inflammation, and fibrosis. Previously, Rho-associated protein kinase (ROCK) 1 has been implicated in lipotoxic signaling in hepatocytes in vitro and high-fat diet-induced lipogenesis in vivo. However, whether ROCK1 plays a role in liver inflammation and fibrosis during NASH is unclear. Here, we hypothesized that pathogenic activation of ROCK1 promotes murine NASH pathogenesis. METHODS AND RESULTS: Patients with NASH had increased hepatic ROCK1 expression compared with patients with fatty liver. Similarly, hepatic ROCK1 levels and activity were increased in mice with NASH induced by a western-like diet that is high in fat, fructose, and cholesterol (FFC). Hepatocyte-specific ROCK1 knockout mice on the FFC diet displayed a decrease in liver steatosis, hepatic cell death, liver inflammation, and fibrosis compared with littermate FFC-fed controls. Mechanistically, these effects were associated with a significant attenuation of myeloid cell recruitment. Interestingly, myeloid cell-specific ROCK1 deletion did not affect NASH development in FFC-fed mice. To explore the therapeutic opportunities, mice with established NASH received ROCKi, a novel small molecule kinase inhibitor of ROCK1/2, which preferentially accumulates in liver tissue. ROCK inhibitor treatment ameliorated insulin resistance and decreased liver injury, inflammation, and fibrosis. CONCLUSIONS: Genetic or pharmacologic inhibition of ROCK1 activity attenuates murine NASH, suggesting that ROCK1 may be a therapeutic target for treating human NASH.

Rho kinase inhibitors ameliorate cognitive impairment in a male mouse model of methamphetamine-induced schizophrenia.

Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Current antipsychotic treatment in SCZ improves positive symptoms but has major side effects and little impact on negative symptoms and cognitive impairment. The pathoetiology of SCZ remains unclear, but is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)-treated male mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced VD impairment. Fasudil also significantly suppressed the increase in the number of c-Fos-positive cells in the infralimbic medial prefrontal cortex (infralimbic mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, another Rho kinase inhibitor, into the infralimbic mPFC or DMS significantly ameliorated METH-induced VD impairment. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the infralimbic mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Oral administration of haloperidol and fasudil ameliorated METH-induced VD impairment, while clozapine had little effect. Oral administration of haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. These results suggest that METH activates Rho kinase in the infralimbic mPFC and DMS, which leads to cognitive impairment in male mice. Rho kinase inhibitors ameliorate METH-induced cognitive impairment, perhaps via the cortico-striatal circuit.

Inhibitors of Rho kinases (ROCK) induce multiple mitotic defects and synthetic lethality in BRCA2-deficient cells.

The trapping of Poly-ADP-ribose polymerase (PARP) on DNA caused by PARP inhibitors (PARPi) triggers acute DNA replication stress and synthetic lethality (SL) in BRCA2-deficient cells. Hence, DNA damage is accepted as a prerequisite for SL in BRCA2-deficient cells. In contrast, here we show that inhibiting ROCK in BRCA2-deficient cells triggers SL independently from acute replication stress. Such SL is preceded by polyploidy and binucleation resulting from cytokinesis failure. Such initial mitosis abnormalities are followed by other M phase defects, including anaphase bridges and abnormal mitotic figures associated with multipolar spindles, supernumerary centrosomes and multinucleation. SL was also triggered by inhibiting Citron Rho-interacting kinase, another enzyme that, similarly to ROCK, regulates cytokinesis. Together, these observations demonstrate that cytokinesis failure triggers mitotic abnormalities and SL in BRCA2-deficient cells. Furthermore, the prevention of mitotic entry by depletion of Early mitotic inhibitor 1 (EMI1) augmented the survival of BRCA2-deficient cells treated with ROCK inhibitors, thus reinforcing the association between M phase and cell death in BRCA2-deficient cells. This novel SL differs from the one triggered by PARPi and uncovers mitosis as an Achilles heel of BRCA2-deficient cells.

Phthalazinone-based lactams and cyclic ureas as ROCK2 selective inhibitors.

Derivatives of lactam, cyclic urea and carbamate were explored as aniline amide replacements in a series of phthalazinone-based ROCK inhibitors. Potent ROCK2 inhibitors such as 22 were identified with excellent overall kinase selectivity as well as good isoform selectivity over ROCK1.

Effects of Y-27632, a Rho-associated Kinase Inhibitor, on Human Corneal Endothelial Cells Cultured by Isolating Human Corneal Endothelial Progenitor Cells.

CONCLUSIONS: Y-27632 enabled the isolation and expansion of HCEPs. It also enhanced the proliferation, viability, and migration of differentiated HCEPs. METHODS: HCEPs were isolated and expanded in a medium with and without 10µM Y-27632, and then differentiated into HCECs in a medium with fetal bovine serum. The characteristics of HCEPs and differentiated HCEPs were confirmed by immunofluorescence staining. The proliferation, viability, morphology, and wound-healing ability of differentiated HCEPs were assessed in the presence of different concentrations of Y-27632. PURPOSE: Human corneal endothelial progenitor cells (HCEPs), which has been selectively isolated and differentiated into human corneal endothelial cells (HCECs), are crucial for repairing corneal endothelial damage. In this study, we evaluated the roles of a Rho-assisted kinase (ROCK) inhibitor, Y-27632, on the isolation and expansion of HCEPs, and assessed the in vitro effects of different concentrations of Y-27632 on the differentiated HCEPs. RESULTS: Y-27632 enabled the isolation and expansion of HCEPs from the corneal endothelium. The differentiated HCEPs showed an optimal increase in proliferation and survival in the presence of 10µM Y-27632. As the concentration of Y-27632 increased, differentiated HCEPs became elongated, and actin filaments were redistributed to the periphery of cells. Y-27632 also caused a concentration-dependent enhancement in the wound-healing ability of differentiated HCEPs.

Neferine, a novel ROCK1-targeting inhibitor, blocks EMT process and induces apoptosis in non-small cell lung cancer.

The compounds derived from Traditional Chinese Medicines have shown various pharmacological activities with unique advantages, especially in the aspect of antitumor. Neferine (Nef), a natural compound, extracted from green seed embryos of Lotus (Nelumbo nucifera Gaertn.) also exerts antitumor effects on cancers. In this study, the effects and mechanisms of Nef on epithelial-to-mesenchymal transition (EMT) process in non-small cell lung cancer (NSCLC) were evaluated. The results showed that Nef had the antitumor effects in vivo and in vitro. Nef significantly suppressed cell viability and induced apoptosis in NSCLC cells, with elevated reactive oxygen species and reduced BCL2/BAX ratio. Nef was also demonstrated to inhibit the invasion, metastasis and EMT process of NSCLC cells, and attenuate EMT-related changes of E-cadherin, N-cadherin and Vimentin at both transcriptional and translational levels. Moreover, we concluded that the inhibitory effects of Nef on EMT was achieved by targeting Rho-associated protein kinase 1, a protein mediating the process of EMT in various cancers. These results showed that Nef had a significant antitumor effect on NSCLC cells by inducing apoptosis and blocking EMT, providing the therapeutical prospect on NSCLC treatment.

[Effect and mechanism of Rho kinase inhibitor on intestinal injury in septic rats].

OBJECTIVE: To explore the effect of Rho kinase inhibitor on intestinal injury in septic rats and its possible mechanism. METHODS: Thirty-two male Sprague-Dawley (SD) rats were randomly divided into sham operation group (Sham group), Rho kinase inhibitor Y-27632 control group (Y+Sham group), sepsis model group [cecal ligation and puncture (CLP) group] and Y-27632 pretreatment group (Y+CLP group), with 8 rats in each group. Rat sepsis model was reproduced by CLP. The rats in the Sham group and Y+Sham group were only separated and moved the cecum without ligation and perforation. The rats in the Y+Sham group and Y+CLP group were pretreated with intraperitoneal injection of Y-27632 solution 5 mg/kg 15 minutes before operation; the rats in the Sham group and CLP group were intraperitoneally injected with the same amount of phosphate buffered saline (PBS). Twenty-four hours after operation, the heart blood was collected and the serum diamine oxidase (DAO) content was determined by enzyme-linked immunosorbent assay (ELISA). Then the small intestine tissue was collected, the pathological changes of the intestinal tissue were observed under the light microscope after hematoxylin-eosin (HE) staining, and Chiu's score was performed. The positive expressions of Rho-related coiled-coil kinase 1 (ROCK1) and nuclear factor-κB (NF-κB) in intestinal tissue were detected by immunohistochemistry. ELISA was used to detect the levels of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in intestinal tissue homogenate. RESULTS: The intestinal tissue structure of the Sham group and Y+Sham group was intact and the mucosa was arranged neatly. Compared with the Sham group, the intestinal mucosa of the CLP group was arranged disorderly, with a large number of inflammatory cells infiltration, and the Chiu's score was significantly increased (3.83±0.27 vs. 0.12±0.11, P < 0.05), indicating that those rats suffered from septic intestinal injury. Compared with the CLP group, the degree of necrosis of intestinal epithelial cells in the Y+CLP group was reduced, a small amount of inflammatory cells infiltration was seen, and the Chiu's score was significantly decreased (2.85±0.21 vs. 3.83±0.27, P < 0.05), indicating that Y-27632 pretreatment could alleviate intestinal injury in septic rats. Compared with the Sham group, the positive expressions of intestinal tissue ROCK1 and NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the CLP group were significantly increased [ROCK1 expression (A value): 0.19 (0.18, 0.22) vs. 0.10 (0.09, 0.11), NF-κB expression (A value): 0.40±0.02 vs. 0.15±0.01, DAO (ng/L): 287.81±23.31 vs. 144.92±17.72, TNF-α (ng/L): 101.08±5.62 vs. 74.81±5.56, all P < 0.05], the level of intestinal homogenate IL-10 was significantly decreased (µg/L: 55.16±5.20 vs. 95.95±7.53, P < 0.05). Compared with the CLP group, the positive expressions of intestinal tissue ROCK1, NF-κB, the contents of serum DAO and intestinal homogenate TNF-α in the Y+CLP group were significantly decreased [ROCK1 expression (A value): 0.15 (0.13, 0.18) vs. 0.19 (0.18, 0.22), NF-κB expression (A value): 0.28±0.01 vs. 0.40±0.02, DAO (ng/L): 243.34±19.76 vs. 287.81±23.31, TNF-α (ng/L): 90.41±8.79 vs. 101.08±5.62, all P < 0.05], while the level of intestinal homogenate IL-10 was significantly increased (µg/L: 66.15±5.74 vs. 55.16±5.20, P < 0.05), indicating that the protective effect of Y-27632 pretreatment on sepsis intestinal injury rats might be related to the regulation of RhoA/ROCK1/NF-κB signaling pathway. CONCLUSIONS: Rho kinase inhibitors can reduce intestinal injury in septic rats, and the mechanism may be related to inhibiting RhoA/ROCK1/NF-κB signaling pathway and reducing intestinal inflammation in septic rats.

Novel ROCK Inhibitors, Sovesudil and PHP-0961, Enhance Proliferation, Adhesion and Migration of Corneal Endothelial Cells.

The loss or dysfunction of human corneal endothelial cells (hCEnCs) is a leading cause of blindness due to corneal failure. Corneal transplantation with a healthy donor cornea has been the only available treatment for corneal endothelial disease. However, the need for way to regenerate the CEnCs has been increased due to the global shortage of donor corneas. The aim of the study is to investigate whether novel Rho-kinase (ROCK) inhibitors can induce the cultivation and regeneration of hCEnCs. Cultured hCEnCs were treated with Y-27632, sovesudil, or PHP-0961 for 24 h. Cellular responses, including cell viability, cytotoxicity, proliferation, and Ki67 expression with ROCK inhibitors were evaluated. We also evaluated wound healing and cell adhesion assays. Porcine corneas were used ex vivo to evaluate the effects of Y-27632, sovesudil, and PHP-0961 on wound healing and regeneration. We performed live/dead cell assays and immunofluorescence staining for SRY (sex determining region Y)-box 2 (SOX2), ß-catenin, and ZO-1 on porcine corneas after ROCK inhibitor treatments. Cell viability, cell proliferation rate, and the number of Ki67-positive cells were higher in Y-27632, sovesudil and PHP-0961 treated cells compared to the control. There was no difference in LDH cytotoxicity test between any groups. Cells treated with Y-27632, sovesudil and PHP-0961 showed faster migration, wound healing, and cell adhesion. In the porcine ex vivo experiments, wound healing, the number of live cells, and SOX2-positive cells were higher in Y-27632, sovesudil and PHP-0961 treated corneas. In all experiments, sovesudil and PHP-0961, the novel ROCK inhibitors, were equal or superior to the results of the ROCK inhibitor positive control, Y-27632. In conclusion, sovesudil and PHP-0961, novel ROCK inhibitors have the capacity to regenerate hCEnCs by enhancing cell proliferation and adhesion between cells.

Novel Dual AChE and ROCK2 Inhibitor Induces Neurogenesis via PTEN/AKT Pathway in Alzheimer's Disease Model.

Alzheimer's disease (AD) is a progressive and complex neurodegenerative disease. Acetylcholinesterase inhibitors (AChEIs) are a major class of drugs used in AD therapy. ROCK2, another promising target for AD, has been associated with the induction of neurogenesis via PTEN/AKT. This study aimed to characterize the therapeutic potential of a novel donepezil-tacrine hybrid compound (TA8Amino) to inhibit AChE and ROCK2 protein, leading to the induction of neurogenesis in SH-SY5Y cells. Experiments were carried out with undifferentiated and neuron-differentiated SH-SY5Y cells submitted to treatments with AChEIs (TA8Amino, donepezil, and tacrine) for 24 h or 7 days. TA8Amino was capable of inhibiting AChE at non-cytotoxic concentrations after 24 h. Following neuronal differentiation for 7 days, TA8Amino and donepezil increased the percentage of neurodifferentiated cells and the length of neurites, as confirmed by ß-III-tubulin and MAP2 protein expression. TA8Amino was found to participate in the activation of PTEN/AKT signaling. In silico analysis showed that TA8Amino can stably bind to the active site of ROCK2, and in vitro experiments in SH-SY5Y cells demonstrate that TA8Amino significantly reduced the expression of ROCK2 protein, contrasting with donepezil and tacrine. Therefore, these results provide important information on the mechanism underlying the action of TA8Amino with regard to multi-target activities.

Rho Kinase Inhibitor Y27632 Improves Recovery After Spinal Cord Injury by Shifting Astrocyte Phenotype and Morphology via the ROCK/NF-κB/C3 Pathway.

Spinal cord injury (SCI) usually results in loss or reduction in motor and sensory functions. Despite extensive research, no available therapy can restore the lost functions after SCI. Reactive astrocytes play a pivotal role in SCI. Rho kinase inhibitors have also been shown to promote functional recovery of SCI. However, the role of Rho kinase inhibitors in reactive astrocytic phenotype switch within SCI remains largely unexplored. In this study, astrocytes were treated with proinflammatory cytokines and/or the Rho kinase inhibitor Y27632. Concomitantly the phenotype and morphology of astrocytes were examined. Meanwhile, the SCI model of SD rats was established, and nerve functions were evaluated following treatment with Y27632. Subsequently, the number of A1 astrocytes in the injured area was observed and analyzed. Eventually, the expression levels of nuclear factor kappa B (NF-κB), C3, and S100A10 were measured. The present study showed that the Rho kinase inhibitor Y27632 improved functional recovery of SCI and elevated the proliferation and migration abilities of the astrocytes. In addition, Y27632 treatment initiated the switch of astrocytes morphology from a flattened shape to a process-bearing shape and transformed the reactive astrocytes A1 phenotype to an A2 phenotype. More importantly, further investigation suggested that Y27632 was actively involved in promoting the functional recovery of SCI in rats by inhabiting the ROCK/NF-κB/C3 signaling pathway. Together, Rho kinase inhibitor Y27632 effectively promotes the functional recovery of SCI by shifting astrocyte phenotype and morphology. Furthermore, the pro-regeneration event is strongly associated with the ROCK/NF-κB/C3 signal pathway.

Inhibition of ROCK ameliorates pulmonary fibrosis by suppressing M2 macrophage polarisation through phosphorylation of STAT3.

BACKGROUND: Emerging evidence provides mechanistic insights into the pathogenesis of pulmonary fibrosis (PF), and rare anti-PF therapeutic method has promising effect in its treatment. Rho-associated coiled-coil kinases (ROCK) inhibition significantly ameliorates bleomycin-induced PF and decreases macrophage infiltration, but the mechanism remains unclear. We established bleomycin and radiation-induced PF to identify the activity of WXWH0265, a newly designed unselective ROCK inhibitor in regulating macrophages. METHODS: Bleomycin-induced PF was induced by intratracheal instillation and radiation-induced PF was induced by bilateral thoracic irradiation. Histopathological techniques (haematoxylin and eosin, Masson's trichrome and immunohistochemistry) and hydroxyproline were used to evaluate PF severity. Western blot, quantitative real-time reverse transcription-polymerase chain reaction and flow cytometry were performed to explore the underlying mechanisms. Bone marrow-derived macrophages (BMDMs) were used to verify their therapeutic effect. Clodronate liposomes were applied to deplete macrophages and to identify the therapeutic effect of WXWH0265. RESULTS: Therapeutic administration of ROCK inhibitor ameliorates bleomycin-induced PF by inhibiting M2 macrophages polarisation. ROCK inhibitor showed no significant anti-fibrotic effect in macrophages-depleted mice. Treatment with WXWH0265 demonstrated superior protection effect in bleomycin-induced PF compared with positive drugs. In radiation-induced PF, ROCK inhibitor effectively ameliorated PF. Fibroblasts co-cultured with supernatant from various M2 macrophages phenotypes revealed that M2 macrophages stimulated by interleukin-4 promoted extracellular matrix production. Polarisation of M2 macrophages was inhibited by ROCK inhibitor treatment in vitro. The p-signal transducer and activator of transcription 3 (STAT3) in lung tissue and BMDMs was significantly decreased in PF in vivo and vitro after treated with ROCK inhibitors. CONCLUSION: Inhibiting ROCK could significantly attenuate bleomycin- and radiation-induced PF by regulating the macrophages polarisation via phosphorylation of STAT3. WXWH0265 is a kind of efficient unselective ROCK inhibitor in ameliorating PF. Furthermore, the results provide empirical evidence that ROCK inhibitor, WXWH0265 is a potential drug to prevent the development of PF.

ROCK inhibition with Y-27632 reduces joint inflammation and damage in serum-induced arthritis model and decreases in vitro osteoclastogenesis in patients with early arthritis.

Rheumatoid arthritis (RA) is a common chronic inflammatory disease affecting primarily peripheral joints, which is only partially controlled with current treatments. RA leads to pain, disability, deformities, and life expectancy shortening. Its pathogenesis is complex involving multiple cell types and signaling pathways that we incompletely understand. One of the pathways we have elucidated starts with WNT5A signaling and contributes to the aggressive phenotype of the RA synoviocytes through RYK-RhoA/ROCK signaling. Now, we have explored the contribution of ROCK to arthritis in vivo, using the K/BxN serum-transfer arthritis model; and to osteoclastogenesis, using the arthritis model and cells from patients with inflammatory arthritis. The mice and cells were treated with the ROCK inhibitor Y-27632 that caused a significant improvement of arthritis and reduction of osteoclastogenesis. The improvement in mouse arthritis was observed in the clinical evaluation and, histologically, in synovial inflammation, cartilage damage, bone erosion, and the abundance of multinucleated TRAP+ cells. Expression of inflammatory mediators in the arthritic joints, as assessed by real-time PCR, was also significantly reduced. The effect on bone was confirmed with in vitro assays using bone marrow precursors of arthritic mice and peripheral blood monocytes of patients with inflammatory arthritis. These assays showed dramatically reduced osteoclastogenesis and bone resorption. Overall, our findings suggest that ROCK inhibition could be part of a therapeutic strategy for RA by its dual action on inflammation and bone erosion.

Safety, efficacy, and patient selection of ripasudil in patients with uncontrolled glaucoma with maximum conventional medical therapy.

Purpose: Ripasudil hydrochloride hydrate (0.4%) is the first Rho-associated protein kinase (ROCK) inhibitor eye drop that lowers intraocular pressure (IOP) by increasing conventional aqueous outflow through the trabecular meshwork and Schlemm's canal. We aimed to evaluate the safety and efficacy of ripasudil in patients using the maximum topical anti-glaucoma medications and with uncontrolled IOP. Methods: In our prospective interventional study, we enrolled 27 eligible and consenting patients (46 eyes) who presented to us between January 2021 and June 2021. Ripasudil 0.4% was added as adjunctive therapy to the ongoing glaucoma treatment. On follow-up visits at 7 days, 15 days, 1 month, 2 months, and 3 months, the visual acuity, IOP with applanation tonometer, anterior segment, and fundus were evaluated. The IOP before and after the use of ripasudil eye drops was compared by paired t-test. Results: Among the 27 patients, 18 were males and 9 were females. A statistically significant reduction in IOP was noted at all time durations (P < 0.00001) with the maximum reduction at 3 months with all patients achieving their target IOP. No patient developed any side effects necessitating the omission of ripasudil. The most common adverse event noted was conjunctival hyperemia (22 patients), which was mild and transient. Conclusion: Ripasudil showed additional IOP-lowering effect with other antiglaucoma medications and exhibited no significant side effects.

Rho kinase inhibitor for primary open-angle glaucoma and ocular hypertension.

BACKGROUND: Glaucoma is a group of optic neuropathies characterized by progressive degeneration of the retinal ganglion cells, axonal loss and irreversible visual field defects. Glaucoma is classified as primary or secondary, and worldwide, primary glaucoma is a leading cause of irreversible blindness. Several subtypes of glaucoma exist, and primary open-angle glaucoma (POAG) is the most common. The etiology of POAG is unknown, but current treatments aim to reduce intraocular pressure (IOP), thus preventing the onset and progression of the disease. Compared with traditional antiglaucomatous treatments, rho kinase inhibitors (ROKi) have a different pharmacodynamic. ROKi is the only current treatment that effectively lowers IOP by modulating the drainage of aqueous humor through the trabecular meshwork and Schlemm's canal. As ROKi are introduced into the market more widely, it is important to assess the efficacy and potential AEs of the treatment. OBJECTIVES: To compare the efficacy and safety of ROKi with placebo or other glaucoma medication in people diagnosed with open-angle glaucoma (OAG), primary open-angle glaucoma (POAG) or ocular hypertension (OHT). SEARCH METHODS: We used standard Cochrane methods and searched databases on 11 December 2020. SELECTION CRITERIA: We included randomized clinical trials examining commercially available ROKi-based monotherapy or combination therapy compared with placebo or other IOP-lowering medical treatments in people diagnosed with (P)OAG or OHT. We included trials where ROKi were administered according to official glaucoma guidelines. There were no restrictions regarding type, year or status of the publication. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. Two review authors independently screened studies, extracted data, and evaluated risk of bias by using Cochrane's RoB 2 tool.  MAIN RESULTS: We included 17 trials with 4953 participants diagnosed with (P)OAG or OHT. Fifteen were multicenter trials and 15 were masked trials. All participants were aged above 18 years. Trial duration varied from 24 hours to 12 months. Trials were conducted in the USA, Canada and Japan. Sixteen trials were funded by pharmaceutical companies, and one trial provided no information about funding sources. The trials compared ROKi monotherapy (netarsudil or ripasudil) or combination therapy with latanoprost (prostaglandin analog) or timolol (beta-blocker) with placebo, timolol, latanoprost or netarsudil. Reported outcomes were IOP and safety. Meta-analyses were applied to 13 trials (IOP reduction from baseline) and 15 trials (ocular AEs).  Of the trials evaluating IOP, seven were at low risk, three had some concerns, and three were at high risk of bias. Three trials found that netarsudil monotherapy may be superior to placebo (mean difference [MD] 3.11 mmHg, 95% confidence interval [CI] 2.59 to 3.62; I2 = 0%; 155 participants; low-certainty evidence). Evidence from three trials found that timolol may be superior to netarsudil with an MD of 0.66 mmHg (95% CI 0.41 to 0.91; I2 = 0%; 1415 participants; low-certainty evidence). Evidence from four trials found that latanoprost may be superior to netarsudil with an MD of 0.97 mmHg (95% CI 0.67 to 1.27; I2 = 4%; 1283 participants; moderate-certainty evidence).  Evidence from three trials showed that, compared with monotherapy with latanoprost, combination therapy with netarsudil and latanoprost probably led to an additional pooled mean IOP reduction from baseline of 1.64 mmHg (95% CI -2.16 to -1.11; 1114 participants). Evidence from three trials showed that, compared with monotherapy with netarsudil, combination therapy with netarsudil and latanoprost probably led to an additional pooled mean IOP reduction from baseline of 2.66 mmHg (95% CI -2.98 to -2.35; 1132 participants). The certainty of evidence was moderate. One trial showed that,  compared with timolol monotherapy, combination therapy with ripasudil and timolol may lead to an IOP reduction from baseline of 0.75 mmHg (95% -1.29 to -CI 0.21; 208 participants). The certainty of evidence was moderate. Of the trials assessing total ocular AEs, three were at low risk, four had some concerns, and eight were at high risk of bias.  We found very low-certainty evidence that netarsudil may lead to more ocular AEs compared with placebo, with 66 more ocular AEs per 100 person-months (95% CI 28 to 103; I2 = 86%; 4 trials, 188 participants). We found low-certainty evidence that netarsudil may lead to more ocular AEs compared with latanoprost, with 29 more ocular AEs per 100 person-months (95% CI 17 to 42; I2 = 95%; 4 trials, 1286 participants).  We found moderate-certainty evidence that, compared with timolol, netarsudil probably led to 21 additional ocular AEs (95% CI 14 to 27; I2 = 93%; 4 trials, 1678 participants). Data from three trials (1132 participants) showed no evidence of differences in the incidence rate of AEs between combination therapy with netarsudil and latanoprost and netarsudil monotherapy (1 more event per 100 person-months, 95% CI 0 to 3); however, the certainty of evidence was low. Similarly, we found low-certainty evidence that, compared with latanoprost, combination therapy with netarsudil and latanoprost may cause 29 more ocular events per 100 person-months (95% CI 11 to 47; 3 trials, 1116 participants). We found moderate-certainty evidence that, compared with timolol monotherapy, combination therapy with ripasudil and timolol probably causes 35 more ocular events per 100 person-months (95% CI 25 to 45; 1 trial, 208 participants). In all included trials, ROKi was reportedly not associated with any particular serious AEs. AUTHORS' CONCLUSIONS: The current evidence suggests that in people diagnosed with OHT or (P)OAG, the hypotensive effect of netarsudil may be inferior to latanoprost and slightly inferior to timolol. Combining netarsudil and latanoprost probably further reduces IOP compared with monotherapy. Netarsudil as mono- or combination therapy may result in more ocular AEs. However, the certainty of evidence was very low or low for all comparisons except timolol. In general, AEs were described as mild, transient, and reversible upon treatment discontinuation. ROKi was not associated with any particular serious AEs. Future trials of sufficient size and follow-up should be conducted to provide reliable information about glaucoma progression, relevant IOP measurements and a detailed description of AEs using similar terminology. This would ensure the robustness and confidence of the results and assess the intermediate- and long-term efficacy and safety of ROKi.

Rho kinase inhibitor induced human dental pulp stem cells to differentiate into neurons.

AIMS: Neurological diseases due to neuron loss have become major public health problems. Current treatment reduces symptoms; however, there is no cure for neurological diseases. Therefore, stem cells may be an alternative therapy. Human dental pulp stem cells (hDPSCs) are an attractive source for cell-based approaches due to their high regenerative potential. The Rho kinase (ROCK) inhibitor Y-27632 promoted the neuronal differentiation of several stem cell types. However, its neuronal-inductive effect on hDPSCs has not been reported. Thus, the aim of our study was to investigate whether Y-27632 can induce the neuronal differentiation of hDPSCs. MAIN METHODS: hDPSCs were isolated from human third molars using an enzymatic method and were subsequently characterized. Cytotoxicity was evaluated using an MTT assay. The optimal concentration to induce neural differentiation was assessed using 1-50 µM Y-27632 as evaluated by Cresyl violet and immunofluorescence staining of neurofilaments and ßIII-tubulin, respectively. Ten µM Y-27632 was used for neuronal induction for 72 h, and differentiation was confirmed based on the expression of neurogenic markers (MAP2, Brn3a, and ChAT) and intracellular calcium activity. KEY FINDINGS: Our findings indicate that Y-27632 was not cytotoxic to hDPSCs and 10 µM Y-27632 was the lowest concentration that induced the morphological changes of hDPSCs into neuronal cells with Cresyl violet-positive staining and significantly enhanced the fluorescence intensity of neurofilament and ßIII-tubulin. The neuronal genes' expression and intracellular calcium activity were upregulated after induction with Y-27632. SIGNIFICANCE: At the optimal concentration and time, Rho kinase inhibitor induces hDPSC differentiation into neuronal cells.