Rho-associated Coiled-coil Forming Protein Kinase Research Articles

Exploring therapeutic targets for molecular therapy of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a chronic and progressive interstitial lung disease with a poor prognosis. Idiopathic pulmonary fibrosis is characterized by repeated alveolar epithelial damage leading to abnormal repair. The intercellular microenvironment is disturbed, leading to continuous activation of fibroblasts and myofibroblasts, deposition of extracellular matrix, and ultimately fibrosis. Moreover, pulmonary fibrosis was also found as a COVID-19 complication. Currently, two drugs, pirfenidone and nintedanib, are approved for clinical therapy worldwide. However, they can merely slow the disease's progression rather than rescue it. These two drugs have other limitations, such as lack of efficacy, adverse effects, and poor pharmacokinetics. Consequently, a growing number of molecular therapies have been actively developed. Treatment options for IPF are becoming increasingly available. This article reviews the research platform, including cell and animal models involved in molecular therapy studies of idiopathic pulmonary fibrosis as well as the promising therapeutic targets and their development progress during clinical trials. The former includes patient case/control studies, cell models, and animal models. The latter includes transforming growth factor-beta, vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor, lysophosphatidic acid, interleukin-13, Rho-associated coiled-coil forming protein kinase family, and Janus kinases/signal transducers and activators of transcription pathway. We mainly focused on the therapeutic targets that have not only entered clinical trials but were publicly published with their clinical outcomes. Moreover, this work provides an outlook on some promising targets for further validation of their possibilities to cure the disease.

Ripasudil as a Potential Therapeutic Agent in Treating Secondary Glaucoma in HTLV-1-Uveitis: An In Vitro Analysis.

Human T-cell leukemia virus type 1 (HTLV-1), a virus that affects 5-10 million people globally, causes several diseases, including adult T-cell leukemia-lymphoma and HTLV-1-associated uveitis (HU). HU is prevalent in Japan and often leads to secondary glaucoma, which is a serious complication. We investigated the efficacy of ripasudil, a Rho-associated coiled coil-forming protein kinase inhibitor, in alleviating changes in human trabecular meshwork cells (hTM cells) infected with HTLV-1. HTLV-1-infected hTM cells were modeled in vitro using MT-2 cells, followed by treatment with varying concentrations of ripasudil. We assessed changes in cell morphology, viability, and inflammatory cytokine levels, as well as NF-κB activation. The results showed that ripasudil treatment changed the cell morphology, reduced the distribution of F-actin and fibronectin, and decreased the levels of certain inflammatory cytokines, such as interleukin (IL)-6, IL-8, and IL-12. However, ripasudil did not significantly affect NF-κB activation or overall cell viability. These findings suggest that ripasudil has the potential to treat secondary glaucoma in patients with HU by modulating cytoskeletal organization and alleviating inflammation in HTLV-1-infected hTM cells. This study lays the foundation for further clinical studies exploring the effectiveness of ripasudil for the treatment of secondary glaucoma associated with HU.

ROCK2-RNA interaction map reveals multiple biological mechanisms underlying tumor progression in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common form of kidney cancer in adults. Despite new therapeutic modalities, the outcomes for RCC patients remain unsatisfactory. Rho-associated coiled-coil forming protein kinase 2 (ROCK2) has previously been shown to be upregulated in RCC, and its expression was negatively correlated with patient survival. However, the precise molecular function of ROCK2 has remained unclear. Herein, using RNA-seq analysis of ROCK2 knockdown and control cells, we identified 464 differentially expressed genes, and 1287 alternative splicing events in 786-O RCC cells. Furthermore, mapping of iRIP-seq reads in 786-O cells showed a biased distribution at 5' UTR, intronic and intergenic regions. By comparing ROCK2-regulated alternative splicing and iRIP-seq data, we found 292 overlapping genes that are enriched in multiple tumorigenic pathways. Taken together, our work defined a complex ROCK2-RNA interaction map on a genomic scale in a human RCC cell line, which deepens our understanding of the molecular function of ROCK2 in cancer development.

Fasudil, a ROCK inhibitor, prevents neuropathic pain in Minamata disease model rats

Methylmercury (MeHg), an environmental toxicant, is known to cause sensory impairment by inducing neurodegeneration of sensory nervous systems. However, in recent years, it has been revealed that neuropathic pain occurs in the chronic phase of MeHg poisoning, that is, in current Minamata disease patients. Our recent study using Minamata disease model rats demonstrated that MeHg-mediated neurodegeneration in the sensory nervous system may induce inflammatory microglia production in the dorsal horn of the spinal cord and subsequent somatosensory cortical rewiring, leading to neuropathic pain. We hypothesized that inhibition of the Rho-associated coiled coil-forming protein kinase (ROCK) pathway could prevent MeHg-induced neuropathic pain because the ROCK pathway is known to be involved in inducing the production of inflammatory microglia. Here, we showed for the first time that Fasudil, a ROCK inhibitor, can prevent neuropathic pain in Minamata disease model rats. In this model, Fasudil significantly suppressed nerve injury-induced inflammatory microglia production in the dorsal horn of the spinal cord and prevented subsequent somatosensory cortical rewiring. These results suggest that the ROCK pathway is involved in the onset and development of neuropathic pain in the chronic phase of Minamata disease, and that its inhibition is effective in pain prevention.

Hydrogen sulphide protects mice against the mutual aggravation of cerebral ischaemia/reperfusion injury and colitis

This study was undertaken to determine whether ischaemia/reperfusion (I/R)-induced brain injury and dextran sulfate sodium (DSS)-induced colitis in mice are related. A cerebral I/R model of mice was established by blocking the bilateral common carotid arteries; 3% DSS in drinking water was administered to mice for 7 days to induce colitis; mice with cerebral I/R and colitis were administered DSS for 7 days from the third day onwards after acute cerebral I/R. Brain damage and intestinal inflammation were also tested. The results revealed that cerebral I/R induced brain damage and a marked increase in glial fibrillary acidic protein (GFAP) expression and upregulation of Rho-associated coiled coil-forming protein kinase (RhoA/ROCK) pathway in mouse hippocampal tissues. However, in the colon tissues of mice with colitis, we found a reduction in GFAP. In addition, the expression of endogenous hydrogen sulphide (H2S) synthase reduced in mice brain tissues with cerebral I/R injury, as well.as in mouse colon tissues with colitis. Interestingly, the cerebral I/R-induced pathological changes in mouse brain tissues were aggravated by colitis, colitis mediated colon inflammation, and pathological changes in intestinal tissues had deteriorated when the mice suffered cerebral I/R 2 days before DSS administration. However, brain injury and colon inflammation in mice suffering from both cerebral I/R and colitis were ameliorated by NaHS, an exogenous H2S donor. Furthermore, we found that NaHS promoted the transformation of astrocytes from “A1” to “A2” type. These findings reveal that cerebral I/R injury and colitis are related, the mechanism is correlated with endogenous H2S deficiency.

Inhibiting the ROCK Pathway Ameliorates Acute Lung Injury in Mice following Myocardial Ischemia/reperfusion

ABSTRACT To clarify the role of Y-27632, a selective inhibitor of Rho-associated coiled-coil forming protein kinase (ROCK), in acute lung injury (ALI) induced by myocardial ischemia/reperfusion (I/R). Mice were randomized into Sham, I/R, and Y-27632 (10, 20 or 30 mg/kg) + I/R groups, and hemodynamics, infarcted area, the protein concentration, neutrophils in bronchoalveolar lavage fluid (BALF), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels were assessed. Pathological changes were evaluated by hematoxylin-eosin (HE) staining; protein and gene expression were measured by Western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR); and apoptosis was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining. ROCK1 and ROCK2 expression was up-regulated in lung tissues of I/R mice compared to sham mice. Y-27632 decreased the protein concentration and the neutrophils in BALF in I/R mice, improved hemodynamics and reduced infarct size (IS)/area at risk (AAR) ratio. In addition, pathological changes in lung tissues of Y-27632-treated mice were mitigated, and these alterations were accompanied by decreases in MDA levels in lung tissues and increases in SOD and GSH-Px levels. Moreover, in I/R group, the number of apoptotic cells in lung tissue was higher than that in sham group, and p53, Caspase-3 and Bax expression was up-regulated; however, following treatment with Y-27632 (10, 20 and 30 mg/kg), these changes were reversed. Inhibition of ROCK pathway by Y-27632 ameliorated ALI in myocardial I/R mice by mitigating oxidative stress, inflammation and cell apoptosis.

Rho-Kinase 1/2 Inhibition Prevents Transforming Growth Factor-β-Induced Effects on Pulmonary Remodeling and Repair.

Transforming growth factor (TGF)-β-induced myofibroblast transformation and alterations in mesenchymal-epithelial interactions contribute to chronic lung diseases such as chronic obstructive pulmonary disease (COPD), asthma and pulmonary fibrosis. Rho-associated coiled-coil-forming protein kinase (ROCK) consists as two isoforms, ROCK1 and ROCK2, and both are playing critical roles in many cellular responses to injury. In this study, we aimed to elucidate the differential role of ROCK isoforms on TGF-β signaling in lung fibrosis and repair. For this purpose, we tested the effect of a non-selective ROCK 1 and 2 inhibitor (compound 31) and a selective ROCK2 inhibitor (compound A11) in inhibiting TGF-β-induced remodeling in lung fibroblasts and slices; and dysfunctional epithelial-progenitor interactions in lung organoids. Here, we demonstrated that the inhibition of ROCK1/2 with compound 31 represses TGF-β-driven actin remodeling as well as extracellular matrix deposition in lung fibroblasts and PCLS, whereas selective ROCK2 inhibition with compound A11 did not. Furthermore, the TGF-β induced inhibition of organoid formation was functionally restored in a concentration-dependent manner by both dual ROCK 1 and 2 inhibition and selective ROCK2 inhibition. We conclude that dual pharmacological inhibition of ROCK 1 and 2 counteracts TGF-β induced effects on remodeling and alveolar epithelial progenitor function, suggesting this to be a promising therapeutic approach for respiratory diseases associated with fibrosis and defective lung repair.

Hyperbaric Oxygen Treatment Improves Intestinal Barrier Function After Spinal Cord Injury in Rats

Intestinal barrier dysfunction is often observed clinically after spinal cord injury (SCI) and seriously affects long-term quality of life. Hyperbaric oxygen (HBO) treatment has been proved to promote barrier function recovery after injury, but the influence of HBO on intestinal barrier function following SCI is unclear. We aimed to investigate the effect and mechanisms of HBO treatment on intestinal barrier function by measuring the level of tight junction (TJ) proteins and the Ras homolog (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway. SCI model was established in rats, and the animals were randomly assigned into three groups: sham-operation group (SH), SCI group and SCI+HBO group. In the SCI+HBO group, the rats inhaled 100% O2 for 1 h at 2.0 atmospheres absolute pressure (ATA) once per day after surgery. Neurological function and intestinal permeability were assessed after surgery, and the jejunum tissue was excised for histological and intestinal barrier function evaluations. The protein levels of TJ and the Rho/ROCK signaling pathway were also measured. The results showed that in the SCI group, intestinal mucosal injury score, intestinal permeability, and levels of Rho and ROCK1 were higher, and TJ proteins occludin and ZO-1 were lower than those in the SH group (P < 0.01). HBO treatment significantly inhibited the expression of Rho and ROCK1, increased occludin and ZO-1 expression, decreased intestinal permeability, and alleviated intestinal mucosal injury as compared with the SCI group (P < 0.05, P < 0.01). The SCI+HBO group showed higher Basso–Beattie–Bresnahan (BBB) scores relative to the SCI group on postoperative days 7 and 14 (P < 0.01). There was a significant negative correlation between BBB score and intestinal mucosal injury score in rats after HBO treatment (P < 0.05). We concluded from this study that HBO treatment promoted the expression of TJ proteins possibly through inhibiting Rho/ROCK signaling pathway, which protected the intestinal barrier function and improved the intestinal permeability after SCI in rats.

Autophagy regulates exosome secretion in rat nucleus pulposus cells via the RhoC/ROCK2 pathway

Our present study investigated whether exosome secretion of nucleus pulposus cells (NPCs) is regulated by autophagy. Different autophagic states of NPCs were induced by rapamycin (Rap), bafilomycin A1 (Baf) and other agents, and it was found that exosomes were secreted in an autophagy-dependent manner. Activation or inhibition of autophagy increased or decreased, respectively, the amount of exosomes that were released into the extracellular space. In addition, in order to confirm that Rap-promoted release of exosomes was mediated by autophagy rather than other pathways, we used autophagy associated gene 5 (ATG5) small-interfering RNA (siRNA) to silence the expression of ATG5 gene, which is indispensable for autophagy. The results showed that siRNA against ATG5 (siATG5) induced an accumulation of intraluminal vesicles (ILVs) in NPCs and a concomitant decrease in the amount of exosomes isolated from supernatant. Ras homolog gene (Rho) and Rho-associated coiled-coil forming protein kinase (ROCK) family molecules are capable of cytoskeletal remodeling and affecting vesicle transport. Therefore, we carried out targeted interventions and evaluated the effects of the RhoC/ROCK2 pathway on the secretion of exosomes within autophagic environment. Knockdown of RhoC and ROCK2 with corresponding siRNA significantly inhibited the secretion of exosomes originating from ILVs in NPCs, even when NPCs were subsequently treated with Rap. Taken together, our findings suggest that autophagy positively regulates expression levels of RhoC and ROCK2, and that the RhoC/ROCK2 pathway exerts a key function on NPCs-derived exosome secretion.

Data on differentially expressed proteins in rock inhibitor-treated human trabecular meshwork cells using SWATH-based proteomics.

Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors represent a novel class of anti-glaucoma drugs because of their ocular hypotensive effects. However, the underlying mechanisms responsible for lowering intraocular pressure (IOP) are not completely clear. The protein profile changes in primary human trabecular meshwork (TM) cells after two days treatment with a ROCK inhibitor were studied using label-free SWATH acquisition. These results provided significant data of key protein candidates underlying the effect of ROCK inhibitor. Using the sensitive label-free mass spectrometry approach with data-independent acquisition (SWATH-MS), we established a comprehensive TM proteome library. All raw data generated from IDA and SWATH acquisitions were uploaded and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS01254).

Effect of miR-133 on apoptosis of trophoblasts in human placenta tissues via Rho/ROCK signaling pathway.

The aim of this study was to explore the role of micro ribonucleic acid (miR)-133 in the apoptosis of human placental trophoblasts through the Ras homolog gene family (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway. The plasma samples were collected from 30 patients with pre-eclampsia (PE) undergoing treatment and 30 healthy subjects (control group) who received physical examination in our hospital. The Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was utilized to measure the expression of miR-133 in PE patients and healthy people. Meanwhile, blood pressure, urine protein content, liver function, and kidney function were detected in patients of both groups as well. Subsequently, the placental trophoblasts were extracted and transfected with inhibitors and miRNA mimics to suppress and overexpress miR-133, respectively. The transfection efficiency was determined by RT-PCR. The levels of interleukin-6 (IL-6), IL-1, and tumor necrosis factor-alpha (TNF-α) were measured in both groups. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay was performed to determine the apoptosis of trophoblasts. Next, the RT-PCR and Western blotting were carried out to detect the expressions of the Rho/ROCK pathway. Furthermore, the influence of miR-133 on the apoptosis of trophoblasts in human placenta tissues through Rho/ROCK was comprehensively observed. In vivo experiments demonstrated that the urinary protein content, miR-133 level, systolic blood pressure, diastolic blood pressure, and liver function and renal function indexes were significantly elevated in pre-eclampsia (PE) patients in comparison with normal subjects (p<0.05). After transfection of mimics and inhibitors, the expression of miR-133 was remarkably up- and down-regulated, respectively. The content of the inflammatory factors in miR-133 mimics group was overtly higher than the other two groups. The TUNEL staining results showed that the number of apoptotic cells significantly increased and decreased in the miR-133 mimics group and miR-133 inhibitors group, respectively. Subsequent experiments indicated that the expressions of apoptosis gene Caspase3, pathway gene, and protein ROCKI were notably up-regulated in miR-133 mimics group. However, they were evidently down-regulated in miR-133 inhibitors group than in the control group. In addition, a consistent trend was observed in the protein expression level. MiR-133 participates in the development and progression of PE through the Rho/ROCK signaling pathway, which may affect the apoptosis of trophoblasts in the placenta tissues.

Upregulation of sphingosine kinase1 contributes toventilator-associated lung injury in a two-hit model.

Ventilator-associated lung injury (VALI) remains a significant medical problem in intensive care units. The present study aimed to investigate the role of sphingosine kinase 1 (SPHK1) in VALI using a two-hit model and explore the potential underlying molecular mechanism. Mice were divided into five groups: i) Non-ventilated group; ii) non-ventilated + lipopolysaccharide (LPS) group; iii) ventilated group; iv) ventilated + LPS group; and v) ventilated + LPS + SPHK1 inhibitor group. Mice were administered LPS (1 mg/kg) via an intraperitoneal injection. After 12 h, the mice were anesthetized and connected to a ventilator (10 ml/kg at 150 breaths/min) for 4 h. SPHK1 inhibitor (50 mg/kg) was injected intraperitoneally 1 h prior to ventilation. Mouse lung vascular endothelial cells were treated with LPS and SPHK1 inhibitor, and then subjected to cyclic stretch for 4 h. The present results suggested that the expression of SPHK1 and sphingosine 1 phosphate was upregulated in the two-hit model of VALI; SPHK1 inhibitor could attenuate VALI in the two-hit model as observed by hematoxylin and eosin staining, and affected the cell count and the protein content levels in the bronchoalveolar lavage fluid. In addition, treatment with SPHK1 inhibitor reduced the wet-to-dry ratio of the lungs and suppressed Evans blue dye leakage into the lung tissue. Furthermore, SPHK1 inhibitor exhibited protective effects on the two-hit model of VALI by inhibiting the Ras homolog family member a-mediated phosphorylation of myosin phosphatase target subunit 1 (MYPT-1) and endothelial hyperpermeability. Additionally, mice were divided into five additional groups: i) Non-ventilated group; ii) non-ventilated + LPS group; iii) ventilated group; iv) ventilated + LPS group; and v) ventilated + LPS + Rho-associated coiled-coil forming protein kinase (ROCK)1 inhibitor group. ROCK1 inhibitor (10 mg/kg) was injected intraperitoneally 1 h prior to ventilation. The present results suggested that ROCK1 inhibitor could attenuate mechanical stretch-induced lung endothelial injury and the phosphorylation of MYPT-1 in vivo and in vitro. Collectively, the present findings indicated that upregulation of SPHK1 may contribute to VALI in a two-hit model.

Upregulated expression of ROCK1 promotes cell proliferation by functioning as a target of miR-335-5p in non-small cell lung cancer.

Lung cancer is regarded as one of the dominant causes in cancer patients among men and women all over the world. Rho-associated coiled-coil forming protein kinase l (ROCK1) is characterized as pivotal downstream effectors of the small GTPase RhoA and reported to participate in tumor metastasis. miR-335-5p acts as tumor suppressor microRNA and is identified to be downregulated in tumor tissues. miR-335-5p/ROCK1 axis has been demonstrated to promote cell proliferation and metastasis in gastric cancer, hepatocellular carcinoma and so on. However, the role it plays in promoting cell proliferation in non-small cell lung cancer (NSCLC) is poorly understood. Here, we demonstrated that the upregulated expression of ROCK1 was highly correlated with downregulated expression of miR-335-5p in NSCLC tissues and cell lines. Mechanistically, Knockdown of ROCK1 inhibited cell proliferation in vitro, accompanied by cell cycle change confirmed by flow analysis. Furthermore, miR-335-5p can downregulate the ROCK1 expression by directly binding to the 3'-untranslated region in posttranscriptional level. In vivo animal model showed similar results. Our findings highlighted the crucial role that miR-335-5p acted as a tumor suppressor to modulate cell proliferation and cell cycle progression via downregulating ROCK1 expression. And this miR-335-5p/ROCK1 axis contributed to NSCLC pathogenesis and might be promising targets for NSCLC therapy.

GLP-1 attenuates Ang II-induced proliferation and migration in rat aorta smooth muscle cells via inhibition of the RhoA/ROCK2 signaling pathway.

Glucagon-like peptide 1 (GLP-1), a neuroendocrine hormone produced by the gastrointestinal tract, plays a significant role in blood glucose regulation; drugs derived from GLP-1 are currently used for the treatment of type 2 diabetes. In addition to regulating glucose homeostasis, the protective effects of GLP-1 on the cardiovascular system are also of interest. However, the vascular protective mechanisms of GLP-1 remain unclear. The present study was designed to evaluate the role of GLP-1 in the proliferation and migration of vascular smooth muscle cells, and the underlying mechanisms. In this study, proliferation, migration, cyclin D1 expression, and phosphorylation of MLC, as well as RhoA and Rho-associated coiled-coil forming protein kinase 2 (ROCK2) expression, were increased in rat aorta smooth muscle cells (RASMCs) following incubation with angiotensin II (Ang II). These effects were significantly attenuated by GLP-1, forskolin (a cAMP activator) and Y-27632 (a ROCK2 inhibitor). However, H89 (a PKA inhibitor) inhibited the action of GLP-1, both in terms of inhibition of RASMC proliferation and migration, and RHOA/ROCK2 expression. These results indicate that GLP-1 inhibits Ang II-induced RASMC proliferation and migration via the cAMP/PKA/RhoA/ROCK2 signaling pathway. Our data suggest that GLP-1 should be considered for use in the clinical treatment of cardiovascular diseases, in addition to its current use in the treatment of diabetes mellitus.

Shen'ge powder decreases the cardiomyocyte hypertrophy in chronic heart failure by activating the Rho protein/Rho-associated coiledcoil forming protein kinase signaling pathway.

The current study aimed to explore the role and the molecular mechanism of Shen'ge powder in cardiomyocyte hypertrophy in chronic heart failure (CHF). Sprague-Dawley rats were selected for the study and divided randomly into four groups: control, model, Shen'ge powder, and fasudil group. An inverted microscope was used to determine the diameter of cardiomyocytes in each group. The survival and apoptotic rate of cardiomyocytes in each group was determined by the tetrazolium dye MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. The messenger RNA levels and protein expression of RhoA, Rho-associated coiled-coil forming protein kinase (ROCK), myosin light-chain phosphatase (MLCP), and myosin light-chain kinase (MLCK) were determined by quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. CHF increased the diameter and apoptotic rate of cardiomyocytes and decreased the survival rate of cardiomyocytes. The levels of RhoA, ROCK, and MLCK were increased significantly in CHF model rats, and the level of MLCP was decreased. After treating with Shen'ge powder, the expression of RhoA, ROCK, and MLCK decreased dramatically and the expression of MLCP increased. Shen'ge powder could reduce cardiomyocyte hypertrophy in CHF by regulating the Rho/ROCK signaling pathway.

Fasudil, a Rho-Associated Coiled Coil-Forming Protein Kinase Inhibitor, Recovers Methylmercury-Induced Axonal Degeneration by Changing Microglial Phenotype in Rats.

Methylmercury (MeHg) is an environmental neurotoxicant that induces neuropathological changes. In this study, we established chronic MeHg-intoxicated rats. These rats survived, and sustained MeHg-induced axonal degeneration, including the dorsal root nerve and the dorsal column of the spinal cord; these changes persisted 12 weeks after MeHg withdrawal. We demonstrated for the first time the restorative effect of Fasudil, a specific inhibitor of Rho-associated coiled coil-forming protein kinase, on axonal degeneration and corresponding neural dysfunction in the established chronic MeHg-intoxicated rats. To investigate the mechanism of this restorative effect, we focused on the expression of Rho protein families. This was supported by our previous study, which demonstrated that cotreatment with Fasudil prevented axonal degeneration by mitigating neurite extension/retraction incoordination caused by MeHg-induced suppression of Rac1 in vitro and in subacute MeHg-intoxicated rats. However, the mechanism of the restorative effect of Fasudil on axonal degeneration in chronic MeHg-intoxicated rats differed from MeHg-mediated neuritic extension/retraction incoordination. We found that the restorative effect of Fasudil was caused by the Fasudil-induced change of microglial phenotype, from proinflammatory to anti-inflammatory; moreover, Fasudil suppressed Rho-associated coiled coil-forming protein kinase activity. Treatment with Fasudil decreased the expression of proinflammatory factors, including tumor necrosis factor-α, inducible nitric oxide synthase, interleukin-1β, and interleukin-6; furthermore, it inactivated the nuclear factor kappa-light-chain-enhancer of activated B cells pathway. Additionally, Fasudil treatment was associated with increased levels of anti-inflammatory factors arginase-1 and interleukin-10. These results suggest that Rho-associated coiled coil-forming protein kinase inhibition may recover MeHg-mediated axonal degeneration and neural dysfunction in chronic MeHg intoxication.

Effect of Simvastatin on the Intestinal Rho/ROCK Signaling Pathway in Rats With Sepsis

BackgroundSimvastatin may alleviate the intestinal barrier dysfunction induced by sepsis. This study aimed to investigate the role of the Ras homolog (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) signaling pathway in the intestinal barrier of simvastatin-treated rats with sepsis. Materials and MethodsMale Wistar rats were pretreated with simvastatin (0.2 μg/g of body weight) for 1 week before cecal ligation and puncture. Twenty-four hours after cecal ligation and puncture, the condition of bacterial translocation was evaluated. Plasma levels of intestinal fatty acid binding protein, D-lactic acid and inflammatory factors, and oxidative stress in the intestine were determined. The intestinal injury scores, as well as the protein levels of Rho, ROCK1, and tight junction proteins ZO-1 and occludin were analyzed. ResultsTreatment with simvastatin alleviated the sepsis-induced increases in the plasma concentration of intestinal fatty acid binding protein and D-lactic acid, as well as the number of colony-forming units in the bacterial culture of the blood, liver, spleen, and kidney. In addition, simvastatin effectively reduced the intestinal levels of tumor necrosis factor α, interleukin-6, high-mobility group box 1, and malondialdehyde and increased the activity of superoxide dismutase in rats with sepsis. Staining with hematoxylin and eosin showed that severe intestinal injury occurred in the sepsis group, which was reduced by the treatment of simvastatin. Furthermore, the expression of Rho and ROCK1 was significantly downregulated and the protein expression levels of ZO-1 and occludin were significantly increased in simvastatin-treated rats (P < 0.05). ConclusionsSimvastatin can ameliorate the intestinal barrier dysfunction caused by sepsis by inhibiting the Rho/ROCK signaling pathway and reducing the levels of inflammatory factors and oxidative stress in the intestine, which also increase the expression of tight junction proteins.

Effect of Different Stimulating Strength of Electroacupuncture on Gastrointestinal Motility and RhoA/ROCK Signaling in Gastric Antral Smooth Muscle in Diabetic Gastroparesis Rats

To observe the effect of different strength of electroacupuncture (EA) stimulation on gastrointestinal motility and Ras homolog gene family member (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) signaling in diabetic gastroparesis (DGP) rats, so as to reveal the underlying mechanisms of EA for improving DGP. Sixty SD rats were randomly and equally divided into blank control, DGP model, weak EA, medium EA, and strong EA groups (n＝12 rats in each). The DGP model was established by intraperitoneal injection of streptozotocin (STZ, 55 mmol/kg, 2%) and high-sugar and high-fat fodder feeding for 8 weeks. EA (0.12, 0.24, 0.36 mA, 20 Hz/100 Hz) was applied to "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Liangmen" (ST 21) for 20 min, once daily for 15 successive days. Blood glucose levels were measured weekly with blood glucose meter and blood glucose test paper. Fecal phenol red excretion method was used to display gastric emptying and small intestinal propulsion function. The expression of RhoA protein in the gastric antral smooth muscle tissue was detected by immunohistochemistry and Western blot (WB), separately, and that of ROCK, myosin phosphatase target subunit 1 (MYPT 1) and phosphorylated (p)-MYPT 1 proteins in gastric antrum detected by WB. Compared with the blank control group, the gastric emptying rate and small intestine propulsion rate of the model group were significantly decreased (P<0.05), and the blood glucose level was remarkably increased (P<0.05). Moreover, the expression levels of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins in the gastric antrum were significantly down-regulated relevant to the control group (P<0.05). After administration of EA, the decreased gastric emptying rate and intestinal propulsion rate, and the down-regulated expression of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins were significantly increased in the strong, medium and weak EA stimulation groups (P<0.05). Comparison among the 3 EA groups showed that the strong stimulation was significantly superior to weak stimulation in up-regulating the expression of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins, and obviously superior to the medium stimulation in up-regulating RhoA and MYPT 1 protein levels (P<0.05), while the medium stimulation was significantly stronger than the weak stimulation in up-regulating the expression of ROCK, MYPT 1 and p-MYPT 1 proteins (P<0.05). There were no significant differences among the 3 EA groups in up-regulating the gastric emptying rate and small intestinal propulsion rate, and between the strong stimulation and medium stimulation in the expression levels of ROCK and p-MYPT 1 proteins (P>0.05). Electroacupuncture stimulation of ST 36-SP 6-ST 21 at 0.12, 0.24 and 0.36 mA can promote the gastrointestinal motility in DGP rats, which may be associated with its effects in enhancing RhoA/ROCK signaling in the gastric antral smooth muscle at different degrees.

The Rho Kinase Isoforms ROCK1 and ROCK2 Each Contribute to the Development of Experimental Pulmonary Fibrosis.

Pulmonary fibrosis is thought to result from dysregulated wound repair after repetitive lung injury. Many cellular responses to injury involve rearrangements of the actin cytoskeleton mediated by the two isoforms of the Rho-associated coiled-coil-forming protein kinase (ROCK), ROCK1 and ROCK2. In addition, profibrotic mediators such as transforming growth factor-β, thrombin, and lysophosphatidic acid act through receptors that activate ROCK. Inhibition of ROCK activation may be a potent therapeutic strategy for human pulmonary fibrosis. Pharmacological inhibition of ROCK using nonselective ROCK inhibitors has been shown to prevent fibrosis in animal models; however, the specific roles of each ROCK isoform are poorly understood. Furthermore, the pleiotropic effects of this kinase have raised concerns about on-target adverse effects of ROCK inhibition such as hypotension. Selective inhibition of one isoform might be a better-tolerated strategy. In the present study, we used a genetic approach to determine the roles of ROCK1 and ROCK2 in a mouse model of bleomycin-induced pulmonary fibrosis. Using ROCK1- or ROCK2-haploinsufficient mice, we found that reduced expression of either ROCK1 or ROCK2 was sufficient to protect them from bleomycin-induced pulmonary fibrosis. In addition, we found that both isoforms contribute to the profibrotic responses of epithelial cells, endothelial cells, and fibroblasts. Interestingly, ROCK1- and ROCK2-haploinsufficient mice exhibited similar protection from bleomycin-induced vascular leak, myofibroblast differentiation, and fibrosis; however, ROCK1-haploinsufficient mice demonstrated greater attenuation of epithelial cell apoptosis. These findings suggest that selective inhibition of either ROCK isoform has the potential to be an effective therapeutic strategy for pulmonary fibrosis.

Study on promotion of bone cancer pain via rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling by negative regulation of microRNA in mice

Objective To observe microRNA (miR-93) regulation in bone cancer pain(BCP) and explore the underlying mechanisms of miR-93 and Rho associated coiled-coil forming protein kinase (ROCK) signaling affecting the biological behaviors of BCP in mice. Methods Male mice were randomly divided into Control group, Sham group, bone cancer pain (BCP group), BCP+PBS group and BCP+Y-27632(ROCK inhibitor) group(n=13). The paw mechanical threshold and paw thermal latency were used to evaluate the behavioral changes on 1 day before surgery and 1, 3, 5, 7, 10, 14 after surgery. L4-L6 dorsal root ganglions were removed on postoperative day 14, followed by Real-time PCR to measure the expression of miR-93. The ROCK2 promoter activity regulated by miR-93 was evaluated by a dual luciferase reporter assay. Western blotting was used to evaluate the protein expression level of ROCK2, phospho-LIM domain kinase (LIMK) and phospho-Cofilin. Results Following intrafemoral carcinoma inoculation, robust mechanical allodynia[Sham group(6.81±1.04) g, BCP group(5.12±0.47) g]and thermal hyperalgesia[Sham group(11.75±1.46) s,BCP group (7.61±1.07) s]in C57BL/6J mice were respectively developed on day 5 and 7, and the symptoms grow progressively with time (P<0.05). The expression level miR-93 is decreased in a time dependent way in BCP group with progressively increased pain. However, the ROCK2 expression level was increased (P<0.05), and subsequently activated the LIMK/Cofilin pathways. Dual luciferase reporter assay demonstrated that miR-93 directly targeted the 3'-UTR of the ROCK2 gene, resulting in inhibition of the post-transcriptional translation of ROCK2. ROCK inhibitor significantly up-regulated paw withdrawal threshold[BCP+PBS group(3.43±0.89) g, BCP+Y-27632 group (5.74±1.02) g], paw withdrawal latency[BCP+PBS group (5.48±1.03) s, BCP+Y-27632 group (9.82±1.24) s]and reduced the protein expression level of ROCK2, phospho-LIMK and phospho-Cofilin (P<0.05). Conclusions Reduced expression of miR-93 can promote BCP through activating ROCK2/LIMK/cofilin pathway. Therefore, miR-93 or ROCK inhibitor may be novel targets for BCP. Key words: Bone cancer pain; Analgesia; MicroRNA-93; Rho associated coiled-coil forming protein kinase2/LIM domain kinase/Cofilin signaling