Rho-associated Coiled-coil Protein Kinase Research Articles

Effects of Rho-Associated Kinase (Rock) Inhibitors (Alternative to Y-27632) on Primary Human Corneal Endothelial Cells.

(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds-AR-13324 (Netarsudil) and its active metabolite, AR-13503-and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM; * p < 0.05); (ii) untreated vs. AR-13503 (10 μM; *** p < 0.001); (iii) Y-27632 vs. AR-13503 (10 μM; ** p < 0.005); (iv) AR-13324 (1 μM) vs. AR-13503 (10 μM; ** p < 0.005); and (v) AR-13324 (0.1 μM) vs. AR-13503 (10 μM; * p < 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans.

Discovery of indoline-based derivatives as effective ROCK2 inhibitors for the potential new treatment of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and devastating lung disease with a median survival of only 3–5 years. Due to the lack of effective therapy, IPF threatens human health. Recently, increasing reports have indicated that Rho-associated coiled-coil protein kinases (ROCKs) play important roles in the development of IPF and might represent a novel target for the treatment of IPF. Herein, a new series of selective ROCK2 inhibitors based on indoline were designed and synthesized. Structural modification resulted in optimized compound 9b with an IC50 value of 6 nM against ROCK2 and the inhibition of collagen gel contraction. Cellular assays demonstrated that 9b could significantly suppress the expression of collagen I and α-SMA, and inhibited ROCK signaling pathway. Oral administration of compound 9b (10 mg/kg) exerted more significant anti-pulmonary fibrosis effects than nintedanib (100 mg/kg) and KD025 (100 mg/kg) in a bleomycin-induced IPF rat model, suggesting that 9b could serve as a potential lead compound for the treatment of IPF.

Fasudil, a ROCK inhibitor, preserves limb integrity in a mouse model of unilateral critical limb ischemia: Possible interplay of inflammatory and angiogenic signaling pathways

Peripheral arterial diseases (PAD) had a great attention owing to devastating consequences of disability and cardiovascular morbidity and mortality. Yet, current therapeutic options are limited to surgical revascularization with no effective pharmacotherapy available. Excessive activity of Rho-associated coiled-coil protein kinase (ROCK) is implicated with several vascular diseases, rendering ROCK inhibition as a potential therapeutic strategy for patients suffering vascular disorders. AimThe current study was dedicated to investigating the vascular protective potential of Fasudil, a ROCK inhibitor, on an experimentally induced unilateral critical limb ischemia (CLI) model in mice and demonstrated the possible underlying mechanisms. MethodsUnilateral CLI was induced by ligation and excision of femoral artery followed by daily i.p. injection of Fasudil (10 mg/kg or 25 mg/kg) up to two weeks post-surgery. Key findingsMice underwent CLI showed decreased antioxidant capacity and increased inflammatory signal, evident by elevation of ERK1/2 in both serum and GC muscles that coincided with increases in VEGFA, HIF-1α and CD34+ cells of GC muscles. CLI resulted in structural damage of GC muscle fibers, with marked apoptosis, declined proliferation and deteriorated peripheral limb function. Treatment with Fasudil restored antioxidant capacity and attenuated VEGFA, HIF-1α, CD34+ cells and inflammatory markers in ischemic limbs. Furthermore, Fasudil preserved histological integrity of ischemic GC muscles, with amelioration of apoptosis, preserved proliferation rate and improvement in peripheral limb function. SignificanceFasudil could protect against experimentally induced unilateral CLI, in a dose-dependent manner, which could pave the way for future clinical application of Fasudil in patients suffering PAD.

LncRNA BCYRN1 promotes cell migration and invasion of non-small cell lung cancer via the miR-30b-3p/ROCK1 axis.

Long non-coding RNA brain cytoplasmic RNA 1 (LncRNA BCYRN1) has been proved to participate in the cancer cell metastasis process, including non-small cell lung cancer (NSCLC). However, the underlying molecular mechanisms involved in the BCYRN1-mediated function remain largely unknown. The qRT-PCR analysis was carried out to examine the relative expressions of BCYRN1, microRNA-30b-3p (miR-30b-3p), and Rho-associated coiled-coil protein kinase 1 (ROCK1). ROCK1 protein level was detected via western blot assay. The migrative and invasive abilities of H520 and A549 cells were evaluated via Transwell assay. The relationships between BCYRN1 and miR-30b-3p or ROCK1 and miR-30b-3p were examined by luciferase reporter assay. The expression levels of BCYRN1 and ROCK1 were upregulated in NSCLC tissues and cells, while miR-30b-3p was downregulated. Higher BCYRN1 expression indicated lymph node metastasis and advanced tumor-node-metastasis (TNM) stage of NSCLC patients. Loss of BCYRN1 suppressed cell migration and invasion. More importantly, miR-30b-3p possessed the binding sites with BCYRN1. Besides, BCYRN1 negatively regulated the expression level of miR-30b-3p. Meanwhile, ROCK1 was proven to be directly targeted by miR-30b-3p. In addition, the silencing of miR-30b-3p also weakened the effect of BCYRN1 knockdown on cell migration and invasion. In vivo, BCYRN1 silencing reduced the growth of A549 cells. LncRNA BCYRN1 was involved in the metastasis of NSCLC through modulating the miR-30b-3p/ROCK1 axis.

Effect of Naoluoxintong on the NogoA/RhoA/ROCK pathway by down-regulating DNA methylation in MCAO rats

Naoluoxintong (NLXT) is a traditional Chinese Medicine (TCM) prescription that is clinically used in the treatment of ischemic stroke (IS). However, its therapeutic mechanism remains unclear. To obtain the mechanism of NLXT by observing the protective effects of NLXT on the NogoA/RhoA/Rock pathway in a rat model of IS by regulating DNA methylation. Rats were divided into five groups using a random number table: normal group, model group, NLXT group, blocker group I (NLXT+SGI-1027) and blocker group II (NLXT+Y27632). The right middle cerebral artery occlusion-reperfusion (MCAO/R) rat model was made, and the regional cerebral blood flow (rCBF) of each group was detected using laser Doppler. The methylation levels of CpG sites of neurite outgrowth inhibitor protein-A (Nogo-A), Nogo receptor (NgR), ras homolog gene family member A (RhoA) and rho-associated coiled-coil protein kinase 2 (ROCK2) genes in rat brain tissue were detected using the bisulfite method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA expression in rat brain tissue. NogoA, RhoA, NgR1, NgR2 and ROCK2 proteins were detected using immunoblotting in rat brain tissue. After the modeling of middle cerebral artery occlusion (MCAO), neurological deficit test was made to ensure the success of the modeling. At each time point after surgery, the rCBF of the other groups decreased compared with the normal group (P<0.01 or P<0.05). Meanwhile, the rCBF increased in blocker group I as well as blocker group II after 3 days (P<0.05). There were differences in the DNA methylation sites of NogoA, RhoA, NgR and ROCK2 genes between the model group and the NLXT group (P<0.05). Compared with the normal group, NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression in the model group increased observably (P<0.01). In comparison with the model group, NogoA and NgR1 gene expression in the blocker group II was prominently observed on the 1st day. NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression remarkably reduced (P<0.01) on the 3rd and 7th days. Compared with the normal group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the model group increased observably (P<0.01). In comparison with the model group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the other groups declined prominently (P<0.01). NLXT can reduce the DNA methylation level of NogoA pathway after IS, thus inhibit the expression of NogoA/RhoA/ROCK pathway from producing anti-cerebral ischemia pharmacological effect.

Novel immunotherapeutic effects of topically administered ripasudil (K-115) on corneal allograft survival

Corneal allograft survival is mediated by the variety of immunological reactions and wound healing process. Our aim was to explore the effects of topical administration of ripasudil, a selective Rho-associated coiled-coil protein kinase inhibitor, on corneal allograft survival. Ripasudil was administered to mice thrice a day after allogeneic corneal transplantation. Corneal graft survival, opacity, neovascularization, re-epithelization, immune cell infiltration, and mRNA levels of angiogenic and pro-inflammatory factors in the grafted cornea and draining lymph nodes (dLNs) were evaluated with slit-lamp microscopy, immunohistochemistry, flow cytometry, and polymerase chain reaction. Graft survival was significantly prolonged with lower graft opacity and neovascularization scores in 0.4% and 2.0% ripasudil-treated groups, and mRNA levels of angiogenic and pro-inflammatory factors in ripasudil-treated grafted corneas were reduced. Moreover, 0.4% and 2.0% ripasudil reduced CD45+-infiltrated leukocyte frequency, Cd11b and Cd11c mRNA levels, and the frequencies of mature dendritic cells, IFNγ-, and IL-17- producing CD4+T cells in the dLNs of recipients. Re-epithelization rate of the grafted cornea was significantly higher in the 0.4% and 2.0% ripasudil groups than in the control. Topically applied ripasudil prolonged graft survival by downregulating neovascularization and inflammation factors, while promoting corneal re-epithelization, suggesting that ripasudil may be useful for suppressing immunological rejection in corneal transplantation.

Design of novel ROCK inhibitors using fragment-based de novo drug design approach.

Rho-associated coiled-coil protein kinase (ROCK) is playing a vital role in the regulation of key cellular events and also responsible for causing several pathological conditions such as cancer, hypertension, Alzheimer's, cerebral vasospasm, and cardiac stroke. Therefore, it has attracted us to target ROCK protein as a potential therapeutic target for combating various diseases. Consequently, we investigated the active site of ROCK I protein and designed novel leads against the target using the de novo evolution drug design approach. Caffeic acid (an aglycone of acteoside) as a scaffold and fragments from 336 reported ROCK inhibitors were used for the design of novel leads. Multiple copy simultaneous search docking was used to identify the suitable fragments to be linked with the scaffold. Basic medicinal chemistry rules, coupled with structural insights generated by docking, led to the design of 7a, 8a, 9a, and 10a as potential ROCK I inhibitors. The designed leads showed better binding than the approved drug fasudil and also interacted with the key hinge region residue Met156 of ROCK I. Further, molecular dynamics (MD) simulation revealed that the protein-ligand complexes were stable and maintained the hydrogen bond with Met156 throughout the MD run. The promising in silico outcomes suggest that the designed compounds could be suitable anti-cancer leads that need to be synthesized and tested in various cancer cell lines. Graphical abstract.

Oxycodone inhibits myocardial cell apoptosis after myocardial ischemia-reperfusion injury in rats via RhoA/ROCK1 signaling pathway.

The purpose of this study was to investigate the effect of oxycodone on myocardial ischemia-reperfusion injury in rats through the Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 1 (ROCK1) signaling pathway. A total of 48 Sprague-Dawley (SD) rats were randomly divided into sham operation group, model group, oxycodone group, and inhibitor group, with 12 rats in each group. The rats in the sham operation group only underwent thoracotomy without ischemia-reperfusion injury, those in the model group were used to prepare the myocardial ischemia-reperfusion model with normal saline intervention, those in the oxycodone group were used to prepare the myocardial ischemia-reperfusion model with oxycodone intervention, and those in the inhibitor group were utilized to prepare the myocardial ischemia-reperfusion model with AG490 intervention. Then, the expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX) were detected by immunohistochemistry, the relative protein expressions of RhoA and ROCK1 were examined via Western blotting, and the messenger ribonucleic acid (mRNA) expressions of Bcl-2 and BAX were measured by quantitative Polymerase Chain Reaction (qPCR). Thereafter, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was adopted for apoptosis detection, and the levels of creatine kinase-muscle/brain (CK-MB), and cardiac Troponin I (cTnI) in serum were detected using an automatic biochemical analyzer. Immunohistochemistry results showed that compared with those in the sham operation group, the positive expression of BAX was remarkably increased (p<0.05), while that of Bcl-2 was significantly decreased (p<0.05) in the model group, oxycodone group, and inhibitor group. Compared with the model group, oxycodone group and inhibitor group had an evidently reduced positive expression of BAX (p<0.05) and an evidently raised positive expression of Bcl-2 (p<0.05). No differences were found in the positive expressions of BAX and Bcl-2 between oxycodone group and inhibitor group (p>0.05). According to Western blotting results, the relative protein expressions of RhoA and ROCK1 in the model group, oxycodone group, and inhibitor group were notably increased compared with those in the sham operation group (p<0.05). In comparison with those in the model group, the relative protein expressions of RhoA and ROCK1 in the oxycodone group and inhibitor group were predominantly reduced (p<0.05). There were no differences in the relative protein expressions of RhoA and ROCK1 between oxycodone group and inhibitor group (p>0.05). Moreover, it was discovered from qRT-PCR results that compared with those in the sham operation group, the mRNA expression of BAX was markedly raised (p<0.05), whereas that of Bcl-2 was decreased predominantly (p<0.05) in the model group, oxycodone group, and inhibitor group. Compared with the model group, oxycodone group, and inhibitor group had an evidently reduced mRNA expression of BAX (p<0.05) and a significantly raised mRNA expression of Bcl-2 (p<0.05). No differences were found in the mRNA expressions of BAX and Bcl-2 between oxycodone group and inhibitor group (p>0.05). In addition, TUNEL assay results manifested that compared with sham operation group, model group, oxycodone group, and inhibitor group had a markedly elevated apoptosis rate (p<0.05). In comparison with the model group, the apoptosis rate in oxycodone group and inhibitor group was remarkably reduced (p<0.05). There was no difference in the apoptosis rate between oxycodone group and inhibitor group (p>0.05). According to biochemical analysis results, the serum levels of CK-MB and cTnI in model group, oxycodone group, and inhibitor group were significantly increased compared with those in the sham operation group, with statistically significant differences (p<0.05). The levels of serum CK-MB and cTnI in the oxycodone group and inhibitor group were substantially lowered in comparison with those in the model group, displaying statistically significant differences (p<0.05). Besides, the levels of serum CK-MB and cTnI in the oxycodone group were not different from those in the inhibitor group (p>0.05). Oxycodone inhibits myocardial cell apoptosis after myocardial ischemia-reperfusion injury by suppressing the RhoA/ROCK1 signaling pathway.

Docking and 3D-QSAR Studies of Hydrazone and Triazole Derivatives for Selective Inhibition of GRK2 over ROCK2

Introduction: G protein-coupled receptor kinase 2 (GRK2) is known to be implicated in heart failure, and therefore serves as an important drug target. GRK2 belongs to the protein kinase A, G, and C family and shares high sequence similarity with its closely related protein, the Rhoassociated coiled-coil protein kinase 2 (ROCK2). Therefore, selective inhibition of GRK2 over ROCK2 is considered crucial for heart failure therapy. Objective: To understand the structural factors for enhancing the inhibitory activity for GRK2 and selectivity over ROCK2, we analyzed and compared molecular interactions using the same set of ligands against both receptors. Methods: We have performed molecular docking and three-dimensional quantitative structure activity relationship (3D-QSAR) studies on a series of hydrazone and triazole derivatives. Results: The presence of hydrophobic substituents at the triazole ring, electronegative substituents between the pyridine and triazole ring and hydrophobic substituents near the benzene ring increases the activity of both kinases. Whereas, having non-bulky substituents near the triazole ring, bulky and hydrophobic substations at the benzene ring and electronegative and H-bond acceptor substituents at the triazole ring showed a higher inhibitory preference for GRK2 over ROCK2. Conclusion: The outcome of this study may be used in the future development of potent GRK2 inhibitors having ROCK2 selectivity.

Rho-kinase ROCK inhibitors reduce oligomeric tau protein

Neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, consist of highly phosphorylated tau proteins. Tau protein binds to microtubules and is best known for its role in regulating microtubule dynamics. However, if tau protein is phosphorylated by activated major tau kinases, including glycogen synthase kinase 3β or cyclin-dependent kinase 5, or inactivated tau phosphatase, including protein phosphatase 2A, its affinity for microtubules is reduced, and the free tau is believed to aggregate, thereby forming neurofibrillary tangles. We previously reported that pitavastatin decreases the total and phosphorylated tau protein using a cellular model of tauopathy. The reduction of tau was considered to be due to Rho-associated coiled-coil protein kinase (ROCK) inhibition by pitavastatin. ROCK plays important roles to organize the actin cytoskeleton, an expected therapeutic target of human disorders. Several ROCK inhibitors are clinically applied to prevent vasospasm postsubarachnoid hemorrhage (fasudil) and for the treatment of glaucoma (ripasudil). We have examined the effects of ROCK inhibitors (H1152, Y-27632, and fasudil [HA-1077]) on tau protein phosphorylation in detail. A human neuroblastoma cell line (M1C cells) that expresses wild-type tau protein (4R0N) by tetracycline-off (TetOff) induction, primary cultured mouse neurons, and a mouse model of tauopathy (rTG4510 line) were used. The levels of phosphorylated tau and caspase-cleaved tau were reduced by the ROCK inhibitors. Oligomeric tau levels were also reduced by ROCK inhibitors. After ROCK inhibitor treatment, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and caspase were inactivated, protein phosphatase 2A was activated, and the levels of IFN-γ were reduced. ROCK inhibitors activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Collectively, these results suggest that ROCK inhibitors represent a viable therapeutic route to reduce the pathogenic forms of tau protein in tauopathies, including Alzheimer's disease.

NANOG improves type I collagen expression in human fetal scleral fibroblasts

Human fetal scleral fibroblasts (HFSFs) are components of the sclera and play important roles in its structure and function. In myopia, scleral remodeling reduces collagen fibers and the sclera begins to thin. NANOG is a key transcription factor essential for pluripotent and self-renewing phenotypes of undifferentiated embryonic stem cells. To determine whether NANOG improves human fetal scleral fibroblast quality and the underlying mechanisms in these cells, we established stable NANOG-overexpressing HFSFs. We studied type I collagen (COL1A 1) and Rho-associated coiled-coil protein kinase 1 (ROCK1) expression in transfected cells. We also investigated POU5F1, SOX2, KLF4, MYC and SALL4 expression in NANOG stably-overexpressed fibroblasts. Our data show that NANOG expression increased proliferation rates in fibroblasts. When compared to controls, expression of COL1A 1 in transfected fibroblasts was increased and the expression of ROCK1 was decreased. Similarly, the expression of POU5F1, SOX2 and KLF4 was downregulated, the expression of MYC was upregulated and there was no significant change in the expression of SALL4 in transfected fibroblasts. Our results suggest that in fibroblasts, NANOG regulates ROCK1 expression and improves COL1A 1 expression to delay scleral remodeling.

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.

Role of inhibiting LIM-kinase2 in improving erectile function through suppression of corporal fibrosis in a rat model of cavernous nerve injury.

We evaluated whether LIM-kinase 2 inhibitor (LIMK2i) could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 (ROCK1)/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury (CNCI). Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups: sham surgery (S), CNCI (I), and CNCI treated with low-dose (L), medium-dose (M), and high-dose (H) LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i (2.5, 5.0, and 10.0 mg kg−1 body weight, respectively) for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson's trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-LIMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg−1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway.

Role of RhoA/ROCK2 signaling pathway in propofol-induced neuroapoptosis in hippocampus of newborn rats

Objective To evaluate the role of Ras homolog family member A (RhoA)/Rho-associated coiled-coil containing protein kinase 2 (ROCK2) signaling pathway in propofol-induced neuroapoptosis in the hippocampus of newborn rats. Methods ExperimentⅠ Primarily cultured hippocampal neurons were seeded in 6-well culture plates at a density of 1×106 cells/ml and divided into 2 groups (n=6 each) using a random number table: solvent control group (C group) and propofol group (P group). Propofol was added with the final concentration of 60 μg/ml in group P. Dimethyl sulfoxide was added with the final concentration of 0.04% in group C. The expression of RhoA and ROCK2 in hippocampal neurons was measured by Western blot at 24 h of incubation.ExperimentⅡ Primarily cultured hippocampal neurons were seeded in 6-well culture plates at a density of 1×106 cells/ml and divided into 3 groups (n=6 each) using a random number table: solvent control group (C group), propofol group (P group) and propofol plus specific RhoA/ROCK2 signaling pathway blocker Y27632 group (P+ Y group). Propofol was added with the final concentration of 60 μg/ml in group P. Propofol at the final concentration of 60 μg/ml and Y27632 at the final concentration of 10 μmol/L were added in group P+ Y.Dimethyl sulfoxide was added with the final concentration of 0.04% in group C. At 24 h of incubation, the neuroapoptosis in hippocampi was detected by flow cytometry, and the expression of activated caspase-3 in hippocampal neurons was measured by Western blot.The apoptotic rate was calculated. Results ExperimentⅠ Compared with group C, the expression of RhoA and ROCK2 in hippocampal neurons was significantly up-regulated in group P (P<0.05). Experiment Ⅱ Compared with group C, the apoptotic rate of hippocampal neurons was significantly increased, and the expression of activated caspase-3 was up-regulated in P and P+ Y groups (P<0.05 or 0.01). Compared with group P, the apoptotic rate of hippocampal neurons was significantly decreased, and the expression of activated caspase-3 was down-regulated in group P+ Y (P<0.05). Conclusion Activation of RhoA/ROCK2 signaling pathway is involved in propofol-induced neuroapoptosis in hippocampi of newborn rats. Key words: Rho factor; Rho-associated kinases; Propofol; Infant, newborn; Apoptosis; Hippocampus; Neurons

Rho-associated coiled-coil forming protein kinase 1 expression in different stages of hepatic fibrosis

Objective To investigate the expression of Rho-associated coiled-coil forming protein kinase 1 (ROCK1) in different stages of hepatic fibrosis. Methods Seventy-two Wistar male rats of 200 to 250 g were selected and randomly divided into control group and model group. The control group was fed normally, and the model group was given 2 ml/kg of carbon tetrachloride (CCl4) intraperitoneally, twice a week. The animals (n=6) were killed at 3rd, and 6th day and 2nd, 4th, 6th, and 8th week. Hematoxylin and eosin (HE) staining was used to observe the pathological changes of live tissues. Immunohistochemistry was used to detect the location and expression of ROCK1. ROCK1 gene transcripts in different periods were quantitatively detected by real-time quantitative polymerase chain reaction (Real-time PCR). Results The positive expression rate of ROCK1 in control group was 11%, 15%, 13%, 11%, 17% and 14%, and that in model group was 14%, 15%, 52%, 65%, 75% and 80% at 3rd, and 6th day and 2nd, 4th, 6th, and 8th week, respectively, with significant differences among them (P=0.001). Real-time PCR revealed that the ROCK1 mRNA expression was 1.018 759, 1.381 913, 1.831 238, 2.515 323, 2.257 623, 2.188 587 in model group at 3rd, and 6th day and 2nd, 4th, 6th, and 8th week when that in control group was corrected to 1. Conclusion The expression level of ROCK1 protein increased gradually in the formation and development of liver fibrosis, suggesting the Rho/ROCK signaling pathway may be another important signaling pathway of liver fibrosis. Key words: Liver fibrosis; Immunohistochemistry; Real-time quantitative polymerase chain reaction; Rho-associated coiled-coil forming protein kinase 1

Vitamin D Reduces Oxidative Stress-Induced Procaspase-3/ROCK1 Activation and MP Release by Placental Trophoblasts.

Increased microparticle (MP) shedding by placental trophoblasts contributes to maternal vascular inflammatory response and endothelial dysfunction in preeclampsia. Vitamin D has beneficial effects in pregnancy; however, its effect on trophoblast MP release has not been investigated. To investigate if vitamin D could protect trophoblasts from oxidative stress-induced MP release. Placental trophoblasts were isolated from uncomplicated and preeclamptic placentas. Effects of vitamin D on MP release induced by oxidative stress inducer CoCl2 were studied. Annexin V+ MPs were assessed by flow cytometry. Expression of caveolin-1, endothelial nitric oxide synthase (eNOS), procaspase-3, cleaved caspase-3, and Rho-associated coiled-coil protein kinase 1 (ROCK1) in trophoblasts and trophoblast-derived MPs were determined by Western blot. Trophoblasts from preeclamptic pregnancies released significantly more MPs than cells from uncomplicated pregnancies (P < 0.01). CoCl2-induced increase in MP release was associated with upregulation of caveolin-1 and downregulation of eNOS expression in trophoblasts (P < 0.05), which could be attenuated by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 could also inhibit CoCl2-induced procaspase-3 cleavage and ROCK1 activation in trophoblasts. Consistently, CoCl2-induced upregulation of procaspase-3, cleaved caspase-3, and ROCK1 expression in trophoblast-derived MPs were also reduced in cells treated with 1,25(OH)2D3. Placental trophoblasts from preeclamptic pregnancies released more MP than cells from uncomplicated pregnancies. Oxidative stress-induced increase in MP shedding is associated with upregulation of caveolin-1 and downregulation of eNOS expression in placental trophoblasts. Inhibition of caspase-3 cleavage and ROCK1 activation, together with upregulation of eNOS expression, could be the potential cellular/molecular mechanism(s) of vitamin D protective effects on placental trophoblasts.

Hydroxyfasudi attenuates lipopolysaccharide induced endothelial dysfunction via suppressing the expression of Rho-associated coiled-coil protein kinase 1, connexin 43 and caveolin 1

Objective: To investigate the effect and mechanism of hydroxyfasudi (HF), a specific Rho kinase inhibitor, on lipopolysaccharide(LPS)induced endothelial dysfunction. Methods: A total of 24 male Sprague Dawley rats were randomly divided into control group(n=6), HF group(n=6), LPS group(n=6) and LPS + HF group(n=6) with random number table method. There was no special treatment in control group. HF (30 mg/kg) was injected intraperitoneally in HF group. LPS (1 mg/kg) were injected intravenously in LPS group. In LPS+ HF group, HF (30 mg/kg) was injected intraperitoneally, followed by intravenous LPS injection (1 mg/kg) 30 minutes later. All rats were sacrificed after 8 hours, and aortic tissue was extracted. RT-PCR was performed to detect mRNA levels of Rho-associated coiled-coil protein kinase (ROCK)1, connexin (Cx)43 and caveolin (Cav)1. The protein levers of ROCK1, Cx43 and Cav-1 were assessed by Western blot and immunohistochemical staining respectively. Results: (1) RT-PCR experiments showed that mRNA levels of ROCK1(2.67±0.03 vs. 1.00±0.04), Cx43(1.73±0.03 vs. 1.00±0.08), and Cav1(1.85±0.04 vs. 1.0±0.03) in LPS group were significantly higher than in control group(all P<0.05). mRNA levels of ROCK1(0.38±0.02), Cx43(0.58±0.02), and Cav1(0.27±0.01) in LPS + HF group were significantly lower than in LPS group(all P<0.05). (2)Western blot analysis showed that protein levels of ROCK1(3.46±0.82 vs. 2.19±0.56), Cx43(0.33±0.09 vs.0.11±0.06), and Cav1(3.45±0.74 vs. 2.25±0.91) in LPS group were significantly higher than in control group(all P<0.05). Protein levels of ROCK1(1.09±0.52), Cx43(0.01±0.06), and Cav1(2.06±0.40) in LPS + HF group were significantly lower than in LPS group(all P<0.05). (3) Immunohistochemical staining showed that protein levels of ROCK1(84.1±0.953.7±2.9), Cx43(99.1±2.1 vs. 46.2±0.8), and Cav1(167.0±6.4 vs. 84.9±1.0) in LPS group were significantly higher than in control group(all P<0.05). Protein levels of ROCK1(30.4±0.6), Cx43(21.4±1.3), and Cav1(55.8±2.8) in LPS + HF group were significantly lower than in LPS group(all P<0.05). Conclusion: HF attenuates LPS induced endothelial dysfunction probably via suppressing the expression of ROCK1, Cx43 and Cav1.

Research of Taurine in improving the proliferation of neural stem cells in fetal rats with intrauterine growth restriction in fetal rats by inhibiting Rho/ROCK signaling pathway

Objective To investigate the effects of Taurine on the proliferation of neural stem cells(NSCs) with intrauterine growth restriction(IUGR) in fetal rats and the expressions of Ras homolog gene family member A(RhoA) and Rho-associated coiled-coil containing protein kinase(ROCK)Ⅱ in the process of proliferation. Methods IUGR fetal rats models were established with low protein diet.NSCs from subventricular zone were isolated and cultured in vitro.The NSCs were divided into 5 groups: normal control group(group Ⅰ), IUGR group(group Ⅱ), IUGR+ Taurine group(group Ⅲ), IUGR+ ROCK blockers group(group Ⅳ), IUGR+ Taurine+ ROCK blockers group(group Ⅴ). The cells were examined by immunofluorescence for counting fatty acid binding protein 7(FABP7)-positive cells.mRNA and protein levels of RhoA and ROCK were detected by quantitative real time polymerase chain reaction (PCR) and Western blot, respectively. Results (1)Data of group Ⅰ were presented as standard, the number of FABP7 positive cells in group Ⅰ, group Ⅱ, group Ⅲ, group Ⅳ, group Ⅴ was (100.0±1.6)%, (63.8±5.2)%, (82.8±4.3)%, (78.6±2.7)%, and (87.6±3.2)%, respectively.Compared with group Ⅰ, FABP7 positive cells in group Ⅱ decreased signi-ficantly by approximately 36%(t=14.98, P 0.05). Conclusions Taurine supplementation can promote the proliferation of NSCs in IUGR fetal rats by inhibiting Rho/ROCK signaling pathway, thus to improve IUGR fetal brain development. Key words: Taurine; Intrauterine growth restriction; Neural stem cell; Ras homolog gene familymember A; Rho-associated coiled-coil containing protein kinase Ⅱ

Vitamin D suppresses oxidative stress-induced microparticle release by human umbilical vein endothelial cells.

Endothelial microparticle (MP) release was increased in numerous cardiovascular diseases including preeclampsia. Oxidative stress is a potent inducer of endothelial dysfunction. In this study, we aimed to investigate if vitamin D could protect endothelial cells (ECs) from MP release induced by oxidative stress. Endothelial cell (from human umbilical vein) oxidative stress was induced by cultivation of cells under lowered oxygen condition (2%O2) for 48 h and cells cultured under standard condition (21%O2) served as control. 1,25(OH)2D3 was used as bioactive vitamin D. Using annexin-V as a marker of released MP assessed by flow cytometry and cytochrome c reduction assay to measure EC superoxide generation, we found that MP release and superoxide generation were significantly increased when cells were cultured under 2%O2, which could be significantly inhibited by 1,25(OH)2D3. To study the potential mechanisms of 1,25(OH)2D3 protective effects on ECs, EC expression of endothelial nitric oxide synthase (eNOS), p-eNOSSer1177, p-eNOSThr495, caveolin-1, extracellular signal-regulated kinase (ERK), p-ERK, Akt, p-AktSer473, Rho-associated coiled-coil protein kinase 1 (ROCK1), and vitamin D receptor were determined. Microparticle expression of eNOS and caveolin-1 were also determined. We found that under lowered oxygen condition, 1,25(OH)2D3 could upregulate EC eNOS, p-eNOSSer1177, and p-AktSer473 expression, but inhibit cleaved ROCK1 expression. The upregulatory and inhibitory effects induced by 1,25(OH)2D3 were dose dependent. Strikingly, we also found that oxidative stress-induced decrease in ratio of eNOS and caveolin-1 expression in MP could be attenuated when 1,25(OH)2D3 was present in culture. These results suggest that upregulation of eNOSSer1177 and AktSer473 phosphorylation and inhibition of ROCK1 cleavage in EC and modulation of eNOS and caveolin-1 expression in MP could be plausible mechanisms of vitamin D protective effects on ECs.

ROCK inhibition abolishes the establishment of the aquiferous system in Ephydatia muelleri (Porifera, Demospongiae)

The Rho associated coiled-coil protein kinase (ROCK) plays crucial roles in development across bilaterian animals. The fact that the Rho/Rock pathway is required to initiate epithelial morphogenesis and thus to establish body plans in bilaterians makes this conserved signaling pathway key for studying the molecular mechanisms that may control early development of basally branching metazoans. The purpose of this study was to evaluate whether or not the main components of this signaling pathway exist in sponges, and if present, to investigate the possible role of the regulatory network in an early branching non-bilaterian species by evaluating ROCK function during Ephydatia muelleri development. Molecular phylogenetic analyses and protein domain predictions revealed the existence of Rho/Rock components in all studied poriferan lineages. Binding assays revealed that both Y-27632 and GSK429286A are capable of inhibiting Em-ROCK activity in vitro. Treatment with both drugs leads to impairment of growth and formation of the basal pinacoderm layer in the developing sponge. Furthermore, inhibition of Em-Rock prevents the establishment of a functional aquiferous system, including the absence of an osculum. In contrast, no effect of ROCK inhibition was observed in juvenile sponges that already possess a fully developed and functional aquiferous system. Thus, the Rho/Rock pathway appears to be essential for the proper development of the freshwater sponge, and may play a role in various cell behaviors (e.g. cell proliferation, cell adhesion and cell motility). Taken together, these data are consistent with an ancestral function of Rho/Rock signaling in playing roles in early developmental processes and may provide a new framework to study the interaction between Wnt signaling and the Rho/Rock pathway.