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Objective:To investigate the effects of fasudil hydrochloride(FH) on Rho-associated kinase 2(ROCK2) protein and ferroptosis in hippocampal area during early brain injury in rats with subarachnoid hemorrhage(SAH).Methods:Total 36 SPF grade Sprague-Dawley rats were divided into 3 groups by random number table method: Sham group, SAH group and SAH+ FH (a ROCK2 protein inhibitor) group (FH goup) with 12 rats in each group.SAH animal model was established by internal carotid artery perforation.The rats in FH group were injected intraperitoneally with FH(15 mg/kg) 30 minutes after successful modeling, and rats in Sham group and SAH group were injected intraperitoneally with the same volume of 0.9% sodium chloride solution.Twenty-four hours after the intervention, shuttle box test was used to observe the learning and memory ability of rats.The Fe 2+ content in rat hippocampus tissue was detected by colorimetry, and the protein levels of ROCK2 and ferroptosis-related long-chain acyl-CoA synthetase 4(ACSL4) and glutathione peroxidase 4(GPX4) in hippocampus were detected by immunohistochemistry and Western blot.Statistical analysis was performed by SPSS 20.0 software.One-way ANOVA was used for multigroup comparison, and LSD test was used for further pairwise comparison. Results:(1)In the shuttle box test, there were statistically significant differences in the number of avoidance reactions and avoidance reaction time of rats among the three groups( F=20.348, 22.316, both P<0.05). The number of avoidance reaction in SAH group was less than that in Sham group ((17.92±2.94) times, (27.13±3.48) times, P<0.05), the time of avoidance reaction in SAH group was longer than that in Sham group ((9.15±2.87) s, (3.68±1.09) s, P<0.05), while the number of avoidance reaction in FH group ((21.63±4.11) times) was more than that in SAH group, and the time of avoidance reaction ((6.08±1.76) s) was shorter than that in SAH group (both P<0.05). (2) The colorimetry results showed that there was a statistically significant difference in the content of Fe 2+ in hippocampus of rats among the three groups( F=7.965, P<0.05). The Fe 2+ content in SAH group was significantly higher than that of Sham group((0.091±0.032) nmol/mg, (0.038±0.024) nmol/mg, P<0.05), and the Fe 2+ content in the FH group ((0.065±0.021) nmol/mg) was lower than that of SAH group ( P<0.05). (3) There were significant differences in the number of ROCK2, ACSL4 and GPX4 positive cells in hippocampus of rats among the three groups in immunohistochemistry ( F=7.602, 14.171, 36.077, all P<0.05). The positive cells of ROCK2 and ACSL4 in SAH group ((21.63±4.72), (55.13±19.41)) were significantly higher than those of Sham group ((11.63±3.62), (23.38±3.74)) (both P<0.05), and the positive cells of ROCK2 and ACSL4 in FH group ((15.88±6.64), (44.75±8.29)) were both lower than those of SAH group(both P<0.05), while the number of GPX4 positive cells in SAH group (25.38±6.30) was significantly lower than that of Sham group (60.25±10.36) ( P<0.05), and the number of GPX4 positive cells in FH group (45.13±7.51) was higher than that of SAH group( P<0.05). (4)The results of Western blot showed that there were significant differences in the expression levels of ROCK2, ACSL4 and GPX4 proteins in the hippocampus of rats among the three groups( F=4.812, 12.573, 10.849, all P<0.05). The protein expression levels of ROCK2 and ACSL4 in SAH group were significantly higher than those in Sham group(both P<0.05), and the protein expression levels of ROCK2 and ACSL4 in FH group were lower than those in SAH group (both P<0.05), while the expression level of GPX4 protein in SAH group (0.27±0.09) was significantly lower than that in Sham group( P<0.05), and the expression level of GPX4 protein in FH group was higher than that of SAH group ( P<0.05). Conclusion:FH can inhibit ferroptosis in the hippocampus and improve the learning and memory ability of rats, and the mechanism may be related with down-regulation of ROCK2 protein.
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@#Endothelial dysfunctionis traditionally considered irreversible, and endothelial keratoplasty(EK)is almost the only treatment available. Recently, however, a surgery called descemetorhexis without endothelial keratoplasty(DWEK)can regenerate the central corneal endothelial cells in patients with Fuchs endothelial corneal dystrophy(FECD), and local Rho-associated kinase inhibitor can enhance its efficacy.
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Objective:To investigate the mechanism of Oxymatrine on epithelial-mesenchymal transition mediated by RhoA/Rho-associated kinase(ROCK) signaling pathway to prevent and treat ulcerative colitis(UC) and its related canceration by detecting the changes of ROCK, E-cadherin and transforming growth factor-β(TGF-β)in colon tissues of mice. Method:Totally 48 male Balb/c mice were randomly divided into normal control group, model group, low, medium and high-dose Oxymatrine groups (25,50,100 mg·kg-1)and Y-27632 group(10 mg·kg-1), with 8 mice in each group. Mice in control group received distilled water, while all the other mice were treated with 3% dextra sulfate sodium for 7 days to induce the ulcerative colitis model. Since the first day of modeling,Y-27632(10 mg·kg-1)and different doses of Oxymatrine(25, 50, 100 mg·kg-1) were intraperitoneally injectedfor 7 days, and equal volume of PBS was intraperitoneally injected in normal group and model group. Body weight loss, stool consistency and fecal blood loss were observed on a daily basis. On the 8thday, mice were put to death,colon was collected and its length was measured; the scores of disease activity index (DAI) were evaluated; part of the colons were fixed and stained with hematoxylin and eosin (HE) for a histopathological analysis; the ultrastructural changes of mucosa tissue in ulcerative colitis were observed by transmission electron microscope. The expression levels of TGF-β in tissue mucosa were tested by enzyme-linked immunosorbentassay(ELISA). The expression levels of Rho-associated kinase-1, Rho-associated kinase-2, E-cadherin and TGF-β in colon were measured by Western blot and Real-time PCR. Result:Compared with normal group, model group showed the infiltration of a large number of inflammatory cells in mucosa and submucosa, disordered gland arrangement, varying degrees of intestinal mucosal defect and even ulcer formation. Under electron microscopy, microvilli were sparse on the surface of intestinal epithelial cells, the gap between cell junctions was widened, goblet cells were reduced and organelles were swollen. The disease activity index,and the expression levels of ROCK-1 and ROCK-2 proteins in the colonic mucosa of model group were increased(Pβ were decreased(PPPβ were increased(PPPβ and E-cadherin protein and mRNA levels were significantly decreased(PConclusion:Oxymatrine may alleviate ulcerative colitis by down-regulating the expression of Rho kinase,up-regulating the expressions of E-cadherin and TGF-β, inducing the apoptosis of intestinal epithelial cells, and mediating epithelial-mesenchymal transition.
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Glaucoma is one of the main causes of blindness in the world,resulting from a slow and progressive loss of retinal ganglion cells (RGCs) and their axons.So far,intraocular pressure reduction is the main treatment modality to control disease progression.However,not all the patients benefit from this therapy,and the pathophysiology of glaucoma is not always associated with an elevated intraocular pressure.These limitations,together with the multifactorial etiology of glaucoma,urge the pressing medical need for novel and alternative treatment strategies.Such new therapies should focus on not only preventing or retarding RGCs death,but also repairing the injured axons.Glaucoma optic nerve protection therapy is becoming a hot spot of current research,especially Rho associated kinase (ROCK) inhibitors as a new drug for the glaucomatic neuroprotection,which can reduce intraocular pressure,inhibit scar formation after anti-glaucoma surgery,improve blood circulation,prevent RGCs death and axonal degeneration and induce axon regeneration.In this review,we focused on the research status and progress of the classical signal pathway of ROCK inhibitor in glaucoma optic nerve protection.
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PURPOSE: To compare the effects of the barrier function in human trabecular meshwork (TM) cells monolayer and the production of nitric oxide (NO) between trabecular outflow drugs, Rho-associated kinase (ROCK) inhibitors, adenosine, and statin. METHODS: Primary cultured TM cells were exposed to 10 or 25 µM Y-27632, 0.1 or 1 µM N6-cyclohexyladenosine (CHA), or 15 or 30 µM simvastatin for 24 hours. NO production and expression of endothelial nitric oxide synthase mRNA were measured by Griess assay and reverse transcription polymerase chain reaction, respectively. Barrier functions of the TM cell monolayer were measured by carboxyfluorescein and trans-endothelial electrical resistance. The expression of matrix metalloproteinase-2 mRNA was assessed with reverse transcription polymerase chain reaction. RESULTS: In TM cells, treatment with each drug increased endothelial nitric oxide synthase mRNA expression. Treatment with 25 µM Y-27632 and 1.0 µM CHA increased NO production significantly (p = 0.035 and p = 0.043, respectively). Treatment with each drug increased the permeability (all p = 0.001) and decreased the trans-endothelial electron resistance of the TM cell monolayer. Treatment with 0.1 µM and 1.0 µM CHA significantly increased matrix metalloproteinase-2 mRNA expression, but simvastatin inhibited its expression. CONCLUSIONS: Since treatment with ROCK inhibitor more greatly increased NO production and permeability than did adenosine or statin, ROCK inhibitor seems to be more effective for lowering intraocular pressure.
Subject(s)
Humans , Adenosine , Electric Impedance , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Intraocular Pressure , Matrix Metalloproteinase 2 , Nitric Oxide Synthase Type III , Nitric Oxide , Permeability , Polymerase Chain Reaction , Reverse Transcription , rho-Associated Kinases , RNA, Messenger , Simvastatin , Trabecular MeshworkABSTRACT
Rho GTPases belong to Ras superfamily, which is reported to involve in cell migration, phagocytosis, contraction and adhesion. ROCK (also known as Rho-associated kinase) is considered to be one of the most important downstream targets of Rho that is widely investigated. Rho/ROCK signal pathway induces cytoskeletal reorganization, cell migration and stress fiber formation, affects endothelial permeability, tissue constriction and growth, involves in diabetic nephropathy, eye disease, cancer, heart disease, nerve injury disease, hypertension, radiation injury and leukemia. As a novel drug research target, Rho/ROCK signal pathway has received more and more attention. This review provides the basic characteristics and physiological effects of Rho/ROCK signal pathway, the relationships between Rho/ROCK signal pathway and diseases, and the therapeutic methods based on the Rho/ROCK signal pathway.
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Freezing and thawing is one of the most widely used tissue engineering techniques for the preservation of ovaries. Many cells and tissues demonstrate changes in functional gene expression after thawing. Several studies have reported the important roles of angiotensin (AT) system during the ovarian follicular growth. AT system consists of ATII, and ATII receptors type I (ATII-RI) and type II (ATII-RII). However, little is known whether frozen-thawed ovaries show any alteration of AT system member gene expression when treated with survival-enhancing factors. We aimed to investigate whether mass freezing and thawing with or without the use of Rho-associated kinase (ROCK) inhibitors up- or down-regulate the expression of ATII, ATII-RI, and ATII-RII genes on frozen-thawed ovarian tissues. Significant changes in the expression of ATII, ATII-RI, and ATII-RII genes were observed on thawed ovaries when compared to fresh control. The treatment with ROCK inhibitors did not significantly alter their expression. In conclusion, freezing and thawing of ovarian tissue may affect the mRNA expression levels of intra-ovarian AT system genes, and modulation of ROCK inhibitor activity may not regulate AT system on the frozenthawed ovarian tissue.
Subject(s)
Female , Angiotensins , Freezing , Gene Expression , Ovary , rho-Associated Kinases , RNA, Messenger , Tissue EngineeringABSTRACT
AIM:To investigate the role of Rho-associated kinase ( ROCK) inhibitor fasudil in the formation of rabbit urethral stricture after injury and to observe the cell activity , migration and extracellular matrix synthesis in the rabbit urethra fibroblasts.METHODS:The rabbit model of urethral stricture was established by microsurgical techniques .The rabbits were divided into sham operation group , operation group and fasudil (3 mg/kg, 10 mg/kg, 30 mg/kg) groups.The diameter of the stenosis was measured by retrograde urethrography 3 months after surgery .The fibroblasts were isolated from urethral scar, and then incubated with fasudil (12.5 μmol/L, 25 μmol/L, 50 μmol/L) in the presence of transforming growth factor-β1 (TGF-β1, 10 μg/L).The untreated cells were used for control .The cell activity was measured by MTT assay.The cell migration ability was tested by the method of Transwell chambers .The protein expression of ROCK , α-smooth muscle actin (α-SMA) , collagen I and collagen III was determined by Western blot analysis .RESULTS:Fasudil significantly reduced formation of urethral stricture after injury (P<0.05).Cultured rabbit fibroblasts with different con-centrations of fasudil inhibited the cell activity and cell migration ability (P<0.05).The protein expression of ROCK,α-SMA, collagen I and collagen III was also inhibited by treatment with fasudil in a dose -dependent manner ( P<0.05 ) . CONCLUSION:Fasudil inhibits the formation of extracellular matrix and reduces the incidence of urethral stricture after injury by down-regulating TGF-β1-induced Rho/ROCK pathway activation in the rabbit urethra fibroblasts .
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Objective To determine the effect of willed movement on the expression of Nogo-A and Rho-associated kinase (ROCK) in adult rats with cerebral ischemia.Methods Cerebral ischemia/reperfusion injury was established by middle cerebral artery occlusion (MCAO) for 2 h,followed by a 24 h reperfusion in 54 adult rats and the degree of their neurological deficit was evaluated using Longa scale.They were then divided randomly into 3 groups,namely the MCAO group,the environmental modification (EM) group,and the willed movement (WM) group.The rats of MCAO group were raised in a regular breeding box,where they could get food and water freely.Meanwhile,those of the other two groups were raised in a homemade box.For the WM group,the water bottle and food were located on the roof of the homemade box.In each group,six rats were killed on day 3,7 and 15 after reperfusion and their neurological deficits were evaluated respectively.Immunohistochemistry assay was employed to examine the expression of Nogo-A and ROCK in the brain tissue around the ischemic foci.Results The rats of the WM group showed lessened neurological deficits on day 15 compared with the model and EM group.Their expression of Nogo-A decreases from(28.92 ± 2.17)/hpf on day 7 to (24.38 ± 2.29)/hpf on day 15 and that of ROCK did from (40.03 ± 2.14)/hpf to (38.08 ± 2.07) / hpf,lower than those of the model and EM group.However,no significant differences were found in the expression of Nogo-A and ROCK between the model group and EM group at any time points.Conclusion Willed movement could promote the functional recovery of neurological deficits in rats with ischemia after reperfusion,which is probably in relation to restrained expression of Nogo-A and Rho-associated in the tissue around the brain ischemic foci.
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PURPOSE: To investigate the effect of ROCK inhibitor Y27632 on the human corneal endothelial cell proliferation in vitro and in vivo. METHODS: Using corneal endothelial cells isolated and cultured from human donor cornea, we compared the effect of Y27632 (10 microM) on the proliferation in vitro by flow cytometry analysis. For the evaluation of the effect of Y27632 (10 mM) in vivo, corneal thickness and wound area were analyzed for the corneal endothelial wound rabbit model induced by transcorneal freezing. RESULTS: Ki67 positive cells were increased in the Y27632 group (9.1 +/- 4.1%) than the control group (8.0 +/- 5.9%), whereas annexin V positive cells in the Y27632 group (2.9 +/- 1.0%) were decreased compared to the control group (4.2 +/- 2.2%). However these were not statistically significant. Wound area after Y27632 application in animal model is concerned, the control group showed significant smaller area (45.6 +/- 0.6 mm2) compared to the Y27632 group (49.3 +/- 0.8 mm2; p = 0.029, Mann-Whitney U test), however, these were not significantly different from the baseline. Corneal thickness was not different between the two groups. CONCLUSIONS: Different from other reports for the effect of Y27632, no significant effect on the proliferation in vitro and wound healing in vivo, regarding human corneal endothelial cell, were found in this study.
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Humans , Amides , Annexin A5 , Cornea , Endothelial Cells , Flow Cytometry , Models, Animal , Pyridines , Tissue Donors , Wound HealingABSTRACT
BACKGROUND AND OBJECTIVES: Recent studies indicate that in response to vasoconstrictor stimuli, the small GTPase RhoA and its down-stream effector, Rho-associated kinase 2 (ROCK)/Rho-kinase, are associated with hypercontraction of the vascular smooth muscle of coronary arteries through augmentation of myosin light chain phosphorylation and Ca2+ sensitization. Expression of ROCK/Rho-kinase mRNA was significantly increased and up-regulated in the spastic coronary artery in a porcine model, and a specific inhibitor of ROCK/Rho-kinase inhibited coronary artery spasm in humans. We therefore explored the role of ROCK2 polymorphisms in the pathogenesis of vasospastic angina (VA). SUBJECTS AND METHODS: We studied 106 patients with VA who exhibited spontaneous or provoked coronary spasm during coronary angiography and compared the prevalence of ROCK2 polymorphisms between this group of patients with VA and controls whose angiograms were normal, and in whom the ergonovine test did not cause spasm (n=107). Five single nucleotide polymorphisms (SNPs) of the ROCK2 gene were selected. SNPs were genotyped by high-resolution melting. Linkage disequilibrium and haplotype analyses were performed using the SHEsis program. RESULTS: The prevalence of genotypes of the 5 interesting SNPs in patients with VA was not different from that in the control group. In haplotype analysis, the haplotype G-T-C-T-G (in order of rs978906, rs2271621, rs2230774, rs1515210, and rs3771106) was significantly associated with a decreased risk of VA (p=0.007). CONCLUSION: The haplotype G-T-C-T-G in the ROCK2 gene had a protective effect against VA, suggesting the involvement of ROCK2 in VA pathogenesis.
Subject(s)
Humans , Coronary Angiography , Coronary Vasospasm , Coronary Vessels , Ergonovine , Freezing , Genotype , GTP Phosphohydrolases , Haplotypes , Linkage Disequilibrium , Muscle Spasticity , Muscle, Smooth, Vascular , Myosin Light Chains , Phosphorylation , Polymorphism, Single Nucleotide , Prevalence , rho-Associated Kinases , RNA, Messenger , SpasmABSTRACT
Objective To study the expression of mRNA of OX40 and OX40L in the sciatic nerve,spleen,peripheral blood mononuclear cells and lymph nodes of EAN under the influence of Rho-kinase inhibitor.Methods All 54 female Lewis rats were divided into 3 groups:the EAN group,the EAN+ Rho-kinase inhibitor group and the complete Freund's adjuvant(CFA)group.The rats were sacrificed at 9,17 and 26 days after immunized.Ox40 and OX40L mRNA were detected by RT-PCR which came from spleens,sciatic nerves,peripheral blood mononuclear cells and lymphonodes.Results In EAN+ Rho-kinase inhibitor group,the mRNA expression of OX40 were 0.266±0.031,0.298±0.024 and 0.113±0.018 at 9.17 and 26 days in the sciatic nerve,the expression were 0.453±0.030,0.496±0.100 and 0.220±0.016 in the lymph nodes.The mRNA expression of OX40L were 0.247±0.018.0.298±0.026 and 0.165±0.013 in the sciatic nerve,the expression were 0.283±0.027,0.306±0.011 and 0.161±0.012 in the lymph nodes.The mRNA expression of OX40 and OX40L in EAN+Rho-kinase inhibitor group was lower than EAN group at the three time points(t=2.24-4.89,P＜0.05),and the demyelination and inflammation cells infiltrating were ameliorated in spinal nerve.CFA group didn't show any clinical manifestation.Conclusion Rho-kinase inhibitor may ameliorate tlIe development of EAN through inhabiting the OX40 and OX40L activation.
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Objective To investigate the role of RhoA/Rho associated kinase-2 (RhoA/ROCK-2) in the development of pulmonary hypertension (PH) in patients with obstructive sleep apnea-hypopnea syndrome (OS-AHS). Methods Thirty patients diagnosed as OSAHS by polysomnograshy(PSG) test in our sleep laberatoty were recruited as the observation group, and fifteen healthy subjects matched in gender, age and body mass index (BMI) were recruited as the controls. Pulmonary arterial pressure was measured by echocardiography. Serum RhoA/ROCK-2 levels were measured. Results The level of PAP was (47.30±12.85)mm Hg in OSAHA patients complicated with PH, (22.31±3.07)mm Hg in OSAHA patients without PH, which were significantly higher than that in the controls (19.47±1.92) mm Hg (W=175.50, P < 0.05). The serum RhoA and ROCK-2 in OSAHA patients with-out PH (10.43±3.10 and 22.31±16.10 μ/L, respectively) were significantly higher those in the controls (2.94±1.20)μg/L and (6.04±0.28)μg/L, respectively) (W=120.00, W= 121.00, respectively, P<0.05), whereas significantly lower than that in OSAHA patients complicated with PH(14.85±8.49)μg/L, (36.81±12.69) μg/L, respectively) (H =29.172, H =30.242, respectively, P <0.05). There was a positive correlation between PAP and AHI in patients with OSAHS, whether complicated with PH or not(r_s=0.793, r_s=0.887,P <0.05), and there was a negative correlation between PAP and LSaO2 in patients with OSAHS (r_s=-0. 562,r_s = -0.751, P <0.05). There were positive correlations between the level of RboA/ROCK-2 and PAP in patients with OSAHS(r_s = 0.793,r_s = 0.887,P < 0.05). Finally, there was a positive correlation between the level of RhoA and ROCK-2 in patients with OSAHS (r_s = 1.000,r_s = 1.000,P < 0.05). Conclusions OSAHS is an inde-pendent risk factor for pulmonary hypertension. The levels of serum RhoA/ROCK-2 in OSAHS patients with PH were increased in the development of the disease. It may play an important role in the process of pulmonary hyper-tension in patients with OSAHS.
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The disability and mortality of cerebrovascular diseases are high,and they cause significant harm to human health.A number of studies have found that a great many factors participated in the pathophysiological processes of cerebrovascular diseases including Rho-associated kinse.This article reviews the mechanisms of action on Rho-associated kinase in the pathophysiological processes of cerebrovascular diseases.
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PURPOSE: A partial bladder outlet obstruction(PBOO) related detrusor hypertrophy is associated with up-regulation of the Rho kinase activity in an experimental animal model, and has been implicated in PBOO induced bladder dysfunction. The effect of a Rho kinase inhibitor on the voiding function in anesthetized rats with PBOO was investigated. MATERIALS AND METHODS: Eighteen Sprague-Dawley rats were divided into control(9 rats) and experimental(9 rats) groups. The experimental group was partially obstructed for 6 weeks, with cystometrograms(CMG) then were performed. The number of voids, and the intercontraction interval (ICI) and peak pressure(PP) were recorded. Rho kinase inhibitors were administered to the experimental group. An additional CMG was performed to observe the effects of Rho kinase inhibition. Bladder tissues were immunohistochemically(IHC) evaluated for the expression of RhoA protein. RESULTS: The bladder weights of the PBOO group were significantly increased compared with the control group(p<0.05). Significant increases in the voiding frequency and PP, but a significant decrease in the ICI was observed in the PBOO group compared to the control group on the CMG (p<0.05). The voiding frequency of the PBOO group was significantly decreased after Rho kinase inhibitor treatment(p<0.05). The Rho kinase inhibitor treated group showed a decrease in the PP and an increase in the ICI compared to the PBOO group. The IHC showed a higher RhoA protein expression in the bladder tissues of the PBOO group. CONCLUSIONS: H-1152, a specific inhibitor of Rho kinase, attenuates the PBOO-related detrusor overactivity in a rat model. The Rho kinase inhibitor appears to be a novel strategy for the management of bladder overactivity.