RhoA/ROCK-1 Signaling Pathway and Oxidative Stress in Coronary Artery Disease Patients

Abstract Introduction: Coronary artery disease (CAD) is an ischemic condition that occurs as a result of partial or complete interruption of blood flow by narrowing or complete blockage of the vessels supplying the heart, which are called coronary arteries. Our objective in this study is to investigate the RhoA/Rho-associated kinase (ROCK)-1 signaling pathway and oxidative stress in CAD patients. Methods: A total of 81 individuals aged between 40-70 years - including 45 patients (15 females and 30 males) who were admitted to the Artvin State Hospital Cardiovascular Surgery Clinic and were diagnosed with CAD and 36 healthy volunteers (15 females and 21 males) - participated in this study. Serum samples were tested for total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein, malondialdehyde (MDA), superoxide dismutase (SOD), RhoA, and ROCK-1 values. Results: Serum RhoA, MDA levels, and ROCK-1 activity in the CAD group were found to be statistically significantly higher than in the control group (P<0.001). Concordantly, serum SOD activity was found to be statistically significantly lower in the CAD group than in the control group (P<0.001). Conclusion: Inhibition of the activity of RhoA/ROCK-1 pathway would be beneficial in treating cardiovascular diseases since this pathway plays an important role in the development of these diseases.

Role of RhoA/ROCK2 signaling pathway in trilobatin-induced reduction of cerebral ischemia-reperfusion injury in rats / 中华麻醉学杂志

Objective:To evaluate role of Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil protein kinase 2 (ROCK2) signaling pathway in trilobatin-induced reduction of cerebral ischemia-reperfusion (I/R) injury in rats.Methods:Eighty clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 230-280 g, were divided into 4 groups ( n=20 each) using a random number table method: sham operation group (group S), cerebral I/R group (group IR), cerebral I/R plus trilobatin group (group T) and cerebral I/R plus trilobatin plus RhoA/ROCK2 signaling pathway agonist AA group (group A). The model of focal cerebral I/R injury was developed by middle cerebral artery occlusion in anesthetized animals.Trilobatin 15 mg/kg was given by gavage twice a day for 3 consecutive days in T and A groups.RhoA/ROCK2 signaling pathway agonist AA 10 mg/kg was intraperitoneally injected before each administration by gavage in group A. Neurobehavioral score was assessed at 24 h of reperfusion, then the rats were sacrificed, and the hippocampal tissues were isolated for determination of the apoptosis rate of hippocampal neurons (by flow cytometry), cerebral infarction volume (by TTC staining), and expression of phosphorylated RhoA (p-RhoA), ROCK2 and cleaved caspase-3 (by Western blot) and for microscopic examination of ultrastructure of hippocampal neurons (with a transmission electron microscope). Results:Compared with group S, the neurobehavioral score, apoptosis rate of hippocampal neurons, and cerebral infarction volume were significantly increased, the expression of p-RhoA, ROCK2 and cleaved caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was aggravated in group IR.Compared with group IR, the neurobehavioral score, apoptosis rate of hippocampal neurons and cerebral infarction volume were significantly decreased, the expression of p-RhoA, ROCK2 and cleaved caspase-3 was down-regulated ( P<0.05), and the pathological damage to hippocampal neurons was alleviated in group T. Compared with group T, the neurobehavioral score, apoptosis rate of hippocampal neurons, and cerebral infarction volume were significantly increased, the expression of p-RhoA, ROCK2 and cleaved caspase-3 was up-regulated ( P<0.05), and the pathological damage to hippocampal neurons was aggravated in group A. Conclusions:RhoA/ROCK2 signaling pathway is involved in trilobatin-induced reduction of cerebral I/R injury, which may be related to inhibition of apoptosis in hippocampal neurons of rats.

Efficacy and safety of Fasudil on vasospasm caused by subarachnoid hemorrhage in elderly patients / 中华老年医学杂志

Objective:To evaluate the efficacy and safety of fasudil on vasospasm caused by subarachnoid hemorrhage in elderly patients.Methods:A total of 100 elderly patients with subarachnoid hemorrhage admitted to our hospital from January 2015 to May 2018 were enrolled as research objects.They were randomly divided into the Fasudil group(n=50, receiving the Rho kinase inhibitor Fasudil therapy)and the Nimodipine group(n=50, receiving Nimodipine therapy). The cerebral vasospasm and cerebral infarction lesions, the ability of daily life, clinical prognostic score, the incidence of symptomatic cerebral vasospasm and adverse reactions during treatment were evaluated and compared between the two groups.Results:After treatment, the incidences of cerebral vasospasm and cerebral infarction in Fasudil group were 2.04%(1/49)and 6.12%(3/49), respectively, which were lower than those in the Nimodipine group[12.50%(6/48)and 20.83%(10/48), respectively]( χ2=6.134 and 6.794, P=0.047 and 0.033). The scores of daily living ability was better in the Fasudil group than in the Nimodipine group(16.09±1.06 vs.22.91±1.66, t=7.721, P=0.026). The incidence of adverse reactions was lower in the Fasudil group than in the Nimodipine group(4.08% or 2/49 vs.16.7% or 8/48, χ2=6.362, P=0.040). There was no statistically significant difference in the proportion of patients with good prognosis between Fasudil group and Nimodipine group. Conclusions:Rho kinase inhibitor Fasudil can effectively prevent and improve cerebral vasospasm caused by subarachnoid hemorrhage, which is beneficial for improving the clinical prognosis and quality of life of the elderly patients with subarachnoid hemorrhage.

Effect of lidocaine on Rho/ROCK signaling pathway during endotoxin-induced acute lung injury in rats / 中华麻醉学杂志

Objective To evaluate the effect of lidocaine on Ras homologue (Rho)/Rho kinase (ROCK) signaling pathway during endotoxin-induced lung injury (LI) in rats.Methods Forty SPF male Wistar rats,aged 5-8 weeks,weighing 200-250 g,were divided into 5 groups (n=8 each) using a random number table method:control group (group C),lipopolysaccharide (LPS) group (group LPS)and lidocaine at 3 different doses groups (L1-3 groups).LI was induced by intraperitoneal LPS 5 mg/kg (0.1 ml).The equal volume of normal saline was given intraperitoneally in group C.Lidocaine 2,4 and 8 mg/kg was intraperitoneally injected at 1 h before LPS administration in L1-3 groups,respectively.The animals were sacrificed at 6 h after LPS or normal saline administration.Broncho-alveolar lavage fluid (BALF) was collected for determination of concentrations of interleukin-1 beta (IL-1β),IL-6 and tumor necrosis factor-alpha (TNF-α) (by enzyme-linked immunosorbent assay).The lung tissues were obtained for examination of the pathological changes which were scored and for measurement of the wet/dry weight ratio (W/D ratio) and activities of myeloperoxidase (MPO) and for determination of the expression of Rho,ROCK1,ROCK2,myosin phosphatase target protein 1 (MYPT1),phosphorylated MYPT1 (p-MYPT1)and ZO-1 (by Western blot).The phosphorylation of MYPT1 was calculated.Results Compared with group C,the activity of MPO,lung injury score,W/D ratio and concentrations of IL-1β,IL-6 and TNF-αin BALF were significantly increased,the expression of Rho,ROCK1 and ROCK2 was up-regulated,the phosphorylation of MYPT-1 was increased,and the expression of ZO-1 was down-regulated in the other four groups (P＜0.05).Compared with group LPS,the activity of MPO,lung injury score,W/D ratio and concentrations of IL-1β,IL-6 and TNF-α in BALF were significantly decreased,the expression of Rho,ROCK1 and ROCK2 was down-regulated,the phosphorylation of MYPT-1 was decreased,and the expression of ZO-1 was up-regulated in L1-L3 groups (P＜0.05).Conclusion Lidocaine can inhibit activation of Rho/ROCK signaling pathway during endotoxin-induced LI in rats,and the effect may be related to the antiinflammatory mechanism of lidocaine.

Comparative Study of ROCK1 and ROCK2 in Hippocampal Spine Formation and Synaptic Function / 神经科学通报·英文版

Rho-associated kinases (ROCKs) are serine-threonine protein kinases that act downstream of small Rho GTPases to regulate the dynamics of the actin cytoskeleton. Two ROCK isoforms (ROCK1 and ROCK2) are expressed in the mammalian central nervous system. Although ROCK activity has been implicated in synapse formation, whether the distinct ROCK isoforms have different roles in synapse formation and function in vivo is not clear. Here, we used a genetic approach to address this long-standing question. Both Rock1 and Rock2 mice had impaired glutamatergic transmission, reduced spine density, and fewer excitatory synapses in hippocampal CA1 pyramidal neurons. In addition, both Rock1 and Rock2 mice showed deficits in long-term potentiation at hippocampal CA1 synapses and were impaired in spatial learning and memory based on the water maze and contextual fear conditioning tests. However, the spine morphology of CA1 pyramidal neurons was altered only in Rock2 but not Rock1 mice. In this study we compared the roles of ROCK1 and ROCK2 in synapse formation and function in vivo for the first time. Our results provide a better understanding of the functions of distinct ROCK isoforms in synapse formation and function.

Metformin Ameliorates Lipotoxic β-Cell Dysfunction through a Concentration-Dependent Dual Mechanism of Action

BACKGROUND: Chronic exposure to elevated levels of free fatty acids contributes to pancreatic β-cell dysfunction. Although it is well known that metformin induces cellular energy depletion and a concomitant activation of AMP-activated protein kinase (AMPK) through inhibition of the respiratory chain, previous studies have shown inconsistent results with regard to the action of metformin on pancreatic β-cells. We therefore examined the effects of metformin on pancreatic β-cells under lipotoxic stress.METHODS: NIT-1 cells and mouse islets were exposed to palmitate and treated with 0.05 and 0.5 mM metformin. Cell viability, glucose-stimulated insulin secretion, cellular adenosine triphosphate, reactive oxygen species (ROS) levels and Rho kinase (ROCK) activities were measured. The phosphorylation of AMPK was evaluated by Western blot analysis and mRNA levels of endoplasmic reticulum (ER) stress markers and NADPH oxidase (NOX) were measured by real-time quantitative polymerase chain reaction analysis.RESULTS: We found that metformin has protective effects on palmitate-induced β-cell dysfunction. Metformin at a concentration of 0.05 mM inhibits NOX and suppresses the palmitate-induced elevation of ER stress markers and ROS levels in a AMPK-independent manner, whereas 0.5 mM metformin inhibits ROCK activity and activates AMPK.CONCLUSION: This study suggests that the action of metformin on β-cell lipotoxicity was implemented by different molecular pathways depending on its concentration. Metformin at a usual therapeutic dose is supposed to alleviate lipotoxic β-cell dysfunction through inhibition of oxidative stress and ER stress.

Mechanism underlying hydrogen-induced improvement of intestinal barrier function in septic mice: RhoA-mDia1 signaling pathway / 中华麻醉学杂志

Objective To investigate the relationship between the mechanism underlying hydrogeninduced improvement of intestinal barrier function and RhoA-mDia1 signaling pathway in septic mice.Methods Sixty male C57BL/6 mice,weighing 20-25 g,aged 6-8 weeks,were divided into 3 groups (n =20 each) using a random number table method:control group (group C),sepsis group (group S),and sepsis plus H2 group (group SH).Sepsis was produced by cecal ligation and puncture (CLP) in mice anesthetized with chloral hydrate.Mice in group SH inhaled air containing 2％ hydrogen for 1 h starting from 1 and 6 h after CLP.At 24 h after CLP,blood samples were obtained by cardiac puncture to measure the activity of serum diamine oxidase (DAO) and to count colony-forming unit (CFU) after bacterial culture,the small intestinal samples were obtained for microscopic examination of the ultrastructure of intestinal epithelial cells (with a transmission electron mnicroscope) and for measurement of the expression and distribution of tight junction protein ZO-1 in intestinal epithelial cells (by immunofluorescence) and expression of RhoA,expression and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) and expression of mDia1 (by Western blot).The p-MYPT1/MYPT1 ratio was calculated.Results Compared with group C,the serum DAO activity and whole blood CFU counts were significantly increased,the expression of RhoA was up-regulated,and the expression of ZO-1 was down-regulated in S and SH groups,the p-MYPT1/MYPT1 ratio was significantly increased,and the expression of mDia1 was down-regulated in group S,and the p-MYPT1/MYPT1 ratio was significantly decreased,and the expression of mDia1 was up-regulated in group SH (P＜0.05).Compared with group S,the serum DAO activity,whole blood CFU counts and p-MYPT1/MYPT1 ratio were significantly decreased,the expression of RhoA was down-regulated,the expression of ZO-1 and mDia1 was up-regulated (P＜0.05),and the ultrastructure changes of intestinal tissues were significantly improved in group SH.Conclusion The mechanism by which hydrogen improves intestinal barrier function is related to inhibiting RhoA activity and increasing mDia1 activity in intestinal epithelial cells of septic mice.

Influence of Rho kinase inhibitor combined furosemide and spironolactone on cardiac function , serum levels of AST , LDH and CK‐MB in patients with acute left heart failure／ / 心血管康复医学杂志

To explore application value of Rho kinase inhibitor (RKI) combined furosemide and spironolactone in patients with acute left heart failure (ALHF).Methods : A total of 94 ALHF patients were randomly and equally divided into diuretic group (received furosemide and spironolactone based on routine treatment ) and triple therapy group (received RKI‐‐fasudil hydrochloride based on diuretic group ) , both groups were continuously treated for 7d.LVESV , LVEDV , LVEF ,serum levels of aspartate transaminase (AST) , lactate dehydrogenase (LDH) and creatine kinase isoenzyme MB (CK‐MB) before and after treatment , therapeutic effects were observed and compared between two groups .Results : Total effective rate of triple therapy group was significantly higher than that of diuretic group (95.75% vs.82.98%) , P＝0.045. Compared with before treatment , there was significant rise in LVEF , and significant reductions in LVESV , LV‐EDV ,serum levels of AST , LDH and CK‐MB in two groups after treatment , P＝0.001 all ;compared with diuretic group after treatment , there was significant rise in LVEF [ (48.27 ± 5.95)% vs.(55.14 ± 6.74)%] , and significant reductions in LVESV [ (86.29 ± 10.41) ml vs.(65.96 ± 9.84) ml] , LVEDV [ (133.71 ± 13.42) ml vs.(120.35 ± 11.25) ml] , serum levels of AST [ (81.23 ± 10.44) U／L vs.(57.58 ± 8.42) U／L] , LDH [ (184.24 ± 13.51) U／Lvs.(124.65 ± 12.42) U／L] and CK‐MB [ (187.84 ± 13.45) U／L vs.(132.54 ± 11.69) U／L] in triple therapy group , P＝0.001 all. There was no significant difference in adverse reactions during treatment between two groups , P＞0.05 both .Conclusion :Rho kinase inhibitor combined furosemide and spironolactone can significantly improve cardiac function and reduce myocar ‐dial damage , and it＇s safe and reliable , which is worth extending .

Research Progress and Forensic Application on the Pathogenesis of Coronary Artery Spasm / 法医学杂志

Coronary artery spasm(CAS)is a hyper-contraction of segmental coronary artery in response to multiple stimuli. At present, it's still in lack of specific diagnostic indicators of sudden cardiac death caused by CAS. This review summarizes current researches on the mechanisms of CAS and describes the roles of vascular endothelial dysfunction and vascular smooth muscle hypersensitivity in the course of CAS. Furthermore, the molecular mechanisms of the endogenous NO and endothelin-1 cause vascular endothelial dysfunction, and the phosphorylation of MLC2, Rho kinase and endoplasmic reticulum stress related to vascular smooth muscle hypersensitivity are discussed. Meanwhile, the possibility of forensic application for the related molecules on the diagnosis of sudden cardiac death caused by CAS are also explored.

ω-3 polyunsaturated fatty acids inhibits RHO-ROCK1 signaling pathway in SGC7901 gastric cancer cells / 中国医师杂志

Objective This study was aimed to investigate the effects of ω-3 polyunstaurated fatty acids (ω-3 PUFAs) on the growth of gastric cancer cells in nude mice,and to find whether the Ros homolog gene Rho-associated coiled-coil containing protein kinase 1 (RHO-ROCK1) signaling pathway is involved.Methods 16 BALB/C nude mice were injected subcutaneously with SGC7901 gastric cancer cells to establish the tumor-bearing mouse model.The mice were randomized:control group (normal saline) and intervention group (ω-3 PUFAs).The mRNA expression of Ros homolog gene family,member A (RHOA),RHOC,and ROCK1 in tumor tissue were detected by quantitative polymerase chain reaction (qPCR).Immunofluorescence and Western blot were used to detect RHOA,RHOC,and ROCK1 protein expression.Results The volume and weight of the tumors in the ω-3 PUFAs group were slightly smaller than that in the control group (P ＞ 0.05).Compared to the control group,hematoxylin and eosin staining showed multifocal tumor necrosis in the ω-3 PUFAs group,while the tumors of the control group showed abundant blood supply.qPCR and Western blot showed that the mRNA and proteins expression of RHOA and ROCK1 in the ω-3 PUFAs group was significantly lower than those in the control group (P ＜ 0.05).The immunofluorescence redults also showed that the expression of these proteins in the ω-3 PUFAs group was slightly lower than that in the control group.Conclusions These results suggested that ω-3 PUFAs may affect the growth of gastric cancer in nude mice by affecting the expression of RHOA,RHOC and ROCK1,thus inhibiting the excessive proliferation of gastric cancer cells and leading to tumor necrosis.

Mechanism of RhoA and its progress in gastric cancer / 肿瘤研究与临床

The morbidity and mortality of gastric cancer is high all over the world, while less specific target of gastric cancer has been found. RhoA is highly expressed in gastric cancer, which is closely related to tumor invasion and metastasis. An increasing evidence has showed that RhoA is a potential target for gene therapy of gastric cancer, and it can predict the invasion and metastasis of tumor cells. This paper reviews the mechanism of RhoA and research progress in gastric cancer.

Role of RhoA/ROCK2 signaling pathway in propofol-induced neuroapoptosis in hippocampus of newborn rats / 中华麻醉学杂志

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 Experiment Ⅰ 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.Experiment Ⅱ 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 concentrauon 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 Experiment Ⅰ 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-rcgulatcd 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.

Antenatal taurine supplementation improves neural axon development in fetal rats with intrauterine growth restriction by inhibiting the activity of Rho-ROCK signaling pathway / 中华围产医学杂志

ObjectiveTo investigate the effects of prenatal taurine supplementation on the Rho-ROCK signaling pathway activity and synaptophysin (Syp) expression in brain tissues of rats with intrauterine growth restriction.MethodsEighteen pregnant Sprague-Dawley rats were randomly divided into control group, fetal growth restriction (FGR) group and taurine group, with six rats in each group. Low-protein diet was given in FGR and taurine groups to establish an FGR model. Taurine 300 mg/(kg·d) was supplemented from gestational day 12 until delivery in taurine group. The mRNA expression levels of neurite growth inhibitor-A(Nogo-A), neurite growth inhibitor receptor (NgR), Rho-A and ROCKⅡin fetal rat brain were detected using reverse transcriptase polymerase chain reaction (n=24), which are the key signaling molecules of the Rho-ROCK signal pathway. The protein expression levels of Nogo-A and NgR were detected by Western blot (n=12). The mean optical density in Nogo-A, NgR and Syp was determined by immunohistochemistry (n=18). One-way analysis of variance and LSD-t test were used for statistical analysis.Results(1) Expression of mRNA: the expression levels of Nogo-A, NgR, Rho-A and ROCKⅡ mRNA in fetal rat brain were 4.09±1.34, 3.01±0.77, 39.89±7.71 and 7.82±1.83, respectively in FGR group, and were significantly higher than in control group (1.00±0.13, 1.00±0.10, 1.02±0.30 and 1.00±0.10) (t=4.735, 5.204, 7.682 and 10.675, allP0.05). (2) Expression of protein by Western blot: the expressions of Nogo-A and NgR protein in fetal rat brain were 1.51±0.09 and 0.31±0.05 in FGR group, 0.82±0.06 and 0.06±0.01 in taurine group, and 1.04±0.10 and 0.09±0.12 in control group. The expression was significantly higher in FGR group than in control group (t=9.644 and 5.285, bothP0.05). (3) Positive expression of protein: the positive expressions of Nogo-A and NgR protein in fetal rat brain were 0.28±0.06 and 0.11±0.02 in FGR group, 0.10±0.02 and 0.04±0.01 in taurine group, and 0.07±0.01 and 0.04±0.01 in control group. The expression was significantly higher in FGR group than in control group (t=9.778 and 7.645, bothP0.05). The positive expression of Syp protein in fetal rat brain was 0.08±0.01 in FGR group, and was significantly lower than in control group (0.16±0.04,t=4.600,P0.05).ConclusionsPrenatal taurine supplementation can improve neural axon development via down-regulating the expressions of the key molecules of Rho-ROCK signal pathway in fetal rat brain tissue.

Fasudil blocks muscle atrophy and C2C12 myoblasts respiratory dysfunction triggered by ROCK1 / 第二军医大学学报

Objective To confirm whether fasudil can block C2C12 myoblasts respiration dysfunction triggered by Rho-associated coiled-coil containing protein kinase 1 (ROCK1), and whether it can block the occurrence of muscle atrophy. Methods C2C12 myoblasts were cultured in vitro, and 2% horse serum was used to induce cell differentiation and maturation. The obtained mature muscle tubule cells were divided into four groups according to the different stimuli: Ad-GFP group, only transfected GPT-adenovirus vector (Adv) in C2C12 myoblasts; Ad-ROCKl group, transfected ROCKl-Adv in C2C12 myoblasts to induce ROCK1 overexpression; Ad-GFPF group, transfected GFP-Adv and given 10 μmol/L fasudil in C2C12 myoblasts; and Ad-ROCKIF group, transfected ROCKl-Adv and given 10 μmol/L fasudil in C2C12 myoblasts. The oxygen consumption rate (OCR) and extracelluar acidification rate (ECAR) of C2C12 myoblasts under different stimulation conditions were evaluated by cell energy metabolism analyzer (Seahorse), so as to determine the effect of ROCK1 overexpression and fasudil stimulation on the respiratory function of C2C12 myoblasts. Mitochondrial fission was measured by MitoTracker® red fluorescent probes. The expressions of ROCK1, mitochondrial related protein 1 (Drpl) and phosphorylated p-Drpl, E3 ubiquitin ligase muscle RING finger-1 protein (MuRFl) and muscle atrophy F-box (MAFbx, Atrogin 1) was measured by Western blotting analysis. Results Seahorse analysis showed that the OCR, ECAR, basal respiration, maximal respiration and respiration required for coupling ATP of C2C12 myoblasts in the Ad ROCK1 group were significantly increased compared with those in the Ad-GFP group (P<0.01); Meanwhile, MitoTracker® staining showed that the mitochondrial fission was increased and the mitochondrial size frequency distribution shifted left in the Ad-ROCK1 group. After exposed to fasudil. the OCR and EACR of C2C12 myoblasts in the Ad-ROCKIF group were significantly decreased versus the Ad-ROCK1 group, and the basal respiration and maximal respiration were significantly increased (P<0.05). Western blotting analysis showed that p-Drpl/Drpl ratio, and the expressions of ROCK1, MuRFl and Atroginl in Ad-ROCKIF group were significantly reduced compared with Ad-ROCKl group (P<0.05). Conclusion Fasudil, an inhibitor of ROCK1, can block the abnormal cell respiration of C2C12 myoblasts caused by overexpressed ROCK 1 in vitro, and can reduce the activity of mitochondrial kinetic protein and the expression of muscle atrophy-related proteins.

Role of ROCK1 in the podocyte injury induced by oxidized low-density lipoprotein / 中华肾脏病杂志

Objective@#To explore the role of ROCK1 in oxidized low-density lipoprotein (ox-LDL) induced podocyte injury and its possible mechanism.@*Methods@#The conditionally immortalized mouse podocyte cells were cultured in vitro and exposed to 20 μg/ml ox-LDL for 24 h. Western blotting was used to analyze the expression level of p-MYPT, nephrin, LC3-Ⅱ, p62, p-ULK1 in groups of control, ox-LDL, ROCK1 siRNA with ox-LDL, wtROCK1 with ox-LDL. Podocytes were incubated with DiI labeled ox-LDL for 4 h and fluorescence microscope was used to analyze lipid distribution.@*Results@#Compared with control group, ox-LDL increased cell cholesterol accumulation, activated ROCK along with decreased nephrin, LC3-Ⅱ(P<0.05), and increased p62, and p-ULK1 expression (P<0.05). Over-expression of ROCK1 significantly decreased the expression of nephrin and LC3-Ⅱ, but up-regulated the levels of p62, p-ULK1 and cell cholesterol accumulation in ox-LDL stimulated podocytes (P<0.05). In contrast, Inhibition of ROCK1 protected podocyte by improved lipophagy.@*Conclusion@#ROCK1 mediated disfunction of lipophagy contributes to the ox-LDL induced podocyte injury.

MicroRNA-124 inhibits rho associated kinase 1 activity to ameliorate the damages of glomerular endothelial cells caused by high glucose / 中华肾脏病杂志

Objective@#To explore the effects of miR-124-ROCK1 signal pathway in the damages of glomerular endothelial cells (GEnCs) induced by high glucose.@*Methods@#Rat glomerular endothelial cells were cultured in different glucose concentrations: normal control group (NG: 5.5 mmol/L), high glucose group (HG: 30.0 mmol/L), and cells were treated with ROCK1 inhibitor Y27632, miR-124-3p mimic, miR-124-3p inhibitor. The expressions of ROCK1 activity, cell apotosis and tight junction proteins were detected by Western blot. The cell tight junction protein ZO-1 in those groups were assessed by laser scanning confocal microscope.@*Results@#High glucose significantly decreased miR-124 expression (P<0.01), ROCK1 activity (P-MYPT1/MYPT1), and cell apoptosis (Cleaved-Caspase3/pro-Caspase3) were found increased while the tight junction proteins ZO-1and Occludin were found decreased in these cells (P<0.05 all P<0.01), However, when pretreated cells with ROCK1 inhibitor Y27632, these injuries were significantly reversed. In cells transfected with miR-124-3p mimic, p-MYPT1/MYPT1 was decreased. p-MYPT1/MYPT1 was however increased in cells transfected with miR-124-3p inhibitor (P<0.05), indicating that miR-124 could directly inhibit ROCK1 activity. The increased ROCK1 activity and apoptosis, as well as the decreased tight junction proteins induced by high glucose were significantly suppressed as miR-124-3p mimic transfected in GEnCs.@*Conclusions@#According to our experiments, high glucose suppressed miR-124 in glomerular endothelial cells, consequenctly activating ROCK1 activity to damage endothelial cells. MiR-124 overexpression could ameliorate these damages induced by high glucose, suggesting that miR-124 might be a new therapeutic target to prevent glomerular endothelial cells injuries in diabetic nephropathy.

Effects of hydrogen on expression of ROCK1 and mDia1 in intestinal tissues of septic mice / 中华麻醉学杂志

Objective To evaluate the effects of hydrogen on the expression of Rho-associated protein kinase 1(ROCK1)and mammalian diaphanous-related formin 1(mDia1)in intestinal tissues of septic mice.Methods Ninety male C57BL/6 mice,weighing 20-25 g,aged 6 weeks,were divided into 3 groups(n=30 each)using a random number table:control group(group C),sepsis group(group S)and sepsis plus hydrogen group(group SH).Sepsis was produced by cecal ligation and puncture(CLP).Group SH inhaled 2% hydrogen for 1 h starting from 1 and 6 h after CLP.Twenty mice in each group were selected and observed for 7-day survival rate.Ten mice in each group were sacrificed at 24 h after CLP,and blood samples were obtained from hearts to measure the activity of serum diamine oxidase(DAO)and to count the colony-forming units after bacterial culture.The small intestinal samples were obtained for microscopic examination of pathological changes and for determination of ROCK1 and mDia1 positive cell rates(using immunohistochemical staining)and expression of intestinal epithelial junctional protein E-cadherin(by immunofluorescent staining).Intestinal damage was assessed and scored.The ratio of ROCK1 to mDia1 positive cell rates(ROCK1/mDia1 ratio)was calculated.Results Compared with group C,the survival rate was significantly decreased,the serum DAO activity,colony counts and intestinal damage scores were increased,ROCK1 and mDia1 positive cell rates were increased,and the expression of E-cadherin was down-regulated in S and SH groups,ROCK1/mDia1 ratio was increased in group S(P0.05).Compared with group S,the survival rate was significantly increased,the serum DAO activity,colony counts and intestinal damage scores were decreased,the ROCK1 positive cell rate was decreased,the mDia1 positive cell rate was increased,ROCK1/mDia1 ratio was decreased,and the expression of E-cadherin was up-regulated in group SH(P<0.05).Conclusion The mechanism by which hydrogen improves intestinal barrier function may be related to down-regulation of ROCK1 expression,up-regulation of mDia1 expression and correction of the imbalance in ROCK1/mDia1 ratio in intestinal tissues of septic mice.

The role of Rho kinase in the protective effects of hydrogen on the damage of Caco-2 epithelial barrier induced by LPS / 天津医药

Objective To investigate the role of Rho kinase (ROCK) in the protective effects of hydrogen on intestinal epithelial barrier function in sepsis. Methods Caco-2 cells were cultured routinely, and divided into 6 groups randomly (n=3):control group (C group), hydrogen-rich medium group (H group), lipopolysaccharide (LPS)-treatment group (L group), hy?drogen+LPS-treatment group (HL group), Rho kinase inhibitor (Y-37632) treatment group (Y group) and Rho kinase inhibi?tor Y-27632+LPS-treatment group (YL group). H group was treated with 0.6 mmol/L hydrogen-rich media. The concentra?tion of LPS and Y-27632 were 50 mg/L and 25μmol/L separately. After the Caco-2 monolayer model was established, the transepithelial electrical resistance (TEER) values were measured regularly. When the TEER value reached 800Ω·cm2, the treatment was administered. Then TEER values were measured at 6 h, 12 h and 24 h, and FITC-dextran permeability was de?tected at 24 h. Cells were seeded on 6-well plates. After cell density reached 80%-90%, treatments were given randomly. The real time-polymerase chain reaction (RT-PCR) was conducted to assess mRNA levels of ZO-1 and ROCK mRNA. ZO-1 and ROCK protein expression levels were detected by Western blot assay. Results Compared with C group, TEER values were elevated in 12 h and 24 h in H group (P protein expression levels of ZO-1 and ROCK between C group and H group (P>0.05). TEER values were elevated at 6 h, 12 h and 24 h in Y group (P 0.05). The mRNA expression of ZO-1 increased and mRNA expression of ROCK decreased in Y group (P <0.05). The TEER values reduced at 6 h, 12 h and 24 h in L group. The FITC-dextran permeability increased significantly, mRNA and protein expressions of ZO-1 significantly decreased, mRNA and protein expressions of ROCK significantly in?creased in L group (all P<0.05). Compared with L group, TEER values increased significantly at 6 h, 12 h and 24 h in YL group, FITC-dextran permeability decreased, mRNA expressions of ZO-1 increased, mRNA expressions of ROCK de?creased in YL group (P<0.05). Compared with L group, TEER values increased at 6 h, 12 h and 24 h in HL group, FITC-dextran permeability reduced markedly, protein expressions of ZO-1 increased at each time point, protein expressions of ROCK decreased at each time point in HL group (P<0.05). Conclusion Hydrogen can protect intestinal barrier function against sepsis, ameliorate the integrity and permeability of intestinal epithelium and increase the expressions of intercellular tight junction proteins. The suppression of Rho kinase over-expression induced by LPS may be involved in these protective effects of hydrogen.

Rho/Rock signaling pathway and the regulation of placental trophoblast infiltration in preeclampsia / 中国医师杂志

Preeclampsia is caused by the dysfunction of placental trophoblast infiltration.The trophoblast immersed in endometrial layer shallow (only up to decidua layer),concurrently resulted in uterine vascular remodeling disorder,and cause placental tissue ischemia,hypoxia,and a series of clinical manifestations of preeclampsia.Rho is a guanosine triphosphate-binding protein whose downstream molecule is Rho-associated coiled-coil forming protein kinase (Rock).Recent studies suggest that Rho/Rock signaling pathway,by regulating the polymerization state of intracellular microfilament skeleton,affects the behavior and function of cells,such as cell adhesion,migration,cell contraction,infiltration and transformation of cancer cells,etc.Thus is involved in the pathogenesis of many diseases.This article summarized the progress of research in the influence of trophoblast infiltration capacity,activation,regulation and the relationship with pregnancy of Rho/Rock signaling pathway,to provide reference for clinical research.

Effect of inhalation of hydrogen gas on Rho/ROCK pathway in lung tissues of septic mice with acute lung injury / 中华麻醉学杂志

Objective To evaluate the effect of inhalation of hydrogen gas (H2) on Rho/Rho kinase (ROCK) pathway in lung tissues of septic mice with acute lung injury.Methods Forty male C57BL/6 mice,weighing 20-25 g,aged 6 weeks,were randomly divided into 4 groups (n=10 each) using a random number table:sham operation group (group Sh),sham operation + inhalation of H2 group (group H2),sepsis group (group S),and sepsis+ inhalation of H2 group (group S+H2).Sepsis was produced by cecal ligation and puncture (CLP).Both H2 and S+H2 groups inhaled 2％ H2 for 1 h starting from 1 and 6 h after CLP.The mice in each group were sacrificed at 24 h after CLP.The bronchoalveolar lavage fluid (BALF) was collected for determination of protein concentrations,polymorphonuclear neutrophil (PMN) count,and concentrations of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) (by enzyme-linked immunosorbent assay).The lung tissues were obtained for examination of the pathological changes which were scored and for determination of the wet/dry weight ratio (W/D ratio),activities of myeloperoxidase (MPO) and superoxide dismutase (SOD),malonaldehyde (MDA) level,the expression of Rho,ROCK1,ROCK2 and activated caspase-3,and phosphorylation of myosin phosphatase target protein 1 (MYPT-1) (by Western blot).Results Compared with group Sh,the concentrations of protein,PMN count,and concentrations of TNF-α and IL-1β in BALF,lung injury score,W/D ratio,and levels of MPO and MDA in lung tissues were significantly increased,the activity of SOD in lung tissues was significantly decreased,the expression of Rho,ROCK1,ROCK2 and activated caspase-3 was significantly upregulated,and the phosphorylation of MYPT-1 in lung tissues was significantly increased in S and S+H2 groups (P＜0.05),and no significant change was found in the parameters mentioned above in group H2 (P＞0.05).Compared with group S,the concentrations of protein,PMN count,and concentrations of TNF-α and IL-1β in BALF,lung injury score,W/D ratio,and levels of MPO and MDA in lung tissues were significantly decreased,the activity of SOD in lung tissues was significantly increased,the expression of Rho,ROCK1,ROCK2 and activated caspase-3 in lung tissues was significantly down-regulated,and the phosphorylation of MYPT-1 in lung tissues was significantly decreased in group S+H2 (P＜0.05).Conclusion The mechanism by which inhalation of H2 attenuates acute lung injury is related to inhibition of Rho/ROCK pathway activation in lung tissues of septic mice.