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1.
Article in English | WPRIM | ID: wpr-878451

ABSTRACT

OBJECTIVES@#The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT.@*METHODS@#Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays.@*RESULTS@#After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (@*CONCLUSIONS@#The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.


Subject(s)
Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Movement , Cell Proliferation , Humans , Neoplasm Invasiveness , Protein Methyltransferases , RNA, Small Interfering , Tongue , Tongue Neoplasms , Transfection , rho-Associated Kinases
2.
Chinese Medical Journal ; (24): 829-839, 2021.
Article in English | WPRIM | ID: wpr-878056

ABSTRACT

BACKGROUND@#MicroRNAs are closely associated with the progression and outcomes of multiple human diseases, including sepsis. In this study, we examined the role of miR-23a in septic injury.@*METHODS@#Lipopolysaccharide (LPS) was used to induce sepsis in a rat model and H9C2 and HK-2 cells. miR-23a expression was evaluated in rat myocardial and kidney tissues, as well as H9C2 and HK-2 cells. A miR-23a mimic was introduced into cells to identify the role of miR-23a in cell viability, apoptosis, and the secretion of inflammatory cytokines. Furthermore, the effect of Rho-associated kinase 1 (ROCK1), a miR-23a target, on cell damage was evaluated, and molecules involved in the underlying mechanism were identified.@*RESULTS@#In the rat model, miR-23a was poorly expressed in myocardial (sham vs. sepsis 1.00 ± 0.06 vs. 0.27 ± 0.03, P < 0.01) and kidney tissues (sham vs. sepsis 0.27 ± 0.03 vs. 1.00 ± 0.06, P < 0.01). Artificial overexpression of miR-23a resulted in increased proliferative activity (DNA replication rate: Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 34.13 ± 3.12 vs. 12.94 ± 1.21 vs. 13.31 ± 1.43 vs. 22.94 ± 2.26, P < 0.05; HK-2 cells: 15.17 ± 1.43 vs. 34.52 ± 3.46 vs. 35.19 ± 3.12 vs. 19.87 ± 1.52, P < 0.05), decreased cell apoptosis (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 11.39 ± 1.04 vs. 32.57 ± 2.29 vs. 33.08 ± 3.12 vs. 21.63 ± 2.35, P < 0.05; HK-2 cells: 15.17 ± 1.43 vs. 34.52 ± 3.46 vs. 35.19 ± 3.12 vs. 19.87 ± 1.52, P < 0.05), and decreased production of inflammatory cytokines, including interleukin-6 (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 59.61 ± 5.14 vs. 113.54 ± 12.30 vs. 116.51 ± 10.69 vs. 87.69 ± 2.97 ng/mL; P < 0.05, F = 12.67, HK-2 cells: 68.12 ± 6.44 vs. 139.65 ± 16.62 vs. 143.51 ± 13.64 vs. 100.82 ± 9.74 ng/mL, P < 0.05, F = 9.83) and tumor necrosis factor-α (Control vs. LPS vs. LPS + Mock vs. LPS + miR-23a: H9C2 cells: 103.20 ± 10.31 vs. 169.67 ± 18.84 vs. 173.61 ± 15.91 vs. 133.36 ± 12.32 ng/mL, P < 0.05, F = 12.67, HK-2 cells: 132.51 ± 13.37 vs. 187.47 ± 16.74 vs. 143.51 ± 13.64 vs. 155.79 ± 15.31 ng/mL, P < 0.05, F = 9.83) in cells. However, ROCK1 was identified as a miR-23a target, and further up-regulation of ROCK1 mitigated the protective function of miR-23a in LPS-treated H9C2 and HK-2 cells. Moreover, ROCK1 suppressed sirtuin-1 (SIRT1) expression to promote the phosphorylation of nuclear factor-kappa B (NF-κB) p65, indicating the possible involvement of this signaling pathway in miR-23a-mediated events.@*CONCLUSION@#Our results indicate that miR-23a could suppress LPS-induced cell damage and inflammatory cytokine secretion by binding to ROCK1, mediated through the potential participation of the SIRT1/NF-κB signaling pathway.


Subject(s)
Animals , Apoptosis/genetics , Cell Line , Cytokines , Inflammation/genetics , Lipopolysaccharides , MicroRNAs/genetics , NF-kappa B , Rats , Sirtuin 1 , rho-Associated Kinases/genetics
3.
Braz. j. med. biol. res ; 53(1): e8883, Jan. 2020. tab, graf
Article in English | LILACS | ID: biblio-1055486

ABSTRACT

Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is one kind of cytoplasmic long non-coding RNA (lncRNA), which has been demonstrated to play a critical function in multiple cancers. However, the detailed mechanism of OIP5-AS1 in the regulation of cervical cancer progression is still obscure. Here, we demonstrated that lncRNA OIP5-AS1 was upregulated in cervical cancer and was correlated with poor prognosis by bioinformatics studies. OIP5-AS1 depletion inhibited cell proliferation and promoted cell apoptosis in cervical cancer cells. Furthermore, we clarified that ROCK1 was the downstream effector of OIP5-AS1 and OIP5-AS1 acted as a molecular sponge of miR-143-3p. Finally, we verified that OIP5-AS1 exerted its function in the regulation of cervical cancer progression via interacting with miR-143-3p to regulate ROCK1 expression. Our study revealed novel mechanisms about how lncRNA OIP5-AS1 executed its function in cervical cancer and thus provided potential therapeutic targets for the disease.


Subject(s)
Humans , Female , Uterine Cervical Neoplasms/pathology , Apoptosis/physiology , MicroRNAs/metabolism , Cell Proliferation/physiology , rho-Associated Kinases/metabolism , RNA, Long Noncoding/metabolism , Gene Expression Regulation, Neoplastic , Up-Regulation , Uterine Cervical Neoplasms/metabolism , Blotting, Western , Apoptosis/genetics , Reverse Transcriptase Polymerase Chain Reaction , MicroRNAs/genetics , Cell Line, Tumor , Cell Proliferation/genetics , rho-Associated Kinases/genetics , RNA, Long Noncoding/genetics
4.
Article in English | WPRIM | ID: wpr-785705

ABSTRACT

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.


Subject(s)
Adenosine Triphosphate , AMP-Activated Protein Kinases , Animals , Blotting, Western , Cell Survival , Electron Transport , Endoplasmic Reticulum , Endoplasmic Reticulum Stress , Fatty Acids, Nonesterified , Insulin , Insulin-Secreting Cells , Metformin , Mice , NADPH Oxidases , Oxidative Stress , Phosphorylation , Polymerase Chain Reaction , Reactive Oxygen Species , rho-Associated Kinases , RNA, Messenger
5.
Article in Chinese | WPRIM | ID: wpr-774300

ABSTRACT

OBJECTIVE@#To investigate the expression change of ROCK1 gene in patients with acute lymphoblastic leukemia (ALL) and its prognostic significance.@*METHODS@#Sixty patients with ALL were selected in our hospital from April 2017 to April 2018, and 60 healthy persons subjected to physical examination were selected as control. The venous blood was taken from the subjects, and then the mononuclear cells were separated. The ROCK1 gene expression level in the samples was detected by RT-PCR, and the expression level of ROCK1 protein was detected by Western blot. The correlation between ROCK1 gene expression and clinical characteristics of ALL patients was analyzed by using statistical methots.@*RESULTS@#The RT-PCR showed that the relative expression level of ROCK1 gene in ALL patients was 1.37 (1.28-1.46), which was significantly higher than that in the control group (P0.05). The standard risk ratio of B-ALL and T-ALL patients with low ROCK1 expression was significantly higher than that in patients with high ROCK1 expression (P<0.05). The high risk ratio of B-ALL and T-ALL patients with low ROCK1 expression was significantly lower than those with high ROCK1 expression (P<0.05). The ratio of CR in the group with low ROCK1 expression patients was significantly higher than that in patients with high ROCK1 expression (P<0.05). The Relapse rate of the group with low ROCK1 expression was significantly lower than that of the group with high ROCK1 expression (P<0.05). Kaplan-Meier survival analysis showed that OS and DFS in ALL patients with low ROCK1 expression were superior to those in ALL patients with high ROCK1 expression (P<0.05). Multiple factor Cox regression analysis showed that age and ROCK1 gene were independent influencing factors for OS (P<0.05); leukocyte count and ROCK1 gene were independent influencing factors for DFS (P<0.05).@*CONCLUSION@#The expression level of ROCK1 gene in ALL patients is high, which may stimulate the genesis of ALL, and the down-regulation of ROCK1 gene expression may help improve the therapeutic effect for ALL patients.


Subject(s)
Acute Disease , Blood Cell Count , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Prognosis , Recurrence , rho-Associated Kinases , Metabolism
6.
Article in Korean | WPRIM | ID: wpr-766613

ABSTRACT

Glaucoma is a progressive degenerative disease of the optic nerve head, characterized by a specific pattern of axonal loss and visual field deterioration. This review aims at introducing the different novel pharmacologic agents for its treatment, as well as their mechanisms. Most glaucoma patients require lifelong care and individualized treatment. Intraocular pressure (IOP), which is regulated by aqueous humor production, outflow via the trabecular meshwork (parasympathomimetics only) and uveoscleral outflow pathways, is currently the only treatable target for glaucoma treatment. Conventional glaucoma medications are categorized as β blockers, α agonists, carbonic anhydrase inhibitors, parasympathomimetics, and prostaglandin analogues. The development of basic research-derived novel classes of pharmacologic agents features novel action mechanisms, which are different from those of conventional medications. New classes of recently approved or clinical trial-tested medications include Rho-kinase inhibitors, nitric oxide donors, adenosine agonists, and prostaglandin analogs targeting E-type prostanoid receptors, etc. Their integration and future development will facilitate the expansion and customization of therapeutic options.


Subject(s)
Adenosine , Aqueous Humor , Axons , Carbonic Anhydrase Inhibitors , Glaucoma , Humans , Intraocular Pressure , Nitric Oxide Donors , Ocular Hypertension , Optic Disk , Parasympathomimetics , Prostaglandins, Synthetic , rho-Associated Kinases , Trabecular Meshwork , Visual Fields
7.
Article in English | WPRIM | ID: wpr-713582

ABSTRACT

The present study was undertaken to investigate the influence of hypothermia on endothelium-independent vascular smooth muscle contractility and to determine the mechanism underlying the relaxation. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Hypothermia significantly inhibited fluoride-, thromboxane A2-, phenylephrine-, and phorbol ester-induced vascular contractions regardless of endothelial nitric oxide synthesis, suggesting that another pathway had a direct effect on vascular smooth muscle. Hypothermia significantly inhibited the fluoride-induced increase in pMYPT1 level and phorbol ester-induced increase in pERK1/2 level, suggesting inhibition of Rho-kinase and MEK activity and subsequent phosphorylation of MYPT1 and ERK1/2. These results suggest that the relaxing effect of moderate hypothermia on agonist-induced vascular contraction regardless of endothelial function involves inhibition of Rho-kinase and MEK activities.


Subject(s)
Animals , Fluorides , Humans , Hypothermia , Isometric Contraction , Male , Muscle, Smooth, Vascular , Nitric Oxide , Phosphorylation , Rats , Relaxation , rho-Associated Kinases
8.
Article in English | WPRIM | ID: wpr-715617

ABSTRACT

In this study, we investigated the effects of pelargonidin, an anthocyanidin found in many fruits and vegetables, on endothelium-independent vascular contractility to determine the underlying mechanism of relaxation. Isometric contractions of denuded aortic muscles from male rats were recorded, and the data were combined with those obtained in western blot analysis. Pelargonidin significantly inhibited fluoride-, thromboxane A2-, and phorbol ester-induced vascular contractions, regardless of the presence or absence of endothelium, suggesting a direct effect of the compound on vascular smooth muscles via a different pathway. Pelargonidin significantly inhibited the fluoride-dependent increase in the level of myosin phosphatase target subunit 1 (MYPT1) phosphorylation at Thr-855 and the phorbol 12,13-dibutyrate-dependent increase in the level of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation at Thr202/Tyr204, suggesting the inhibition of Rho-kinase and mitogen-activated protein kinase kinase (MEK) activities and subsequent phosphorylation of MYPT1 and ERK1/2. These results suggest that the relaxation effect of pelargonidin on agonist-dependent vascular contractions includes inhibition of Rho-kinase and MEK activities, independent of the endothelial function.


Subject(s)
Animals , Anthocyanins , Aorta , Blotting, Western , Endothelium , Fluorides , Fruit , Humans , Isometric Contraction , Male , Muscle, Smooth, Vascular , Muscles , Myosin-Light-Chain Phosphatase , Phosphorylation , Phosphotransferases , Protein Kinases , Rats , Relaxation , rho-Associated Kinases , Vasoconstriction , Vegetables
9.
Rev. chil. cardiol ; 36(3): 209-220, dic. 2017. tab, graf
Article in Spanish | LILACS | ID: biblio-899588

ABSTRACT

Resumen: Objetivo: Determinar algunos mecanismos moleculares por los cuales la activación de ROCK cardíaca post infarto del miocardio (IAM) participa en el remodelado y en deterioro de la función sistólica. Métodos: Determinación simultánea de niveles de proteínas blanco de ROCK cardíaca, de función sistólica in vivo del ventrículo izquierdo (VI) y de fibrosis e hipertrofia cardíaca en ratas con IAM en condiciones de inhibición de ROCK con fasudil. Resultados : Siete días post IAM la masa ventricular relativa aumentó significativamente en un 30% en el grupo MI y se redujo con fasudil. La disfunción sistólica VI mejoró significativamente con fasudil mientras que la activación de ROCK cardíaca se redujo a niveles del grupo control. El inhibidor de ROCK también redujo significativamente los niveles cardíacos elevados de las isoformas ROCK1 y ROCK2, de MHC-β y del colágeno miocárdico. En el grupo con IAM aumentaron significativamente los niveles de fosforilación de ERK 42 y ERK 44 (en 2 veces y en 63%, respectivamente), mientras que en el grupo IAM tratado con fasudil estos niveles fueron similares a los del grupo control. El IAM aumentó significativamente los niveles fosforilados del factor de transcripción GATA-4, que se normalizaron con el inhibidor de ROCK. Conclusiones: La disfunción sistólica post IAM se asoció fuertemente con la activación del ROCK cardíaca y con la fosforilación de proteínas río abajo de ROCK que promueven remodelado cardíaco como β-MHC y la vía ERK / GATA-4.


Abstracts: Objective: to determine some molecular mechanisms by which cardiac ROCK activation after myocardial infarction (MI) intervene in cardiac systolic function decline and remodeling. Methods: simultaneous measurement of different cardiac ROCK target proteins levels, in vivo left ventricular (LV) systolic function, myocardial fibrosis, and hypertrophy in rats with MI under ROCK inhibition with fasudil were performed. Results: seven days after MI the relative ventricular mass increased significantly by 30% in the MI groupand was reduced with fasudil. LV systolic dysfunction improved significantly with fasudil whereas at the same time cardiac ROCK activation was reduced to sham levels. The ROCK inhibitor also reduced increased cardiac levels of both ROCK1 and ROCK2 isoforms, β-MHC levels and myocardial collagen volume fraction decline. MI significantly increased phosphorylation levels of ERK 42 and ERK 44 by 2-fold and 63% respectively whereas in the fasudil-treated MI group these levels were similar to those in the sham group. MI significantly increased phosphorylated levels of the transcription factor GATA-4 which were normalyzed by the ROCK inhibitor. Conclusion: LV systolic dysfunction after MI was strongly associated to cardiac ROCK activation and subsequent phosphorylation of ROCK target proteins that promote ventricular remodeling, such as β-MHC and the ERK/GATA-4 pathway. ROCK inhibition with fasudil significantly improved systolic function, diminished myocardial fibrosis, and normalized β-MHC and ERK/GATA-4 phosphorylation levels.


Subject(s)
Animals , Rats , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , Protein Kinase Inhibitors/pharmacology , rho-Associated Kinases/antagonists & inhibitors , Myocardial Infarction/drug therapy , Organ Size/drug effects , Phosphorylation , Blotting, Western , Ventricular Function, Left/drug effects , Rats, Sprague-Dawley , Cardiomegaly/drug therapy , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Ventricular Remodeling/drug effects , Disease Models, Animal , Myocardial Infarction/enzymology
10.
National Journal of Andrology ; (12): 110-119, 2017.
Article in Chinese | WPRIM | ID: wpr-812801

ABSTRACT

Objective@#To screen lentiviral vectors carrying siRNA which can specifically down-regulate the gene expression of the sphingosine-1-phosphate receptor 3 (S1PR3) in the corpus cavernosum smooth muscle (CCSM) cells of rats with spontaneous hypertension (SHT) and investigate the influence of the vectors on the signaling pathways of ROCK1, ROCK2 and eNOS in the CCSM cells of SHT rats.@*METHODS@#Using the S1PR3 mRNA sequence of the rat as an interfering target, we designed and synthesized three pairs of siRNA sequences (siRNA1, 2 and 3) targeting S1PR3 and one pair of negative control, and then constructed and packaged them into lentiviral vectors. We cultured the CCSM cells of SHT and Wistar-Kyoto (WKY) rats in vitro and randomly divided them into groups A (SHT untransfected control), B (SHT transfected and carrying negative control virus), C (SHT transfected and carrying siRNA1 targeting S1PR3), D (SHT transfected and carrying siRNA2 targeting S1PR3), E (SHT transfected and carrying siRNA3 targeting S1PR3), and F (WKY untransfected control). With the multiplicity of infection (MOI) = 60, we transfected the CCSM cells of the SHT rats with the lentiviral vector and then determined the expression of the green fluorescent protein (GFP) as well as the mRNA and protein expressions of S1PR3, ROCK1, ROCK2 and eNOS in the CCSM cells of the SHT and WKY rats by RT-PCR and Western blot.@*RESULTS@#Gene sequencing proved the successful construction of the lentiviral vector. The transfection efficiency of the CCSM cells of the rats was >80% in groups B, C, D and E. Compared with group A, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 exhibited no significant difference in group B but were remarkably decreased in groups C, D, E and F (P0.05) but remarkably lower than those in group F (P0.05) but markedly increased in groups A, B, C and D (P< 0.05), while those of eNOS remarkably decreased in groups A, B, C, D and E (P< 0.05).@*CONCLUSIONS@#The three constructed lentiviral vectors carrying siRNA targeting different loci of the S1PR3 gene could significantly inhibit the expression of S1P3 as well as RhoA/Rho kinase signaling pathways in the CCSM cells of SHT rats, and the vector carrying siRNA3 exhibited the highest inhibitory effect.


Subject(s)
Animals , Down-Regulation , Gene Expression , Genetic Vectors , Green Fluorescent Proteins , Metabolism , Lentivirus , Genetics , Male , Myocytes, Smooth Muscle , Metabolism , Nitric Oxide Synthase Type III , Metabolism , Penis , Metabolism , RNA, Messenger , RNA, Small Interfering , Genetics , Metabolism , Random Allocation , Rats , Rats, Inbred WKY , Receptors, Lysosphingolipid , Genetics , Metabolism , Signal Transduction , Sphingosine-1-Phosphate Receptors , Transfection , rho-Associated Kinases , Metabolism
11.
Article in English | WPRIM | ID: wpr-80651

ABSTRACT

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)
Adenosine , Electric Impedance , Humans , 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 Meshwork
12.
Article in Chinese | WPRIM | ID: wpr-340547

ABSTRACT

<p><b>OBJECTIVE</b>To study the possible effect of antepartum taurine supplementation in regulating the activity of Rho family factors and promoting the proliferation of neural stem cells in neonatal rats with fetal growth restriction (FGR), and to provide a basis for antepartum taurine supplementation to promote brain development in children with FGR.</p><p><b>METHODS</b>A total of 24 pregnant Sprague-Dawley rats were randomly divided into three groups: control, FGR, and taurine (n=8 each ). A rat model of FGR was established by food restriction throughout pregnancy. RT-PCR, immunohistochemistry, and Western blot were used to measure the expression of the specific intracellular markers for neural stem cells fatty acid binding protein 7 (FABP7), Rho-associated coiled-coil containing protein kinase 2 (ROCK2), ras homolog gene family, member A (RhoA), and Ras-related C3 botulinum toxin substrate (Rac).</p><p><b>RESULTS</b>The FGR group had significantly lower OD value of FABP7-positive cells and mRNA and protein expression of FABP7 than the control group, and the taurine group had significantly higher OD value of FABP7-positive cells and mRNA and protein expression of FABP7 than the FGR group (P<0.05). The FGR group had significantly higher mRNA expression of RhoA and ROCK2 than the control group. The taurine group had significantly higher mRNA expression of RhoA and ROCK2 than the control group and significantly lower expression than the FGR group (P<0.05). The FGR group had significantly lower mRNA expression of Rac than the control group. The taurine group had significantly higher mRNA expression of Rac than the FGR and control groups (P<0.05). The FGR group had significantly higher protein expression of RhoA and ROCK2 than the control group. The taurine group had significantly lower protein expression of RhoA and ROCK2 than the FGR group (P<0.05).</p><p><b>CONCLUSIONS</b>Antepartum taurine supplementation can promote the proliferation of neural stem cells in rats with FGR, and its mechanism may be related to the regulation of the activity of Rho family factors.</p>


Subject(s)
Animals , Animals, Newborn , Body Weight , Brain , Cell Proliferation , Fatty Acid-Binding Protein 7 , Female , Fetal Growth Retardation , Drug Therapy , Male , Neural Stem Cells , Physiology , Rats , Rats, Sprague-Dawley , Taurine , Pharmacology , rho-Associated Kinases , Genetics , rhoA GTP-Binding Protein , Genetics
13.
National Journal of Andrology ; (12): 393-400, 2016.
Article in Chinese | WPRIM | ID: wpr-262340

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expressions of sphingosine-1-phosphate receptors 1-3 (S1P1- 3) in the corpus cavernosum of castrated male rats and its relationship with the NOS/NO/cGMP and RhoA/Rho kinase signaling pathways.</p><p><b>METHODS</b>We equally randomized 18 eight-week-old healthy male SD rats into a sham-operation control, a castration, and a testosterone replacement (TR) group and harvested the bilateral testes and epididymides from the rats in the latter two groups, followed by 4 weeks of subcutaneous injection of testosterone propionate at 3 mg per kilogram of the body weight per day for those in the TR group and that of plant oil for those in the control and castration groups. At the age of 12 weeks, we measured the serum testosterone (T) level and maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP) of the animals and determined the expressions of SlP1-3, eNOS, P-eNOS, ROCK1, and ROCK2 in the corpus cavernosum by Western blot and immunohistochemistry.</p><p><b>RESULTS</b>The serum T level was significantly decreased in the rats of the castration group as compared with those of the control and TR groups ([0.41 ± 0.04] vs [16.01 ± 1.02] and [15.84 ± 1.32] nmol/L, P < 0.01), with no statistically significant difference between the latter two groups. The ICPmax/MAP at 0 V, 3 V, and 5 V electric stimulation was remarkably lower in the rats of the castration group (0.088 ± 0.014, 0.323 ± 0.014, and 0.432 ± 0.012) than in those of the control group (0.155 ± 0.011, 0.711 ± 0. 010, and 0.819 ± 0.024) and TR group (0.153 ± 0.012, 0.696 ± 0.017, and 0.763 ± 0.027) (P < 0.01), with no significant difference between the latter two groups. With GAPDH as internal control, the animals of the castration group showed markedly reduced expressions of S1P1 ([49.99 ± 3.39]%), eNOS ([46.82 ± 3.81]%) , and P-eNOS ([45.42 ± 4.35]%) in comparison with those in the control group ([72.57 ± 3.06], [89.76 ± 3.98], and [82.53 ± 8.92] and TR group ([71.77 ± 4.43], [87.19 ± 4.23], and [79.82 ± 7.38]%) (P < 0.01) , while the expressions of S1P2, S1P3, ROCK1, and ROCK2 were significantly upregulated in the castration group ([82.35 ± 4.13], [61.03 ± 5.14], [74.50 ± 4.02], and [69.83 ± 5.75]%) as compared with those in the control group ([41.67 ± 1.68], [31.66 ± 2.67], [35.69 ± 5.56], and [39.85 ± 7.17]%) and TR group ([42.80 ± 3.87], [32.25 ± 4.22], 38.06 ± 5.21], and [42.36 ± 4.44]%) (P < 0.01).</p><p><b>CONCLUSION</b>Androgen deficiency induces significant reduction of ICPmax/ MAP in male rats, which is possibly associated with the decline of S1P1 in the corpus cavernosum, inhibition of the eNOS/NO/cGMP signaling pathway, increased expressions of S1P2 and S1P3, and activation of the RhoA/Rho kinase signaling pathway.</p>


Subject(s)
Animals , Male , Nitric Oxide Synthase Type III , Metabolism , Orchiectomy , Penis , Metabolism , Random Allocation , Rats , Rats, Sprague-Dawley , Receptors, Lysosphingolipid , Metabolism , Testosterone , Blood , Pharmacology , rho-Associated Kinases , Metabolism
14.
Yonsei Medical Journal ; : 321-327, 2016.
Article in English | WPRIM | ID: wpr-147359

ABSTRACT

PURPOSE: Increased lipoprotein-associated phospholipase A2 (Lp-PLA2) activity and Rho kinase activity may be associated with atherosclerosis. The principal aim of this study was to examine whether darapladib (a selective Lp-PLA2 inhibitor) could reduce the elevated Lp-PLA2 and Rho kinase activity in atherosclerosis. MATERIALS AND METHODS: Studies were performed in male Sprague-Dawley rats. The atherosclerosis rats were prepared by feeding them with a high-cholesterol diet for 10 weeks. Low-dose darapladib (25 mg.kg-1.d-1) and high-dose darapladib (50 mg.kg-1.d-1) interventions were then administered over the course of 2 weeks. RESULTS: The serum levels of triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hs-CRP), and Lp-PLA2, significantly increased in atherosclerosis model groups, as did Rho kinase activity and cardiomyocyte apoptosis (p0.05). CONCLUSION: Darapladib, a Lp-PLA2 inhibitor, leads to cardiovascular protection that might be mediated by its inhibition of both Rho kinase and Lp-PLA2 in atherosclerosis.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/antagonists & inhibitors , Animals , Atherosclerosis/blood , Benzaldehydes , C-Reactive Protein/metabolism , Cholesterol/blood , Cholesterol, HDL/blood , Cholesterol, LDL/blood , Dose-Response Relationship, Drug , Male , Oximes , Phospholipase A2 Inhibitors/administration & dosage , Rats , Rats, Sprague-Dawley , Triglycerides/blood , rho-Associated Kinases/metabolism
15.
Article in English | WPRIM | ID: wpr-250311

ABSTRACT

Emerging evidence indicates that microglia activation plays an important role in spinal cord injury (SCI) caused by trauma. Studies have found that inhibiting the Rho/Rho-associated protein kinase (ROCK) signaling pathway can reduce inflammatory cytokine production by microglia. In this study, Western blotting was conducted to detect ROCK2 expression after the SCI; the ROCK Activity Assay kit was used for assay of ROCK pathway activity; microglia morphology was examined using the CD11b antibody; electron microscopy was used to detect microglia phagocytosis; TUNEL was used to detect tissue cell apoptosis; myelin staining was performed using an antibody against myelin basic protein (MBP); behavioral outcomes were evaluated according to the methods of Basso, Beattie, and Bresnahan (BBB). We observed an increase in ROCK activity and microglial activation after SCI. The microglia became larger and rounder and contained myelin-like substances. Furthermore, treatment with fasudil inhibited neuronal cells apoptosis, alleviated demyelination and the formation of cavities, and improved motor recovery. The experimental evidence reveals that the ROCK inhibitor fasudil can regulate microglial activation, promote cell phagocytosis, and improve the SCI microenvironment to promote SCI repair. Thus, fasudil may be useful for the treatment of SCI.


Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine , Pharmacology , Therapeutic Uses , Animals , Apoptosis , Male , Microglia , Metabolism , Myelin Basic Protein , Metabolism , Myelin Sheath , Metabolism , Phagocytosis , Protein Kinase Inhibitors , Pharmacology , Therapeutic Uses , Rats , Rats, Sprague-Dawley , Spinal Cord Injuries , Drug Therapy , rho-Associated Kinases , Metabolism
16.
Article in English | WPRIM | ID: wpr-728550

ABSTRACT

The present study was undertaken to investigate the influence of cardamonin on vascular smooth muscle contractility and to determine the mechanism(s) involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Cardamonin significantly relaxed fluoride-, phenylephrine-, and phorbol ester-induced vascular contractions, suggesting that it has an anti-hypertensive effect on agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, cardamonin significantly inhibited the fluoride-induced increase in pMYPT1 level and phenylephrine-induced increase in pERK1/2 level, suggesting inhibition of Rho-kinase and MEK activity and subsequent phosphorylation of MYPT1 and ERK1/2. This study provides evidence that the relaxing effect of cardamonin on agonist-induced vascular contraction regardless of endothelial function involves inhibition of Rho-kinase and MEK activity.


Subject(s)
Animals , Fluorides , Humans , Isometric Contraction , Male , Muscle, Smooth, Vascular , Nitric Oxide , Phosphorylation , Rats , rho-Associated Kinases
17.
Article in English | WPRIM | ID: wpr-647620

ABSTRACT

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)
Angiotensins , Female , Freezing , Gene Expression , Ovary , rho-Associated Kinases , RNA, Messenger , Tissue Engineering
18.
Article in Chinese | WPRIM | ID: wpr-815049

ABSTRACT

OBJECTIVE@#To explore the therapeutic effect of Fasudil-modified splenic mononuclear cells (MNCs) in experimental autoimmune encephalomyelitis (EAE) and the possible mechanisms.
@*METHODS@#C57BL/6 female mice were immunized with myelin oligodendrocyte glycoprotein peptide 35-55 to establish active immunity EAE model. Splenic MNCs were isolated on the 9th day after immunization and treated with or without Fasudil for 72 h in vitro. These cells were collected for analysis of the variance of T cell subtypes, the level of cytokines and the activity of Rho kinase (ROCK). MNCs (5×107 cells) were resuspended in 500 µL of phosphate buffer solution (PBS) and transferred into EAE model (intraperitoneal injection), which was divided into a PBS-MNCs group and a Fasudil-MNCs group. Changes of body weight and clinical symptom scores were observed.
@*RESULTS@#Splenic encephalitogenic MNCs from EAE mice on the 9th day after immunization could establish passive transfer EAE model. But Fasudil-treated MNCs did not trigger EAE development. Compared with the PBS-MNCs group, the loss of body weight was less in the Fasudil-MNCs group. The in vitro experiment indicated that Fasudil could suppress the activity of ROCK on MNCs (P<0.01), decrease the percentage of CD4+ T cells with the expression of interferon-γ (IFN-γ) and interleukin-17 (IL-17) (IFN-γ: P<0.01; IL-17: P<0.05), while increase the secretion of CD4+ T cells with the expression of transforming growth factor-β (TGF-β) and IL-10 (all P<0.001) . Furthermore, Fasudil could inhibit the release of IL-17 (P<0.001) and enhance the level of IL-10 (P<0.05).
@*CONCLUSION@#Fasudil-modified cell therapy affects the occurrence and development of EAE by inhibiting the inflammatory reaction of helper T cell 1 (Th1) and Th17 while enhancing the immunoregulative effect of Th2.


Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine , Animals , Encephalomyelitis, Autoimmune, Experimental , Female , Interferon-gamma , Interleukin-10 , Interleukin-17 , Mice , Mice, Inbred C57BL , Myelin-Oligodendrocyte Glycoprotein , Spleen , T-Lymphocytes , Transforming Growth Factor beta , rho-Associated Kinases
19.
Article in Chinese | WPRIM | ID: wpr-814997

ABSTRACT

OBJECTIVE@#To explore the effect of ROCK inhibitor Y-27632 on the matrix metalloproteinase 2 and 9 (MMP2 and MMP9) gene expression and activity in tumor necrosis factor α (TNF-α)-treated human umbilical vein endothelial cell (HUVEC).
@*METHODS@#HHUVEC was divided into 3 groups, a control group, a TNF-α group, and a TNF-α plus Y-27632 group. The expressions of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), MMP2 and MMP9 were examined by real-time PCR. The MMP2/9 activity was measured by gelatin zymography.
@*RESULTS@#Compared to the control group, the mRNA expressions of ICAM-1, VCAM-1, MMP2 and MMP9 were increased TNF-α-treated cells, which were suppressed by ROCK inhibitor (P<0.01). The MMP2/9 activity was elevated in TNF-α-treated cells, which was reversed by ROCK inhibitor (P<0.05).
@*CONCLUSION@#ROCK inhibitor can suppress TNF-α-induced inflammation in endothelial cells through down-regulation of MMP2/9.


Subject(s)
Amides , Cells, Cultured , Down-Regulation , Endothelial Cells , Humans , Intercellular Adhesion Molecule-1 , Matrix Metalloproteinase 2 , Matrix Metalloproteinase 9 , Pyridines , Signal Transduction , Tumor Necrosis Factor-alpha , Umbilical Veins , Vascular Cell Adhesion Molecule-1 , rho-Associated Kinases
20.
National Journal of Andrology ; (12): 483-490, 2016.
Article in Chinese | WPRIM | ID: wpr-304714

ABSTRACT

<p><b>Objective</b>To investigate the potential role of the RhoA/Rock signaling pathway in the formation of prostate cancer and the effects of the Rock inhibitor fasudil on the invasion, migration and apoptosis of human prostate cancer cells.</p><p><b>METHODS</b>Human prostate cancer cell lines PC3 and DU145 were treated with fasudil at the concentrations of 5, 10, 20, 40, 80, and 160 μmol/L, respectively, and those as negative controls cultured in the Ham's-F12 medium, all for 24 hours. Then, MTT assay was used to measure the cell inhibition rate and half maximal inhibitory concentration (IC50) value of fasudil, with 1/4 of IC50 as the medication dose for further investigation. The expressions of RhoA, RockⅠ, and RockⅡ proteins in the PC3 and DU145 cells were detected by Western blot and immunohistochemistry, and the invasion, migration and apoptosis of the cells were determined using the Transwell chamber, scratch wound healing assay and flow cytometry.</p><p><b>RESULTS</b>Fasudil inhibited the proliferation of the PC3 cells from (9.29±1.23)% at 5 μmol/L to (81.37±3.97)% at 160 μmol/L and that of DU145 from (7.59±1.54)% to (76.53±2.67)%, both in a dose-dependent manner (P<0.05 ). Significantly fewer PC3 and DU145 cells migrated into the lower compartment in the experimental group (39.2±8.4 and 34.2±6.7) than in the negative control (116.8±9.3 and 112.5±10.8) (P<0.05 ). The wound healing rates of the PC3 and DU145 cells were remarkably lower in the former ([37.26±1.17]% and [32.38±2.73]%) than in the latter ([78.12±4.16]% and [69.47±6.71]%) (P<0.05 ). Annexin V-FITC/PI double staining showed markedly increased apoptosis rates of PC3 and DU145 cells treated with fasudil ([31.88±2.49]% and [28.65±2.99]%) as compared with the negative controls ([7.51±2.28]% and [7.13±1.61]%) (P<0.05 ). The expressions of RockⅠ and RockⅡ were significantly reduced in the fasudil-treated cells in comparison with those of the control group (P<0.05 ) while that of RhoA showed no significant difference between the two groups (P>0.05 ).</p><p><b>CONCLUSIONS</b>The RhoA/Rock signaling pathway may play an important role in the formation of prostate cancer. Fasudil can significantly inhibit the proliferation, migration, and invasion and promote the apoptosis of human prostate cancer PC3 and DU145 cells by reducing RhoA/Rho kinase activity.</p>


Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine , Pharmacology , Apoptosis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Humans , Male , Prostatic Neoplasms , Drug Therapy , Pathology , Signal Transduction , rho-Associated Kinases
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