Expression Of ROCK1 Research Articles

LINC00536 Promotes Breast Cancer Progression by Regulating ROCK1 via Sponging of miR-214-5p.

Accumulating evidence has shown that long noncoding RNAs (lncRNAs) play a significant role in regulating gene expression and participating in the progression of various malignancies. In our study, by analyzing data from The Cancer Genome Atlas (TCGA), LINC00536 was found to be highly expressed in breast cancer (BC) tissues, but its function and clinical significance in BC are still unknown. Therefore, we aimed to explore the role and molecular mechanism of LINC00536 in BC. We collected human BC tissue specimens and validated that LINC00536 was overexpressed in BC tissues. Increased LINC00536 expression was associated with advanced TNM stage, larger tumor diameter, lymph node metastasis and poor prognosis in patients with BC. Univariate and multivariate Cox regression analyses showed that high LINC00536 expression was an independent prognostic risk factor for overall survival in BC patients. Furthermore, quantitative reverse transcription PCR (qRT-PCR) showed that LINC00536 was upregulated in BC cell lines. Then, we confirmed that LINC00536 silencing-inhibited BC cell proliferation, migration, and invasion and led to cell cycle arrest in vitro. Animal experiments showed that knockdown of LINC00536 expression suppressed tumorigenesis in vivo. Mechanistically, LINC00536 serves as a ceRNA for miR-214-5p, increasing the expression of ROCK1, which acts as a tumor promoter in BC. Rescue assays revealed that miR-214-5p inhibition or ROCK1 overexpression could neutralize the suppressive effects of LINC00536 knockdown on cell proliferation, migration and invasion. Our data indicated that LINC00536 accelerates BC progression by regulating the miR-214-5p/ROCK1 pathway, which might provide a new perspective to investigate the development process of BC.

Klf4 deficiency exacerbates myocardial ischemia/reperfusion injury in mice via enhancing ROCK1/DRP1 pathway-dependent mitochondrial fission

RationalExcessive mitochondrial fission is considered key process involved in myocardial ischemia/reperfusion (I/R) injury. However, the upstream mechanism remains largely unclear. Decreased level of Kruppel Like Factor 4 (KLF4) has been implicated in the pathogenesis of mitochondrial dysfunction and heart's adaption to stress. However, the role of Klf4 in I/R process is not fully elucidated. This study aims to investigate how Klf4 regulates mitochondrial dynamics and further clarify its underlying mechanism during cardiac I/R injury. MethodsLoss-of-function and gain-of-function strategies were applied to investigate the role of Klf4 in cardiac I/R injury via genetic ablation or intra-myocardial adenovirus injection. Mitochondrial dynamics was analyzed by confocal microscopy in vitro and transmission electron microscopy in vivo. Chromatin immunoprecipitation and luciferase reporter assay were performed to explore the underlying mechanisms. ResultsKLF4 was downregulated in I/R heart. Cardiac-specific Klf4 knockout significantly exacerbated cardiac dysfunction in I/R mice. Mechanistically, Klf4 deficiency aggravated mitochondrial apoptosis, reduced ATP generation and boosted ROS overproduction via enhancing DRP1-dependent mitochondrial fission. ROCK1 was identified as a kinase regulating DRP1 activity at Ser616. Klf4 deficiency upregulated the expression of ROCK1 at transcriptional level, thus increasing S616-DRP1-mediated mitochondrial fission during I/R. Finally, reconstitution of Klf4 inhibited mitochondrial fission, restored mitochondrial function and alleviated I/R injury. ConclusionOur study provides the first evidence that Klf4 deficiency exacerbates myocardial I/R injury through regulating ROCK1 expression at transcriptional level to induce DRP1-mediated mitochondrial fission. Targeting mitochondrial dynamics by restoring Klf4 might be potentially cardio-protective strategies attenuating I/R injury.

Correction for: Long non-coding RNA DLEUI promotes papillary thyroid carcinoma progression by sponging miR-421 and increasing ROCK1 expression.

Correction for: Long non-coding RNA DLEUI promotes papillary thyroid carcinoma progression by sponging miR-421 and increasing ROCK1 expression.

Inhibition of interaction between ROCK1 and Rubicon restores autophagy in endothelial cells and attenuates brain injury after prolonged ischemia.

Acute ischemic stroke (AIS) induces cerebral endothelial cell death resulting in the breakdown of the blood-brain barrier (BBB). Endothelial cell autophagy acts as a protective mechanism against cell death. Autophagy is activated in the very early stages of ischemic stroke and declines after prolonged ischemia. Previous studies have shown that Rubicon can inhibit autophagy. The current study aimed to investigate whether continuous long-term ischemia can inhibit autophagy in endothelial cells after ischemic stroke by regulating the function of Rubicon and its underlying mechanism. Wild-type male C57BL/6J mice were subjected to transient middle cerebral artery occlusion (tMCAO). ROCK1, ROCK2, and NOX2 inhibitors were injected into male mice 1h before the onset of tMCAO. Disease severity and BBB permeability were evaluated. bEnd.3 cells were cultured in vitro and subjected to oxygen-glucose deprivation (OGD). bEnd.3 cells were pretreated with or without ROCK1, ROCK2, or NOX2 inhibitors overnight and then subjected to OGD. Cell viability and permeability were also evaluated. The expression of Rubicon, ROCK1, and autophagy-related proteins were analyzed. Increased BBB permeability was correlated with Rubicon expression in tMCAO mice and Rubicon was upregulated in endothelial cells subjected to OGD. Autophagy was inhibited in endothelial cells after long-term OGD treatment and knockdown of Rubicon expression restored autophagy and viability in endothelial cells subjected to 6-h OGD. ROCK1 inhibition decreased the interaction between Beclin1 and Rubicon and restored cell viability and autophagy suppressed by 6-h OGD treatment in endothelial cells. Additionally, ROCK1 inhibition suppressed Rubicon, attenuated BBB disruption, and brain injury induced by prolonged ischemia in 6-h tMCAO mice. Prolonged ischemia induced the death of brain endothelial cells and the breakdown of the BBB, thus aggravating brain injury by increasing the interaction of ROCK1 and Rubicon with Beclin1 while inhibiting canonical autophagy. Inhibition of ROCK1 signaling in endothelial cells could be a promising therapeutic strategy to prolong the therapeutic time window in AIS.

Zearalenone-Induced Mechanical Damage of Intestinal Barrier via the RhoA/ROCK Signaling Pathway in IPEC-J2 Cells.

Zearalenone (ZEN) is a widespread contaminant of cereals and agricultural products which causes food safety issues. Ingesting food or feed contaminated with ZEN can disrupt the intestinal epithelial barrier function. The RhoA/ROCK signaling pathway plays a key role in regulating the epithelial barrier function, but studies on such roles have rarely focused on the intestine. The aim of this experiment was to investigate the exact mechanism of ZEN-induced intestinal barrier damage and whether the RhoA/ROCK signaling pathway is involved. The results showed that ZEN significantly induced alkaline phosphatase (AP) activity and FITC–dextran (4 kDa) passage across the epithelial barrier, which significantly reduced the transepithelial resistance (TEER). Meanwhile, ZEN could induce the significantly down-regulated mRNA expression of tight junction proteins (occludin, claudin-1, ZO-1, and claudin-3) and redistribution of ZO-1 immunofluorescence. Further studies demonstrated that ZEN exposure activated the RhoA/ROCK signaling pathway, significantly up-regulated the mRNA expression of ROCK1, the main effector of the signaling pathway, the protein expression of phosphorylated myosin light chain (MLC) and myosin light chain kinase (MLCK), and relatively increased the activity of ATP in cells, simultaneously remodeling the cytoskeleton (F-actin). Overall, our study indicated that ZEN induced intestinal barrier dysfunction by activating the RhoA/ROCK signaling pathway.

Transcription Factor p300 Regulated miR-451b Weakens the Cigarette Smoke Extract-Induced Cellular Stress by Targeting RhoA/ROCK2 Signaling

Background A previous study identified miR-451b as a potential biomarker in smoker with or without chronic obstructive pulmonary disease (COPD). However, the function and molecular mechanisms of miR-451b in the pathogenesis of COPD remain elusive. Methods Macrophages and lung fibroblasts were exposed to 10% cigarette smoke extract (CSE) solution for 24 h. Expression miR-451b and its potential transcription factor p300 were detected. The association between p300 and miR-451b, miR-451b and RhoA was validated by luciferase reporter assay. The release of IL-12 and TNF-αby macrophages was measured by ELISA assay, and Transwell assay was performed to analyze its migration and invasion. Collagen protein of fibroblasts was detected by Western blotting. Results Results showed that p300 and miR-451b was downregulated, while RhoA was upregulated in CSE-induced macrophages and lung fibroblasts. The stimulation of CSE promoted the degradation of p300 by ubiquitination, and RhoA was confirmed as the target gene of miR-451b. MiR-451b overexpression significantly decreased the release of IL-12 and TNF-α, downregulated the expression of RhoA, ROCK2, and p65, and suppressed cell migration and invasion in CES-induced macrophages. In addition, miR-451b overexpression decreased the expression of RhoA, ROCK2, COL1A1, and COL2A1 in lung fibroblasts. Conclusions Our data suggest that p300/miR-451b protects against CSE-induced cell stress possibly through downregulating RhoA/ROCK2 pathway.

Renoprotection and Its Mechanism of Fasudil in Nitric Oxide Synthase-Deficient Hypertensive Rats

Abstract Background To investigate whether fasudil can improve renal microcirculation and protect the kidney in NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertensive rats by inhibiting Rho/Rho associated coiled-coil containing protein kinase (ROCK) signal pathway. Methods The nitric oxide-deficient hypertension was induced by l-NAME (60 mg/l·d) which was provided in drinking water for 8 weeks in Wistar rats. Then they were randomly divided into l-NAME and l-NAME + fasudil groups. Blood, urine, and left kidney samples were collected after fasudil intervention for 6 weeks. Renal artery blood flow, renal function markers in serum and urine, morphological changes of renal interlobular artery, and expression levels of related proteins were detected. Results Compared with normal control rats, the levels of serum creatinine (SCr) and blood urea nitrogen (BUN) increased while creatinine clearance (CCr) decreased in l-NAME rats. Fasudil treatment significantly decreased SCr and BUN, and increased CCr compared with l-NAME group. Compared with normal controls, the intima of renal interlobular artery increased, the lumen narrowed, the expression of ROCK-1, phosphorylated myosin phosphatase 1/myosin phosphatase 1 (p-MYPT1/MYPT1) increased while endothelial nitric oxide synthase (eNOS) protein levels decreased in l-NAME group (all P < 0.05). Fasudil treatment reduced intimal hyperplasia and lumen stenosis of renal interlobular artery, downregulated ROCK-1 and p-MYPT protein levels and upregulated eNOS expression in l-NAME rats (all P < 0.05). Conclusions Fasudil treatment improves renal function and structure of renal interlobar artery in nitric oxide-deficient hypertensive rats induced by l-NAME.

Regulation of RhoA/ROCK1 signaling pathway by miR 26b in sepsis induced acute lung injury

Purpose: To investigate the role of miR-26b in the regulation of RhoA/ ROCK1 signaling pathway in acute lung injury (ALI) caused by sepsis.
 Methods: Thirty male rats were randomized into sham group (SG), cecal ligation and puncture (CLP) group (CG) and miR-26b mimic group (MG). Hematoxylin and eosin (H & E) staining assay was performed to determine the pathological characteristics of rat lung tissues in each group, while enzyme-linked immunosorbent assay (ELISA) was conducted to determine TNF-α and IL-1β levels. The miR-26b expression was evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR), while RhoA and Rock1 protein levels were assessed using western blotting.
 Results: The CG had significant lung injury in comparison with the SG. There were significant elevation in TNF-α and IL-1β levels (p < 0.05). RhoA and ROCK1 levels in lung tissue were noticeably elevated in CG (p < 0.05). After treatment, lung injury in MG was reduced in contrast to CG. The MG showed statistically significant decrease (p < 0.05) in the levels of TNF-α and IL-1β, while the lung tissue mRNA expression and the RhoA and ROCK1 expression levels were significantly reduced in MG (p < 0.05).
 Conclusion: The MiR-26b mimics plays an important role in the treatment of ALI induced by sepsis in rats by regulating RhoA/ROCK1 signaling pathway. Thus, the findings of this study provide a theoretical basis for clinical studies on the use of miR-26b in the therapy of sepsis.

MicroRNA-148a Controls Epidermal and Hair Follicle Stem/Progenitor Cells by Modulating the Activities of ROCK1 and ELF5

Skin and hair development is regulated by complex programs of gene activation and silencing and microRNA-dependent modulation of gene expression to maintain normal skin and hair follicle development, homeostasis, and cycling. In this study, we show that miR-148a, through its gene targets, plays an important role in regulating skin homeostasis and hair follicle cycling. RNA and protein analysis of miR-148a and its gene targets were analyzed using a combination of invitro and invivo experiments. We show that the expression of miR-148a markedly increases during telogen (bulge and hair germ stem cell compartments). Administration of antisense miR-148a inhibitor into mouse skin during the telogen phases of the postnatal hair cycle results in accelerated anagen development and altered stem cell activity in the skin. We also show that miR-148a can regulate colony-forming abilities of hair follicle bulge stem cells as well as control keratinocyte proliferation/differentiation processes. RNA and protein analysis revealed that miR-148a may control these processes by regulating the expression of Rock1 and Elf5 invitro and invivo. These data provide an important foundation for further analyses of miR-148a as a crucial regulator of these genes target in the skin and hair follicles and its importance in maintaining stem/progenitor cell functions during normal tissue homeostasis and regeneration.

Haptoglobin treatment contributes to regulating nitric oxide signal and reduces oxidative stress in the penis: A preventive treatment for priapism in sickle cell disease.

Background: In sickle cell disease (SCD), reduced bioavailability of endothelial NO and cGMP results in reduced expression of phosphodiesterase type 5 (PDE5), thus impairing the penile erection control mechanism and resulting in prolonged penile erection (priapism). In SCD, reduced NO bioavailability is associated with excess plasma hemoglobin due to intravascular hemolysis and increased oxidative stress. Haptoglobin is the plasma protein responsible for reducing plasma hemoglobin levels, but in SCD, haptoglobin levels are reduced, which favors the accumulation of hemoglobin in plasma. Therefore, we aimed to evaluate the effects of haptoglobin treatment on functional and molecular alterations of erectile function, focusing on the contractile and relaxant mechanisms of corpus cavernosum (CC), as well as oxidative stress. Methods: SCD mice were treated with haptoglobin (400 mg/kg, subcutaneous) or vehicle of Monday, Wednesday and Friday for a period of 1 month. Corpus cavernosum strips were dissected free and placed in organ baths. Cumulative concentration-response curves to the acetylcholine, sodium nitroprusside, phenylephrine and KCL, as well as to electrical field stimulation (EFS), were obtained in CC. Protein expressions of eNOS, phosphorylation of eNOS at Ser-1177, nNOS, PDE5, ROCK1, ROCK2, gp91phox, 3-nitrotyrosine, and 4-HNE were measured by western blot in CC. Results: Increased CC relaxant responses to acetylcholine, sodium nitroprusside and electrical-field stimulation were reduced by haptoglobin in SCD mice. Reduced CC contractile responses to phenylephrine and KCl were increased by haptoglobin in SCD mice. Haptoglobin prevented downregulated eNOS, p-eNOS (Ser-1177), PDE5, and ROCK2 protein expressions and reduced protein expressions of reactive oxygen species markers, NADPH oxidase subunit gp91phox, 3-nitrotyrosine and 4-HNE in penises from SCD mice. Haptoglobin treatment did not affect ROCK1 and nNOS protein expressions in penises from SCD mice. Basal cGMP production was lower in the SCD group, which was normalized by haptoglobin treatment. Conclusion: Treatment with haptoglobin improved erectile function due to up-regulation of eNOS-PDE5 expression and down-regulation of the gp91phox subunit of NADPH oxidase and oxidative/nitrosative stress in the penises of SCD mice. Treatment with haptoglobin also increased contractile activity due to up-regulation of ROCK2. Therefore, haptoglobin treatment may be an additional strategy to prevent priapism in SCD.

A novel coumarin derivative DBH2 inhibits proliferation and induces apoptosis of chronic myeloid leukemia cells

With the development of tyrosine kinase inhibitor (TKI) resistance, finding the novel effective chemotherapeutic agent is of seminal importance for chronic myelogenous leukemia (CML) treatment. This study aims to find the effective anti-leukemic candidates and investigate the possible underlying mechanism. We synthesized the novel coumarin derivatives and evaluated their anti-leukemic activity. Cell viability assay revealed that compound DBH2 exhibited the potent inhibitory activity on the proliferation of CML K562 cells and TKI resistant K562 cells. Morphological observation and flow cytometry confirmed that DBH2 could selectively induce cell apoptosis and cell cycle arrest at G2/M phase of the K562 cells, which was further confirmed on the bone marrow cells from CML transgenic model mice and CD34+ bone marrow leukemic cells from CML patients. Treatments of DBH2 in combination with imatinib could prolong the survival rate of SCL-tTA-BCR/ABL transgenic model mice significantly. Quantitative RT-PCR revealed that DBH2 inhibited the expression of STAT3 and STAT5 in K562 cells, and caspase-3 knockout alleviated the DBH2 induced apoptosis. Furthermore, DBH2 could induce the expression of PARP1 and ROCK1 in K562 cells, which may play the important role in caspase-dependent apoptosis. Our results concluded that coumarin derivative DBH2 serves as a promising candidate for the CML treatment, especially in the combination with imatinib for the TKI resistant CML, and STAT/caspase-3 pathway was involved in the molecular mechanism of anti-leukemic activity of DBH2.

Retraction Note: Knockdown of long non‑coding RNA TUG1 depresses apoptosis of hippocampal neurons in Alzheimer's disease by elevating microRNA‑15a and repressing ROCK1 expression.

Retraction Note: Knockdown of long non‑coding RNA TUG1 depresses apoptosis of hippocampal neurons in Alzheimer's disease by elevating microRNA‑15a and repressing ROCK1 expression.

Protective Effect of Rho-Associated Protein Kinase (ROCK) Activated by Bone Marrow Mesenchymal Stem Cells on Bone Strength and Osteoblasts in Rats with Osteoporosis

This study assesses BMSCs’ effect on bone strength and osteoblast activity in rats with osteoporosis. 40 SD female rats were assigned into normal (A) group, model (B) group, puerarin (C) group, and BMSCs (D) group followed by analysis of bone strength by small animal bone strength analyzer and the expression of ROCK1 protein in tibia by immunohistochemistry. The bone strength of group B was lower than group A (P < 0.05), and higher in groups C and D than group B (P < 0.05) with further higher in group D than group C (P < 0.05). Group A showed obvious mesh cancellous bone trabecular bone and continuity, compact structure, and group B cells appeared loose and irregular distribution, parts cancellous bone trabeculae decrease and part of the fracture, and arranged in sparse irregular distribution. The decrease of trabecular bone in group C and D is not more obvious than group B and the morphology in group D got better improved than in group C. Group B showed lower proliferation of osteoblasts and ALP activity than group A (P < 0.05) and the proliferation and ALP activity in groups C and D was increased significantly compared with group B (P < 0.05). ROCK1 expression was significantly lower in group B than group A (P < 0.05) higher in groups C and D than group B (P < 0.05). Bone marrow mesenchymal stem cells (BMSCs) can effectively increase the bone strength and activity of osteoblasts in rats with osteoporosis, and promote the activation of ROCK signal.

MicroRNA-148a induces apoptosis and prevents angiogenesis with bevacizumab in colon cancer through direct inhibition of ROCK1/c-Met via HIF-1α under hypoxia.

Angiogenesis and antiapoptosis effects are the major factors influencing malignancy progression. Hypoxia induces multiple mechanisms involving microRNA (miRNA) activity. Vascular endothelial growth factor (VEGF) is correlated with angiogenesis. An antiapoptotic factor, myeloid leukemia 1 (Mcl-1) is the main regulator of cell death. This study examined the role of miR-148a in inhibiting VEGF and Mcl-1 secretion by directly targeting ROCK1/c-Met by downregulating HIF-1α under hypoxia. The protein expression of ROCK1 or Met/HIF-1α/Mcl-1 in HCT116 and HT29 cells (all P < 0.05) was significantly reduced by miR-148a. The tube-formation assay revealed that miR-148a significantly suppressed angiogenesis and synergistically enhanced the effects of bevacizumab (both P < 0.05). The MTT assay revealed the inhibitory ability of miR-148a in HCT116 and HT29 cells (both P < 0.05). miR-148a and bevacizumab exerted synergistic antitumorigenic effects (P < 0.05) in an animal model. Serum miR-148a expression of metastatic colorectal cancer (mCRC) patients with a partial response was higher than that of mCRC patients with disease progression (P = 0.026). This result revealed that miR-148a downregulated HIF-1α/VEGF and Mcl-1 by directly targeting ROCK1/c-Met to decrease angiogenesis and increase the apoptosis of colon cancer cells. Furthermore, serum miR-148a levels have prognostic/predictive value in patients with mCRC receiving bevacizumab.

Development of Olaparib-Resistance Prostate Cancer Cell Lines to Identify Mechanisms Associated with Acquired Resistance.

Simple SummaryPARP inhibitors (PARPi; olaparib) are presently in clinical trials for advanced prostate cancer (PC). Resistance mechanisms are not fully understood in PC compared to ovarian and breast cancers. Our study aimed to identify new molecular mechanisms that affect acquired olaparib-resistance. We developed new resistant PC cell line models derived from original PC cell lines. We identified that DNA repair, autophagy, and the Rho-associated coiled-coil containing protein kinase 2 (ROCK2) could be potential targets to reverse the acquired olaparib-resistance.Background: Poly (ADP-ribose) polymerase inhibitors (PARPi) were initially deployed to target breast and ovarian tumors with mutations in DNA damage response genes. Recently, PARPi have been shown to be beneficial in the treatment of prostate cancer (PC) patients having exhausted conventional therapeutics. Despite demonstrating promising response rates, all patients treated with PARPi eventually develop resistance. However, PARPi resistance in PC is not well understood, and further studies are required to understand PARPi resistance in PC to propose strategies to circumvent resistance. Methods: Starting from well-established olaparib-sensitive PC cell lines (LNCaP, C4-2B and DU145), we derived olaparib-resistant (OR) PC cell lines and performed a microarray analysis. Results: The olaparib IC50 values of OR cell lines increased significantly as compared to the parental cell lines. Gene expression analyses revealed that different pathways, including DNA repair, cell cycle regulation and autophagy, were affected by acquired resistance. A total of 195 and 87 genes were significantly upregulated and downregulated, respectively, in all three OR cell lines compared to their parental counterparts. Among these genes, we selected BRCC3, ROCK2 and ATG2B for validation. We showed that ROCK2 expression, basal autophagy and homologous recombination (HR) efficiency were increased in all OR cell lines. Conclusions: Our study provides a new in vitro model to study PARPi resistance in PC and suggests new possible targets to reverse resistance and prolong the benefits of PARPi treatment.

Long Noncoding RNA KCNMB2-AS1 Increases ROCK1 Expression by Sponging microRNA-374a-3p to Facilitate the Progression of Non-Small-Cell Lung Cancer [Retraction

[This retracts the article DOI: 10.2147/CMAR.S270646.].

High Glucose Aggravates Retinal Endothelial Cell Dysfunction by Activating the RhoA/ROCK1/pMLC/Connexin43 Signaling Pathway.

PurposeThis research aims to explore the mechanism underlying the relationship between RhoA/ROCK signaling and Connexin43 (Cx43) in retinal endothelial cell dysfunction and to evaluate the protective effect of ROCK inhibitors against retinal endothelial cell dysfunction in diabetic retinopathy (DR) models.MethodsTUNEL staining, hematoxylin and eosin staining, a retinal digestion assay, and Evans blue assay were conducted to explore the effect of fasudil in alleviating retinal dysfunction induced by DR. ELISA, the CCK-8 assay, and flow cytometry were conducted to study inflammation, viability, and apoptosis of mouse retinal microvascular endothelial cells treated with high glucose and ROCK inhibitors. The qRT–PCR and Western blotting were used to evaluate the expression of RhoA, ROCK1, ROCK2, MLC, pMLC, and Cx43. Co-immunoprecipitation was used to verify the interaction between pMLC and Cx43. Immunofluorescence and scrape-loading and dye transfer were used to evaluate the expression and function of Cx43.ResultsMarked endothelial cell dysfunction resulting from the activation of RhoA/ROCK1 signaling was found in in vivo and in vitro models of DR. Via interaction with pMLC, which is downstream of RhoA/ROCK1, a significant downregulation of Cx43 was observed in retinal endothelial cells. Treatment with ROCK inhibitors ameliorated retinal endothelial dysfunction in vitro. The ROCK inhibitor, fasudil, significantly alleviated retinal dysfunction as shown by a decrease of retinal acellular capillaries, an improvement of vascular permeability, and a reduction of cell apoptosis in vivo.ConclusionsOur study highlights a novel mechanism that high glucose could activate RhoA/ROCK1/pMLC signaling, which targets the expression and localization of Cx43 and is responsible for cell viability, apoptosis, and inflammation, resulting in retinal endothelial cell injury. ROCK inhibitors markedly ameliorate endothelial cell dysfunction, suggesting their therapeutic potential for diabetic retinopathy.

Mechanical Stretch Promotes Macrophage Polarization and Inflammation via the RhoA-ROCK/NF-κB Pathway.

Macrophages play an essential role in the pathogenesis of most inflammatory diseases. Recent studies have shown that mechanical load can influence macrophage function, leading to excessive and uncontrolled inflammation and even systemic damage, including cardiovascular disease and knee osteoarthritis. However, the molecular mechanism remains unclear. In this study, murine RAW264.7 cells were treated with mechanical stretch (MS) using the Flexcell-5000T Tension System. The expression of inflammatory factors and cytokine release were measured by RT-qPCR, ELISA, and Western blotting. The protein expression of NF-κB p65, Iκb-α, p-Iκb-α, RhoA, ROCK1, and ROCK2 was also detected by Western blotting. Then, Flow cytometry was used to detect the proportion of macrophage subsets. Meanwhile, Y-27632 dihydrochloride, a ROCK inhibitor, was added to knockdown ROCK signal transduction in cells. Our results demonstrated that MS upregulated mRNA expression and increased the secretion levels of proinflammatory factors iNOS, IL-1β, TNF-α, and IL-6. Additionally, MS significantly increased the proportion of CD11b+CD86+ and CD11b+CD206+ subsets in RAW264.7 macrophages. Furthermore, the protein expression of RhoA, ROCK1, ROCK2, NF-κB p65, and IκB-α increased in MS-treated RAW264.7 cells, as well as the IL-6 and iNOS. In contrast, ROCK inhibitor significantly blocked the activation of RhoA-ROCK and NF-κB pathway, decreased the protein expression of IL-6 and iNOS, reduced the proportion of CD11b+CD86+ cells subpopulation, and increased the proportion of CD11b+CD206+ cell subpopulation after MS. These data indicate that mechanical stretch can regulate the RAW264.7 macrophage polarization and enhance inflammatory responses in vitro, which may contribute to activation the RhoA-ROCK/NF-κB pathway.

Micro RNA 200a contributes to the smooth muscle cells growth in aged-related erectile dysfunction via regulating Rho/ROCK pathway.

Aged-related erectile dysfunction (A-ED) is generally regarded as degeneration of penile erectile tissue due to age, male hormone deficiency and concomitant cardiovascular disease. Current pathological studies of A-ED are still limited. In this study, aged rats were divided into AE group (aged rats with ED) and YN group (young normal rats) for evaluating the roles of miRNA-200a and RhoA/ROCK signalling pathway in A-ED. Apo-morphine test, ICP measurement and pathological results were compared between these two groups. After transfection of miRNA-200a into Corpus cavernosum smooth muscle cells (CCSMCs), the expression of miRNA-200a, RhoA, ROCK1 and ROCK2 in the AE group were significantly increased. Additionally, miRNA-200a, RhoA, ROCK1 and ROCK2 were upregulated at a high level after transfecting the miRNA-200a mimics. Therefore, we speculated that miRNA-200a is a positive regulator, which may inhibit the growth of CCSMCs by activating the Rho/ROCK pathway in vitro.

Targeting Effect of Betulinic Acid Liposome Modified by Hyaluronic Acid on Hepatoma Cells In Vitro

Betulinic acid (BA) is a natural pentacyclic triterpenoid with broad-spectrum anticancer activity, which has great development potential as an anti-cancer drug. In this study, a novel hyaluronic acid (HA)-modified BA liposome (BA-L) was developed for use in targeted liver cancer therapy. The size, polymer dispersity index (PDI), zeta potential, and entrapment efficiency were measured. Cell viability, cell migration and clonogenicity, cellular uptake, immunohistochemistry of CD44, and protein expression of ROCK1/IP3/RAS were also investigated. BA, BA-L, and HA-BA-L had no inhibitory effect on the activity of LO2 normal hepatocytes, but they inhibited the proliferation of HepG2 and SMMC-7721 cells in a dose- and time-dependent manner, with HA-BA-L exhibiting the most prominent inhibitory effect. Compared with the BA-L group, the expression of CD44 in HepG2 cells in the HA-BA-L group was decreased. The results of WB showed that BA, BA-L, and HA-BA-L downregulated the expression of ROCK1, IP3, and RAS in HepG2 cells, and the expression level in the HA-BA-L group was significantly decreased. The easily prepared HA-BA-L was demonstrated to be an excellent CD44-mediated intracellular delivery system capable of targeting effects. Further mechanistic research revealed that the inhibition of HA-BA-L on HepG2 cells may be mediated by blocking the ROCK1/IP3/RAS signaling pathways.