Expression Levels Of RhoA Research Articles

SiRNA Mediated Downregulation of RhoA Expression Reduces Oxidative Induced Apoptosis in Retinal Ganglion Cells.

Glaucoma is the second leading cause of blindness. Apoptosis of retinal ganglion cells (RGCs) is an important mechanism of glaucomatous optic injury. Rho kinase expression is significantly increased in apoptotic RGCs. This study aimed to investigate the role of RhoA, a Rho GTPase, on the survival of RGCs and further to explore its potential therapeutic applications. RGCs were treated with siRhoA for 24 hours in vitro. Knockdown of RhoA was confirmed with quantitative RT-PCR. Oxidative stress was induced by treating the RGCs with 200 μM of H2O2 for 1 hour, and apoptosis of RGCs was quantified with TUNEL assay in situ, and with flow cytometry. The mRNA expression levels of RhoA, Nogo receptor, caspase 3 and Bcl-2 were evaluated by quantitative RT-PCR, and the protein levels of RhoA, ROCK1, ROCK2, Nogo receptor, caspase 3 and Bcl-2 were evaluated by Western blot. We found siRhoA treatment efficiently downregulated the expression of RhoA in RGCs and protected against H2O2-induced injury in RGCs in vitro. Apoptosis of RGC cells under oxidative stress was quantified in situ using TUNEL assay and confirmed with flow cytometry (FCM). With the knockdown of RhoA, the expression of ROCK1, ROCK2, Nogo Receptor, Casepase-3 were decreased, while the expression of Bcl-2 was increased in both mRNA and protein level. Our data indicated that siRhoA prevented H2O2-induced apoptosis in RGC cells by modulating the RhoA/ROCK pathway. The results suggested that siRhoA may exert potentially effective neuroprotection for RGCs by reducing injury.

Potential role of RhoA GTPase regulation in type interferon signaling in systemic lupus erythematosus

ObjectiveSystemic lupus erythematosus (SLE) is an autoimmune disorder characterized by abnormal activation of the type I interferon (IFN) pathway, which results in tissue inflammation and organ damage. We explored the role of the RhoA GTPase in the type I IFN activation pathway to provide a potential basis for targeting GTPase signaling for the treatment of SLE.MethodsTotal RNA was extracted from peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy controls, and the mRNA expression levels of RhoA and IFN-stimulated genes were measured by SYBR Green quantitative reverse transcriptase-polymerase chain reaction. IFN-a-stimulated response element (ISRE)-luciferase reporter gene assays and Western blotting were conducted to assess the biologic function of RhoA. An enzyme-linked immunoassay (ELISA) measured C-X-C motif chemokine ligand 10 (CXCL10) protein expression.ResultsOur studies demonstrate that the expression of RhoA in the PBMCs of SLE subjects was significantly higher than in healthy controls and positively correlated with type I IFN scores and type I IFN-stimulated gene (ISGs) expression levels. SiRNA-mediated knockdown of RhoA and the RhoA/ROCK inhibitor Y27632 reduced the activity of the type I IFN-induced ISRE, the signal transducer and activator of transcription 1 (STAT-1) phosphorylation, and the expression of CXCL10 and 2′-5′-oligoadenylate synthetase 1 (OAS1). Finally, we verified that Y27632 could significantly down-regulate the OAS1 and CXCL10 expression levels in the PBMCs of SLE patients.ConclusionOur study shows that RhoA positively regulates the activation of the type I IFN response pathway. Reducing the expression level of RhoA inhibits the abnormal activation of the type I IFN system, and the RhoA/ROCK inhibitor Y27632 decreases aberrant type I IFN signaling in SLE PBMCs, suggesting the possibility of targeting the RhoA GTPase for the treatment of SLE.

Ulinastatin protects against myocardial ischemia/reperfusion injury in rats via Rho/ROCK signaling pathway.

We aimed to study the influences of ulinastatin on diseased myocardial tissues, cardiomyocyte apoptosis and inflammatory reaction in rats with myocardial ischemia/reperfusion injury (IRI) via the Ras homolog (Rho)/Rho-associated kinase (ROCK) signaling pathway and its mechanism. The rats were randomly divided into three groups: control group (C group), IR model group (IR group) and IR model + ulinastatin treatment group (UR group). The pathological changes in myocardial tissues were detected via HE staining, the markers of myocardial injury were examined using kits, and apoptosis was determined through TUNEL assay. Moreover, ELISA was applied to measure the expressions of TNF-α, interleukin-6 (IL-6) and IL-8 in cardiac tissues, and Western blotting was performed to detect the protein expression levels of RhoA, ROCK2 and MLCP. The myocardial infarction area in the IR group was markedly larger than that in the C group (P<0.01) but was significantly reduced after ulinastatin treatment (P<0.05), and the IR group had higher levels of AST, cTnI, CK-MB and LDH than C group, but the levels of those indexes were significantly reduced after ulinastatin treatment.The cardiomyocyte apoptosis was increased in the IR group compared with that in the C group, while it was decreased in the UR group in comparison with that in the IR group. Besides, the UR group exhibited lowered expression levels of the Rho/ROCK signaling pathway-related proteins compared with the IR group. Ulinastatin may ameliorate the prognosis of rats with myocardial IRI via the Rho/ROCK signaling pathway.

Effects of combined electroacupuncture and medication therapy on the RhoA/ROCK-2 signaling pathway in the striatal region of rats afflicted by cerebral ischemia

ObjectiveTo investigate the effects of electroacupuncture(EA), gastrodin(Gas), and their combination on the signaling pathways involving Ras homologous gene family member A (RhoA) and Rho-associated frizzled helix protein kinase (ROCK-2) within the striatal region of rats subjected to cerebral ischemia. Additionally, we aim to elucidate the therapeutic effects and potential underlying mechanisms associated with the concurrent application of electroacupuncture and medication in the treatment of cerebral ischemia. MethodsRats were randomly assigned to one of five groups, namely, the sham operation (Sham) group, model group, EA group, Gas group, and the EA combined with Gas group (referred to as the "EA+Gas group"). Each group consisted of ten rats. Following the induction of cerebral ischemia, the EA group and EA+Gas group received EA stimulation at the Baihui(GV20) and Zusanli(ST36) acupoints for 30 min per session, administered once daily for 14 consecutive days. The Gas group and EA+Gas group were intraperitoneally injected with Gas at a dosage of 10 mg/kg, also administered once daily for 14 consecutive days. Nissl staining was employed to observe morphological alterations in the striatal nerve cells of rats in each group. Immunohistochemistry and western blot techniques were employed to evaluate the expression levels of striatal RhoA and ROCK-2 proteins. ResultsIn comparison to the Sham group, the model group exhibited a substantial reduction in the number of striatal nerve cells on the ischemic side, accompanied by notable changes in cell morphology, characterized by reduced cytoplasm, defective and atrophied cytosol, solidified nuclei, loosely arranged cells, and enlarged intercellular spaces. Additionally, there was a notable increase in the positive expression of RhoA and ROCK-2. In contrast, when compared to the model group, the EA, Gas, and EA+Gas groups demonstrated an elevated number of normal nerve cells within the ischemic striatal region, with a significant improvement in cell count and morphology. Furthermore, positive expression levels of RhoA and ROCK-2 were notably reduced in these groups. Compared with the EA group or the GAS group, the number of normal nerve cells in the striatum on the ischemic side of the EA+GAS group was further increased, and the positive expression level of RhoA and ROCK-2 were both further reduced. ConclusionThe protective mechanism underlying the therapeutic efficacy of EA combined with Gas against cerebral ischemic striatal injury in rats may be associated with the inhibition of the activation of the RhoA/ROCK-2 signaling pathway. Importantly, the therapeutic effects observed with the combination of electroacupuncture and medication were superior to those achieved with EA alone or the sole administration of Gas.

MiR-155 enhances phagocytosis of alveolar macrophages through the mTORC2/RhoA pathway.

Alveolar macrophage phagocytosis is significantly reduced in Chronic obstructive pulmonary disease, and cigarette smoke extract is one of the chief reasons for this decrease. Nevertheless, the specific underlying mechanism remains elusive. In this study, the role and possible mechanism of miR-155-5p/mTORC2/RhoA in the phagocytosis of mouse alveolar macrophages (MH-S) were explored. Our results revealed that cigarette smoke extract intervention reduced MH-S cell phagocytosis and miR-155-5p expression. Meanwhile, the dual-luciferase reporter assay validated that Rictor is a target of miR-155-5p. On the one hand, transfecting miR-155-5p mimic, mimic NC, miR-155-5p inhibitor, or inhibitor NC in MH-S cells overexpressing miR-155-5p increased the Alveolar macrophage phagocytotic rate, up-regulated the expression level of RhoA and p-RhoA, and down-regulated that of mTOR and Rictor mRNA and protein. On the other hand, inhibiting the expression of miR-155-5p lowered the phagocytotic rate, up-regulated the expression of mTOR, Rictor mRNA, and protein, and down-regulated the expression of RhoA and p-RhoA, which taken together, authenticated that miR-155-5p participates in macrophage phagocytosis via the mTORC2/RhoA pathway. Finally, confocal microscopy demonstrated that cells overexpressing miR-155-5p underwent cytoskeletal rearrangement during phagocytosis, and the phagocytic function of cells was enhanced, signaling that miR-155-5p participated in macrophage skeletal rearrangement and enhanced alveolar macrophage phagocytosis by targeting the expression of Rictor in the mTORC2/RhoA pathway.

Zhuidu Formula suppresses the migratory and invasive properties of triple-negative breast cancer cells via dual signaling pathways of RhoA/ROCK and CDC42/MRCK

Ethnopharmacological relevanceZhuidu Formula (ZDF) is composed of triptolide, cinobufagin and paclitaxel, which are the active ingredients of Tripterygium wilfordii Hook. F, dried toad skin and Taxus wallichiana var. chinensis (Pilg) Florin, respectively. Modern pharmacological studies show that triptolide, cinobufagin, and paclitaxel are well-known natural compounds that exert anti-tumor effects by interfering with DNA synthesis, inducing tumor cell apoptosis, and inhibiting the dynamic balance of the tubulin. However, the mechanism by which the three compounds inhibit triple-negative breast cancer (TNBC) metastasis is unknown. ObjectiveThe objective of this investigation was to examine the inhibitory essences of ZDF on the metastasis of TNBC and elucidate its potential mechanism. Materials and methodsCell viability of triptolide (TPL), cinobufagin (CBF), and paclitaxel (PTX) on MDA-MB-231 cells was assessed employing a CCK-8 assay. The drug interactions of the three drugs on MDA-MB-231 cells were determined in vitro utilizing the Chou-Talalay method. MDA-MB-231 cells were identified for migration, invasion and adhesion in vitro through the implementation of the scratch assay, transwell assay and adhesion assay, respectively. The formation of cytoskeleton protein F-actin was detected by immunofluorescence assay. The expressions of MMP-2 and MMP-9 in the supernatant of the cells were determined by ELISA analysis. The Western blot and RT-qPCR were employed to explore the protein expressions associated with the dual signaling pathways of RhoA/ROCK and CDC42/MRCK. The anti-tumor efficacy of ZDF in vivo and its preliminary mechanism were investigated in the mouse 4T1 TNBC model. ResultsThe results demonstrated that ZDF could significantly reduce the viability of the MDA-MB-231 cell, and the combination index (CI) values of actual compatibility experimental points were all less than 1, demonstrating a favorable synergistic compatibility relationship. It was found that ZDF reduces RhoA/ROCK and CDC42/MRCK dual signaling pathways, which are responsible for MDA-MB-231cell migration, invasion, and adhesion. Additionally, there has been a significant reduction in the manifestation of cytoskeleton-related proteins. Furthermore, the expression levels of RhoA, CDC42, ROCK2, and MRCKβ mRNA and protein were down-regulated. ZDF significantly decreased the protein expressions of vimentin, cytokeratin-8, Arp2 and N-WASP, and inhibited actin polymerization and actomyosin contraction. Furthermore, MMP-2 and MMP-9 levels in the high-dose ZDF group were decreased by 30% and 26%, respectively. ZDF significantly reduced the tumor volume and protein expressions of ROCK2 and MRCKβ in tumor tissues without eliciting any perceptible alterations in the physical mass of the mice, and the reduction was more pronounced than that of the BDP5290 treated group. ConclusionThe current investigation demonstrates that ZDF exhibits a proficient inhibitory impact on TNBC metastasis by regulating cytoskeletal proteins through the dual signaling pathways of RhoA/ROCK and CDC42/MRCK. Furthermore, the findings indicate that ZDF has significant anti-tumorigenic and anti-metastatic characteristics in breast cancer animal models.

HIF-1α dependent RhoA as a novel therapeutic target to regulate rheumatoid arthritis fibroblast-like synoviocytes migration in vitro and in vivo.

The purpose of this work is to investigate how the Rho family of GTPases A (RhoA) mediates the pathogenesis of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). The expression of RhoA in the synovial tissues of RA and Healthy people (Control) was detected using immunohistochemistry methods. The expression of RhoA and hypoxia-inducible factor-1α (HIF-1α) is inhibited by small interfering RNAs (siRNAs). The inhibition effect on RA-FLS migration was further investigated. The protein expression level of HIF-1α, RhoA, focal adhesion kinase (FAK), and myosin light chain (MLC) was also analysed using western blotting (WB). DBA1 mice were immunised with the mixture of bovine type II collagen and Freund's adjuvant to establish collagen induced arthritis (CIA) mouse model. Lip-siRhoA is administered through joint injection every two days. Micro-computed tomography (micro-CT) was used to detect mouse ankle joint destruction and evaluate the bone loss of the periarticular side. Destruction of the ankle articular cartilage was tested by histology. Expressions of P-RhoA, P-FAK and P-MLC in the ankle joint was detected by immunohistochemistry assay. The expression level of RhoA in the synovial tissues of RA patients was significantly higher than that in control group. Hypoxia was able to up-regulate the expression of RhoA. Whereas, HIF-1α siRNA (siHIF-1α) could down-regulate the expression of RhoA. Additionally, both of siHIF-1α and RhoA siRNA (siRhoA) delivered by liposome (Lip-siHIF-1α and Lip-siRhoA) were found to suppress FAK and MLC phosphorylation in vitro. In CIA mouse model, Lip-siRhoA was demonstrated to ameliorate the destruction of ankle joint and reduce the severity of ankle joint cartilage damage by micro-CT and histological staining, respectively. Therefore, inhibition of FLS cell migration can protect articular bone from destruction. Furthermore, the expression of P-RhoA, P-FAK and P-MLC was evaluated and found to be down-regulated by Lip-siRhoA in vivo. The results demonstrated that under hypoxic environment, HIF-1α dependent RhoA pathway played an important role on cytoskeleton remodelling and RA-FLS migration. Through down-regulating RhoA expression, it could effectively treat RA in vitro and in vivo. Our study provides new evidence for the potential clinical application of RhoA as a candidate for the treatment of RA.

Effects of berberine hydrochloride on left ventricular structure and function in rats with myocardial hypertrophy

Background Inhibition of Rho/ROCK signalling pathway related proteins can alleviate left ventricular hypertrophy. Berberine hydrochloride (BBR) can effectively inhibit left ventricular hypertrophy. The purpose of this study is to explore the relationship between BBR and Rho/ROCK signalling pathway. Methods Isoproterenol (ISO) was used to induce left ventricular hypertrophy in rats. Two-dimensional speckle tracking technique (2D-STE) was used to evaluate rats in each group (group A: normal control group; Group B: isoproterenol model group; Group C: ISO + 5mg/kg BBR group; Group D: ISO + 10mg/kg BBR group) Heart structure and systolic function. HE staining and Masson staining were used to observe the pathological changes in four groups of rats. The expression levels of RhoA, ROCK-1, TGF-β1 and PTEN protein in myocardial tissues were detected by immunohistochemistry. Result The interventricular septum (IVS) of rats in groups C and D was thinner than that in group B (4.05 ± 0.16 mm vs. 3.50 ± 0.29 mm vs. 4.41 ± 0.23 mm, respectively, p < 0.05), and the global radial strain (GRS) of rats in groups C and D was higher than that in group B, especially in group D (26.05 ± 1.41 vs. 30.64 ± 1.63 vs. 19.40 ± 1.05, respectively, p < 0.05). Compared with group B, the expression levels of RhoA, ROCK-1 and TGF-β1 in groups C and D decreased, while the expression level of PTEN increased, especially in group D (all p < 0.05). Conclusion BBR can improve the cardiac structure and systolic function of rats with left ventricular hypertrophy, and the improvement mechanism may be related to Rho/ROCK signalling pathway.

Dysregulation of miR-193a serves as a potential contributor to MS pathogenesis via affecting RhoA and Rock1

Multiple sclerosis (MS) is one of the most common neurological diseases that cause chronic inflammation of the central nervous system and demyelination of the myelin sheath. At present, microRNAs (miRNAs) are considered not only a diagnostic and prognostic indicator of diseases but also a new goal in gene therapy. This study aims to find a simple, non-invasive, valuable biomarker for early detection and potential treatment of MS. In the present study, 30 patients with MS were included. The qRT-PCR method was performed to evaluate the expression level of miR-193a, RhoA, and ROCK1. Besides, western blotting was performed to determine the expression level of RhoA and ROCK1 at protein levels. Moreover, we aimed to clarify the possible correlation between miR-193a-5p and its-regulated target genes so that miR-193a-5p mimic was transfected into MS-derived cultured PBMSs, and the expression level of RhoA and ROCK1 were then evaluated by qRT-PCR and Western blotting. In the final step, the correlation between miR-193a-5p and clinicopathological features of patients was investigated. Results showed that miR-193a was decreased while RhoA and ROCK1 were up-regulated in PBMCs obtained from patients with MS compared to the control group. It was also revealed that miR-193a transfection reduced RhoA and ROCK1 expression at mRNA and protein levels. The results from the Chi-square analysis showed that down-regulation of miR-193a was associated with increased CRP level, CSF IgG positivity, and MSSS (Multiple Sclerosis Severity Score), suggesting miR-193a is a potential diagnostic and prognostic indicator. We implied that miR-193a could modulate RhoA and ROCK 1 expression in MS patients, in which its down-regulation leads to increased expression of RhoA and ROCK1 and poor prognosis of patients with MS. Therefore, miR-193a and its associated targets could serve potential prognostic, diagnostic, and therapeutic efficacy in MS patients.

Inhibition of Rho/ROCK signaling pathway participates in the cardiac protection of exercise training in spontaneously hypertensive rats

Exercise training (ExT) is capable of improving the heart function of spontaneously hypertensive rats (SHRs), but the underlying molecular mechanisms remain elusive. This study was aimed to investigate whether inhibition of RhoA/ROCK signaling pathway contributes to the cardiac protection by low-intensity ExT in SHRs. The results demonstrated that, compared with Wistar-Kyoto (WKY) rats, SHRs obviously exhibited higher blood pressure, increased heart weight index and thickness of left ventricular wall, decreased left ventricular function, damaged myocardial construction, and increased collagen fiber of left ventricle (P < 0.05 or P < 0.01). Meanwhile, the mRNA and protein expression levels of RhoA and ROCK in the heart of SHRs were significantly increased, compared with those of WKY rats (P < 0.05 or P < 0.01). Interestingly, the pathological changes of heart aforementioned were all improved in SHR-ExT rats compared with SHR-Sed rats (P < 0.05 or P < 0.01), indicating the cardiac protection of exercise training. In addition, the cardiac protective effect of exercise training could be blocked by LPA, an activator of Rho/ROCK signaling, and the protective effect in SHR rats could be mimicked by Fasudil, an inhibitor of Rho/ROCK signaling. The results strongly suggest that low-intensity ExT can protect heart against structure and function through inhibiting Rho/ROCK signaling pathway in hypertensive rats.

Effect of electroacupuncture on colonic motility and RhoA and ROCK protein expression in IBS-D rats

To observe the effect of electroacupuncture (EA) of "Tianshu"(ST25) and "Shangjuxu"(ST37)on gastrointestinal motility, psychological abnormality and expression of RhoA and ROCK protein in rats with diarrhea predominant irritable bowel syndrome (IBS-D), so as to explore its underlying mechanism in improving IBS-D. Thirty-six male rats were randomly and equally divided into control, model, EA and medication groups (n=9 in each group). The IBS-D model with psychological abnormality was established by gavage of dinitrobenzene sulfonic acid (DNBS) + chronic restraint stress. EA (2 Hz/100 Hz, 0.3 mA) was applied to bilateral ST25 and ST37 for 20 min, once a day for 7 days. Rats of the medication group received gavage of pinaverium bromide solution (15 mg/kg), once a day for 7 days. The rats' food intake in 24 h, body mass and colonic contraction waves were recorded. The anhedonia-related behavior was measured using the sucrose consumption test. The elevated plus maze test (the open-arm residence time ratio) was used to assess the anxiety-like behavior. The small intestinal propulsion rate test was used to assess the intestinal motility. The expression levels of RhoA and ROCK proteins in the colonic tissue were measured by Western blot. After modeling, the body mass, food consumption, sucrose preference index, the open-arm residence time ratio andlatency of colonic contraction waves were significantly decreased (P<0.05), and the number of contraction waves, intestinal propulsive rate, and the expression levels of RhoA and ROCK proteins considerably increased (P<0.01，P<0.05) in the model group relevant to the control group. Following the interventions, the decrease of body mass, food consumption, sucrose preference index, open-arm residence time ratio and latency of contraction waves, and the increase of the contraction waves, intestinal propulsive rate, and the expression levels of RhoA and ROCK proteins were all reversed by both EA and medication (P<0.05，P<0.01). The effect of EA was significantly superior to that of medication in increasing the sucrose pre-ference index (P<0.05). EA can improve both colonic motility and psychological disorders in IBS-D rats with psychological disorder, which may be related to its function in down-regulating the expression of colonic RhoA and ROCK proteins.

RHOA protein expression correlates with clinical features in gastric cancer: a systematic review and meta-analysis

BackgroundGastric cancer (GC) is one of the most fatal cancers worldwide and is generally only detected after it has progressed to an advanced stage. Since there is a lack of comprehensive data on RHOA protein expression of patients with GC, this study utilized a systematic review and meta-analysis to address the limitation. The objective of this meta-analysis was to link GC clinical features with RHOA protein high- vs. low-expressing patients with GC.MethodsThe PubMed and Web of Science were used for a systematic literature review of GC related to RHOA. The included studies were obtained from two literature databases from past to Aug 31, 2021, by searching keywords. This meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The odds ratios (ORs) and 95% confidential intervals (CIs) for clinical features were estimated according to the high and low protein expression levels of RhoA. The mean effect sizes of ORs were obtained using the random-effects and fixed-effects models of meta-analysis. Heterogeneity of the studies was assesed by using statistics: τ2, I2; and Q values. The symmetry of funnel plots were inspected for publication bias.ResultsFinally, 10 studies including 1,389 patients with GC (735 RHOA-positive and 654 RHOA-negative) were eligible for our meta-analysis to estimate associations between the protein expression and clinical features (e.g., Union for International Cancer Control [UICC] stage progression, differentiation, Lauren histological classification, and vascular invasion). In our meta-analysis, RHOA positive expression was determined to have a statistically significant association with UICC stage progression (P = 0.02) and poorly differentiated status (P = 0.02). The association between RHOA positivity and Lauren subtypes was not statistically significant (P = 0.07).ConclusionsThis meta-analysis suggested that RhoA protein expression in patients with GC was associated with clinical features: UICC stage progression and poorly differentiated status. Our findings are inconclusive but indicate that high RHOA protein expressing patients with GC could predict advanced UICC stages. A large prospective cohort study is required for validation in future.

Simvastatin alleviates epithelial-mesenchymal transition and oxidative stress of high glucose-induced lens epithelial cells in vitro by inhibiting RhoA/ROCK signaling.

Diabetic cataracts (DC) is one of the main causes of blindness among patients with diabetes mellitus. The aim of the present study was to examine the effect of simvastatin on lens epithelial cells in DC and the underlying mechanism. The viability of SRA01/04 cells treated with different concentrations of simvastatin was detected using a Cell Counting Kit-8 assay before and after high glucose (HG) treatment. The expression levels of E-cadherin, N-cadherin, Vimentin and α-smooth muscle actin (α-SMA), proteins associated with epithelial-mesenchymal transition (EMT), in addition to RhoA, Rho-associated kinases (ROCK)1 and ROCK2, proteins related to RhoA/ROCK signaling, were also measured in SRA01/04 cells treated with HG and simvastatin, with or without U46619, using western blot analysis. DCFH-DA dyes, superoxide dismutase (SOD) and glutathione (GSH)/glutathione disulfide (GSSG) kits were used to measure the levels of oxidative stress parameters in SRA01/04 cells treated with HG and simvastatin with or without U46619. The cell viability of SRA01/04 cells treated with simvastatin was found to be significantly elevated after HG treatment. The protein expression levels of E-cadherin were increased but those of N-cadherin, Vimentin and α-SMA decreased after HG and simvastatin treatment, and this was reversed by U46619. The levels of SOD and GSH-GSSG were found to be increased whereas reactive oxygen species levels were decreased, effects that were reversed by U46619. Additionally, the protein expression levels of RhoA, ROCK1 and ROCK2 were markedly decreased. These findings provided evidence that simvastatin increased HG-induced SRA01/04 cell viability and exerted inhibitory effects on EMT and oxidative stress that occurs during DC.

Dietary patterns in association with the expression of pro-metastatic genes in primary breast cancer.

Metastasis is a major leading cause of mortality in female breast cancer (BrCa). Cellular motility is a pathological process of metastasis remarked by the overexpression of cortactin (CTTN), Ras homolog family member-A (RhoA), and Rho-associated kinase (ROCK) genes.Their balanceisresponsible forupholding the integrity of healthy epithelial cell junctions. This study aimed to explore the associations between a posteriori dietary patterns and theexpression levels of pro-metastatic genes in primary BrCa. In this consecutive case series, 215 eligible women, newly diagnosed with histologically confirmednon-metastatic BrCa (stage I-IIIA), were recruited from Hospitals in Tabriz, NorthwesternIran (2015-2017). The tumoral expression levels of genes were quantified using real-time reverse transcription-polymerase chain reaction. Dietary data assessment was carried out using a validated food frequency questionnaire. Three dietary patterns were identified using principal component analysis (KMO = 0.699). Adherence to the "vegan" pattern (vegetables, fruits, legumes, nuts, seeds, and whole grains) was inversely associated with the expression levels of RhoA (ORAdj.T3vs.T1 = 0.24, 95%CI 0.07-0.79) and ROCK (ORAdj.T3vs.T1 = 0.26, 95%CI 0.08-0.87). In addition, the highest adherence to the "prudent" pattern (spices, seafood, dairy, and vegetable oils) decreased the odds of overexpressions at RhoA (ORAdj.T3vs.T1 = 0.26, 95%CI 0.08-0.84) and ROCK genes (ORAdj.T3vs.T1 = 0.29, 95%CI 0.09-0.95). The highest adherence to "Western" pattern (meat, processed meat, hydrogenated fat, fast food, refined cereals, sweets, and soft drinks) was a risk factor associated with the overexpression of RhoA (ORAdj.T3vs.T1 = 3.15, 95%CI 1.12-8.85). Adherence to healthy dietary patterns was significantly associated with the downregulation of pro-metastatic genes. Findings provided new implications to advance the nutrigenomic knowledge to prevent the odds ofover-regulations in pro-metastatic genes of theprimary BrCa.

Mechanism study of the protective effects of selective cyclooxygenase-2 enzyme inhibitors on the liver of rats with type 2 diabetes mellitus combined with nonalcoholic steatohepatitis via Rho/ROCK pathway

Objective: To investigate whether the selective cyclooxygenase-2 enzyme inhibitors celecoxib has protective effect on the liver of rats with type 2 diabetes mellitus (T2DM) combined with nonalcoholic steatohepatitis (NASH) via inhibiting the expression of Rho/ROCK pathway. Methods: Forty male SD rats were randomly divided into four groups: type 2 diabetes mellitus combined with nonalcoholic steatohepatitis (T2DM-NASH) group, T2DM-NASH + celecoxib group, control group, and control+celecoxib group. The T2DM-NASH and T2DM-NASH + celecoxib groups were fed with high-sugar and fat diet, and the control group and control + celecoxib group were fed with basal diet (25 kJ/kg). Four weeks later, streptozotocin (STZ, 30 mg/kg) was intraperitoneally injected into the NASH group and T2DM-NASH + celecoxib group to induce T2DM model, and the control group and control + celecoxib group were intraperitoneally injected with isovolumic citric acid-sodium citrate buffer. Four weeks after STZ injection, the T2DM-NASH + celecoxib group and the control + celecoxib group were gavaged with celecoxib (10 mg·kg·d) dissolved in normal saline for 4 weeks, and the remaining two groups of rats were gavaged with isovolumic normal saline for 4 weeks. Animals were sacrificed at the end of the 12- weeks, and the liver tissue was collected. Liver pathological changes were observed by HE staining. The expressions of RhoA, RhoA, ROCK1 and ROCK2 proteins in liver were detected by immunohistochemistry and western blot. The expressional condition of RhoA, ROCK1 and ROCK2 mRNA in liver were detected by real-time quantitative PCR. The differences were compared between protein and mRNA expression among the groups by analysis of variance and t-test. Results: Compared with the control group and the control + celecoxib group, the liver tissue of the T2DM-NASH group and the T2DM-NASH + celecoxib group had severe steatosis, and there was partial inflammatory cell infiltration under the light microscope. The expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were significantly increased (P < 0.05) in each liver tissue, while liver steatosis was reduced to certain extent in T2DM-NASH + celecoxib group than T2DM-NASH group, and the expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were decreased in each liver tissue of T2DM-NASH group (P < 0.05). Conclusion: The selective cyclooxygenase-2 enzyme inhibitors celecoxib has a protective effect on the liver of rats with T2DM-NASH, and its effect may be achieved by inhibiting the expression of Rho/ROCK pathway.

RhoA, ROCK-1, and ROCK-2 Gene Expression and Polymorphisms in Cholesteatoma Patients.

Background: The aim of this study was to evaluate the gene polymorphism and expressions of Rho-A, ROCK-1, and ROCK-2 in cholesteatoma.Methods: In this study, 120 healthy control group patients and 120 cholesteatoma patients were enrolled. Venous blood was taken from all of the cholesteatoma and control group patients. The genotyping of ROCK-1(G/T)rs35996865, ROCK-2(A/C)rs10178332, and Rho-A(A/T)rs2177268 polymorphisms was performed using predesigned TaqMan SNP Genotyping Assays. Assays-on-Demand SNP genotyping kit was used for the real-time polymerase chain reaction. The expression levels of Rho-A(Hs00357608_m1), ROCK-1(Hs01127699_m1), and ROCK-2(Hs00178154_m1) genes were determined.Results: The expression of Rho-A, ROCK-1, and ROCK-2 was lower in cholesteatoma patients than in the control group. There was no difference in Rho-AAT/TT and ROCK-1GT/TT variation in cholesteatoma patients compared to the control. However, ROCK-2 AC/CC variance was lower in cholesteatoma patients.Conclusion: The expression of Rho-A, ROCK-1, and ROCK-2 genes may be decreased in cholesteatoma. Furthermore, since ROCK-2 AC/CC genotype is also lower in cholesteatoma, having C allele seems to decrease the risk of developing this disease.

RhoA/ROCK pathway involved in effects of Sanjiao acupuncture on learning and memory and synaptic plasticity in Alzheimer's disease mice

To observe the effect of Sanjiao acupuncture(triple energizer acupuncture)on the small G protein guanosine triphosphate enzyme subfamily protein RhoA/Rho kinase (ROCK) pathway in Alzheimer's disease mice, and explore its effect on learning and memory function and neurosynaptic plasticity. Forty SAMP8 senile dementia mice were randomly divided into model, Sanjiao acupuncture (acupuncture), non acupoint acupuncture (non-acupoint) and fasudil groups, with 10 mice in each group, another 10 SAMR normal aging mice were selected as normal aging group. Mice in the acupuncture group were treated with acupuncture intervention on "Danzhong"(CV18), "Zhongwan"(CV13), "Qihai"(BL24) and bilateral "Xuehai"(SP10) and "Zusanli" (ST36). Mice in the non-acupoint group were treated with acupuncture at each of the left and right non-acupoints under the ribs and mice in the fasudil group were intraperitoneally injected with fasudil (25 mg/kg). The mice in each group were given medicine or acupuncture on the second day after grouping for 28 continuously days, once a day. Morris water maze test was used to test the learning and memory ability of mice. HE staining was used to observe the pathological changes of neurons in hippocampus. The number of hippocampal neuron dendritic spine was detected by FD fast Golgi staining kit. The contents of β-amyloid 42 (Aβ42) and phosphorylated tau protein (p-tau) in hippocampus were detected by ELISA. Western blot was used to detect the protein relative expression levels of RhoA, ROCK, F-actin and p-cofilin in hippocampus. Compared with those in the normal aging group, the hippocampal neurons of the model group were disorderly arranged, the number of neuron was reduced, the escape latency, hippocampal Aβ42 and p-tau contents, RhoA and ROCK protein expressions increased (P<0.05), the number of crossing the original platform, the number of neuronal dendritic spines, expressions of F-actin and p-cofilin decreased (P<0.05). After the interventions, there was no statistically significant difference in the above indicators in the non-acupoint group relevant to the model group (P>0.05). The acupuncture group and fasudil group improved the hippocampal pathological damage. The escape latency, hippocampal Aβ42 and p-tau contents, the expressions of RhoA and ROCK protein decreased (P<0.05), and the number of crossing the original platform, the number of hippocampal neuron dendritic spines, expressions of F-actin and p-cofilin increased (P<0.05) in both of the acupuncture and fasudil groups in contrast to the model and non-acupoint groups. Compared with the acupuncture group, there was no significant difference in the above indicators in the fasudil group (P>0.05). Sanjiao acupuncture may inhibit the activation of the RhoA/ROCK pathway, so as to improve the learning and memory function of AD mice, increase the number of hippocampal neuron dendritic spines, and promote synaptic plasticity.

Islet Transplantation Reverses Podocyte Injury in Diabetic Nephropathy or Induced by High Glucose via Inhibiting RhoA/ROCK/NF-κB Signaling Pathway.

Objective Abnormal signaling pathways play a crucial role in the mechanisms of podocyte injury in diabetic nephropathy. They also affect the recovery of podocytes after islet transplantation (IT). However, the specific signaling abnormalities that affect the therapeutic effect of IT on podocytes remains unclear. The purpose of this study was to assess whether the RhoA/ROCK/NF-κB signaling pathway is related to podocyte restoration after IT. Methods A mouse model of diabetic nephropathy was established in vivo using streptozotocin. The mice were then subsequently reared for 4 weeks after islet transplantation to determine the effect of IT. Islet cells, CCG-1423 (RhoA Inhibitor), and fasudil (ROCK inhibitor) were then cocultured with podocytes in vitro to assess their protective effects on podocyte injury induced by high glucose (HG). Protein expression levels of RhoA, ROCK1, synaptopodin, IL-6, and MCP-1 in kidney tissues were then measured using immunohistochemistry and Western blotting techniques. Results Islet transplantation reduced the expression levels of RhoA/ROCK1 and that of related inflammatory factors such as IL-6 and MCP-1 in the kidney podocytes of diabetic nephropathy. In the same line, islet cells reduced the expression of RhoA, ROCK1, and pp65 in immortalized podocytes under high glucose (35.0 mmol/L glucose) conditions. Conclusions Islet transplantation can reverse podocyte injury in diabetes nephropathy by inhibiting the RhoA/ROCK1 signaling pathway. Islet cells have a strong protective effect on podocytes treated with high glucose (35.0 mmol/L glucose). Discovery of signaling pathways affecting podocyte recovery is helpful for individualized efficacy evaluation and targeted therapy of islet transplantation patients.

Salidroside and isorhamnetin attenuate urotensin II-induced inflammatory response in vivo and in vitro: Involvement in regulating the RhoA/ROCK II pathway.

Urotensin II (UII), a vital vasoconstrictor peptide, causes an inflammatory response in the pathogenesis of atherosclerosis. Previous studies have reported that the Ras homolog gene family, member A (RhoA)/Rho kinases (ROCK) pathway modulates the inflammatory response of the atherosclerotic process. However, to the best of our knowledge, whether the RhoA/ROCK pathway mediates the inflammatory effect of UII has not been previously elucidated. Salidroside and isorhamnetin are two early developed antioxidant Tibetan drugs, both displaying cardioprotective effects against atherosclerosis. Therefore, the aim of the present study was to investigate the protective effects of salidroside, isorhamnetin or combination of these two drugs on the UII-induced inflammatory response in vivo (rats) or in vitro [primary vascular smooth muscle cells (VSMCs)], as well as to examine the role of the RhoA/ROCK pathway in these processes. The levels of inflammatory markers were measured via ELISA. The mRNA and protein expression levels of RhoA and ROCK II were detected using reverse transcription-quantitative PCR assay and western blot analysis. It was demonstrated that salidroside, isorhamnetin and both in combination decreased the levels of the serum pro-inflammatory cytokines TNF-α and IL-1β, as well as increased the levels of the anti-inflammatory cytokine IL-10 and macrophage migration inhibitory factor in rats with subacute infusion of UII and in the culture supernatant from primary VSMCs-exposed to UII. Moreover, salidroside, isorhamnetin and both in combination attenuated the mRNA and protein expression levels of RhoA and ROCK II in vivo and in vitro, at concentrations corresponding to human therapeutic blood plasma concentrations. Thus, these drugs could inhibit the RhoA/ROCK II pathway under UII conditions. The combination of salidroside and isorhamnetin did not display a stronger inhibitory effect on the inflammatory response and the RhoA/ROCK II pathway compared with salidroside and isorhamnetin in isolation. Collectively, the results indicated that salidroside, isorhamnetin and both in combination inhibited the RhoA/ROCK II pathway, which then attenuated the inflammatory response under UII-induced conditions, resulting in cardioprotection in atherosclerosis.

GDF-5 induces epidermal stem cell migration via RhoA-MMP9 signalling.

The migration of epidermal stem cells (EpSCs) is critical for wound re‐epithelization and wound healing. Recently, growth/differentiation factor‐5 (GDF‐5) was discovered to have multiple biological effects on wound healing; however, its role in EpSCs remains unclear. In this work, recombinant mouse GDF‐5 (rmGDF‐5) was found via live imaging in vitro to facilitate the migration of mouse EpSCs in a wound‐scratch model. Western blot and real‐time PCR assays demonstrated that the expression levels of RhoA and matrix metalloproteinase‐9 (MMP9) were correlated with rmGDF‐5 concentration. Furthermore, we found that rmGDF‐5 stimulated mouse EpSC migration in vitro by regulating MMP9 expression at the mRNA and protein levels through the RhoA signalling pathway. Moreover, in a deep partial‐thickness scald mouse model in vivo, GDF‐5 was confirmed to promote EpSC migration and MMP9 expression via RhoA, as evidenced by the tracking of cells labelled with 5‐bromo‐2‐deoxyuridine (BrdU). The current study showed that rmGDF‐5 can promote mouse EpSC migration in vitro and in vivo and that GDF‐5 can trigger the migration of EpSCs via RhoA‐MMP9 signalling.