RhoA mRNA Expression Research Articles

Neddylation of RhoA impairs its protein degradation and promotes renal interstitial fibrosis progression in diabetic nephropathy.

Diabetic nephropathy (DN) is a common and serious complication of diabetes, characterized by chronic fibro-inflammatory processes with an unclear pathogenesis. Renal fibrosis plays a significant role in the development and progression of DN. While recent research suggests that the neddylation pathway may influence fibrotic processes, its specific dysregulation in DN and the underlying mechanisms remain largely unexplored. This study identified the neddylation of RhoA as a novel post-translational modification that regulates its expression and promotes renal fibrosis in DN. We here demonstrated that two key components of the neddylation pathway-NEDD8-activating enzyme E1 subunit 1 (NAE1) and NEDD8-are significantly upregulated in human chronic kidney disease (CKD) specimens compared to healthy kidneys, implicating neddylation in CKD-associated fibrosis. Our findings further revealed that both pharmacological inhibition of neddylation using MLN4924 and genetic knockdown of NAE1 mitigate renal fibrosis in mouse models of streptozotocin-induced diabetes and unilateral ureteral obstruction (UUO). Immunoprecipitation-mass spectrometry (IP-MS) and subsequent function assays demonstrated a direct interaction between RhoA and NEDD8. Importantly, neddylation inhibition reduced RhoA protein expression, highlighting a potential therapeutic target. Additionally, a positive correlation was noted between elevated NEDD8 mRNA levels and RhoA mRNAexpression in human CKD specimens. RhoA overexpression counteracted the antifibrotic effects of neddylation inhibition, underscoring its critical role in fibrosis progression. Mechanistically, we unveiled that neddylation enhances RhoA protein stability by inhibiting its ubiquitination-mediated degradation, which subsequently activates the ERK1/2 pathway. Collectively, this study provides novel insights into NAE1-dependent RhoA neddylation as a key contributor to renal fibrosis in DN. The NAE1 protein mediates RhoA protein hyper-neddylation and subsequent stabilization of the RhoA protein, which, in turn, contributes to the development of renal fibrosis and inflammation through an ERK1/2-dependent mechanism. Consequently, targeting neddylation inhibition represents a viable therapeutic approach for the treatment of renal fibrosis in DN.

Butyric acid alleviates LPS-induced intestinal mucosal barrier damage by inhibiting the RhoA/ROCK2/MLCK signaling pathway in Caco2 cells.

Butyric acid (BA) can potentially enhance the function of the intestinal barrier. However, the mechanisms by which BA protects the intestinal mucosal barrier remain to be elucidated. Given that the Ras homolog gene family, member A (RhoA)/Rho-associated kinase 2 (ROCK2)/Myosin light chain kinase (MLCK) signaling pathway is crucial for maintaining the permeability of the intestinal epithelium, we further investigated whether BA exerts a protective effect on epithelial barrier function by inhibiting this pathway in LPS-induced Caco2 cells. First, we aimed to identify the optimal treatment time and concentration for BA and Lipopolysaccharide (LPS) through a CCK-8 assay. We subsequently measured Trans-epithelial electrical resistance (TEER), FITC-Dextran 4 kDa (FD-4) flux, and the mRNA expression of ZO-1, Occludin, RhoA, ROCK2, and MLCK, along their protein expression levels, and average fluorescence intensity following immunofluorescence staining. We then applied the ROCK2 inhibitor Y-27632 and reevaluated the TEER, FD-4 flux, and mRNA, and protein expression of ZO-1, Occludin, RhoA, ROCK2, and MLCK, as well as their distribution in Caco2 cells. The optimal treatment conditions were determined to be 0.2 mmol/L BA and 5 μg/mL LPS for 24 hours. Compared with LPS treatment alone, BA significantly mitigated the reduction in the TEER, decreased FD-4 flux permeability, increased the mRNA expression of ZO-1 and Occludin, and normalized the distribution of ZO-1 and Occludin in Caco2 cells. Furthermore, BA inhibited the expression of RhoA, ROCK2, and MLCK, and normalized their localization within Caco2 cells. Following treatment with Y-27632, the epithelial barrier function, along with the mRNA and protein expression and distribution of ZO-1 and Occludin were further normalized upon inhibition of the pathway. These findings contribute to a deeper understanding of the potential mechanisms through which BA attenuates LPS-induced impairment of the intestinal epithelial barrier.

The Reduction of Traumatic Spinal Cord Secondary Injury by Anti-RhoA siRNA Functionalized Nucleic Acid Nanoparticles (NANPs)

Primary injury of the spinal cord is caused by a mechanical traumatic event which is rapidly followed by a secondary injury cascade that may evolve for several months leading to biological and functional changes. During the secondary injury, many pathophysiological pathways and process are activated including inflammation, oxidative stress, demyelination, excitotoxicity, axon degeneration, and cell death. The RhoA/Rho kinase pathway significantly contributes to spinal degeneration and regeneration and therefore represents a potential therapeutic target. Nucleic acid nanoparticles (NANPs) offer easy rational and programable design with the potential to carry on multiple synergistic therapeutic nucleic acid functional motifs. In this context we designed, synthesized, and assembled several representative NANPs decorated with multiple copies of siRNAs targeting RhoA. Subsequently we assessed NANPs' physicochemical properties, toxicity, and immunorecognition upon delivery with the nanocarrier PLGA-g-PEI (PgP), developed with the aim to select for the most immunoquiescent type of formulations. In addition, we observed that L1 neural cell adhesion molecule conjugated PgP (L1-PgP) efficiently delivered NANP-siRhoA in cultured neuroblastoma (B35) cells. RhoA mRNA expression was significantly reduced by all L1-PgP/ NANP-siRhoA relative to the untreated control, while no significant differences were observed between the different NANP-siRhoAs.

3D spheroids versus 2D-cultured human adipose stem cells to generate smooth muscle cells in an internal anal sphincter-targeting cryoinjured mouse model

BackgroundThe efficacy of cell implantation via 3D-spheroids to treat basal tone in fecal incontinence remains unclear. To address this, in this study, we aimed to identify cell differentiation and assess the development of a contractile phenotype corresponding to smooth muscle cells (SMCs) following implantation of 3D-spheroid and 2D-cultured human adipose stem cells (hASCs) in an in vivo internal anal sphincter (IAS)-targeted mouse model.MethodsWe developed an IAS-targeted in vivo model via rapid freezing (at − 196 °C) of the dorsal layers of the region of interest (ROI) of the IAS ring posterior quarter, between the submucosal and muscular layers, following submucosal dissection (n = 60 rats). After implantation of tetramethylindocarbocyanine perchlorate (Dil)-stained 3D and 2D-cells into randomly allocated cryoinjured rats, the entire sphincter ring or only the cryoinjured ROI was harvested. Expression of SMC markers, RhoA/ROCKII and its downstream molecules, and fibrosis markers was analyzed. Dil, α-smooth muscle actin (α-SMA), and RhoA signals were used for cell tracking.ResultsIn vitro, 3D-spheroids exhibited higher levels of SMC markers and RhoA/ROCKII-downstream molecules than 2D-hASCs. The IAS-targeted cryoinjured model exhibited substantial loss of SMC layers of the squamous epithelium lining of the anal canal, as well as reduced expression of SMC markers and RhoA-related downstream molecules. In vivo, 3D-spheroid implantation induced SMC markers and contractile molecules weakly at 1 week. At 2 weeks, the mRNA expression of aSma, Sm22a, Smoothelin, RhoA, Mypt1, Mlc20, Cpi17, and Pp1cd increased, whereas that of fibrosis markers reduced significantly in the 3D-spheroid implanted group compared to those in the sham, non-implanted, and 2D-hASC implanted groups. Protein levels of RhoA, p-MYPT1, and p-MLC20 were higher in the 3D-spheroid-implanted group than in the other groups. At 2 weeks, in the implanted groups, the cryoinjured tissues (which exhibited Dil, α-SMA, and RhoA signals) were restored, while they remained defective in the sham and non-implanted groups.ConclusionsThese findings demonstrate that, compared to 2D-cultured hASCs, 3D-spheroids more effectively induce a contractile phenotype that is initially weak but subsequently improves, inducing expression of RhoA/ROCKII-downstream molecules and SMC differentiation associated with IAS basal tone.

The role of the RHOA/ROCK pathway in the regulation of myometrial stages throughout pregnancy

BackgroundControlling uterine contractile activity is essential to regulate the duration of pregnancy. During most of the pregnancy, the uterus does not contract (i.e., myometrial quiescence). The myometrium recovers its contractile phenotype at around 36 weeks (i.e., myometrial activation) through several mechanisms. The RHOA/ROCK pathway plays a vital role in facilitating muscular contractions by calcium sensitization in humans. Yet, the role of this pathway during different myometrial stages, including quiescence, has not been elucidated. Objectivewe aimed to study the role of the RHOA/ROCK pathway in the regulation of the different myometrial stages throughout pregnancy. Specifically, we hypothesized that the inhibition of the components of the RHOA/ROCK pathway play an important role in maintaining uterine quiescence. Study designMyometrial samples were obtained from pregnant individuals who underwent cesarean section. Pregnant individuals who delivered preterm without labor (myometrial quiescence), preterm with labor (nonphysiological myometrial stimulation), term not in labor (activation), and term in labor (physiological myometrial stimulation) were included. The mRNA and protein expression of RHOA, ROCK I, ROCK II, RND1-3, and ROCK activity through pMYTP1 were evaluated. ResultsWe found that the human myometrium constitutively expressed RHOA/ROCK pathway components throughout pregnancy. No changes in the components of the RHOA/ROCK pathway were found during quiescence. Moreover, the RHOA protein and ROCK activity increased in the myometrium during labor, supporting the hypothesis that this pathway participates in maintaining the contractile activity of the myometrium. This study provides insight into the role of the RHOA/ROCK pathway in controlling myometrial contractile activity during pregnancy.

High Expression of AhR and Environmental Pollution as AhR-Linked Ligands Impact on Oncogenic Signaling Pathways in Western Patients with Gastric Cancer-A Pilot Study.

The vast majority of gastric cancer (GC) cases are adenocarcinomas including intestinal and diffuse GC. The incidence of diffuse GC, often associated with poor overall survival, has constantly increased in Western countries. Epidemiological studies have reported increased mortality from GC after occupational exposure to pro-carcinogens that are metabolically activated by cytochrome P450 enzymes through aryl hydrocarbon receptor (AhR). However, little is known about the role of AhR and environmental AhR ligands in diffuse GC as compared to intestinal GC in Western patients. In a cohort of 29, we demonstrated a significant increase in AhR protein and mRNA expression levels in GCs independently of their subtypes and clinical parameters. AhR and RHOA mRNA expression were correlated in diffuse GC. Further, our study aimed to characterize in GC how AhR and the AhR-related genes cytochrome P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) affect the mRNA expression of a panel of genes involved in cancer development and progression. In diffuse GC, CYP1A1 expression correlated with genes involved in IGF signaling, epithelial-mesenchymal transition (Vimentin), and migration (MMP2). Using the poorly differentiated KATO III epithelial cell line, two well-known AhR pollutant ligands, namely 2-3-7-8 tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (BaP), strongly increased the expression of CYP1A1 and Interleukin1β (IL1B), and to a lesser extend UGT1, NQO1, and AhR Repressor (AhRR). Moreover, the increased expression of CYP1B1 was seen in diffuse GC, and IHC staining indicated that CYP1B1 is mainly expressed in stromal cells. TCDD treatment increased CYP1B1 expression in KATO III cells, although at lower levels as compared to CYP1A1. In intestinal GC, CYP1B1 expression is inversely correlated with several cancer-related genes such as IDO1, a gene involved in the early steps of tryptophan metabolism that contributes to the endogenous AhR ligand kynurenine expression. Altogether, our data provide evidence for a major role of AhR in GC, as an environmental xenobiotic receptor, through different mechanisms and pathways in diffuse and intestinal GC. Our results support the continued efforts to clarify the identity of exogenous AhR ligands in diffuse GC in order to define new therapeutic strategies.

Heraclenin promotes the osteogenic differentiation of bone marrow stromal cells by activating the RhoA/ROCK pathway.

Osteoporosis is a devastating skeletal disease, the pathogenesis of which is related to abnormal bone metabolism, featured by the imbalance between osteoblastic bone formation and osteoclastic bone resorption. Stem cell-based therapies have been demonstrated to improve osteoporosis treatment. Previously, the linear furanocoumarin heraclenin was reported to enhance osteoblast differentiation and mineralization in mouse mesenchymal stem cells (MSCs), suggesting its potential for osteogenic differentiation and bone regeneration. Our study was designed to confirm the promotive role of heraclenin on osteogenic differentiation of human bone MSCs (BMSCs) and explore the underlying mechanisms. Human BMSCs were treated for 24, 48, and 72h with heraclenin (5, 10, 20, 40, and 80 μM), and cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. To further evaluate the cytotoxicity of heraclenin, cell suspension obtained from BMSCs treated with heraclenin (5, 10, and 20 μM) for 72h was subjected to a MUSE™ cell analyzer for cell viability and count assay. BMSCs were incubated in osteogenic induction medium for 7 days. Then, osteogenic differentiation and mineralization of BMSCs were assessed through alkaline phosphatase (ALP) and Alizarin Red S staining. The expression of osteogenesis markers including ALP, osteocalcin (OCN), osterix (OSX), and runt-related transcription factor 2 (RUNX2) was detected via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The effects of heraclenin on the RhoA/ROCK pathway were estimated through western blotting. Y-27632, the ROCK inhibitor, was used to confirm the role of the RhoA/ROCK pathway in heraclenin-mediated osteogenic differentiation of BMSCs. Heraclenin (5-80 μM) was non-toxic on human BMSCs. Heraclenin treatment (5-20 μM) dose-dependently enhanced ALP activity and calcium deposition. Furthermore, heraclenin promoted ALP, OCN, OSX, and RUNX2 mRNA and protein expression. Mechanically, heraclenin treatment increased RhoA and ROCK1 mRNA expression, stimulated the translocation of ROCK from the cytosolic to the membrane fraction, and elevated the protein levels of phosphorylated cofilin (p-cofilin) and active RhoA. Additionally, treatment with Y-27632 overturned the promotion of heraclenin on ALP activity, calcium deposition, the expression of osteogenesis markers, and the RhoA/ROCK signaling pathway. Heraclenin facilitates the osteogenic differentiation of human BMSCs through the activation of the RhoA/ROCK pathway.

MiR-140-3p Normalizes Vascular Smooth Muscle Cell Behavior by Inhibiting the RhoA/ROCK Signaling Pathway in Lower-extremity Arteriosclerosis Obliterans.

Excessive vascular smooth muscle cell (VSMC) proliferation and migration are the main contributors to the symptoms of lower-extremity arteriosclerosis obliterans (ASO). Previous studies suggested that microRNAs (miRNAs) regulate VSMC activity. Nevertheless, the molecular mechanisms by which they do so are unclear. The present study aimed to identify the biological processes accounting for the effects of miR-140-3p on VSMCs in ASO. The expression levels of miR-140-3p in clinical samples were analyzed by real-time polymerase chain reaction. An ASO cell model was established to investigate the expression of miR-140-3p on VSMCs. The transwell® assays and MTT assays were used to assess migration and proliferation. The interaction between RhoA and miR-140-3p was verified using the Dualluciferase reporter assay. Western blot technique was used to identify RhoA, RhoA-associated protein kinase 1 (ROCK1), and ROCK2. We discovered that miR-140-3p inhibited the proliferation, migration, and invasion but promoted the apoptosis of VSMCs, and RhoA was its downstream target gene. RhoA, ROCK1, and ROCK2 were upregulated in vascular tissues damaged by ASO compared to normal, healthy arteries. MiR-140-3p also decreased RhoA, ROCK1, and ROCK2 mRNA and protein expression. Overall, the present work partially elucidated the mechanism by which miR-140-3p regulates VSMC function and offered novel insights into potential therapeutic approaches for patients with lower-extremity arteriosclerosis obliterans.

FAM120A promotes angiogenesis in pancreatic cancer by increasing the mRNA expression of RHOA

FAM120A promotes angiogenesis in pancreatic cancer by increasing the mRNA expression of RHOA

Abstract 2038: Statin Targeted Treatment Against Intimal Hyperplasia Using Unique Chitosan-PLGA Nanoparticles

Introduction: Statins have beneficial pleiotropic effects, including reducing intimal hyperplasia (IH) after intervention for peripheral arterial disease. Using a unique nanoparticle (NP) to locally maximize statin’s pleiotropic benefits, we hypothesized chitosan-functionalized polylactic-co-glycolic nanoparticles loaded with simvastatin (SL-cNPs) would: 1) be taken up by endothelial cells (ECs) and vascular smooth muscle cells (VSMCs); 2) affect EC and VSMC function; and 3) reduce IH compared to systemic simvastatin. Methods: cNPs were loaded with simvastatin (2% w/w ) and encapsulated with lipophilic tracer, DiD. In vitro , human aortic ECs and VSMCs (passage 3-5) were cultured with 0.2mg SL-cNPs or basal media (30 min). After 24 hours, uptake of SL-cNPs was assessed by immunostaining and flow cytometry. Functional effect of SL-cNPs was determined using qPCR for mRNA expression of RhoA, RhoB and Ras. In vivo, male Sprague-Dawley rats (n=6-9/group) underwent carotid artery balloon injury in the following groups: 1) intraluminal therapy using normal saline control, empty cNPs or SL-cNPs; and 2) oral simvastatin (10mg/kg/day, 7 days pre-operative until sacrifice) plus intraluminal saline or SL-cNPs. Rats were sacrificed (day 14), perfusion fixed, arteries harvested, and IH quantified with morphometric analysis. Student’s t-test and one-way ANOVA was performed (p <0.05 for significance). Results: In vitro , SL-cNPs were readily taken up by ECs (79.6%, 38,606/48,501) and VSMCs (46.4%, 25,774/55,549). SL-cNPs induced a compensatory increase in expression of EC RhoA (1.3-fold) and VSMC RhoA (1.7-fold) and RhoB (5.4-fold) (p<0.05). In vivo, oral simvastatin plus intraluminal SL-cNPs reduced IH compared to controls (0.44 ± 0.04 vs 0.34 ± 0.03, p<0.05). Although oral simvastatin and SL-cNPs alone trended toward IH reduction, the effect was not significant (0.35 ± 0.03 and 0.377 ± 0.03, respectively). No difference existed between oral simvastatin and SL-cNP therapy. Conclusion: cNPs can be used as a novel vehicle to locally deliver statins to vascular cells. Although only the combination of oral simvastatin and SL-cNPs effectively reduced IH, different routes and/or concentration of SL-cNPs may allow for a more robust effect on IH prevention.

Mechanisms of electroacupuncture relieves uterine smooth muscle spasm in dysmenorrhea rats based on Rho/ROCK signaling pathway.

To investigate the effect of electroacupuncture (EA) on Rho/Rho-associated coiled-coil-forming kinases (ROCK) signaling pathway of uterus tissue in rats with dysmenorrhea, so as to explore the underlying mechanism of EA treating primary dysmenorrhea (PD) and uterine smooth muscle spasm, and to observe whether there is a difference in the effect of meridian acupoints in Conception Vessel (CV) and Governer Vessel (GV). Sixty female SD rats were randomly divided into saline, model, CV, GV, and non-acupoint groups, with 12 rats in each group. The dysmenorrhea model was established by subcutaneous injection of estradiol diphenhydrate combined with intraperitoneal injection of oxytocin (OT). EA (2 Hz) was applied to "Qihai" (CV6) and "Zhongji" (CV3) for CV group, "Mingmen" (GV4) and "Yaoshu" (GV2) for GV group, "non-acupoint 1" and "non-acupoint 3" on the left side for non-acupoint group, and manual acupuncture was applied to "Guanyuan" (CV4) for CV group, "Yaoyangguan" (GV3) for GV group, "non-acupoint 2" on the left side for non-acupoint group. The treatment was conducted for 20 min each time, once daily for 10 days. The writhing score was evaluated. The smooth myoelectric signals of rats' uterus in vivo were recorded by multi-channel physiological recorder. The uterine histopathological changes were observed by HE staining. The contents of prostaglandin F2α (PGF2α), OT and calcium ion (Ca2+) in uterine tissue of rats were detected by ELISA. The protein and mRNA expression levels of smooth muscle 22-α (SM22-α), RhoA and ROCKⅡ in uterine tissue were detected by Western blot and fluorescence quantitative PCR, respectively. Compared with the saline group, the writhing score of rats in the model group was increased (P<0.01), the amplitude voltage of uterine smooth muscle in vivo was elevated (P<0.01), the contents of PGF2α, OT and Ca2+, the protein and mRNA expression of SM22-α, RhoA and ROCK Ⅱ in uterine tissue were all increased (P<0.01). Compared with the model and the non-acupoint groups, the writhing scores of the CV and the GV groups were decreased (P<0.01, P<0.05), the amplitude voltage of uterine smooth muscle was decreased (P<0.01), the contents of PGF2α, OT and Ca2+ in uterine tissue were decreased (P<0.01, P<0.05), and the protein expression and mRNA expression of SM22-α, RhoA and ROCKⅡ in uterine tissue were decreased (P<0.01, P<0.05). HE staining showed extensive exfoliation of uterine intima with severe edema and increased glandular secretion in the model group, which was alleviated in the CV and GV groups. EA at acupoints of CV and GV can significantly reduce the writhing score, uterine smooth muscle amplitude voltage, pathological injury degree of uterus, and relieve spasm of uterine smooth muscle in dysmenorrhea rats, which may be related to its effect in regulating PGF2α and OT contents, inhibiting the Rho/ROCK signaling pathway, and reducing the SM22-α, RhoA, ROCKⅡ protein and mRNA expression, and Ca2+ content in uterine tissue.

Arsenic exposure induces small intestinal toxicity in mice by barrier damage and inflammation response via activating RhoA/ROCK and TLR4/Myd88/NF-κB signaling pathways

Numerous studies have shown that arsenic (As) is an important hazardous metalloid that is commonly considered to have systemic toxicity. The main pathway of arsenic exposure is oral; however, many of the events that occur during its passage through the gastrointestinal tract are unclear, and there are few reports on the effect of arsenic on small intestinal mucosal barrier. This study aimed to investigate arsenic-induced mucosal barrier damage in the small intestine of mice induced by oral exposure and its potential mechanisms. In the present study, histomorphometric and immunohistochemical analyses showed that arsenic-treated mice exhibited signs of irregularly arranged and atrophied small intestinal villi, reduced villus lengths, inflammatory cells infiltration, along with up-regulated expression of inflammatory factors TNF-α, IL-6 and IL-1β in the small intestine of mice. The myeloperoxidase (MPO) activity was also increased in As-exposed mice. Transmission electron microscopy (TEM) analysis demonstrated that intestinal epithelial tight junctions (TJs) were impaired in the small intestines of mice in As group. In addition, arsenic down-regulated mRNA levels of TJ-related genes (ZO-1, ZO-2, occludin, claudin-1, and claudin-7) and protein levels of ZO-1, occludin and claudin-1 were significantly reduced in arsenic-treated groups, while arsenic also increased levels of TLR4, Myd88, NF-κB, RhoA, and ROCK mRNA and protein expression. In summary, these results indicate that the small intestine toxicity in mice evoked by arsenic was correlated with the activation of TLR4/Myd88/NF-κB and RhoA/ROCK pathways.

THE INHIBITORY EFFECTS OF VALPROIC ACID ON RHOA-MEDIATED VASCULAR SMOOTH MUSCLE CELL CONTRACTION

Objective: Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Dysfunction of vascular smooth muscle cells (VSMCs) is well-established to contribute to the pathogenesis of various vascular diseases. Growing evidence indicates that increased VSMC contractility plays a primary role in the development of pathological artery spasms. Nevertheless, effect of VPA on VSMC contractility, and its mechanism of action remain unknown. Here, we investigated the mechanism by which VPA inhibits VSMC contractility and vessel contraction in rat VSMCs and endothelium-deprived aortas. Design and method: We performed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), RhoA activity assay, transfection of constitutively active (CA)-RhoA gene, and western blot analyses. We also conducted ex vivo studies using isolated rat aortas. Results: VPA inhibited phenylephrine (PE)-induced phosphorylation of myosin light chain (MLC) at Ser19 (p-MLC-Ser19) in VSMCs. VPA did not alter expressions of MLC kinase and Rho-associated kinase 1, the upstream kinases responsible for MLC phosphorylation. In addition, VPA did not affect phosphorylation of myosin phosphatase target subunit 1, a regulatory subunit of myosin phosphatase. VPA decreased RhoA mRNA expression and its protein levels in a time- and dose-dependent manner, and as expected, VPA decreased amount of active GTP-bound RhoA in a dose-dependent manner. Ectopic expression of CA-RhoA gene significantly reversed VPA-inhibited p-MLC-Ser19 and p-CPI-17-Thr38. Finally, VPA significantly attenuated PE-induced vessel contraction in endothelium-deprived rat aortas, and similar to the in vitro results, VPA decreased RhoA expression and levels of p-MLC-Ser19 and p-CPI-17-Thr38 in rat aortas. Conclusions: We demonstrate that VPA mitigates PE-induced VSMC contractility and vessel contraction by decreasing expressions of RhoA and p-MLC-Ser19 in rat VSMCs and aortas. These results suggest that VPA may be useful in the treatment of vasospastic diseases including coronary artery vasospasm and post-subarachnoid hemorrhage cerebral vasospasm.

Human Umbilical Cord Mesenchymal Stem Cells Improve the Status of Hypoxic/Ischemic Cerebral Palsy Rats by Downregulating NogoA/NgR/Rho Pathway

Human umbilical cord mesenchymal stem cells (hUCMSC) have shown promising potential in ameliorating brain injury, but the mechanism is unclear. We explore the role of NogoA/NgR/Rho pathway in mediating hUCMSC to improve neurobehavioral status and alleviate brain injury in hypoxia/ischemia-induced CP (cerebral palsy) rat model in order to promote the clinical application of stem cell therapy in CP. The injury model of HT22 cells was established after 3 h hypoxia, and then co-cultured with hUCMSC. The rat model of CP was established by ligation of the left common carotid artery for 2.5 h. Subsequently, hUCMSC was administered via the tail vein once a week for a total of four times. The neurobehavioral status of CP rats was determined by behavioral experiment, and the pathological brain injury was determined by pathological staining method. The mRNA and protein expressions of NogoA, NgR, RhoA, Rac1, and CDC42 in brain tissues of rats in all groups and cell groups were detected by real-time quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunofluorescence. The CP rats exhibited obvious motor function abnormalities and pathological damage. Compared with the control group, hUCMSC transplantation could significantly improve the neurobehavioral situation and attenuate brain pathological injury in CP rats. The relative expression of NogoA, NgR, RhoA mRNA, and protein in brain tissues of rats in the CP group was significantly higher than the rats in the sham and CP+hUCMSC group. The relative expression of Rac1, CDC42 mRNA, and protein in brain tissues of rats in the CP group was significantly lower than the rats in the sham and CP+hUCMSC group. The animal experiment results were consistent with the experimental trend of hypoxic injury of HT22 cells. This study confirmed that hUCMSC can efficiently improve neurobehavioral status and alleviate brain injury in hypoxia/ischemia-induced CP rat model and HT22 cell model through downregulating the NogoA/NgR/Rho pathway.

Investigating the Anticancer Activity of G-Rh1 Using In Silico and In Vitro Studies (A549 Lung Cancer Cells)

Ginsenoside Rh1 (G-Rh1), a possible bioactive substance isolated from the Korean Panax ginseng Meyer, has a wide range of pharmacological effects. In this study, we have investigated the anticancer efficacy of G-Rh1 via in silico and in vitro methodologies. This study mainly focuses on the two metastatic regulators, Rho-associated protein kinase 1 (ROCK1) and RhoA, along with other standard apoptosis regulators. The ROCK1 protein is a member of the active serine/threonine kinase family that is crucial for many biological processes, including cell division, differentiation, and death, as well as many cellular processes and muscle contraction. The abnormal activation of ROCK1 kinase causes several disorders, whereas numerous studies have also shown that RhoA is expressed highly in various cancers, including colon, lung, ovarian, gastric, and liver malignancies. Hence, inhibiting both ROCK1 and RhoA will be promising in preventing metastasis. Therefore, the molecular level interaction of G-Rh1 with the ROCK1 and RhoA active site residues from the preliminary screening clearly shows its inhibitory potential. Molecular dynamics simulation and principal component analysis give essential insights for comprehending the conformational changes that result from G-Rh1 binding to ROCK1 and RhoA. Further, MTT assay was employed to examine the potential cytotoxicity in vitro against human lung cancer cells (A549) and Raw 264.7 Murine macrophage cells. Thus, G-Rh1 showed significant cytotoxicity against human lung adenocarcinoma (A549) at 100 µg/mL. In addition, we observed an elevated level of reactive oxygen species (ROS) generation, perhaps promoting cancer cell toxicity. Additionally, G-Rh1 suppressed the mRNA expression of RhoA, ROCK1, MMP1, and MMP9 in cancer cell. Accordingly, G-Rh1 upregulated the p53, Bax, Caspase 3, caspase 9 while Bcl2 is downregulated intrinsic pathway. The findings from our study propose that the anticancer activity of G-Rh1 may be related to the induction of apoptosis by the RhoA/ROCK1 signaling pathway. As a result, this study evaluated the functional drug-like compound G-Rh1 from Panax ginseng in preventing and treating lung cancer adenocarcinoma via regulating metastasis and apoptosis.

Effects of RhoA on depression-like behavior in prenatally stressed offspring rats

Depression is a common mental disease that can lead to suicide when severe. Exposure to prenatal stress (PS) can lead to depression-like behavior in offspring, but the mechanism is unclear. RhoA (Ras homology family member A) plays an important role in stress-induced changes in synaptic plasticity, participating in the development of depression by activating the downstream effector ROCK (Rho-associated protein kinase). This study explored the influence in the expression of RhoA and downstream molecules ROCK1/2 in prenatally stressed rats, and the effect of RhoA inhibitor simvastatin on depression-like behavior induced by PS. Depression-like behavior in offspring was detected by sucrose preference test, forced swimming test, and open-field test. The mRNA and protein expression of RhoA and ROCK1/2 in the hippocampus and prefrontal cortex of offspring rats were detected by qRT-PCR and western blotting, respectively. Our results showed that PS causes depression-like behavior in offspring rats, associated with elevated expression of RhoA, ROCK1/2 in the hippocampus and prefrontal cortex. After administration of simvastatin to PS rats, the expression of RhoA and ROCK2 was significantly reduced, alleviating depression-like behavior. Our study demonstrated that RhoA participates in the depression-like behavior in prenatally stressed offspring rats, which may be a potential target for antidepressant therapy.

HNRNPC regulates RhoA to induce DNA damage repair and cancer-associated fibroblast activation causing radiation resistance in pancreatic cancer.

Pancreatic cancer (PC) is one of the most lethal types of cancer due to its asymptomatic nature in the early stages and consequent late diagnosis. Its mortality rate remains high despite advances in treatment strategies, which include a combination of surgical resection and adjuvant therapy. Although these approaches may have a positive effect on prognosis, the development of chemo‐ and radioresistance still poses a significant challenge for successful PC treatment. Heterogeneous nuclear ribonucleoprotein C1/C2 (HNRNPC) and RhoA have been implicated in the regulation of tumour cell proliferation and chemo‐ and radioresistance. Our study aims to investigate the mechanism for HNRNPC regulation of PC radiation resistance via the RhoA pathway. We found that HNRNPC and RhoA mRNA and protein expression levels were significantly higher in PC tissues compared to adjacent non‐tumour tissue. Furthermore, high HNRNPC expression was associated with poor patient prognosis. Using HNRNPC overexpression and siRNA interference, we demonstrated that HNRNPC overexpression promoted radiation resistance in PC cells, while HNRNPC knockdown increased radiosensitivity. However, silencing of RhoA expression was shown to attenuate radiation resistance caused by HNRNPC overexpression. Next, we identified RhoA as a downstream target of HNRNPC and showed that inhibition of the RhoA/ROCK2‐YAP/TAZ pathway led to a reduction in DNA damage repair and radiation resistance. Finally, using both in vitro assays and an in vivo subcutaneous tumour xenograft model, we demonstrated that RhoA inhibition can hinder the activity of cancer‐related fibroblasts and weaken PC radiation resistance. Our study describes a role for HNRNPC and the RhoA/ROCK2‐YAP/TAZ signalling pathways in mediating radiation resistance and provides a potential therapeutic target for improving the treatment of PC.

Moxibustion pretreatment inhibits RhoA/ROCK signaling to prevent lung inflammation in asthmatic rats: 艾灸预处理降低RhoA/ROCK信号表达预防哮喘大鼠肺炎

Objective: To determine whether pretreatment moxibustion prevents asthma by down-regulating the lung RhoA/ROCK pathway in rats with bronchial asthma and benignly mediating the lung inflammatory response. Methods: Twenty Sprague Dawley (SD) rats were randomly divided into normal control group (C), asthma model group (M), suspended moxibustion 40 min +asthma group (SM40), and suspended moxibustion 10 min +asthma group (SM10). Ovalbumin was used as a sensitizer. The two moxibustion groups completed moxibustion treatment lasted 40 min or 10 min respectively 30 min before modeling onset, and was repeated five times in each modeling cycle, for a total of 15 times. Samples were harvested on day 30. Results: Lung impairment was significant in the M group, whereas pretreatment with SM10 and SM40 dramatically attenuated the injury. After modeling, mRNA expression of RhoA and ROCK2 in the lung tissue was significantly higher than that in C group (both P < 0.001), resulting in significant increase in protein levels of IL-17A (P < 0.001). Significant decrease in RhoA and ROCK2 mRNA expression was seen in the SM10 (P<0.001, P<0.01) and SM40 (both P<0.001) groups compared to that with M rats. The differential trend in the SM40 group was more evident than that in the SM10 group. Regarding IL-10 or IL-17A protein concentration, an upregulation or down-regulation was observed in both SM10 (P<0.05, P < 0.01) and SM40 groups (both P < 0.001) compared to that with the M group. Conclusions: Moxibustion pretreatment significantly prevented pulmonary inflammation in asthmatic rats, potentially via inhibition of the RhoA/ROCK pathway. The efficacty of moxibustion appeared to be significantly associated with the duration of intervention with moxibustion.

Cancer-associated fibroblasts as cellular vehicles in endometrial cancer cell migration.

Cell motility is a critical step in the metastasis cascade. However, the role of cancer-associated fibroblasts (CAFs) in facilitating endometrial cancer (EC) cell motility remains unclear. The present study aimed to investigate the role of CAFs in EC motility in a 3D environment. A co-culture model was established using an EC cell line (ECC-1) and CAFs on a Matrigel® matrix and compared to the respective individual monocultures. It was demonstrated that endometrial CAFs increased the motility of the EC cell line, compared with the monoculture. Using live cell imaging, CAFs were observed to form cell projections that served as contact guidance for ECC-1 cell locomotion in the spheroid formation process. These effects were specific to CAFs, as fibroblasts isolated from benign endometrial tissue samples did not form cell projections. Molecular analysis revealed that RhoA/Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) signaling activation partly contributed to CAF-mediated ECC-1 cell migration. The presence of Matrigel® increased the mRNA expression of RhoA, and the mRNA and protein expression levels of its downstream effectors, ROCK1 and p-MLC, respectively, in the ECC-1 and CAF co-culture, as well as the ECC-1 and CAF monocultures. Interestingly, high phosphorylation levels of myosin light chain mediated the activation of RhoA/ROCK1 signaling in the ECC-1 and CAF co-culture. The ROCK1 inhibitor Y-27632 attenuated the motility of tumor cells in ECC-1 and CAF co-cultures. However, similar treatment led to a significant inhibition in the motility of the CAF monoculture, but not the ECC-1 monoculture. Moreover, tumor spheroid formation was inhibited due to a reduction in stress fiber formation in ECC-1 and CAF co-cultures. Altogether, these findings suggest that the regulation of the RhoA/ROCK1 signaling pathway is required for CAFs to serve as cellular vehicles in order for EC cells to migrate and form spheroids in a 3D environment.

Effect of Naoluoxintong on the NogoA/RhoA/ROCK pathway by down-regulating DNA methylation in MCAO rats

Naoluoxintong (NLXT) is a traditional Chinese Medicine (TCM) prescription that is clinically used in the treatment of ischemic stroke (IS). However, its therapeutic mechanism remains unclear. To obtain the mechanism of NLXT by observing the protective effects of NLXT on the NogoA/RhoA/Rock pathway in a rat model of IS by regulating DNA methylation. Rats were divided into five groups using a random number table: normal group, model group, NLXT group, blocker group I (NLXT+SGI-1027) and blocker group II (NLXT+Y27632). The right middle cerebral artery occlusion-reperfusion (MCAO/R) rat model was made, and the regional cerebral blood flow (rCBF) of each group was detected using laser Doppler. The methylation levels of CpG sites of neurite outgrowth inhibitor protein-A (Nogo-A), Nogo receptor (NgR), ras homolog gene family member A (RhoA) and rho-associated coiled-coil protein kinase 2 (ROCK2) genes in rat brain tissue were detected using the bisulfite method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect NogoA, RhoA, NgR1, NgR2 and ROCK2 mRNA expression in rat brain tissue. NogoA, RhoA, NgR1, NgR2 and ROCK2 proteins were detected using immunoblotting in rat brain tissue. After the modeling of middle cerebral artery occlusion (MCAO), neurological deficit test was made to ensure the success of the modeling. At each time point after surgery, the rCBF of the other groups decreased compared with the normal group (P<0.01 or P<0.05). Meanwhile, the rCBF increased in blocker group I as well as blocker group II after 3 days (P<0.05). There were differences in the DNA methylation sites of NogoA, RhoA, NgR and ROCK2 genes between the model group and the NLXT group (P<0.05). Compared with the normal group, NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression in the model group increased observably (P<0.01). In comparison with the model group, NogoA and NgR1 gene expression in the blocker group II was prominently observed on the 1st day. NogoA, NgR1, NgR2, RhoA and ROCK2 gene expression remarkably reduced (P<0.01) on the 3rd and 7th days. Compared with the normal group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the model group increased observably (P<0.01). In comparison with the model group, NogoA, RhoA, NgR1, NgR2 and ROCK2 protein expression in the other groups declined prominently (P<0.01). NLXT can reduce the DNA methylation level of NogoA pathway after IS, thus inhibit the expression of NogoA/RhoA/ROCK pathway from producing anti-cerebral ischemia pharmacological effect.