Rho-associated Protein Kinase 1 Research Articles

Inhibition of the FOXO1–ROCK1 axis mitigates cardiomyocyte injury under chronic hypoxia in Tetralogy of Fallot by maintaining mitochondrial quality control

IntroductionPersistent chronic myocardial hypoxia causes disturbances in mitochondrial quality control (MQC), ultimately leading to increased cardiomyocyte injury in patients with Tetralogy of Fallot (TOF). The present study aimed to identify the key effector molecules of cardiomyocyte injury under chronic hypoxia in TOF. MethodsClinical data from TOF patients were collected and whole transcriptome sequencing was performed on myocardial samples. Chronic hypoxia models were established in cardiac-specific knockout mice and cardiomyocytes, and a series of molecular experiments were used to determine the specific mechanisms involved. ResultsClinical cohort data and whole-transcriptome sequencing analysis of myocardial samples from TOF patients revealed that forkhead box O1 (FOXO1) plays an important role in chronic hypoxic cardiomyocyte injury. In a model of chronic hypoxia established in FOXO1 cardiac-specific knockout mice and FOXO1 gene-deficient cardiomyocytes, the AMPK signaling pathway regulates the expression of FOXO1, which in turn disrupts MQC by regulating the transcriptional activation of Rho-associated protein kinase 1 (ROCK1), and increasing the production of mitochondrial ROS, thereby exacerbating damage to cardiomyocytes. Excessive reactive oxygen species (ROS) production during MQC dysfunction further activates Cox7a2L to increase the assembly of the respiratory chain supercomplex. In addition, we found that miR-27b-3p partially binds to the 3′ untranslated region of FOXO1 to exert a protective effect. ConclusionsMaintenance of MQC under chronic hypoxia is achieved through a series of injury-protection mechanisms, suggesting that FOXO1 inhibition may be crucial for future mitigation of chronic hypoxic cardiomyocyte injury in TOF.

DDR1 Drives Malignant Progression of Gastric Cancer by Suppressing HIF-1α Ubiquitination and Degradation.

The extracellular matrix (ECM) has been demonstrated to be dysregulated and crucial for malignant progression in gastric cancer (GC), but the mechanism is not well understood. Here, that discoidin domain receptor 1 (DDR1), a principal ECM receptor, is recognized as a key driver of GC progression is reported. Mechanistically, DDR1 directly interacts with the PAS domain of hypoxia-inducible factor-1α (HIF-1α), suppresses its ubiquitination and subsequently strengthens its transcriptional regulation of angiogenesis. Additionally, DDR1 upregulation in GC cells promotes actin cytoskeleton reorganization by activating HIF-1α/ Ras Homolog Family Member A (RhoA)/Rho-associated protein kinase 1 (ROCK1) signaling, which in turn enhances the metastatic capacity. Pharmacological inhibition of DDR1 suppresses GC progression and angiogenesis in patient-derived xenograft (PDX) and organoid models. Taken together, this work first indicates the effects of the DDR1-HIF-1α axis on GC progression and reveals the related mechanisms, providing experimental evidence for DDR1 as a therapeutic target for GC.

Rock2 heterozygosity improves recognition memory and endothelial function in a mouse model of 16p11.2 deletion autism syndrome

Rho-associated protein kinase-2 (ROCK2) is a critical player in many cellular processes and was incriminated in cardiovascular and neurological disorders. Recent evidence has shown that non-selective pharmacological blockage of ROCKs ameliorates behavioral alterations in a mouse model of 16p11.2 haploinsufficiency. We had revealed that 16p11.2-deficient mice also display cerebrovascular abnormalities, including endothelial dysfunction. To investigate whether genetic blockage of ROCK2 also exerts beneficial effects on cognition and angiogenesis, we generated mice with both 16p11.2 and Rock2 haploinsufficiency (16p11.2df/+;Rock2+/−). We find that Rock2 heterozygosity on a 16p11.2df/+ background significantly improved recognition memory. Furthermore, brain endothelial cells from 16p11.2df/+;Rock2+/− mice display improved angiogenic capacity compared to cells from 16p11.2df/+ littermates. Overall, this study implicates Rock2 gene as a modulator of 16p11.2-associated alterations, highlighting its potential as a target for treatment of autism spectrum disorders.

SMP30 Interacts with ROCK1 to Inhibit HCC Invasiveness by Effecting the Epithelial-Mesenchymal Transition.

The senescence marker protein 30 (SMP30) is a calcium-binding protein whose expression decreases with age, and is closely associated with hepatocellular carcinoma (HCC) development. The primary goal of this study was to examine the mechanistic effect of SMP30 on HCC migration and invasion. Bioinformatic and immunohistochemical approaches were used to examine the expression of SMP30 in HCC tissues and its relationship to patient survival. We investigated the effects of SMP30 expression on HCC cell proliferation, migration, invasion, and cell cycle dynamics. cDNA microarray technology was used to determine the gene expression profile of SK-Hep-1 cells following recombinant SMP30 overexpression to identify genes downstream of SMP30 that regulate HCC cell migration and invasion. We identified SMP30 interacting proteins by affinity purification-mass spectrometry (AP-MS) and co-immunoprecipitation/western blotting (COIP-WB). SMP30 expression was lower in HCC tissues compared with normal liver tissues, and its expression positively correlated with overall survival in HCC patients. Additionally, SMP30 overexpression effectively blocked the migratory and invasive properties of SK-Hep-1 cells, but did not affect either proliferation rates or cell cycle. cDNA microarray results confirmed that many of the differentially expressed genes identified are involved in the process of epithelial-mesenchymal transition (EMT). AP-MS and COIP-WB experiments confirmed that Rho-associated protein kinase 1 (ROCK1) interacts with SMP30 in SK-Hep-1 cells, and ROCK1 is known to intimately regulate the EMT process. SMP30 inhibits HCC metastasis by influencing the expression of EMT-related proteins after interacting with ROCK1.

Silk Sericin is a Potent, Multifunctional Biomaterial for Endothelialization of Tissue‐Engineered Heart Valves

AbstractEndothelialization is important for offering an anticoagulant surface, which is crucial for the construction of tissue‐engineered heart valves. Silk sericin is extensively investigated in the biomedical field due to its remarkable biological activities and diverse functional groups, favoring chemical modifications for the formation of versatile constructs. Herein, a sericin covalently modified valve (SCMV) is successfully fabricated by coupling maleylated silk sericin (MSS) with thiolated decellularized heart valve (DHV) through a Michael addition reaction. The results demonstrate that SCMV exhibits a smooth surface, higher hydrophilicity, and excellent resistance to platelets adhesion compared to DHV. Additionally, SCMV promotes endothelial cells (ECs) adhesion under both static and flow conditions and enhances their survival in a mouse subcutaneous implant model. The study also discovered that MSS‐stimulated ECs exhibit increased RhoA activation, expression of rho‐associated protein kinase 2 (ROCK2) and phosphorylated myosin light chain 2 (MLC2), actin polymerization, and cell adhesion; whereas actin organization and cell adhesion are significantly reduced by treatment with Y‐27632, a ROCK inhibitor. Furthermore, several integrin subunits, including α1, α2, α3, α5, α6, β1, β3, and β5, are involved in this regulatory process. Collectively, the findings highlight the potential application of silk sericin in promoting endothelialization of DHV while uncovering novel mechanisms underlying its regulatory effect on ECs adhesion.

ROCK1 inhibition improves wound healing in diabetes via RIPK4/AMPK pathway.

Refractory wounds are a severe complication of diabetes mellitus that often leads to amputation because of the lack of effective treatments and therapeutic targets. The pathogenesis of refractory wounds is complex, involving many types of cells. Rho-associated protein kinase-1 (ROCK1) phosphorylates a series of substrates that trigger downstream signaling pathways, affecting multiple cellular processes, including cell migration, communication, and proliferation. The present study investigated the role of ROCK1 in diabetic wound healing and molecular mechanisms. Our results showed that ROCK1 expression significantly increased in wound granulation tissues in diabetic patients, streptozotocin(STZ)-induced diabetic mice, and db/db diabetic mice. Wound healing and blood perfusion were dose-dependently improved by the ROCK1 inhibitor fasudil in diabetic mice. In endothelial cells, fasudil and ROCK1 siRNA significantly elevated the phosphorylation of adenosine monophosphate-activated protein kinase at Thr172 (pThr172-AMPKα), the activity of endothelial nitric oxide synthase (eNOS), and suppressed the levels of mitochondrial reactive oxygen species(mtROS) and nitrotyrosine formation. Experiments using integrated bioinformatics analysis and coimmunoprecipitation established that ROCK1 inhibited pThr172-AMPKα by binding to receptor-interacting serine/threonine kinase 4 (RIPK4). These results suggest that fasudil accelerated wound repair and improved angiogenesis at least partially through the ROCK1/RIPK4/AMPK pathway. Fasudil may be a potential treatment for refractory wounds in diabetic patients.

Discovery of dual rho-associated protein kinase 1 (ROCK1)/apoptosis signal–regulating kinase 1 (ASK1) inhibitors as a novel approach for non-alcoholic steatohepatitis (NASH) treatment

In the current study we suggest a novel approach to curb non-alcoholic steatohepatitis (NASH) progression, and we suggest privileged scaffolds for the design of novel compounds for this aim. NASH is an advanced form of non-alcoholic fatty liver disease that can further progress into fibrosis, cirrhosis, and hepatocellular carcinoma. It is a widely emerging disease affecting 25% of the global population and has no current approved treatments. Protein kinases are key regulators of cellular pathways, of which, Rho-associated protein kinase 1 (ROCK1) and apoptosis signal–regulating kinase 1 (ASK1) play an important role in the progression of NASH and they stand out as promising targets for NASH therapy. Interestingly, their kinase domains are found to be similar in sequence and topology; therefore, dual inhibition of ROCK1 and ASK1 is expected to be amenable and could achieve a more favourable outcome. To reach this goal, a training set of ROCK1 and ASK1 protein structures co-crystalized with type 1 (ATP-competitive) inhibitors was constructed to manually generate receptor-based pharmacophore models representing ROCK1 and ASK1 inhibitors’ common pharmacophoric features. The models produced were assessed using a test set of both ROCK1 and ASK1 actives and decoys, and their performance was evaluated using different assessment metrics. The best pharmacophore model obtained, showing a Mathew’s correlation coefficient (MCC) of 0.71, was then used to screen the ZINC purchasable database retrieving 6178 hits that were filtered accordingly using several medicinal chemistry and pharmacokinetics filters returning 407 promising compounds. To confirm that these compounds are capable of binding to the target kinases, they were subjected to molecular docking simulations at both protein structures. The results were then assessed individually and filtered, setting the spotlight on various privileged scaffolds that could be exploited as the nucleus for designing novel ROCK1/ASK1 dual inhibitors.

Caspase-4 has a role in cell division in epithelial cells through actin depolymerization

In addition to its role in pyroptosis and inflammatory cytokine maturation, caspase-4 (CASP4) also contributes to the fusion of phagosomes with lysosomes and cell migration. However, its role in cell division remains elusive. In this study, we demonstrate that CASP4 is indispensable for proper cell division in epithelial cells. Knockout of CASP4 (CASP4 KO) in HepG2 cells led to delayed cell proliferation, increased cell size, and increased multinucleation. In mitosis, CASP4 KO cells showed multipolar spindles, asymmetric spindle positioning, and chromosome segregation errors, ultimately increasing DNA content and chromosome number. We also found that phalloidin, a marker of filamentous actin, increased in CASP4 KO cells owing to suppressed actin depolymerization. Moreover, the levels of actin polymerization-related proteins, including Rho-associated protein kinase1 (ROCK1), LIM kinase1 (LIMK1), and phosphorylated cofilin, significantly increased in CASP4 KO cells. These results suggest that CASP4 contributes to proper cell division through actin depolymerization.

ROCK1 is a multifunctional factor maintaining the primordial follicle reserve and follicular development in mice.

The follicle is the basic structural and functional unit of the ovary in female mammals. The excessive depletion of follicles will lead to diminished ovarian reserve or even premature ovarian failure, resulting in diminished ovarian oogenesis and endocrine function. Excessive follicular depletion is mainly due to loss of primordial follicles. Our analysis of published human ovarian single-cell sequencing results by others revealed a significant increase in rho-associated protein kinase 1 (ROCK1) expression during primordial follicle development. However, the role of ROCK1 in primordial follicle development and maintenance is not clear. This study revealed a gradual increase in ROCK1 expression during primordial follicle activation. Inhibition of ROCK1 resulted in reduced primordial follicle activation, decreased follicular reserve, and delayed development of growing follicles. This effect may be achieved through the HIPPO pathway. The present study indicates that ROCK1 is a key molecule for primordial follicular reserve and follicular development.NEW & NOTEWORTHY ROCK1, one of the Rho GTPases, plays an important role in primordial follicle reserve and follicular development. ROCK1 was primarily expressed in the cytoplasm of oocytes and granulosa cell in mice. Inhibition of ROCK1 significantly reduced the primordial follicle reserve and delayed growing follicle development. ROCK1 regulates primordial follicular reserve and follicle development through the HIPPO signaling pathway. These findings shed new lights on the physiology of sustaining female reproduction.

Artesunate Induces Ferroptosis in Hepatic Stellate Cells and Alleviates Liver Fibrosis via the ROCK1/ATF3 Axis.

Development of fibrosis in chronic liver disease requires activation of hepatic stellate cells (HSCs) and leads to a poor outcome. Artesunate (Art) is an ester derivative of artemisinin that can induce ferroptosis in HSCs, and activated transcriptional factor 3 (ATF3) is an ATF/CREB transcription factor that is induced in response to stress. In this study, we examined the role of the Rho-associated protein kinase 1 (ROCK1)/ATF3 axis in Art-induced ferroptosis in HSCs. HSC activation and ferroptosis were studied in vitro by western blotting, polymerase chain reaction, immunofluorescence, and other assays. ATF3 electrophoretic mobility and ROCK1 protein stability were assayed by western blotting. Immunoprecipitation was used to detect the interaction of ROCK1 and ATF3, as well as ATF3 phosphorylation. A ubiquitination assay was used to verify ROCK1 degradation. Atf3-interfering and Rock1-overexpressing mice were constructed to validate the anti-hepatic fibrosis activity of Art in vivo. Art induced ferroptosis in HSCs following glutathione-dependent antioxidant system inactivation resulting from nuclear accumulation of unphosphorylated ATF3 mediated by ROCK1-ubiquitination in vitro. Art also decreased carbon tetrachloride-induced liver fibrosis in mice, which was reversed by interfering with Atf3 or overexpressing Rock1. The ROCK1/ATF3 axis was involved in liver fibrosis and regulation of ferroptosis, which provides an experimental basis for further study of Art for the treatment of liver fibrosis.

Whole-genome DNA methylation profiling reveals epigenetic signatures in developing muscle in Tan and Hu sheep and their offspring.

The Tan sheep is a popular local breed in China because of its tenderness and flavor. The Hu sheep breed is also famous for its high litter size, and its muscle growth rate is faster than that of Tan sheep. However, the epigenetic mechanism behind these muscle-related phenotypes is unknown. In this study, the longissimus dorsi tissue from 18 6 month-old Tan sheep, Hu sheep, and Tan-Hu F2 generation (6 sheep per population) were collected. After genomic DNA extraction, whole-genome bisulfite sequencing (WGBS) and bioinformatics analysis were performed to construct genome-wide DNA methylome maps for the Tan sheep, Hu sheep and their Tan-Hu F2 generation. Distinct genome-wide DNA methylation patterns were observed between Tan sheep and Hu sheep. Moreover, DNA methylated regions were significantly increased in the skeletal muscle from Tan sheep vs. the F2 generation compared to the Hu sheep vs. F2 generation and the Tan sheep vs. Hu sheep. Compared with Hu sheep, the methylation levels of actin alpha 1 (ACTA1), myosin heavy chain 11 (MYH11), Wiskott-Aldrich syndrome protein (WAS), vav guanine nucleotide exchange factor 1 (VAV1), fibronectin 1 (FN1) and Rho-associated protein kinase 2 (ROCK2) genes were markedly distinct in the Tan sheep. Furthermore, Gene Ontology analysis indicated that these genes were involved in myotube differentiation, myotube cell development, smooth muscle cell differentiation and striated muscle cell differentiation. The findings from this study, in addition to data from previous research, demonstrated that the ACTA1, MYH11, WAS, VAV1, FN1, and ROCK2 genes may exert regulatory effects on muscle development.

HDAC3 deficiency protects against acute lung injury by maintaining epithelial barrier integrity through preserving mitochondrial quality control

Sepsis is one common cause of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which is closely associated with high mortality in intensive care units (ICU). Histone deacetylase 3 (HDAC3) serves as an important epigenetic modifying enzyme which could affect chromatin structure and transcriptional regulation. Here, we explored the effects of HDAC3 in type II alveolar epithelial cells (AT2) on lipopolysaccharide (LPS)-induced ALI and shed light on potential molecular mechanisms. We generated ALI mouse model with HDAC3 conditional knockout mice (Sftpc-cre; Hdac3f/f) in AT2 and the roles of HDAC3 in ALI and epithelial barrier integrity were investigated in LPS-treated AT2. The levels of HDAC3 were significantly upregulated in lung tissues from mice with sepsis and in LPS-treated AT2. HDAC3 deficiency in AT2 not only decreased inflammation, apoptosis, and oxidative stress, but also maintained epithelial barrier integrity. Meanwhile, HDAC3 deficiency in LPS-treated AT2 preserved mitochondrial quality control (MQC), evidenced by the shift of mitochondria from fission into fusion, decreased mitophagy, and improved fatty acid oxidation (FAO). Mechanically, HDAC3 promoted the transcription of Rho-associated protein kinase 1 (ROCK1) in AT2. In the context of LPS stimulation, the upregulated ROCK1 elicited by HDAC3 could be phosphorylated by Rho-associated (RhoA), thus disturbing MQC and triggering ALI. Furthermore, we found that forkhead box O1 (FOXO1) was one of transcription factors of ROCK1. HDAC3 directly decreased the acetylation of FOXO1 and promoted its nuclear translocation in LPS-treated AT2. Finally, HDAC3 inhibitor RGFP966 alleviated epithelial damage and improved MQC in LPS-treated AT2. Altogether, HDAC3 deficiency in AT2 alleviated sepsis-induced ALI by preserving mitochondrial quality control via FOXO1-ROCK1 axis, which provided a potential strategy for the treatment of sepsis and ALI.

Rhubarb Anthraquinones Ameliorates Inflammatory Lung Injury by Enhancing Alveolar Epithelium Tight Junction Proteins through RhoA/ROCK1 Signalling

Background: Leakage of the alveolar epithelial barrier can lead to pulmonary oedema, leading to organ dysfunction. Objectives: Combination anthraquinone (CA) and free anthraquinone (FA) were extracted from Rhubarb, and the prevention and therapeutic effects of Rhubarb anthraquinones (RA) on lipopolysaccharide (LPS)-induced lung injury were investigated. Materials and Methods: The CA and FA were extracted from Rhubarb by water solvent extraction and ethanol solvent extraction. The extracted RA content was determined using spectrophotometry and high-performance liquid chromatography (HPLC). A mouse model of inflammatory lung injury was established by LPS induction to study the mechanism of RA activity. Inflammatory factors were measured using an enzyme-linked immunosorbent assay. Changes in alveolar epithelial leakage were assessed using a permeability assay. Changes in lung injury were evaluated by histopathological and ultrastructural observations. Tight junction (TJ) markers and Ras homolog gene family member A (RhoA)/Rho-associated protein kinase 1 (ROCK1) pathway-related proteins were measured using immunohistochemistry and Western blotting. As an agonist of RhoA/Rock signalling, U46619 was used to further verify whether RA acts through this pathway. Results: RA could inhibit pulmonary inflammation by decreasing the level of inflammatory factors. The alveolar epithelial permeability was reduced, and the pathological injury of the lung tissue was alleviated after administration with RA. Furthermore, the expression of TJ proteins was up regulated and RhoA/ROCK1 signalling was inhibited in the presence of RA. The effects of RA on TJ proteins were partially reversed by U46619. Conclusion: RA effectively protects mice against inflammatory lung injury by enhancing alveolar epithelial TJ via RhoA/ROCK1 signalling.

Schisandrin B inhibits tumor progression of hepatocellular carcinoma by targeting the RhoA/ROCK1 pathway.

Schisandrin B (Sch. B) performs various pharmacological properties, including anticancer activities. However, the pharmacological mechanisms of Sch. B in hepatocellular carcinoma (HCC) are not fully elucidated. We investigated the impact and mechanism on progression in HCC, and to provide new experimental evidence for HCC treatment. To determine the inhibitory effect of Sch. B on HCC in vivo, 32 Balb/c nude mice were used to prepare the tumor-bearing mice model by subcutaneously inoculating HCC cells (Huh-7). As tumor volume grew to 100 mm3, mice were randomly divided into Saline (control group), 100 mg/kg Sch. B group (Sch. B-L), 200 mg/kg Sch. B group (Sch. B-M), and 400 mg/kg Sch. B group (Sch. B-H) (n=8). Saline or different concentration Sch. B was used to treat mice via gavage administration for 21 days. After mice were euthanized, tumor weight and volume were evaluated. Cell apoptosis was detected by TUNEL. Ki-67 and PCNA were detected by immunohistochemical staining. The RhoA and Rho-associated protein kinase 1 (ROCK1) were determined by western blot. In vitro experiment, Huh-7 cell were treated by Sch. B at 40, 30, 20, 10, 5, 1, and 0 µM to detect cell proliferation by Cell Counting Kit-8 (CCK-8). Huh-7 cells were divided as a control group, Sch. B group, and Sch. B + RhoA overexpression (Sch. B + RhoA) group. RhoA and ROCK1 were examined. The colony formation assay and flow cytometry were used to detect cell proliferation and apoptosis. The wound healing and Transwell assays were used for cell metastasis detection. Our results showed 100, 200 and 400 mg/kg Sch. B significantly reduced tumor weight and volume. And 200 and 400 mg/kg Sch. B increased apoptosis, and reduced Ki-67 and PCNA levels, inhibited the RhoA and ROCK1 in vivo (P<0.05). In vitro experiment, Sch. B inhibited Huh-7 cell proliferation at concentration more than 10 μM (P<0.05). Sch. B decreased cell duplication, promoted apoptosis and blocked migration and invasion of Huh-7 (P<0.05). Sch. B inhibited RhoA and ROCK1 level as compared with control group (P<0.05). RhoA overexpression reversed the effect of Sch. B (P<0.05). Sch. B inhibits Huh-7 cells progression via RhoA/ROCK1 pathway. The results provide new evidence for the clinical treatment of HCC.

Apigenin Attenuates Hippocampal Microglial Activation and Restores Cognitive Function in Methotrexate-Treated Rats: Targeting the miR-15a/ROCK-1/ERK1/2 Pathway

Microglial activation underpins the methotrexate (MTX)-induced neurotoxicity; however, the precise mechanism remains unclear. This study appraised the potential impact of apigenin (Api), a neuroprotective flavonoid, in MTX-induced neurotoxicity in rats in terms of microglial activation through targeting the miR-15a/Rho-associated protein kinase-1 (ROCK-1)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Male Sprague Dawley rats were randomly divided into 4 groups: Normal control (saline i.p. daily and i.v. on days 8 and 15); Api control (20 mg/kg, p.o.) daily for 30 days; MTX-alone (75 mg/kg, i.v.) on days 8 and 15, then four i.p. injections of leucovorin (LCV): 6 mg/kg after 18 h, then three doses (3 mg/kg) every 8 h post-MTX; and Api co-treated (20 mg/kg/day, p.o.) throughout the model for 30 days, with administration of MTX and LCV as in group 3. MTX administration elevated hippocampal ionized calcium-binding adaptor protein-1 (Iba-1) immunostaining, indicating microglial activation. This was accompanied by neuroinflammation, oxidative stress, and enhanced apoptosis manifested by elevated hippocampal interleukin-1β, malondialdehyde, and caspase-3, and decreased reduced glutathione levels. Concurrently, abated miR-15a expression, overexpression of its target ROCK-1, diminished downstream ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation, and decreased hippocampal brain-derived neurotrophic factor (BDNF) levels were observed. Api mitigated the MTX-induced neurotoxicity by reversing the biochemical, histopathological, and behavioral derangements tested by novel object recognition and Morris water maze tests. Conclusively, Api lessens MTX-induced neuroinflammation, oxidative stress, and apoptosis and boosts cognitive function through inhibiting microglial activation via modulating the miR-15a/ROCK-1/ERK1/2/CREB/BDNF pathway.Graphical Graphical abstract showing the effects of methotrexate and apigenin co-treatment in MTX-induced neurotoxicity model.On the left, methotrexate (MTX) administration to rats resulted in hippocampal miR-15a downregulation, which triggered an enhanced expression of its target ROCK-1, consequently inhibiting the downstream ERK1/2/CREB/BDNF pathway, instigating a state of microglial activation, neuroinflammation, oxidative stress, and apoptosis. On the other hand, apigenin (Api) co-treatment restored miR-15a, inhibited ROCK-1 expression, and activated the ERK1/2/CREB/BDNF pathway, leading to diminished hippocampal microglial activation, neuroinflammation, and apoptosis, and restoration of the redox balance, along with improvement in memory and cognitive function of the MTX-treated rats.

Microenvironmental Stiffness Directs Chondrogenic Lineages of Stem Cells from the Human Apical Papilla via Cooperation between ROCK and Smad3 Signaling.

Cell-based cartilage tissue engineering faces a great challenge in the repair process, partly due to the special physical microenvironment. Human stem cell from apical papilla (hSCAP) shows great potential as seed cells because of its versatile differentiation capacity. However, whether hSCAP has potent chondrogenic differentiation ability in the physical microenvironment of chondroid remains unknown. In this study, we fabricated poly(dimethylsiloxane) (PDMS) substrates with different stiffnesses and investigated the chondrogenic differentiation potential of hSCAPs. First, we found that hSCAPs cultured on soft substrates spread more narrowly accompanied by cortical actin organization, a hallmark of differentiated chondrocytes. On the contrary, stiff substrates were favorable for cell spreading and stress fiber formation. More importantly, the increased chondrogenic differentiation of hSCAPs seeded on soft substrates was confirmed by characterizing increased extracellular proteoglycan aggregation through Alcian blue staining and Safranin O staining and enhanced markers toward chondrogenic differentiation including SRY-box transcription factor 9 (Sox9), type II collagen (Col2), and aggrecan in both normal α-minimum essential medium (αMEM) and specific chondrogenic medium (CM) culture conditions. Then, we investigated the mechanosensing/mechanotransduction governing the chondrogenic differentiation of hSCAPs in response to different stiffnesses and found that stiffness-sensitive integrin β1 and focal adhesion kinase (FAK) were essential for mechanical signal perception and were oriented at the start of mechanotransduction induced by matrix stiffness. We next showed that the increased nuclear accumulation of Smad3 signaling and target Sox9 facilitated the chondrogenic differentiation of hSCAPs on the soft substrates and further verified the importance of Rho-associated protein kinase (ROCK) signaling in regulating chondrogenic differentiation and its driving factors, Smad3 and Sox9. By using SIS3, the specific inhibitor of p-Smad3, and miRNA targeting Rho-associated protein kinase 1 (ROCK-1), we finally confirmed the importance of ROCK/Smad3/Sox9 axis in the chondrogenic differentiation of hSCAPs in response to substrate stiffness. These results help us to increase the understanding of how microenvironmental stiffness directs chondrogenic differentiation from the aspects of mechanosensing, mechanotransduction, and cell fate decision, which will be of great value in the application of hSCAPs in cartilage tissue engineering.

Dasabuvir suppresses esophageal squamous cell carcinoma growth in vitro and in vivo through targeting ROCK1

Esophageal squamous cell carcinoma (ESCC) is an upper gastrointestinal cancer with high morbidity and mortality. New strategies are urgently needed to prolong patients’ survival. Through screening FDA-approved drugs, we found dasabuvir, a drug approved for hepatitis C virus (HCV) treatment, suppressed ESCC proliferation. Dasabuvir could inhibit the growth of ESCC cells in a time and dose-dependent manner and arrested cell cycle at the G0/G1 phase. The antitumor activity was further validated in vivo using patient-derived xenograft tumor models. In terms of mechanism, we unveil that dasabuvir is a Rho-associated protein kinase 1 (ROCK1) inhibitor. Dasabuvir can bind to ROCK1 and suppress its kinase activity, thus downregulating the phosphorylation of ERK1/2 by ROCK1 and the expression of cyclin-dependent kinase 4 (CDK4) and cyclin D1. These results provide evidence that dasabuvir suppresses ESCC growth in vivo and in vitro through blocking ROCK1/ERK signaling pathway.

Regulation of oxidized LDL-induced proliferation and migration in human vascular smooth muscle cells by a novel circ_0007478/miR-638/ROCK2 ceRNA network.

Circular RNAs (circRNAs) have been implicated in the pathogenesis of atherosclerosis (AS) and the migration and proliferation of vascular smooth muscle cells (VSMCs) under oxidized low-density lipoprotein (ox-LDL). Here, we defined the exact action of human circ_0007478 in VSMC migration and proliferation induced by ox-LDL. Human VSMCs (HVSMCs) were exposed to ox-LDL. Circ_0007478, microRNA (miR)-638, and rho-associated protein kinase 2 (ROCK2) levels were gauged by quantitative real-time PCR (qRT-PCR) and western blot. Cell viability and proliferation were assessed by MTT and EdU assays, respectively. Transwell assays were used to detect cell migration and invasion. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to evaluate the direct relationship between miR-638 and circ_0007478 or ROCK2. Our data indicated that circ_0007478 expression was augmented in AS serum samples and ox-LDL-treated HVSMCs. Depletion of circ_0007478 attenuated HVSMC proliferation, migration, and invasion induced by ox-LDL. Mechanistically, circ_0007478 targeted miR-638 by directly pairing to miR-638. Reduction of miR-638 reversed the effects of circ_0007478 depletion on ox-LDL-evoked proliferation, migration, and invasion in HVSMCs. ROCK2 was a direct miR-638 target and miR-638-mediated inhibition of ROCK2 relieved ox-LDL-evoked HVSMC proliferation, migration, and invasion. Furthermore, circ_0007478 was identified as a competing endogenous RNA (ceRNA) for miR-638 to modulate ROCK2 expression. Our present study establishes an undescribed ceRNA regulatory network, in which circ_0007478 targets miR-638 to upregulate ROCK2, thereby contributing to ox-LDL-induced proliferation and migration in HVSMCs.

MORN4 protects cardiomyocytes against ischemic injury via MFN2-mediated mitochondrial dynamics and mitophagy

The imbalance of mitochondrial fission and fusion dynamics causes ischemic cardiomyocyte apoptosis and heart injury by affecting mitophagy. Regulation of mitochondrial dynamics is an important therapeutic strategy for ischemic heart diseases. Considering the important roles of MORN motifs in heart diseases and chloroplast fission, we aimed to investigate the possible role of MORN repeat-containing protein 4 (MORN4) in the progression of myocardial infarction (MI), ischemic cardiomyocyte apoptosis, mitochondrial dynamics, and mitophagy. We found that in the MI mouse, MORN4 knockdown remarkably accelerated cardiac injury and fibrosis with deteriorating cardiac dysfunction. Sphingosylphosphorylcholine (SPC) alleviated ischemic cardiomyocyte apoptosis and heart injury through increased level of MORN4, indicating a vital function of MORN4 in heart with SPC used to clarify the molecular mechanisms underlying the functions of MORN4. Mechanistically, we found that MORN4 directly binds to MFN2 and promotes the phosphorylation of MFN2 S442 through Rho-associated protein kinase 2 (ROCK2), which mediates beneficial mitophagy induced by mitochondrial dynamics, while SPC promoted the binding of MORN4 and MFN2 and the process. Taken together, our data reveal a new perspective role of MORN4 in ischemic heart injury, and report that SPC could regulate myocardial mitochondrial homeostasis by activating the MORN4-MFN2 axis during the ischemic situation, this finding provides novel targets for improving myocardial ischemia tolerance and rescue of acute myocardial infarction.

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.