Sevoflurane-induced overexpression of extrasynaptic α5-GABAAR via the RhoA/ROCK2 pathway impairs cognitive function in aged mice.

Perioperative neurocognitive disorder (PND) is a serious neurologic complication in aged patients and might be associated with sevoflurane exposure. However, the specific pathogenesis is still unclear. The distribution of α5-GABAAR, a γ-aminobutyric acid type A receptor (GABAAR) subtype, at extrasynaptic sites is influenced by the anchor protein radixin, whose phosphorylation is regulated via the RhoA/ROCK2 signaling pathway and plays a crucial role in cognition. However, whether sevoflurane affects the ability of radixin phosphorylation to alter extrasynaptic receptor expression is unknown. Aged mice were exposed to sevoflurane to induce cognitive impairment. Both total proteins and membrane proteins were extracted for analysis. Cognitive function was evaluated using the Morris water maze and fear conditioning test. Western blotting was used to determine the expression of ROCK2 and the phosphorylation of radixin. Furthermore, the colocalization of p-radixin and α5-GABAAR was observed. To inhibit ROCK2 activity, either an adeno-associated virus (AAV) or fasudil hydrochloride was administered. Aged mice treated with sevoflurane exhibited significant cognitive impairment accompanied by increased membrane expression of α5-GABAAR. Moreover, the colocalization of α5-GABAAR and p-radixin increased after treatment with sevoflurane, and this change was accompanied by an increase in ROCK2 expression and radixin phosphorylation. Notably, inhibiting the RhoA/ROCK2 pathway significantly decreased the distribution of extrasynaptic α5-GABAAR and improved cognitive function. Sevoflurane activates the RhoA/ROCK2 pathway and increases the phosphorylation of radixin. Excess α5-GABAAR is anchored to extrasynaptic sites and impairs cognitive ability in aged mice. Fasudil hydrochloride administration improves cognitive function.

miR-101a-3p/ROCK2 axis regulates neuronal injury in Parkinson's disease models.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons in substantia nigra pars compacta (SNpc). This study focuses on deciphering the role of microRNA (miR)-101a-3p in the neuronal injury of PD and its regulatory mechanism. METHODS: We constructed a mouse model of PD by intraperitoneal injection of 1-methyl 4-phenyl 1, 2, 3, 6-tetrahydropyridine hydrochloride (MPTP), and used 1-methyl-4-phenylpyridinium (MPP+) to treat Neuro-2a cells to construct an in-vitro PD model. Neurological dysfunction in mice was evaluated by swimming test and traction test. qRT-PCR was utilized to examine miR-101a-3p expression and ROCK2 expression in mouse brain tissues and Neuro-2a cells. Western blot was conducted to detect the expression of α-synuclein protein and ROCK2 in mouse brain tissues and Neuro-2a cells. The targeting relationship between miR-101a-3p and ROCK2 was determined by dual-luciferase reporter gene assay. The apoptosis of neuro-2a cells was assessed by flow cytometry. RESULTS: Low miR-101a-3p expression and high ROCK2 expression were found in the brain tissues of PD mice and MPP+-treated Neuro-2a cells; PD mice showed decreased neurological disorders, and apoptosis of Neuro-2a cells was increased after MPP+ treatment, both of which were accompanied by increased accumulation of α-synuclein protein. After miR-101a-3p was overexpressed, the neurological function of PD mice was improved, and the apoptosis of Neuro-2a cells induced by MPP+ was alleviated, and the accumulation of α-synuclein protein was reduced; ROCK2 overexpression counteracted the protective effect of miR-101a-3p. Additionally, ROCK2 was identified as the direct target of miR-101a-3p. CONCLUSION: MiR-101a-3p can reduce neuronal apoptosis and neurological deficit in PD mice by inhibiting ROCK2 expression, suggesting that miR-101a-3p is a promising therapeutic target for PD.

M2 Microglia-Derived Exosomes Protect Against Glutamate-Induced HT22 Cell Injury via Exosomal miR-124-3p.

As one of the most serious complications of sepsis, sepsis-associated encephalopathy has not been effectively treated or prevented. Exosomes, as a new therapeutic method, play a protective role in neurodegenerative diseases, stroke and traumatic brain injury in recent years. The purpose of this study was to investigate the role of exosomes in glutamate (Glu)-induced neuronal injury, and to explore its mechanism, providing new ideas for the treatment of sepsis-associated encephalopathy. The neuron damage model induced by Glu was established, and its metabolomics was analyzed and identified. BV2 cells were induced to differentiate into M1 and M2 subtypes. After the exosomes from both M1-BV2 cells and M2-BV2 cells were collected, exosome morphological identification was performed by transmission electron microscopy and exosome-specific markers were also detected. These exosomes were then cocultured with HT22 cells. CCK-8 method and LDH kit were used to detect cell viability and toxicity. Cell apoptosis, mitochondrial membrane potential and ROS content were respectively detected by flow cytometry, JC-1 assay and DCFH-DA assay. MiR-124-3p expression level was detected by qRT-PCR and Western blot. Bioinformatics analysis and luciferase reporter assay predicted and verified the relationship between miR-124-3p and ROCK1 or ROCK2. Through metabolomics, 81 different metabolites were found, including fructose, GABA, 2, 4-diaminobutyric acid, etc. The enrichment analysis of differential metabolites showed that they were mainly enriched in glutathione metabolism, glycine and serine metabolism, and urea cycle. M2 microglia-derived exosomes could reduce the apoptosis, decrease the accumulation of ROS, restore the mitochondrial membrane potential and the anti-oxidative stress ability in HT22 cells induced by Glu. It was also found that the protective effect of miR-124-3p mimic on neurons was comparable to that of M2-EXOs. Additionally, M2-EXOs might carry miR-124-3p to target ROCK1 and ROCK2 in neurons, affecting ROCK/PTEN/AKT/mTOR signaling pathway, and then reducing Glu-induced neuronal apoptosis. M2 microglia-derived exosomes may protect HT22 cells against Glu-induced injury by transferring miR-124-3p into HT22 cells, with ROCK being a target gene for miR-124-3p.

A phase II study of belumosudil for chronic graft-versus-host disease in patients who failed at least one line of systemic therapy in China.

BACKGROUND: Chronic graft-versus-host disease (cGVHD) is an immune-related disorder that is the most common complication post-allogenic hematopoietic stem cell transplant. Corticosteroids with or without calcineurin inhibitors (CNIs) remain the mainstay of cGVHD treatment for first-line therapy. However, for many patients, cGVHD symptoms cannot be effectively managed and thus require second-line therapy. Currently, there is no approved treatment for second-line cGVHD treatment in China. In this study, belumosudil, a highly selective and potent rho-associated coiled-coil-containing protein kinase-2 inhibitor demonstrated to be effective for cGVHD in the United States and other Western countries, is investigated in patients with cGVHD in China for its overall benefit-risk balance. METHODS: This multicenter, open-label phase II study evaluated the safety, efficacy, and pharmacokinetics of oral belumosudil 200 mg once daily in cGVHD patients who had been treated with at least one line of systemic therapy in China. The primary endpoint was overall response rate (ORR); each individual patient's response was assessed by the investigator using the 2014 National Institutes of Health consensus criteria. Secondary endpoints were duration of response (DOR), time to response (TTR), changes in Lee Symptom Scale (LSS) score, organ response rate, corticosteroid dose change, CNI dose change, failure-free survival, time-to-next-treatment, overall survival, and safety. RESULTS: Thirty patients were enrolled in the study with a median follow-up time of 12.9 months. ORR was 73.3% (95% confidence interval: 54.1-87.7%) and all responders achieved partial response. Median DOR among responders was not reached and median TTR was 4.3 weeks (range: 3.9-48.1). Fifteen patients (50.0%) achieved clinically meaningful response in terms of reduction in LSS score by ≥ 7 points from baseline. Corticosteroid and CNI dose reductions were reported in 56.7% (17/30) and 35.0% (7/20) of patients, respectively. Most treatment-emergent adverse events (TEAEs) were mild to moderate in severity, with 11 patients (36.7%) experiencing grade ≥ 3 TEAEs. The most common grade ≥ 3 TEAE was pneumonia (n = 5, 16.7%). CONCLUSIONS: Belumosudil treatment demonstrated a favorable benefit-risk balance in treating cGVHD patients who previously have had standard corticosteroid therapy in China where approved second-line setting is absent. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT04930562.

Therapeutic potential of Lingjiao Gouteng decoction in acute alcohol intoxication and alcohol-induced brain injury involving the RhoA/ROCK2/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Alcohol misuse persists as a prevalent societal concern and precipitates diverse deleterious consequences, entailing significant associated health hazards including acute alcohol intoxication (AAI). Binge drinking, a commonplace pattern of alcohol consumption, may incite neurodegeneration and neuronal dysfunction. Clinicians tasked with managing AAI confront a dearth of pharmaceutical intervention alternatives. In contrast, natural products have garnered interest due to their compatibility with the human body and fewer side effects. Lingjiao Gouteng decoction (LGD), a classical traditional Chinese medicine decoction, represents a frequently employed prescription in cases of encephalopathy, although its efficacy in addressing acute alcoholism and alcohol-induced brain injury remains inadequately investigated. AIM OF THE STUDY: To investigate the conceivable therapeutic benefits of LGD in AAI and alcohol-induced brain injury, while delving into the underlying fundamental mechanisms involved. MATERIALS AND METHODS: We established an AAI mouse model through alcohol gavage, and LGD was administered to the mice twice at the 2 h preceding and 30 min subsequent to alcohol exposure. The study encompassed the utilization of the loss of righting reflex assay, histopathological analysis, enzyme-linked immunosorbent assays, and cerebral tissue biochemical assays to investigate the impact of LGD on AAI and alcohol-induced brain injury. These assessments included a comprehensive evaluation of various biomarkers associated with the inflammatory response and oxidative stress. Finally, RT-qPCR, Western blot, and immunofluorescence staining were carried out to explore the underlying mechanisms through which LGD exerts its therapeutic influence, potentially through the regulation of the RhoA/ROCK2/NF-κB signaling pathway. RESULTS: Our investigation underscores the therapeutic efficacy of LGD in ameliorating AAI, as evidenced by discernible alterations in the loss of righting reflex assay, pathological analysis, and assessment of inflammatory and oxidative stress biomarkers. Furthermore, the results of RT-qPCR, Western blot, and immunofluorescence staining manifest a noteworthy regulatory effect of LGD on the RhoA/ROCK2/NF-κB signaling pathway. CONCLUSIONS: The present study confirmed the therapeutic potential of LGD in AAI and alcohol-induced brain injury, and the protective effects of LGD against alcohol-induced brain injury may be intricately linked to the RhoA/ROCK2/NF-κB signaling pathway.

LPAR6 Participates in Neuropathic Pain by Mediating Astrocyte Cells via ROCK2/NF-κB Signal Pathway.

Neuropathic pain (NPP) is a common type of chronic pain. Glial cells, including astrocytes (AS), are believed to play an important role in the progression of NPP. AS cells can be divided into various types based on their expression profiles, among which A1 and A2 types have clear functions. A1-type AS cells are neurotoxic, while A2-type AS cells exert neuroprotective functions. Some types of lysophosphatidic acid receptors (LPAR) have been shown to play a role in NPP. However, it remains unclear how AS cells and LPAR6 affect the occurrence and progression of NPP. In this study, we established a mouse model of chronic constriction injury (CCI) to simulate NPP. It was found that the expression of LPAR6 in AS cells of the spinal dorsal horn was increased in the CCI model, and the thresholds of mechanical and thermal pain were elevated after knocking out LPAR6, indicating that LPAR6 and AS cells participated in the occurrence of NPP. The experiment involved culturing primary AS cells and knocking down LPAR6 by Lentivirus. The results showed that the NF-κB signal pathway was activated and the number of A1-type AS cells increased in the CCI model. However, LPAR6 knockdown inhibited the NF-κB signal pathway and A1-type AS cells. The results of the mRNA sequencing and immunoprecipitation test indicate an interaction between LPAR6 and ROCK2. Inhibiting ROCK2 by Y-27632 increased mechanical and thermal pain thresholds and alleviated NPP at the molecular level. The study presents evidence that LPAR6 activates the NF-κB pathway through ROCK2 and contributes to the progression of NPP by increasing A1-type AS and decreasing A2-type AS. This suggests that LPAR6 could be a potential therapeutic target for alleviating NPP. Clinical applications that are successful can offer new therapeutic options, enhance the quality of life for patients, and potentially uncover new mechanisms for pain modulation.

Circ_0114428 knockdown inhibits ROCK2 expression to assuage lipopolysaccharide-induced human pulmonary alveolar epithelial cell injury through miR-574-5p.

BACKGROUND: Sepsis-induced acute lung injury (ALI) accounts for about 40% of ALI, accompanied by alveolar epithelial injury. The study aimed to reveal the role of circular RNA_0114428 (circ_0114428) in sepsis-induced ALI. METHODS: Human pulmonary alveolar epithelial cells (HPAEpiCs) were treated with lipopolysaccharide (LPS) to mimic a sepsis-induced ALI cell model. RNA expression of circ_0114428, miR-574-5p and Rho-associated coiled-coil containing protein kinase 2 (ROCK2) was detected by qRT-PCR. Protein expression was checked by Western blotting. Cell viability, proliferation and apoptosis were investigated by cell counting kit-8, 5-Ethynyl-29-deoxyuridine (EdU) and flow cytometry analysis, respectively. The levels of pro-inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA). Oxidative stress was analyzed by lipid peroxidation Malondialdehyde (MDA) and Superoxide Dismutase (SOD) activity detection assays. The interplay among circ_0114428, miR-574-5p and ROCK2 was identified by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation assays. RESULTS: Circ_0114428 and ROCK2 expression were significantly increased, but miR-574-5p was decreased in blood samples from sepsis patients and LPS-stimulated HPAEpiCs. LPS treatment led to decreased cell viability and proliferation and increased cell apoptosis, inflammation and oxidative stress; however, these effects were relieved after circ_0114428 knockdown. Besides, circ_0114428 acted as a miR-574-5p sponge and regulated LPS-treated HPAEpiC disorders through miR-574-5p. Meanwhile, ROCK2 was identified as a miR-574-5p target, and its silencing protected against LPS-induced cell injury. Importantly, circ_0114428 knockdown inhibited ROCK2 production by interacting with miR-574-5p. CONCLUSION: Circ_0114428 knockdown protected against LPS-induced HPAEpiC injury through miR-574-5p/ROCK2 axis, providing a novel therapeutic target in sepsis-induced ALI.

Discovery of a novel ROCK2 ATP competitive inhibitor by DNA-encoded library selection.

Neurodegeneration disorders, such as Alzheimer's disease (AD), have garnered significant attention due to their impact on individuals and society as a whole. Understanding the mechanisms behind these disorders and developing effective therapy strategies is of utmost importance. One potential therapeutic target that has emerged is Rho-associated coiled-coil containing protein kinase 2 (ROCK2), as its accumulation and activity have been closely linked to memory loss. In this report, we present the findings of a recent discovery involving a new molecule that has the ability to competitively inhibit ROCK2 activity. This molecule was identified through the utilization of a DNA-encoded library (DEL) screening platform. Following selection against ROCK2, an off-DNA compound was synthesized and examined to ascertain its inhibitory properties, selectivity, mechanism of action, and binding mode analysis. From the screening, compound CH-2 has demonstrated an IC50 value of 28 nM against ROCK2, while exhibiting a 5-fold selectivity over ROCK1. Further analysis through molecular docking has provided insights into the specific binding modes of this compound. Our findings suggest that DEL selection offers a rapid method for identifying new inhibitors. Among these, the CH-2 compound shows promise as a potential ROCK2 inhibitor and warrants further investigation.

The role of KDM4A-mediated histone methylation on temozolomide resistance in glioma cells through the HUWE1/ROCK2 axis.

Temozolomide (TMZ) resistance presents a significant challenge in the treatment of gliomas. Although lysine demethylase 4A (KDM4A) has been implicated in various cancer-related processes, its role in TMZ resistance remains unclear. This study aims to elucidate the contribution of KDM4A to TMZ resistance in glioma cells and its potential implications for glioma prognosis. We assessed the expression of KDM4A in glioma cells (T98G and U251MG) using qRT-PCR and Western blot assays. To explore the role of KDM4A in TMZ resistance, we transfected siRNA targeting KDM4A into drug-resistant glioma cells. Cell viability was assessed using the CCK-8 assay and the TMZ IC50 value was determined. ChIP assays were conducted to investigate KDM4A, H3K9me3, and H3K36me3 enrichment on the promoters of ROCK2 and HUWE1. Co-immunoprecipitation confirmed the interaction between HUWE1 and ROCK2, and we examined the levels of ROCK2 ubiquitination following MG132 treatment. Notably, T98G cells exhibited greater resistance to TMZ than U251MG cells, and KDM4A displayed high expression in T98G cells. Inhibiting KDM4A resulted in decreased cell viability and a reduction in the TMZ IC50 value. Mechanistically, KDM4A promoted ROCK2 transcription by modulating H3K9me3 levels. Moreover, disruption of the interaction between HUWE1 and ROCK2 led to reduced ROCK2 ubiquitination. Inhibition of HUWE1 or overexpression of ROCK2 counteracted the sensitization effect of si-KDM4A on TMZ responsiveness in T98G cells. Our findings highlight KDM4A's role in enhancing TMZ resistance in glioma cells by modulating ROCK2 and HUWE1 transcription and expression through H3K9me3 and H3K36me3 removal.

Inhibition of microRNA-34c reduces detrusor ROCK2 expression and urinary bladder inflammation in experimental cystitis.

Interstitial cystitis (IC), also called painful bladder syndrome (PBS), is 2 to 5 times more common in women than in men, yet its cause and pathogenesis remain unclear. In our study using the cyclophosphamide (CYP)-induced mouse model of cystitis, histological evaluation of the urinary bladder (UB) lamina propria (LP) showed immune cell infiltrations, indicating moderate to severe inflammation. In this study, we noticed a differential expression of a subset of microRNAs (miRs) in the UB cells (UBs) of CYP-induced cystitis as compared to the control. UB inflammatory scores and inflammatory signaling were also elevated in CYP-induced cystitis as compared to control. We identified eight UBs miRs that exhibited altered expression after CYP induction and are predicted to have a role in inflammation and smooth muscle function (miRs-34c-5p, -34b-3p, -212-3p, -449a-5p, -21a-3p, -376b-3p, -376b-5p and - 409-5p). Further analysis using ELISA for inflammatory markers and real-time PCR (RT-PCR) for differentially enriched miRs identified miR-34c as a potential target for the suppression of UB inflammation in cystitis. Blocking miR-34c by antagomir ex vivo reduced STAT3, TGF-ß1, and VEGF expression in the UBs, which was induced during cystitis as compared to control. Interestingly, miR-34c inhibition also downregulated ROCK2 but elevated ROCK1 expression in bladder and detrusor cells. Thus, the present study shows that targeting miR-34c can mitigate the STAT3, TGF-ß, and VEGF, inflammatory signaling in UB, and suppress ROCK2 expression in UBs to effectively suppress the inflammatory response in cystitis. This study highlights miR-34c as a potential biomarker and/or serves as the basis for new therapies for the treatment of cystitis.

Unraveling the rationale and conducting a comprehensive assessment of KD025 (Belumosudil) as a candidate drug for inhibiting adipogenic differentiation-a systematic review.

Rho-associated kinases (ROCKs) are crucial during the adipocyte differentiation process. KD025 (Belumosudil) is a newly developed inhibitor that selectively targets ROCK2. It has exhibited consistent efficacy in impeding adipogenesis across a spectrum of in vitro models of adipogenic differentiation. Given the novelty of this treatment, a comprehensive systematic review has not been conducted yet. This systematic review aims to fill this knowledge void by providing readers with an extensive examination of the rationale behind KD025 and its impacts on adipogenesis. Preclinical evidence was gathered owing to the absence of clinical trials. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed, and the study's quality was assessed using the Joanna Briggs Institute (JBI) Checklist Critical Appraisal Tool for Systematic Reviews. In various in vitro models, such as 3T3-L1 cells, human orbital fibroblasts, and human adipose-derived stem cells, KD025 demonstrated potent anti-adipogenic actions. At a molecular level, KD025 had significant effects, including decreasing fibronectin (Fn) expression, inhibiting ROCK2 and CK2 activity, suppressing lipid droplet formation, and reducing the expression of proadipogenic genes peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Additionally, KD025 resulted in the suppression of fatty acid-binding protein 4 (FABP4 or AP2) expression, a decrease in sterol regulatory element binding protein 1c (SREBP-1c) and Glut-4 expression. Emphasis must be placed on the fact that while KD025 shows potential in preclinical studies and experimental models, extensive research is crucial to assess its efficacy, safety, and potential therapeutic applications thoroughly and directly in human subjects.

Philadelphia chromosome-negative myeloproliferative chronic neoplasms: is clonal hematopoiesis the main determinant of autoimmune and cardio-vascular manifestations?

In this article, we reviewed the possible mechanisms linking the clonal hematopoiesis of indeterminate potential (CHIP) to chronic myeloproliferative neoplasms (MPNs), autoimmune diseases (ADs), and cardiovascular diseases (CADs). CHIP is characterized by the presence of clonal mutations with an allelic frequency >2% in the peripheral blood without dysplasia, overt hematological neoplasms, or abnormalities in blood cell count. The prevalence may reach 20% of elderly healthy individuals and is considered a risk factor for myelodysplastic neoplasms and acute leukemia. In MPNs, CHIP is often associated with mutations such as JAK2V617F or DNMT3A, TET2, or ASXL1, which exhibit a 12.1- and 1.7-2-fold increase in CADs. Specifically, JAK2-mutated cells produce excessive cytokines and reactive oxygen species, leading to proinflammatory modifications in the bone marrow microenvironment. Consequently, the likelihood of experiencing thrombosis is influenced by the variant allele frequency (VAF) of the JAK2V617F mutation, which also appears to be correlated with anti-endothelial cell antibodies that sustain thrombosis. However, DNMT3A mutations induce pro-inflammatory T-cell polarization and activate the inflammasome complex, while TET2 downregulation leads to endothelial cell autophagy and inflammatory factor upregulation. As a result, in patients with TET2 and DNMT3A-related CHIP, the inflammasome hyperactivation represents a potential cause of CADs. CHIP also occurs in patients with large and small vessel vasculitis, while ADs are more frequently associated with MPNs. In these diseases, monocytes and neutrophils play a key role in the formation of neutrophil extracellular trap (NET) as well as anti-endothelial cell antibodies, resulting in a final procoagulant effect. ADs, such as systemic lupus erythematosus, psoriasis, and arthritis, are also characterized by an overexpression of the Rho-associated coiled-coil containing protein kinase 2 (ROCK2), a serine/threonine kinase that can hyperactivate the JAK-STAT pathway. Interestingly, hyperactivation of ROCK2 has also been observed in myeloid malignancies, where it promotes the growth and survival of leukemic cells. In summary, the presence of CHIP, with or without neoplasia, can be associated with autoimmune manifestations and thrombosis. In the presence of these manifestations, it is necessary to consider a "disease-modifying therapy" that may either reduce the clonal burden or inhibit the clonally activated JAK 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.

OBJECTIVE: To 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. METHODS: Rats 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. RESULTS: In 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. CONCLUSION: The 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.

A 3D biomimetic model of lymphatics reveals cell-cell junction tightening and lymphedema via a cytokine-induced ROCK2/JAM-A complex.

Impaired lymphatic drainage and lymphedema are major morbidities whose mechanisms have remained obscure. To study lymphatic drainage and its impairment, we engineered a microfluidic culture model of lymphatic vessels draining interstitial fluid. This lymphatic drainage-on-chip revealed that inflammatory cytokines that are known to disrupt blood vessel junctions instead tightened lymphatic cell-cell junctions and impeded lymphatic drainage. This opposing response was further demonstrated when inhibition of rho-associated protein kinase (ROCK) was found to normalize fluid drainage under cytokine challenge by simultaneously loosening lymphatic junctions and tightening blood vessel junctions. Studies also revealed a previously undescribed shift in ROCK isoforms in lymphatic endothelial cells, wherein a ROCK2/junctional adhesion molecule-A (JAM-A) complex emerges that is responsible for the cytokine-induced lymphatic junction zippering. To validate these in vitro findings, we further demonstrated in a genetic mouse model that lymphatic-specific knockout of ROCK2 reversed lymphedema in vivo. These studies provide a unique platform to generate interstitial fluid pressure and measure the drainage of interstitial fluid into lymphatics and reveal a previously unappreciated ROCK2-mediated mechanism in regulating lymphatic drainage.

ZNF143 inhibits hepatocyte mitophagy and promotes non-alcoholic fatty liver disease by targeting increased lncRNA NEAT1 expression to activate ROCK2 pathway.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorders worldwide. The mitophagy is suggested to be repressed in NAFLD, but the mechanism remains to be elucidated. METHODS: NAFLD cell and mouse models were established by treating with free fatty acid (FFA) and feeding a high fat diet (HFD), respectively. QRT-PCR, Western blotting, or IHC measured the expression of ZNF143, lncRNA NEAT1, ROCK2, and lipid formation/mitophagy-related proteins. Cell viability and mitophagy were evaluated by MTT and immunofluorescence. The chloroform-methanol extraction method measured triglyceride and total cholesterol levels. ELISA detected ALT and AST levels. The interactions among ZNF143, lncRNA NEAT1 and SND1 were analysed by ChIP, dual-luciferase reporter, pull-down, and RIP. The lipid droplets were determined by Oil-red O and HE staining. RESULTS: ZNF143 and lncRNA NEAT1 were upregulated in hepatic cells treated with FFA (p < 0.01 and p < 0.001). Knockdown of ZNF143 or lncRNA NEAT1 inhibited lipid droplets formation, while promoting mitophagy (p < 0.01 and p < 0.001). ZNF143 promoted lncRNA NEAT1 transcriptional expression through binding to its promoter. LncRNA NEAT1 increased ROCK2 mRNA stability by targeting SND1. LncRNA NEAT1 or ROCK2 overexpression reversed the effect of ZNF143 or lncRNA NEAT1 knockdown on hepatic steatosis and mitophagy (p < 0.01 and p < 0.001). ZNF143 or lncRNA NEAT1 knockdown inhibited HFD-induced steatosis and promoted mitophagy in vivo (p < 0.01 and p < 0.001). CONCLUSION: The upregulation of lncRNA NEAT1 caused by ZNF143 promoted NAFLD through inhibiting mitophagy via activating ROCK2 pathway by targeting SND1, providing potential targets for NAFLD therapy.

The RhoA-ROCK1/ROCK2 Pathway Exacerbates Inflammatory Signaling in Immortalized and Primary Microglia.

Neuroinflammation is a unifying factor among all acute central nervous system (CNS) injuries and chronic neurodegenerative disorders. Here, we used immortalized microglial (IMG) cells and primary microglia (PMg) to understand the roles of the GTPase Ras homolog gene family member A (RhoA) and its downstream targets Rho-associated coiled-coil-containing protein kinases 1 and 2 (ROCK1 and ROCK2) in neuroinflammation. We used a pan-kinase inhibitor (Y27632) and a ROCK1- and ROCK2-specific inhibitor (RKI1447) to mitigate a lipopolysaccharide (LPS) challenge. In both the IMG cells and PMg, each drug significantly inhibited pro-inflammatory protein production detected in media (TNF-α, IL-6, KC/GRO, and IL-12p70). In the IMG cells, this resulted from the inhibition of NF-κB nuclear translocation and the blocking of neuroinflammatory gene transcription (iNOS, TNF-α, and IL-6). Additionally, we demonstrated the ability of both compounds to block the dephosphorylation and activation of cofilin. In the IMG cells, RhoA activation with Nogo-P4 or narciclasine (Narc) exacerbated the inflammatory response to the LPS challenge. We utilized a siRNA approach to differentiate ROCK1 and ROCK2 activity during the LPS challenges and showed that the blockade of both proteins may mediate the anti-inflammatory effects of Y27632 and RKI1447. Using previously published data, we show that genes in the RhoA/ROCK signaling cascade are highly upregulated in the neurodegenerative microglia (MGnD) from APP/PS-1 transgenic Alzheimer's disease (AD) mice. In addition to illuminating the specific roles of RhoA/ROCK signaling in neuroinflammation, we demonstrate the utility of using IMG cells as a model for primary microglia in cellular studies.

ROCK2-RNA interaction map reveals multiple biological mechanisms underlying tumor progression in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common form of kidney cancer in adults. Despite new therapeutic modalities, the outcomes for RCC patients remain unsatisfactory. Rho-associated coiled-coil forming protein kinase 2 (ROCK2) has previously been shown to be upregulated in RCC, and its expression was negatively correlated with patient survival. However, the precise molecular function of ROCK2 has remained unclear. Herein, using RNA-seq analysis of ROCK2 knockdown and control cells, we identified 464 differentially expressed genes, and 1287 alternative splicing events in 786-O RCC cells. Furthermore, mapping of iRIP-seq reads in 786-O cells showed a biased distribution at 5' UTR, intronic and intergenic regions. By comparing ROCK2-regulated alternative splicing and iRIP-seq data, we found 292 overlapping genes that are enriched in multiple tumorigenic pathways. Taken together, our work defined a complex ROCK2-RNA interaction map on a genomic scale in a human RCC cell line, which deepens our understanding of the molecular function of ROCK2 in cancer development.

Calycosin Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Mice through the miR-375-3p/ROCK2 Axis.

Objective: Septic patients are especially vulnerable to acute lung injury (ALI). Calycosin (CAL) has various promising pharmacological activities. This paper aims to expound on the role of CAL in mice with sepsis-induced ALI and the associated mechanisms.Methods: Mouse models of sepsis-induced ALI were established using lipopolysaccharide (LPS). Pulmonary histopathological changes were observed by HE staining. Cell apoptosis was assessed by TUNEL staining. Pulmonary edema was evaluated by measuring wet/dry weight. Bronchoalveolar lavage fluid (BALF) was collected to count inflammatory cells. In vitro LPS models were established using MLE-12 cells. miR-375-3p expression was determined by RT-qPCR. Cell viability and apoptosis were assessed by MTT assay and flow cytometry. Levels of inflammatory cytokines were determined by ELISA. The target relationship between miR-375-3p and ROCK2 was analyzed by the dual-luciferase assay. ROCK2 protein level was determined by Western blot.Results: miR-375-3p was weakly-expressed in mice with sepsis-induced ALI, and CAL treatment elevated miR-375-3p expression. CAL treatment mitigated pulmonary tissue damage and edema, decreased apoptosis and inflammatory cells, downregulated levels of pro-inflammatory cytokines, and upregulated levels of anti-inflammatory cytokines in mice with sepsis-induced ALI. CAL treatment increased MLE-12 cell viability and decreased apoptosis and inflammation in MLE-12 cells. Inhibition of miR-375-3p partially abrogated CAL-mediated protective action on MLE-12 cells. miR-375-3p attenuated LPS-induced MLE-12 cell injury by targeting ROCK2.Conclusion: CAL upregulates miR-375-3p to target ROCK2, thus protecting against sepsis-induced ALI in mice.

RUNX1T1 modulates myogenic differentiation by regulating the calcium signaling pathway and the alternative splicing of ROCK2.

RUNX1T1 (Runt-related transcription factor 1, translocated to 1) plays a wide-ranging and diverse role in cellular development, including hematopoiesis and adipogenesis. However, little is known about the function of RUNX1T1 in the skeletal muscle development. Here, we assessed the impact of RUNX1T1 on the proliferation and myogenic differentiation of goat primary myoblasts (GPMs). It was observed that RUNX1T1 is highly expressed during the early stages of myogenic differentiation and the fetal stage. Moreover, the knockdown of RUNX1T1 promotes the proliferation and inhibits myogenic differentiation and mitochondrial biogenesis of GPMs. RNA sequencing analysis revealed that significantly differentially expressed genes in RUNX1T1 knockdown cells were enriched in the calcium signaling pathway. Additionally, we discovered that RUNX1T1 regulates alternative splicing (AS) events involved in myogenesis. We also show that silencing RUNX1T1 blocked the Ca2+ -CAMK signaling pathway and reduced the expression levels of muscle-specific isoforms of recombinant rho associated coiled coil containing crotein kinase 2 (ROCK2) during myogenic differentiation, partially explaining why RUNX1T1 deficiency leads to the impairment of myotube formation. These findings suggest that RUNX1T1 is a novel regulator of myogenic differentiation that regulates the calcium signaling pathway and AS of ROCK2. Overall, our results highlight the critical role of RUNX1T1 in myogenesis and broaden our understanding of myogenic differentiation.

Flavonoids from Citrus paradise cv. Changshan-huyou exerts protective effect on ischemia-induced cerebral injury in mice via inhibiting RhoA-ROCK2 signaling pathway. / èƒ¡æŸšé»„é ®é€šè¿‡æŠ‘åˆ¶RhoA-ROCK2信号通路发挥脑缺血保护作用.

OBJECTIVES: To investigate the protective effect and mechanism of total flavonoids from Citrus paradise cv. Changshan-huyou extracts (TFC) on oxygen-glucose deprivation (OGD) of primary neurons and chronic ischemia-induced cerebral injury in mice. METHODS: Primary hippocampal neurons of 18-day fetal rats were isolated and cultured for 1 week, then treated with 0.25, 0.50 and 1.00 mg/mL TFC. After oxygen-glucose deprivation for 1 h, cells were reperfused for 6 and 24 h, respectively. The cytoskeleton was observed by phalloidin staining. In animal study, 6-week ICR male mice were randomly divided into sham operation group, model group, low-dose (10 mg/kg), medium-dose (25 mg/kg) and high-dose (50 mg/kg) TFC treatment groups, with 20 mice in each group. After 3 weeks, chronic cerebral ischemia was induced by unilateral common carotid artery ligation in all groups except sham operation group. Mice were treated with different concentrations of TFC in the three TFC treatment groups for 4 weeks. Open field test, novel object recognition test and Morris water maze test were used to evaluate anxiety, learning and memory of these mice. Nissl, HE and Golgi stainings were used to detect neuronal degeneration and dendritic spine changes in the cortex and the hippocampus. The expression levels of Rho-associated kinase (ROCK) 2, LIM kinase (LIMK) 1, cofilin and its phosphorylation, as well as the expression of globular actin (G-actin) and filamentous actin (F-actin) protein in hippocampus of mice were detected by Western blotting. RESULTS: Neurons subjected to OGD showed that neurites displayed shortening and breakage; while treatment with TFC reversed OGD-induced neurite injury, especially in the 0.50 mg/mL TFC group. Compared with the sham operation group, the mice in the model group showed a significant decline in anxiety and cognitive ability (P<0.01), whereas treatment with TFC significantly reversed anxiety and cognitive deficits (P<0.05). Improvement in the medium-dose TFC group was the most obvious. Histopathological analysis indicated that the number of Nissl bodies and dendritic spines in hippocampus and cortex were decreased in the model group (all P<0.01). However, after treatment with medium dose of TFC, the number of Nissl bodies and dendritic spines (all P<0.05) was significantly recovered. Compared with the sham operation group, the phosphorylation level of ROCK2 in the brain tissue of the model group was significantly increased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly decreased (P<0.05), and the relative content ratio of G-actin/F-actin was significantly increased (P<0.05). After administration of TFC, the phosphorylation level of ROCK2 in brain tissue of each group was significantly decreased (P<0.05), while the phosphorylation levels of LIMK1 and cofilin were significantly up-regulated (P<0.05) and the relative content ratio of G-actin/F-actin was significantly decreased (P<0.05). CONCLUSIONS: TFC protects from ischemia-induced cytoskeletal damage, reduces neuronal dendritic spine injury and protects mice against chronic cerebral ischemia through RhoA-ROCK2 signaling pathway, indicating that TFC might be a potential candidate for treatment of chronic ischemic cerebral injury.