Fluoride-induced hypertension by regulating RhoA/ROCK pathway and phenotypic transformation of vascular smooth muscle cells: In vitro and in vivo evidence.

Fluoride exposure has been implicated as a potential risk factor for hypertension, but the underlying mechanisms remain unclear. This study investigated the role of the RhoA/ROCK signaling pathway in fluoride-induced hypertension. Male Wistar rats were divided into different groups and exposed to varying concentrations of sodium fluoride (NaF) or sodium chloride (NaCl) via drinking water. The rats' blood pressure was measured, and their aortic tissue was utilized for high-throughput sequencing analysis. Additionally, rat and A7r5 cell models were established using NaF and/or Fasudil. The study evaluated the effects of fluoride exposure on blood pressure, pathological changes in the aorta, as well as the protein/mRNA expression levels of phenotypic transformation indicators (a-SMA, calp, OPN) in vascular smooth muscle cells (VSMCs), along with the RhoA/ROCK signaling pathway (RhoA, ROCK1, ROCK2, MLC/p-MLC). The results demonstrated that fluoride exposure in rats led to increased blood pressure. High-throughput sequencing analysis revealed differential gene expression associated with vascular smooth muscle contraction, with the RhoA/ROCK signaling pathway emerging as a key regulator. Pathological changes in the rat aorta, such as elastic membrane rupture and collagen fiber deposition, were observed following NaF exposure. However, fasudil, a ROCK inhibitor, mitigated these pathological changes. Both in vitro and in vivo models confirmed the activation of the RhoA/ROCK signaling pathway and the phenotypic transformation of VSMCs from a contractile to a synthetic state upon fluoride exposure. Fasudil effectively inhibited the activities of ROCK1 and ROCK2 and attenuated the phenotypic transformation of VSMCs. In conclusion, fluoride has the potential to induce hypertension through the activation of the RhoA/ROCK signaling pathway and phenotypic changes in vascular smooth muscle cells. These results provide new insights into the mechanism of fluoride-induced hypertension.

Exercise Attenuate Diaphragm Atrophy in COPD Mice via Inhibiting the RhoA/ROCK Signaling.

Background: Exercise is an indispensable component of pulmonary rehabilitation with strong anti-inflammatory effects. However, the mechanisms by which exercise prevents diaphragmatic atrophy in COPD (chronic obstructive pulmonary disease) remain unclear. Methods: Forty male C57BL/6 mice were assigned to the control (n=16) and smoke (n=24) groups. Mice in the smoke group were exposed to the cigarette smoke (CS) for six months. They were then divided into model and exercise training groups for 2 months. Histological changes were observed in lung and diaphragms. Subsequently, agonist U46639 and antagonist Y27632 of RhoA/ROCK were subjected to mechanical stretching in LPS-treated C2C12 myoblasts. The expression levels of Atrogin-1, MuRF-1, MyoD, Myf5, IL-1ß, TNF-α, and RhoA/ROCK were determined by Western blotting. Results: Diaphragmatic atrophy and increased RhoA/ROCK expression were observed in COPD mice. Exercise training attenuated diaphragmatic atrophy, decreased the expression of MuRF-1, and increased MyoD expression in COPD diaphragms. Exercise also affects the upregulation of RhoA/ROCK and inflammation-related proteins. In in vitro experiments with C2C12 myoblasts, LPS remarkably increased the level of inflammation and protein degradation, whereas Y27632 or combined with mechanical stretching prevented this phenomenon considerably. Conclusion: RhoA/ROCK plays an important role in the prevention of diaphragmatic atrophy in COPD.

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.

Oligomeric Tau-induced oxidative damage and functional alterations in cerebral endothelial cells: Role of RhoA/ROCK signaling pathway.

Despite of yet unknown mechanism, microvascular deposition of oligomeric Tau (oTau) has been implicated in alteration of the Blood-Brain Barrier (BBB) function in Alzheimer's disease (AD) brains. In this study, we employed an in vitro BBB model using primary mouse cerebral endothelial cells (CECs) to investigate the mechanism underlying the effects of oTau on BBB function. We found that exposing CECs to oTau induced oxidative stress through NADPH oxidase, increased oxidative damage to proteins, decreased proteasome activity, and expressions of tight junction (TJ) proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5. These effects were suppressed by the pretreatment with Fasudil, a RhoA/ROCK signaling inhibitor. Consistent with the biochemical alterations, we found that exposing the basolateral side of CECs to oTau in the BBB model disrupted the integrity of the BBB, as indicated by an increase in FITC-dextran transport across the model, and a decrease in trans endothelial electrical resistance (TEER). oTau also increased the transmigration of peripheral blood mononuclear cells (PBMCs) in the BBB model. These functional alterations in the BBB induced by oTau were also suppressed by Fasudil. Taken together, our findings suggest that targeting the RhoA/ROCK pathway can be a potential therapeutic strategy to maintain BBB function in AD.

The Roles of RhoA/ROCK/NF-κB Pathway in Microglia Polarization Following Ischemic Stroke.

Ischemic stroke is the leading cause of death and disability worldwide. Nevertheless, there still lacks the effective therapies for ischemic stroke. Microglia are resident macrophages of the central nervous system (CNS) and can initiate immune responses and monitor the microenvironment. Microglia are activated and polarize into proinflammatory or anti­inflammatory phenotype in response to various brain injuries, including ischemic stroke. Proinflammatory microglia could generate immunomodulatory mediators, containing cytokines and chemokines, these mediators are closely associated with secondary brain damage following ischemic stroke. On the contrary, anti-inflammatory microglia facilitate recovery following stroke. Regulating the activation and the function of microglia is crucial in exploring the novel treatments for ischemic stroke patients. Accumulating studies have revealed that RhoA/ROCK pathway and NF-κB are famous modulators in the process of microglia activation and polarization. Inhibiting these key modulators can promote the polarization of microglia to anti-inflammatory phenotype. In this review, we aimed to provide a comprehensive overview on the role of RhoA/ROCK pathway and NF-κB in the microglia activation and polarization, reveal the relationship between RhoA/ROCK pathway and NF-κB in the pathological process of ischemic stroke. In addition, we likewise discussed the drug modulators targeting microglia polarization.

RVG29 Peptide-Modified Exosomes Loaded with Mir-133b Mediate the RhoA-ROCK Pathway to Improve Motor and Neurological Symptoms in Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Drug delivery to the brain through the blood-brain barrier (BBB) is a significant challenge in PD treatment. Exosomes, which can efficiently traverse the BBB, which many drugs cannot penetrate, are ideal natural carriers for drug delivery. In this study, the BBB shuttle peptide was modified on the exosome surfaces. Three types of exosomes were constructed, each modified with a distinct peptide (RVG29, TAT, or Ang2) and loaded with miR-133b. The safety and brain-targeting capabilities of these peptide-modified exosomes were then evaluated. Finally, the mechanism by which RVG29-Exo-133b regulates the RhoA-ROCK signaling pathway was investigated. The findings indicate that the three peptide-modified exosomes were adequately tolerated, safe, and effectively assimilated in vivo and ex vivo, with RVG29 exhibiting superior targeting to the brain. Furthermore, RVG29-Exo-133b decreased the phosphorylation level of the Tau protein by targeting the RhoA-ROCK signaling pathway. It also enhanced the motor function in mice with PD, thereby reducing the degree of depression, improving dopaminergic neuron function, and attenuating 6-OHDA-induced nerve damage. In this study, we developed a stable drug delivery mechanism that targets the intracerebral region using exosomes. Furthermore, a novel strategy was developed to manage PD and can potentially serve as a preclinical basis for utilizing exosomes in the diagnosis and treatment of neurodegenerative conditions.

CTNNAL1 promotes the structural integrity of bronchial epithelial cells through the RhoA/ROCK1 pathway.

Adhesion molecules play critical roles in maintaining the structural integrity of the airway epithelium in airways under stress. Previously, we reported that catenin alpha-like 1 (CTNNAL1) is downregulated in an asthma animal model and upregulated at the edge of human bronchial epithelial cells (HBECs) after ozone stress. In this work, we explore the potential role of CTNNAL1 in the structural adhesion of HBECs and its possible mechanism. We construct a CTNNAL1 ‒/‒ mouse model with CTNNAL1-RNAi recombinant adeno-associated virus (AAV) in the lung and a CTNNAL1-silencing cell line stably transfected with CTNNAL1-siRNA recombinant plasmids. Hematoxylin and eosin (HE) staining reveals that CTNNAL1 ‒/‒ mice have denuded epithelial cells and structural damage to the airway. Silencing of CTNNAL1 in HBECs inhibits cell proliferation and weakens extracellular matrix adhesion and intercellular adhesion, possibly through the action of the cytoskeleton. We also find that the expressions of the structural adhesion-related molecules E-cadherin, integrin ß1, and integrin ß4 are significantly decreased in ozone-treated cells than in vector control cells. In addition, our results show that the expression levels of RhoA/ROCK1 are decreased after CTNNAL1 silencing. Treatment with Y27632, a ROCK inhibitor, abolished the expressions of adhesion molecules induced by ozone in CTNNAL1-overexpressing HBECs. Overall, the findings of the present study suggest that CTNNAL1 plays a critical role in maintaining the structural integrity of the airway epithelium under ozone challenge, and is associated with epithelial cytoskeleton dynamics and the expressions of adhesion-related molecules via the RhoA/ROCK1 pathway.

Mertk Reduces Blood-Spinal Cord Barrier Permeability Through the Rhoa/Rock1/P-MLC Pathway After Spinal Cord Injury.

Disruption of the blood-spinal cord barrier (BSCB) is a critical event in the secondary injury following spinal cord injury (SCI). Mertk has been reported to play an important role in regulating inflammation and cytoskeletal dynamics. However, the specific involvement of Mertk in BSCB remains elusive. Here, we demonstrated a distinct role of Mertk in the repair of BSCB. Mertk expression is decreased in endothelial cells following SCI. Overexpression of Mertk upregulated tight junction proteins (TJs), reducing BSCB permeability and subsequently inhibiting inflammation and apoptosis. Ultimately, this led to enhanced neural regeneration and functional recovery. Further experiments revealed that the RhoA/Rock1/P-MLC pathway plays a key role in the effects of Mertk. These findings highlight the role of Mertk in promoting SCI recovery through its ability to mitigate BSCB permeability and may provide potential targets for SCI repair.

Oligomeric Amyloid-ß and Tau Alter Cell Adhesion Properties and Induce Inflammatory Responses in Cerebral Endothelial Cells Through the RhoA/ROCK Pathway.

Dysfunction of cerebral endothelial cells (CECs) has been implicated in the pathology of Alzheimer's disease (AD). Despite evidence showing cytotoxic effects of oligomeric amyloid-ß (oAß) and Tau (oTau) in the central nervous system, their direct effects on CECs have not been fully investigated. In this study, we examined the direct effects of oAß, oTau, and their combination on cell adhesion properties and inflammatory responses in CECs. We found that both oAß and oTau increased cell stiffness, as well as the p-selectin/Sialyl-LewisX (sLeX) bonding-mediated membrane tether force and probability of adhesion in CECs. Consistent with these biomechanical alterations, treatments with oAß or oTau also increased actin polymerization and the expression of p-selectin at the cell surface. These toxic oligomeric peptides also triggered inflammatory responses, including upregulations of p-NF-kB p65, IL-1ß, and TNF-α. In addition, they rapidly activated the RhoA/ROCK pathway. These biochemical and biomechanical changes were further enhanced by the treatment with the combination of oAß and oTau, which were significantly suppressed by Fasudil, a specific inhibitor for the RhoA/ROCK pathway. In conclusion, our data suggest that oAß, oTau, and their combination triggered subcellular mechanical alterations and inflammatory responses in CECs through the RhoA/ROCK pathway.

Rhoa/ROCK, mTOR and Secretome-Based Treatments for Ischemic Stroke: New Perspectives.

Ischemic stroke triggers a complex cascade of cellular and molecular events leading to neuronal damage and tissue injury. This review explores the potential therapeutic avenues targeting cellular signaling pathways implicated in stroke pathophysiology. Specifically, it focuses on the articles that highlight the roles of RhoA/ROCK and mTOR signaling pathways in ischemic brain injury and their therapeutic implications. The RhoA/ROCK pathway modulates various cellular processes, including cytoskeletal dynamics and inflammation, while mTOR signaling regulates cell growth, proliferation, and autophagy. Preclinical studies have demonstrated the neuroprotective effects of targeting these pathways in stroke models, offering insights into potential treatment strategies. However, challenges such as off-target effects and the need for tissue-specific targeting remain. Furthermore, emerging evidence suggests the therapeutic potential of MSC secretome in stroke treatment, highlighting the importance of exploring alternative approaches. Future research directions include elucidating the precise mechanisms of action, optimizing treatment protocols, and translating preclinical findings into clinical practice for improved stroke outcomes.

Deoxynivalenol Induces Intestinal Epithelial Barrier Damage through RhoA/ROCK Pathway-Mediated Apoptosis and F-Actin-Associated Tight Junction Disruption.

Deoxynivalenol (DON) poses a serious global food safety risk due to its high toxicity and contamination rate. It disrupts the intestinal epithelial barrier, allowing exogenous toxins to enter the circulation and resulting in sepsis and systemic toxicity. In this research, 32 male Kunming mice and Porcine Small Intestinal Epithelial (IPEC-J2) cells were treated with DON at 0-4.8 mg/kg (7 d) and 0-12 µM (24 h), respectively. Histopathological results revealed that DON disrupted the intestinal epithelial barrier, causing apoptosis and tight junction (TJ) injury. Immunofluorescence and protein expression results showed that DON-induced p53-dependent mitochondrial pathway apoptosis and fibrillar actin (F-actin)-associated TJ injury and that the RhoA/ROCK pathway were activated in mice jejunal tissue and IPEC-J2 cells. Pretreatment with RhoA or ROCK inhibitors (Rosin or Y-27632) maintained DON-induced apoptosis and F-actin-associated TJ injury in IPEC-J2 cells. Thus, DON induces damage to the intestinal epithelial barrier through the RhoA/ROCK pathway-mediated apoptosis and F-actin-associated TJ disruption.

Exploration of the Mechanisms Underlying Yu's Enema Formula in Treating Ulcerative Colitis by Blocking the RhoA/ROCK Pathway based on Network Pharmacology, High-performance Liquid Chromatography Analysis, and Experimental Verification.

BACKGROUND: The traditional Chinese medicine formula, Yu's Enema Formula (YEF), has demonstrated potential in the treatment of Ulcerative Colitis (UC). OBJECTIVE: This study aimed to unveil the anti-UC mechanisms of YEF. METHODS: Utilizing public databases, we obtained YEF and UC-related targets. GO and KEGG analyses were conducted via clusterProfiler and Reactome. The STRING database facilitated the construction of the PPI network, and hub targets were selected using cytoHubba. We used R software for differential expression and correlation analyses, and molecular docking was performed with PyMOL and AutoDock. HPLC analysis identified the compounds in YEF. For in vivo validation, a UC rat model was employed. RESULTS AND DISCUSSION: 495 YEF-UC overlapping targets were identified. GO and KEGG analyses indicated enrichment in exogenous stimuli response, peptide response, positive MAPK cascade regulation, interleukin- related signaling, and the TLR4 cascade. Hub targets included CTNNB1, JUN, MAPK1, MAPK3, SRC, STAT3, TLR4, TP53, and RELA, which were often interconnected. Molecular docking revealed quercetin's strong binding affinity with CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, consistent with HPLC analysis. In vivo experiments suggested that YEF has the potential to alleviate UC symptoms and protect the intestinal mucosal barrier by inhibiting the RhoA/ROCK pathway. CONCLUSION: YEF may safeguard the intestinal mucosal barrier in UC by targeting CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, while blocking the RhoA/ROCK pathway.

RhoA/ROCK Pathway Is Upregulated in Experimental Autoimmune Myocarditis and Is Inhibited by Simvastatin at the Stage of Myosin Light Chain Phosphorylation.

Many studies have proven the involvement of the RhoA/ROCK pathway in autoimmune and cardiovascular diseases and the beneficial effects of its downregulation. Here, we examined whether the effect of simvastatin on experimental autoimmune myocarditis (EAM) may be through targeting the Ras homolog family member A/Rho-associated coiled-coil containing kinases (RhoA/ROCK) pathway and whether previously shown downregulation of metalloproteinase 9 (MMP-9) could be associated with MLC phosphorylation. Two doses of simvastatin were administered to experimental rats with autoimmune myocarditis by gastric gavage for 3 weeks, at the stage of development of the inflammatory process. Immunohistochemical staining for RhoA and ROCK1 was evaluated semi-quantitatively with H-score. The RhoA staining showed no significant differences in expression between the groups, but the ROCK1 expression was significantly upregulated in the hearts of the EAM group and was not downregulated by simvastatin. The Western blotting analysis of the last downstream product of the RhoA/ROCK axis, phosphorylated myosin light chain (phospho-MYL9), revealed that protein content increased in EAM hearts and it was prevented by the highest dose of simvastatin. Our findings suggest that the RhoA/ROCK pathway is upregulated in EAM, and simvastatin in EAM settings inhibits the RhoA/ROCK pathway at the stage of phosphorylation of myosin light chains and provides a new insight into the molecular pathology of autoimmune myocarditis.

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.

Activation of RhoA/ROCK2 signaling by hypoxia-inducible factor 1α in promoting tumor growth and metastasis in human colon cancer.

Background: Recent evidence strongly suggests the profound role of the tumor microenvironment in cancer development and progression. A hypoxic microenvironment is widely acknowledged to be a typical feature of solid tumors, and altered hypoxia-inducible factor 1α (HIF-1α) expression has been associated with the formation and the progression of many solid tumors; however, the underlying mechanism of this relationship remains obscure. Methods: Clinical colorectal cancer tissue samples were collected to detect the differential expression of HIF-1α, Ras homolog family member A (RhoA), and Rho-associated, coiled-coil containing protein kinase 2 (ROCK2). With hypoxic stress, small interfering RNA (siRNA) targeting HIF-1α, lentivirus transfection of RhoA was used to study the mechanism of HIF-1α and RhoA/ROCK2 signaling pathways in the growth and metastasis of colon cancer. Results: According to Cell Counting Kit 8, wound-healing, and Transwell assays, HIF-1α expression activated the RhoA/ROCK2 pathway within colon cancer cell lines, accelerating their growth and expansion. In cells transfected with LV-RhoA, inactivating the RhoA/ROCK2 pathway with the specific inhibitor Y-27632 decreased tumor growth and metastasis under hypoxic conditions, while activating the RhoA/ROCK2 pathway restored these biological properties. The Western blot results showed that the expression levels of pMYPT1, cyclin D1, and MMP2 in the siRNA + LV-RhoA group were also significantly increased compared with those in the siRNA group. Conclusions: For the first time, this study demonstrated that HIF-1α can promote the growth and metastasis of colon cancer via directly affecting RhoA/ROCK2 signaling and thus represents a novel therapeutic target for colon cancer.

Piezo2 Contributes to Traumatic Brain Injury by Activating the RhoA/ROCK1 Pathways.

Traumatic brain injury (TBI) can lead to short-term and long-term physical and cognitive impairments, which have significant impacts on patients, families, and society. Currently, treatment outcomes for this disease are often unsatisfactory, due at least in part to the fact that the molecular mechanisms underlying the development of TBI are largely unknown. Here, we observed significant upregulation of Piezo2, a key mechanosensitive ion channel protein, in the injured brain tissue of a mouse model of TBI induced by controlled cortical impact. Pharmacological inhibition and genetic knockdown of Piezo2 after TBI attenuated neuronal death, brain edema, brain tissue necrosis, and deficits in neural function and cognitive function. Mechanistically, the increase in Piezo2 expression contributed to TBI-induced neuronal death and subsequent production of TNF-α and IL-1ß, likely through activation of the RhoA/ROCK1 pathways in the central nervous system. Our findings suggest that Piezo2 is a key player in and a potential therapeutic target for TBI.

"Baihui" (DU20)-penetrating "Qubin" (GB7) acupuncture on blood-brain barrier integrity in rat intracerebral hemorrhage models via the RhoA/ROCK II/MLC 2 signaling pathway.

BACKGROUND: Blocking the RhoA/ROCK II/MLC 2 (Ras homolog gene family member A/Rho kinase II/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the RhoA/ROCK II/MLC 2 signaling pathway changes the pathogenic processes of the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH. METHODS: Scalp acupuncture (SA) therapy was performed on rats with ICH at the acupuncture point "Baihui"-penetrating "Qubin," and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the RhoA/ROCK II/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging. RESULTS: We found that ROCK II acts as a promoter of the RhoA/ROCK II/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the pre-intervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK II, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at "Baihui"-penetrating "Qubin" and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the RhoA/ROCK II/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats. CONCLUSION: This study found that these experimental data indicated that SA at "Baihui"-penetrating "Qubin" could preserve BBB integrity and neurological function recovery after ICH by inhibiting RhoA/ROCK II/MLC 2 signaling pathway activation and by regulating endothelial cell-related proteins.

Unveiling the Therapeutic Potential of Herba Epimedii: Enhancing Bone Healing Through Cytoskeletal Regulation of RhoA/Rock1 Pathway.

Herba Epimedii is widely used to promote bone healing, and their active ingredients are total flavonoids of Epimedium (TFE). Ras homolog gene family member A / Rho-associated protein kinase (RhoA/Rock), an important pathway regulating the cytoskeleton, has been proven to affect bone formation. However, whether TFE promotes bone healing via this pathway remains unclear. In this study, the therapeutic effects of TFE were estimated using micro-computed tomography and hematoxylin and eosin staining of pathological sections. F-actin in osteoblasts was stained to investigate the protective effects of TFE on the cytoskeleton. Its regulatory effects on the RhoA/Rock1 pathway were explored using RT-qPCR and Western blot analysis. Besides, flow cytometry, alkaline phosphatase and nodule calcification staining were performed to evaluate the effects on osteogenesis. The bone healing in rats was improved, the cytoskeletal damage in osteoblasts was reduced, the RhoA/Rock1 pathway was downregulated, and osteogenesis was enhanced after TFE treatment. Thus, TFE can promote bone formation at least partially by regulating the expression of key genes and proteins in the cytoskeleton. The findings of this study provided evidence for clinical applications and would contribute to a better understanding of Epimedium's mechanisms in treating bone defects.

Berberine promotes lacteal junction zippering and ameliorates diet-induced obesity through the RhoA/ROCK signaling pathway.

BACKGROUND: Obesity has emerged as a global epidemic. Recent research has indicated that diet-induced obesity can be prevented by promoting lacteal junction zippering. Berberine, which is derived from natural plants, is found to be promising in weight reduction, but the underlying mechanism remains unspecified. PURPOSE: To determine whether berberine protects against obesity by regulating the lacteal junction and to explore potential molecular mechanisms. METHODS: Following the induction of the diet-induced obese (DIO) model, mice were administered low and high doses of berberine for 4 weeks. Indicators associated with insulin resistance and lipid metabolism were examined. Various methods, such as Oil Red O staining, transmission electron microscopy imaging, confocal imaging and others were used to observe the effects of berberine on lipid absorption and the lacteal junction. In vitro, human dermal lymphatic endothelial cells (HDLECs) were used to investigate the effect of berberine on LEC junctions. Western Blot and immunostaining were applied to determine the expression levels of relevant molecules. RESULTS: Both low and high doses of berberine reduced body weight in DIO mice without appetite suppression and ameliorated glucolipid metabolism disorders. We also found that the weight loss effect of berberine might contribute to the inhibition of small intestinal lipid absorption. The possible mechanism was related to the promotion of lacteal junction zippering via suppressing the ras homolog gene family member A (RhoA)/Rho-associated kinase (ROCK) signaling pathway. In vitro, berberine also promoted the formation of stable mature junctions in HDLECs, involving the same signaling pathway. CONCLUSION: Berberine could promote lacteal junction zippering and ameliorate diet-induced obesity through the RhoA/ROCK signaling pathway.

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

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