Comprehensive combined analysis of physician-initiated phase II and III clinical trials on a cultured human corneal endothelial cell product for treating bullous keratopathy.

To evaluate the efficacy and safety of a cultured human corneal endothelial cell (cHCEC) product in eyes with bullous keratopathy (BK). Combined analysis of multicenter phase II and III clinical trials. This analysis involved 15 BK eyes in the phase II trial and 12 BK eyes in the phase III trial that underwent cHCEC transplant therapy. Safety was assessed in all the cases. Efficacy was assessed in 17 cases with exclusion of the low- and medium-dose groups in the phase II trial. The primary endpoint was a corneal endothelial cell density of 1000 cells/mm2 or more at 24 weeks post-transplant, which was attained in 94.1% of the eyes (16 of 17), with a 95% CI of 71.3-99.9%. Additionally, 82.4% of the eyes (14 of 17) met the secondary endpoint of reduction in corneal thickness to less than 630 µm without corneal epithelial edema within the same time frame, with a 95% CI of 56.6-96.2%. The mean decrease in corneal thickness from baseline to 24 weeks post-transplant was -187.4 µm (95% CI, -240.2 µm to -134.5 µm). Furthermore, all the eyes exhibited improvement in best-corrected visual acuity from baseline to 24 weeks post-transplant (95% CI, 80.5-100.0%). By 24 weeks post-transplant, 88.9% of the patients (24 of 27) had experienced adverse events, which were mostly local, mild, and transient. The cHCEC product of this study reconstitutes the corneal endothelial layer with high cellular density and restores corneal thickness and improves visual acuity.

Depletion of the Rho GTPases Cdc42, Rac1 or RhoA reduces PDGF-induced STAT1 and STAT3 signaling.

This study investigates the role of Rho GTPases, specifically Cdc42, Rac1, and RhoA, in platelet-derived growth factor receptors (PDGFRα and PDGFRß) signaling. Signal transducer and activator of transcription (STAT) proteins, essential for cellular processes such as proliferation and immune response, are activated downstream of PDGFRs. Dysregulation of these pathways is linked to various diseases, including cancer. The current study examines the effects of Rho GTPase depletion on PDGFR phosphorylation, STAT protein stability, and downstream signaling. Results indicate that depletion of Cdc42, Rac1, or RhoA impairs PDGFR phosphorylation and reduces STAT1 and STAT3 signaling, without significantly affecting AKT and ERK1/2 pathways. The findings highlight the critical regulatory roles of Rho GTPases in PDGFR-mediated STAT signaling.

Regulation and signaling of the LIM domain kinases.

The LIM domain kinases (LIMKs) are important actin cytoskeleton regulators. These proteins, LIMK1 and LIMK2, are nodes downstream of Rho GTPases and are the key enzymes that phosphorylate cofilin/actin depolymerization factors to regulate filament severing. They therefore perform an essential role in cascades that control actin depolymerization. Signaling of the LIMKs is carefully regulated by numerous inter- and intra-molecular mechanisms. In this review, we discuss recent findings that improve the understanding of LIM domain kinase regulation mechanisms. We also provide an up-to-date review of the role of the LIM domain kinases, their architectural features, how activity is impacted by other proteins, and the implications of these findings for human health and disease.

Corrigendum: Nonmuscle myosin heavy chain IIA-mediated exosome release via regulation of the rho-associated kinase 1/myosin light chains/actin pathway.

[This corrects the article DOI: 10.3389/fphar.2020.598592.].

Preliminary identification of somatic mutations profile in ACL injury.

Anterior cruciate ligament (ACL) injury is a common orthopedic disease with a high incidence, long recovery time, and often requiring surgical treatment. However, the susceptibility factors for ACL injury are currently unclear, and there is a lack of analysis on the differences in the ligament itself. Previous studies have focused on germline mutations, with less research on somatic mutations. To determine the role of somatic mutations in ACL injuries, we recruited seven patients between the ages of 20 and 39 years diagnosed with ACL injuries, collected their peripheral blood, injured ligament ends, and healthy ligament ends tissues, and performed exome sequencing with gene function enrichment analysis. We detected multiple gene mutations and gene deletions, which were only present in some of the samples. Unfortunately, it was not possible to determine whether these somatic mutations are related to ligament structure or function, or are involved in ACL injury. However, this study provides valuable clues for future in-depth research.

Y-27632 enables long-term expansion of mouse submandibular gland epithelial cells via inactivation of TGF-ß1/CTGF/p38 and ROCK2/JNK signaling pathway.

OBJECTIVES: This study aimed to investigate the effects of Y-27632 on the long-term maintainence of mouse submandibular epithelial cells (SG-Epis) in vitro and to elucidate the underlying mechanisms. METHODS: The role of the Rho-associated kinase (ROCK) inhibitor Y-27632 in maintaining SG-Epis and its underlying mechanisms were evaluated by examining the in vitro expansion of mouse SG-Epis. Changes in key cellular characteristics, such as proliferation, long-term expansion, and mRNA and protein expression, were assessed in the presence or absence of Y-27632. RESULTS: Treatment with Y-27632 significantly enhanced the proliferative potential of SG-Epis, preserving Krt8 and Krt14 expression over 17 passages. In the absence of Y-27632, SG-Epis lost their epithelial morphology. However, Y-27632 treatment maintained the epithelial morphology and downregulated mRNA levels of Tgf-ß1, Ctgf, and Rock2. Treatment with TGF-ß1 indicated that TGF-ß/CTGF/p38 signaling is responsible for the maintenance of SG-Epis, while RNA interference studies revealed that ROCK2/c-Jun N-terminal kinase (JNK) signaling is also crucial for SG-Epis proliferation and maintenance. CONCLUSIONS: The TGF-ß1/CTGF/p38 and ROCK2/JNK signaling pathways are responsible for SG-Epis proliferation, and Y-27632 treatment effectively inactivates these pathways, enabling long-term in vitro maintenance of SG-Epis. The culture method utilizing Y-27632 provides an effective approach for the in vitro expansion of SG-Epis.

[Effects of inhibition of Rho/ROCK pathway on proliferation and migration of airway smooth muscle cells and related mechanisms]. / 抑制Rho/ROCKé€šè·¯å¯¹æ°”é“å¹³æ»‘è‚Œç»†èƒžå¢žæ®–ä¸Žè¿ç§»çš„å½±å“åŠç›¸å ³æœºåˆ¶.

OBJECTIVES: To investigate the effects and molecular mechanisms of inhibition of the Ras homolog gene (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) pathway on the proliferation and migration of airway smooth muscle cells involving myocardin (MYOCD). METHODS: Human airway smooth muscle cells were infected with the adenoviral vector Ad-ZsGreen-shRNA-hROCK1 in vitro. The cells were randomly divided into four groups: ROCK1 gene silencing control (shNC) group, shNC + arachidonic acid (AA, Rho/ROCK pathway activator) group, ROCK1 gene silencing (shROCK1) group, and shROCK1 + AA group (n=3 each). Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of ROCK1 and MYOCD mRNA and protein. ELISA was employed to measure the levels of globular actin and filamentous actin, while immunofluorescent staining and scratch assays were utilized to assess cell proliferation and migration. RESULTS: Compared to the shNC + AA group, the shROCK1 + AA group exhibited decreased levels of ROCK1 and MYOCD mRNA and protein expression, reduced expression levels of globular actin and filamentous actin, and diminished cell proliferation and migration capabilities (P<0.05). CONCLUSIONS: Inhibition of the Rho/ROCK pathway suppresses the proliferation and migration of airway smooth muscle cells, which may be associated with the downregulation of MYOCD.

The New Era of Therapeutic Strategies for the Management of Retinitis Pigmentosa: A Narrative Review of the Pathomolecular Mechanism for Gene Therapies.

Retinitis pigmentosa, or RP, is a group of inherited retinal degenerations involving progressive loss of photoreceptor cells- rods and cones- ultimately causing severe vision loss and blindness. RP, although a very common ailment, continues to be an incurable disease with little to be done medically. However, with the breakthroughs in gene therapy and stem cell transplantation in recent years, a new door has been opened to the treatment of RP. This narrative review summarizes the pathomolecular mechanisms of RP, focusing on the genetic and molecular abnormalities that lead to the process of retinal degeneration. In this section, we talk about the current theories of how RP develops, gene mutations, oxidative stress, and inflammation. We also delve into new therapeutic approaches such as gene therapy, stem cell transplantation and genome surgery, which are designed to either replace or repair the damaged photoreceptors to restore vision and ultimately enhance the life of the RP patient. Another topic covered is the obstacles and research frontiers of these revolutionary treatments. This article is intended to give a complete overview of the molecular processes of RP and the promising treatment strategies that could change the way this devastating disease is treated.

Evaluation of Rho kinase inhibitor effects on neuroprotection and neuroinflammation in an ex-vivo retinal explant model.

BACKGROUND: Glaucoma is a leading cause of blindness, affecting retinal ganglion cells (RGCs) and their axons. By 2040, it is likely to affect 110 million people. Neuroinflammation, specifically through the release of proinflammatory cytokines by M1 microglial cells, plays a crucial role in glaucoma progression. Indeed, in post-mortem human studies, pre-clinical models, and ex-vivo models, RGC degeneration has been consistently shown to be linked to inflammation in response to cell death and tissue damage. Recently, Rho kinase inhibitors (ROCKis) have emerged as potential therapies for neuroinflammatory and neurodegenerative diseases. This study aimed to investigate the potential effects of three ROCKis (Y-27632, Y-33075, and H-1152) on retinal ganglion cell (RGC) loss and retinal neuroinflammation using an ex-vivo retinal explant model. METHODS: Rat retinal explants underwent optic nerve axotomy and were treated with Y-27632, Y-33075, or H-1152. The neuroprotective effects on RGCs were evaluated using immunofluorescence and Brn3a-specific markers. Reactive glia and microglial activation were studied by GFAP, CD68, and Iba1 staining. Flow cytometry was used to quantify day ex-vivo 4 (DEV 4) microglial proliferation and M1 activation by measuring the number of CD11b+, CD68+, and CD11b+/CD68+ cells after treatment with control solvent or Y-33075. The modulation of gene expression was measured by RNA-seq analysis on control and Y-33075-treated explants and glial and pro-inflammatory cytokine gene expression was validated by RT-qPCR. RESULTS: Y-27632 and H-1152 did not significantly protect RGCs. By contrast, at DEV 4, 50 µM Y-33075 significantly increased RGC survival. Immunohistology showed a reduced number of Iba1+/CD68+ cells and limited astrogliosis with Y-33075 treatment. Flow cytometry confirmed lower CD11b+, CD68+, and CD11b+/CD68+ cell numbers in the Y-33075 group. RNA-seq showed Y-33075 inhibited the expression of M1 microglial markers (Tnfα, Il-1ß, Nos2) and glial markers (Gfap, Itgam, Cd68) and to reduce apoptosis, ferroptosis, inflammasome formation, complement activation, TLR pathway activation, and P2rx7 and Gpr84 gene expression. Conversely, Y-33075 upregulated RGC-specific markers, neurofilament formation, and neurotransmitter regulator expression, consistent with its neuroprotective effects. CONCLUSION: Y-33075 demonstrates marked neuroprotective and anti-inflammatory effects, surpassing the other tested ROCKis (Y-27632 and H-1152) in preventing RGC death and reducing microglial inflammatory responses. These findings highlight its potential as a therapeutic option for glaucoma.

A comparative anticancer analysis of iron oxide nanoparticles of Hippophae rhamnoides and Cichorium intybus found in the Karakoram Range of Gilgit Baltistan against liver cancer targeting the RhoA gene.

OBJECTIVE: The current research work focused on the evaluation of of H. rhamnoides and C. intybus Fe2O3 NPs against liver cancer cell line (HepG2) by performing antiproliferative assay targeting the RhoA gene and apoptotic pathway genes and proteins. METHODS: Fe2O3 NPs were synthesized using extracts of H. rhamnoides and C. intybus and characterized by UV-Vis spectroscopy, FTIR, SEM/EDS and XRD. MTT assay was used to study cytotoxicity against the HepG2 cells. Real-time qPCR and ELISA were used for the gene and protein analysis. RESULTS: An absorbance peak at 300 nm for H. rhamnoides and 289 nm for C. intybus nanoparticles were observed by UV-Vis analysis. The FTIR bands of H. rhamnoide Fe2O3 NPs suggested the presence of aldehydes, alcohols and polyols whereas bands of C. intybus Fe2O3 NPs suggested the presence of carboxyl groups, hydroxyl groups, alkynes and amines. The size of Fe2O3 NPs was found to be 27 ± 5nm for H. rhamnoides and 84 ± 4nm for C. intybus. The IC50 value of 41.69 µM for H. rhmnoides and 71.04 µM for C. intybus Fe2O3 NPs compared to plant extract (78.10 and 96.03 µM for H. rhamnoides and C. intybus, respectively) were found against HepG2 cells. The gene expression and protein levels of RhoA were decreased whereas those of bax, caspase 3, caspase 8 and caspase 9 were found increased. CONCLUSION: Nanoparticles and extract of H. rhamnoides were found more effective as compared to C. intybus, which was evident by the results of cytotoxicity and analysis of studied genes and proteins.

RHO Variants and Autosomal Dominant Retinitis Pigmentosa: Insights from the Italian Genetic Landscape.

Autosomal dominant retinitis pigmentosa (AD-RP) is caused by several genes, among which RHO is one of the most investigated. This article will be focused on RHO and its role in explaining AD-RP cases in the Italian population, taking advantage of the experience of the Genomic Medicine Laboratory UILDM at the Santa Lucia Foundation IRCCS. The retrospective evaluation of the distribution of RHO variants in the Italian patients with a clinical suspicion of RP pointed out eight variants. Of them, four variants (c.632A>T, c.1040C>T, c.1030C>T, c.383_392del) were pathogenic and made it possible to confirm the diagnosis of AD-RP in nine affected patients, highlighting a lower frequency (17%) of RHO variants compared to previous studies (30-40%). In addition, this study identified four variants classified as Variants of Uncertain Significance (VUS). In conclusion, the experience of the Genomic Medicine Laboratory provides an overview of the distribution of RHO variants in the Italian population, highlighting a slightly lower frequency of these variants in our cases series compared to previous reports. However, further studies on RHO variants are essential to characterize peculiar RP phenotypes and extend the spectrum of disease associated with this gene.

Cytoarchitecture of Breast Cancer Cells under Diabetic Conditions: Role of Regulatory Kinases-Rho Kinase and Focal Adhesion Kinase.

Diabetes greatly reduces the survival rates in breast cancer patients due to chemoresistance and metastasis. Reorganization of the cytoskeleton is crucial to cell migration and metastasis. Regulatory cytoskeletal protein kinases such as the Rho kinase (ROCK) and focal adhesion kinase (FAK) play a key role in cell mobility and have been shown to be affected in cancer. It is hypothesized that diabetes/high-glucose conditions alter the cytoskeletal structure and, thus, the elasticity of breast cancer cells through the ROCK and FAK pathway, which can cause rapid metastasis of cancer. The aim of the study was to investigate the role of potential mediators that affect the morphology of cancer cells in diabetes, thus leading to aggressive cancer. Breast cancer cells (MDA-MB-231 and MCF-7) were treated with 5 mM glucose (low glucose) or 25 mM glucose (high glucose) in the presence of Rho kinase inhibitor (Y-27632, 10 mM) or FAK inhibitor (10 mM). Cell morphology and elasticity were monitored using atomic force microscopy (AFM), and actin staining was performed by fluorescence microscopy. For comparative study, normal mammary breast epithelial cells (MCF-10A) were used. It was observed that high-glucose treatments modified the cytoskeleton of the cells, as observed through AFM and fluorescence microscopy, and significantly reduced the elasticity of the cells. Blocking the ROCK or FAK pathway diminished the high-glucose effects. These changes were more evident in the breast cancer cells as compared to the normal cells. This study improves the knowledge on the cytoarchitecture properties of diabetic breast cancer cells and provides potential pathways that can be targeted to prevent such effects.

Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.

Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.

ARHGEF3 Regulates Hair Follicle Morphogenesis.

During embryogenesis, cells arrange into precise patterns that enable tissues and organs to develop specialized functions. Despite its critical importance, the molecular choreography behind these collective cellular behaviors remains elusive, posing a major challenge in developmental biology and limiting advances in regenerative medicine. By using the mouse hair follicle as a mini-organ system to study the formation of bud-like structures during embryonic development, our work uncovers a crucial role for the Rho GTPase regulator ARHGEF3 in hair follicle morphogenesis. We demonstrate that Arhgef3 expression is upregulated at the onset of hair follicle placode formation. In Arhgef3 knockout animals, we observed defects in placode compaction, leading to impaired hair follicle downgrowth. Through cell culture models, we show that ARHGEF3 promotes F-actin accumulation at the cell cortex and P-cadherin enrichment at cell-cell junctions. Collectively, our study identifies ARHGEF3 as a new regulator of cell shape rearrangements during hair placode morphogenesis, warranting further exploration of its role in other epithelial appendages that arise from similar developmental processes.

Roles of small GTPases in cardiac hypertrophy (Review).

Cardiac hypertrophy results from the heart reacting and adapting to various pathological stimuli and its persistent development is a major contributing factor to heart failure. However, the molecular mechanisms of cardiac hypertrophy remain unclear. Small GTPases in the Ras, Rho, Rab, Arf and Ran subfamilies exhibit GTPase activity and play crucial roles in regulating various cellular responses. Previous studies have shown that Ras, Rho and Rab are closely linked to cardiac hypertrophy and that their overexpression can induce cardiac hypertrophy. Here, we review the functions of small GTPases in cardiac hypertrophy and provide additional insights and references for the prevention and treatment of cardiac hypertrophy.

Patterns of Fitness and Gene Expression Epistasis Generated by Beneficial Mutations in the rho and rpoB Genes of Escherichia coli during High-Temperature Adaptation.

Epistasis is caused by genetic interactions among mutations that affect fitness. To characterize properties and potential mechanisms of epistasis, we engineered eight double mutants that combined mutations from the rho and rpoB genes of Escherichia coli. The two genes encode essential functions for transcription, and the mutations in each gene were chosen because they were beneficial for adaptation to thermal stress (42.2 °C). The double mutants exhibited patterns of fitness epistasis that included diminishing returns epistasis at 42.2 °C, stronger diminishing returns between mutations with larger beneficial effects and both negative and positive (sign) epistasis across environments (20.0 °C and 37.0 °C). By assessing gene expression between single and double mutants, we detected hundreds of genes with gene expression epistasis. Previous work postulated that highly connected hub genes in coexpression networks have low epistasis, but we found the opposite: hub genes had high epistasis values in both coexpression and protein-protein interaction networks. We hypothesized that elevated epistasis in hub genes reflected that they were enriched for targets of Rho termination but that was not the case. Altogether, gene expression and coexpression analyses revealed that thermal adaptation occurred in modules, through modulation of ribonucleotide biosynthetic processes and ribosome assembly, the attenuation of expression in genes related to heat shock and stress responses, and with an overall trend toward restoring gene expression toward the unstressed state.

Conserved signaling modules regulate filamentous growth in fungi: a model for eukaryotic cell differentiation.

Eukaryotic organisms are composed of different cell types with defined shapes and functions. Specific cell types are produced by the process of cell differentiation, which is regulated by signal transduction pathways. Signaling pathways regulate cell differentiation by sensing cues and controlling the expression of target genes whose products generate cell types with specific attributes. In studying how cells differentiate, fungi have proved valuable models because of their ease of genetic manipulation and striking cell morphologies. Many fungal species undergo filamentous growth-a specialized growth pattern where cells produce elongated tube-like projections. Filamentous growth promotes expansion into new environments, including invasion into plant and animal hosts by fungal pathogens. The same signaling pathways that regulate filamentous growth in fungi also control cell differentiation throughout eukaryotes and include highly conserved mitogen-activated protein kinase (MAPK) pathways, which is the focus of this review. In many fungal species, mucin-type sensors regulate MAPK pathways to control filamentous growth in response to diverse stimuli. Once activated, MAPK pathways reorganize cell polarity, induce changes in cell adhesion, and promote the secretion of degradative enzymes that mediate access to new environments. However, MAPK pathway regulation is complicated because related pathways can share components with each other yet induce unique responses (i.e. signal specificity). In addition, MAPK pathways function in highly integrated networks with other regulatory pathways (i.e. signal integration). Here, we discuss signal specificity and integration in several yeast models (mainly Saccharomyces cerevisiae and Candida albicans) by focusing on the filamentation MAPK pathway. Because of the strong evolutionary ties between species, a deeper understanding of the regulation of filamentous growth in established models and increasingly diverse fungal species can reveal fundamentally new mechanisms underlying eukaryotic cell differentiation.

Mechanisms of Rho GTPases in regulating tumor proliferation, migration and invasion.

The occurrence of most cancers is due to the clonal proliferation of tumor cells, immune evasion, and the ability to spread to other body parts. Rho GTPases, a family of small GTPases, are key regulators of cytoskeleton reorganization and cell polarity. Additionally, Rho GTPases are key proteins that induce the proliferation and metastasis of tumor cells. This review focuses on the complex regulatory mechanisms of Rho GTPases, exploring their critical role in promoting tumor cell proliferation and dissemination. Regarding tumor cell proliferation, attention is given to the role of Rho GTPases in regulating the cell cycle and mitosis. In terms of tumor cell dissemination, the focus is on the role of Rho GTPases in regulating cell migration and invasion. Overall, this review elucidates the mechanisms of Rho GTPases members in the development of tumor cells, aiming to provide theoretical references for the treatment of mammalian tumor diseases and related applications.

EspH utilizes phosphoinositide and Rab binding domains to interact with plasma membrane infection sites and Rab GTPases.

Enteropathogenic E. coli (EPEC) is a Gram-negative bacterial pathogen that causes persistent diarrhea. Upon attachment to the apical plasma membrane of the intestinal epithelium, the pathogen translocates virulence proteins called effectors into the infected cells. These effectors hijack numerous host processes for the pathogen's benefit. Therefore, studying the mechanisms underlying their action is crucial for a better understanding of the disease. We show that translocated EspH interacts with multiple host Rab GTPases. AlphaFold predictions and site-directed mutagenesis identified glutamic acid and lysine at positions 37 and 41 as Rab interacting residues in EspH. Mutating these sites abolished the ability of EspH to inhibit Akt and mTORC1 signaling, lysosomal exocytosis, and bacterial invasion. Knocking out the endogenous Rab8a gene expression highlighted the involvement of Rab8a in Akt/mTORC1 signaling and lysosomal exocytosis. A phosphoinositide binding domain with a critical tyrosine was identified in EspH. Mutating the tyrosine abolished the localization of EspH at infection sites and its capacity to interact with the Rabs. Our data suggest novel EspH-dependent mechanisms that elicit immune signaling and membrane trafficking during EPEC infection.

Peripheral Rho-associated protein kinase activation mediates acupuncture analgesia.

Background: Acupuncture has been proven effective for various types of pain, and peripheral molecular signals around acupuncture-treated areas have been suggested to contribute to the analgesic effects of acupuncture. However, the underlying mechanism from these peripheral molecular signals to central ones remains unclear. The purpose of this study was to investigate whether peripheral Rho-associated protein kinase (ROCK) activation induced by acupuncture treatment mediates acupuncture analgesia, and also to investigate the relationship between ROCK activation and extracellular signal-regulated kinase (ERK), which has previously been proven to mediate acupuncture analgesia and other related molecular changes during acupuncture. Methods: Acupuncture was treated at the bilateral GB34 acupoints of C57BL/6 mice, after which changes in ROCK activation and the location of its expression in the skin were analyzed. To verify the role of ROCK in acupuncture analgesia, we administrated ROCK inhibitor Y-27632 (0.3 µg/ul) into the skin before acupuncture treatment with formalin and complete Freund adjuvant (CFA) induced pain models, then the nociceptive responses were analyzed. Results: Acupuncture treatment produced ROCK2 activation in the skin after 30 and 60 min, and the histological analyses revealed that ROCK2 was activated in the fibroblast of the dermis. The acupuncture-induced ROCK2 expression was significantly attenuated by the ERK inhibitor, whereas phospho-ERK expression was not inhibited by ROCK inhibitor. In both the formalin- and CFA-induced mouse pain models, acupuncture analgesia was blocked by ROCK inhibitor administration. Conclusion: Acupuncture treatment-induced ROCK2 expression is a downstream effector of phospho-ERK in the skin and plays a crucial role in acupuncture analgesia.