Esculin alleviates LPS-induced acute lung injury via inhibiting neutrophil recruitment and migration.

OBJECTIVES: Acute lung injury (ALI) poses a serious threat to human health globally, particularly with the Coronavirus 2019 (COVID-19) pandemic. Excessive recruitment and infiltration of neutrophils is the major etiopathogenesis of ALI. Esculin, also known as 6,7-dihydroxycoumarin, is a remarkable compound derived from traditional Chinese medicine Cortex fraxini. Accumulated evidence indicates that esculin has potent anti-inflammatory effects, but its pharmaceutical effect against ALI and potential mechanisms are still unclear. METHODS: This study evaluated the protective effect of esculin against ALI by histopathological observation and biochemical analysis of lung tissues and bronchoalveolar lavage fluid (BALF) in lipopolysaccharide (LPS)-challenged ALI mice in vivo. The effects of esculin on N-formyl-met-leu-phe (fMLP)-induced neutrophil migration and chemotaxis were quantitatively assessed using a Transwell assay and an automated cell imaging system equipped with a Zigmond chamber, respectively. The drug affinity responsive target stability (DARTS) assay, in vitro protein binding assay and molecular docking were performed to identify the potential therapeutic target of esculin and the potential binding sites and pattern. RESULTS: Esculin significantly attenuated LPS-induced lung pathological injury, reduced the levels of pro-inflammatory cytokines in both BALF and lung, and suppressed the activation of NF-κB signaling. Esculin also significantly reduced the number of total cells and neutrophils as well as myeloperoxidase (MPO) activity in the BALF. Esculin impaired neutrophil migration and chemotaxis as evidenced by the reduced migration distance and velocity. Furthermore, esculin remarkably inhibited Vav1 phosphorylation, suppressed Rac1 activation and the PAK1/LIMK1/cofilin signaling axis. Mechanistically, esculin could interact with ß2 integrin and then diminish its ligand affinity with intercellular adhesion molecule-1 (ICAM-1). CONCLUSIONS: Esculin inhibits ß2 integrin-dependent neutrophil migration and chemotaxis, blocks the cytoskeletal remodeling process required for neutrophil recruitment, thereby contributing to its protective effect against ALI. This study demonstrates the new therapeutic potential of esculin as a novel lead compound.

Effect of manipulation on cartilage in rats with knee osteoarthritis based on the Rho-associated protein kinase/LIM kinase 1/Cofilin signaling pathways.

OBJECTIVE: To investigate the effect of manipulation treatment on knee osteoarthritis rats and the effect on Rho-associated protein kinase (ROCK)/LIM-kinase1 (LIMK1)/Cofilin signaling pathway. METHOD: Fifty Specific pathogen Free Sprague-Dawley rats were randomly divided into five groups ( = 8 each): blank group, model group, manipulation group, celecoxib group, and manipulation combined with celecoxib group (MC group). The osteoarthritis model was established by injecting 0.2 mL 4% papain into the articular disc of the rats. After successfully establishing the model, we treated the manipulation group with pushing manipulation using one-finger-meditation to the Neixiyan (EX-LE4), Waixiyan (EX-LE5), Xuehai (SP10), Liangqiu (ST34), and Zusanli (ST36) acupoints for 10 min each time. Also, the celecoxib group was gavaged with 24 mg•kg•d celecoxib, while the MC group was treated using both of these two methods. After four weeks, the cartilage of the right femur was removed for hematoxylin-eosin staining of the cartilage tissue. The expressions of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in serum were observed using the enzyme-linked immunosorbent assay. Besides, we detected the expressions of ROCK, LIMK1, Phospho-LIM-kinase1 (Phospho-LIMK1), Cofilin, and Phospho-Cofilin by Western blot. RESULTS: Compared to the model group, the manipulation group, celecoxib group, and MC group all exhibited superior results concerning pathological morphologic changes of cartilage, as observed by hematoxylin-eosin staining and calculated using the Mankin score. Besides, in contrast to the blank group, the model group exhibited elevated serum levels of IL-1ß and TNF-α ( 0.01), while the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage were all higher ( 0.01). Also, the serum levels of IL-1ß and TNF-α in each treatment group were lower (0.01) than in the model group. Moreover, there were lower expressions of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the manipulation group and the MC group (< 0.01). Compared with the model group, the expression of ROCK, LIMK1, Phospho-LIMK1, Cofilin, and Phospho-Cofilin in cartilage in the celecoxib group were not statistically different ( > 0.05). CONCLUSION: In this study, we established that manipulation has a better curative effect than celecoxib. Manipulation inhibits the development of cytoskeleton damage in cartilage and slows articular degeneration by regulating the expression of related proteins in the cytoskeletal signaling pathway.

Shuxuetong injection and its peptides enhance angiogenesis after hindlimb ischemia by activating the MYPT1/LIMK1/Cofilin pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxuetong (SXT) injection is formulated by leech and earthworm, has been widely used in the treatment of thrombotic cardiovascular and cerebrovascular diseases with remarkable clinical efficacy. AIM OF THE STUDY: The purpose of this study is to investigate the protective mechanism of SXT injection on the mice model of hindlimb ischemia, and to evaluate the angiogenic effects of SXT injection and its main active substances. MATERIALS AND METHODS: Hindlimb ischemia was induced by left femoral artery ligation. After operation, the mice were injected with saline, 10 mg/kg/d cilostazol, 37.5 mg/kg/d SXT injection, 75 mg/kg/d SXT injection and 150 mg/kg/d SXT injection via tail vein for 4 weeks. Ischemia severity was assessed using laser Doppler perfusion imaging system. Tissue recovery and capillary density were evaluated by histological and immunofluorescent staining. Vascular endothelial growth factor-A (VEGF-A) and platelet-derived growth factor (PDGF-BB) expression were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) analyses. Human umbilical vein endothelial cells (HUVECs) proliferation was measured using a BrdU kit and the viability of HUVECs was performed by MTT assay. Migration of HUVECs was performed by the wound healing method and a modified transwell assay. Capillary tube formation by HUVECs was examined by using Matrigel assay. Western blotting was used to detect the expressions of p-Cofilin, p-MYPT1, and p-LIMK1. RESULTS: SXT injection treatment significantly restored the blood flow and reduced tissue injury in mouse gastrocnemius muscle. SXT injection treatment increased capillary density and promoted angiogenesis in hindlimb ischemia. Moreover, SXT injection enhanced the expression of VEGF-A and PDGF-BB at both mRNA and protein levels in ischemic tissue of mice. SXT injection and its main active peptides dramatically increased the migration and capillary tube formation of HUVECs. SXT injection and its peptides enhanced protein expressions of the phosphorylation of MYPT1, Cofilin, and LIMK1. DSYVGDEAQSKR, YNELRVAPEEHP, and IQFLPEGSPVTM may act as the active components of SXT injection. CONCLUSION: SXT injection promoted angiogenesis and improved function recovery in hindlimb ischemia mice by regulation of VEGF-A/PDGF-BB. Moreover, SXT injection and its active peptides induced cell migration and tube formation in HUVECs through activating the MYPT1/LIMK1/Cofilin pathway. This study provided experimental basis for SXT injection in the treatment of ischemic diseases and revealed the effective substance of SXT injection in regulating angiogenesis, providing better evidence for the clinical application of SXT injection.

Protein domain-based prediction of drug/compound-target interactions and experimental validation on LIM kinases.

Predictive approaches such as virtual screening have been used in drug discovery with the objective of reducing developmental time and costs. Current machine learning and network-based approaches have issues related to generalization, usability, or model interpretability, especially due to the complexity of target proteins' structure/function, and bias in system training datasets. Here, we propose a new method "DRUIDom" (DRUg Interacting Domain prediction) to identify bio-interactions between drug candidate compounds and targets by utilizing the domain modularity of proteins, to overcome problems associated with current approaches. DRUIDom is composed of two methodological steps. First, ligands/compounds are statistically mapped to structural domains of their target proteins, with the aim of identifying their interactions. As such, other proteins containing the same mapped domain or domain pair become new candidate targets for the corresponding compounds. Next, a million-scale dataset of small molecule compounds, including those mapped to domains in the previous step, are clustered based on their molecular similarities, and their domain associations are propagated to other compounds within the same clusters. Experimentally verified bioactivity data points, obtained from public databases, are meticulously filtered to construct datasets of active/interacting and inactive/non-interacting drug/compound-target pairs (~2.9M data points), and used as training data for calculating parameters of compound-domain mappings, which led to 27,032 high-confidence associations between 250 domains and 8,165 compounds, and a finalized output of ~5 million new compound-protein interactions. DRUIDom is experimentally validated by syntheses and bioactivity analyses of compounds predicted to target LIM-kinase proteins, which play critical roles in the regulation of cell motility, cell cycle progression, and differentiation through actin filament dynamics. We showed that LIMK-inhibitor-2 and its derivatives significantly block the cancer cell migration through inhibition of LIMK phosphorylation and the downstream protein cofilin. One of the derivative compounds (LIMKi-2d) was identified as a promising candidate due to its action on resistant Mahlavu liver cancer cells. The results demonstrated that DRUIDom can be exploited to identify drug candidate compounds for intended targets and to predict new target proteins based on the defined compound-domain relationships. Datasets, results, and the source code of DRUIDom are fully-available at: https://github.com/cansyl/DRUIDom.

RhoA/Rock2/Limk1/cofilin1 pathway is involved in attenuation of neuronal dendritic spine loss by paeonol in the frontal cortex of D-galactose and aluminum­induced Alzheimer's disease­like rat model.

Alzheimer's disease (AD) has become the most prevalent neurodegenerative disorder. Given the pathogenesis of AD is unclear, there is currently no drug approved to halt or delay the progression of AD. Therefore, it is pressing to explore new targets and drugs for AD. In China, polyphenolic Chinese herbal medicine has been used for thousands of years in clinical application, and no toxic effects have been reported. In the present study, using D­galactose and aluminum­induced rat model, the effects of paeonol on AD were validated via the Morris water maze test, open field test, and elevated plus maze test. Neuronal morphology in frontal cortex was assessed using ImageJ's Sholl plugin and RESCONSTRUCT software. RhoA/Rock2/Limk1/cofilin1 signaling pathway­related molecules were determined by Western blotting. Cofilin1 and p­cofilin1 were analyzed by immunofluorescence. Results showed that pre­treatment with paeonol attenuated D­galactose and aluminum­induced behavioral dysfunction and AD­like pathological alterations in the frontal cortex. Accompanied by these changes were the alterations in the dendrite and dendritic spine densities, especially the mushroom­type and filopodia­type spines in the apical dendrites, as well as actin filaments. In addition, the activity and intracellular distribution of cofilin1 and the molecules RhoA/Rock2/Limk1 that regulate the signaling pathway for cofilin1 phosphorylation have also changed. Our data suggests that paeonol may be through reducing Aß levels to alleviate the loss of fibrillar actin and dendrites and dendritic spines via the Rho/Rock2/Limk1/cofilin1 signaling pathway in the frontal cortex, and ultimately improving AD­like behavior.

6-Hydroxythiobinupharidine Inhibits Migration of LM8 Osteosarcoma Cells by Decreasing Expression of LIM Domain Kinase 1.

BACKGROUND/AIM: Osteosarcoma is the most malignant type of bone tumor. Patients with osteosarcoma metastases have a poorer prognosis than those without metastases. Thus, the prognosis of osteosarcoma patients with metastases must be improved. MATERIALS AND METHODS: The present study investigated the inhibitory effects of 6-hydroxythiobinupharidine isolated from Nuphar pumilum on migration of LM8 murine osteosarcoma cells by a migration assay and also examined the expression of proteins related to actin dynamics by western blot. The present study also developed an automatic cell counting system using machine learning to count migrated cells by Fiji and Trainable Weka Segmentation. RESULTS: 6-Hydroxythiobinupharidine inhibited migration of LM8 osteosarcoma cells in a dose-dependent manner, and decreased protein expression of Lin11, Isl-1, and Mec-3 domain kinase 1 (LIMK1) and the levels of phosphorylated Cofilin. CONCLUSION: 6-Hydroxythiobinupharidine suppressed migration of LM8 osteosarcoma cells by decreasing expression of LIMK1. 6-Hydroxythiobinupharidine could be potentially used as an anti-metastatic compound.

Baicalin regulates depression behavior in mice exposed to chronic mild stress via the Rac/LIMK/cofilin pathway.

BACKGROUND: Depression is a common disease that endangers people's physical and mental health. Traditional Chinese medicine has advantages in treating the emotional and cognitive symptoms of depressive disorders. OBJECTIVE: To study the effects of baicalin on the behavior and to clarify the underlying mechanism through evaluation of the Rac1-LIMK1-cofilin pathway. METHODS: A chronic mild stress (CMS) model of depression was used. Baicalin was administered to the mice for the intervention, and the positive control group was treated with fluoxetine. Behavioral tests were conducted to observe the degree of depressive disorders. Synaptophysin (SYP), postsynaptic density protein-95 (PSD95), brain-derived neurotrophic factor (BDNF), tyrosine kinase receptors (TrkB), Rac1 and cofilin expression was determined using Western blot analysis, and mRNA was quantified using real-time PCR. RESULTS: Mice in the CMS group showed an increase in depression-like behavior (p < 0.01), while mice in the baicalin and fluoxetine groups showed a decrease in depression-like behavior (p < 0.01), compared with the control group. Electron microscopy showed ultrastructural changes in the hippocampal CA3 area of the CMS group, which were alleviated by baicalin treatment. SYP, PSD95, BDNF, TrkB, Rac1 and cofilin protein expression levels were decreased in the CMS group compared with the control group, while these levels were increased in the baicalin and fluoxetine groups (p < 0.01). There was no significant difference among the baicalin and fluoxetine groups (p > 0.05). CONCLUSION: Baicalin markedly alleviated depression-like behavioral changes, exerted effects on SYP, PSD95, BDNF, and TrkB expression, activated the Rac1-cofilin pathway, and subsequently improve synaptic plasticity.

Targeting ROCK/LIMK/cofilin signaling pathway in cancer.

Rho-associated coiled-coil-containing protein kinase (ROCK)/Lin11, Isl-1 and Mec-3 kinase (LIMK)/cofilin-signaling cascades are stimulated by receptor tyrosine kinases, G protein-coupled receptors, integrins and its ligands, growth factors, hormones, fibronectin, collagen, and laminin. Activated signaling cascades can cause transit from normal cells to cancer cells by modulating actin/filament dynamics. In various cancers including breast, prostate, and colorectal cancers, high expression or activity of each cascade protein is significantly associated with poor survival rate of patients as well as aggressive metastasis. Silencing ROCK, LIMK, or cofilin can abrogate their activities and inhibit cancer cell growth, invasion, and metastasis. Therefore ROCK/LIMK/cofilin signaling proteins might be good candidates to develop cancer prevention strategies or therapeutics. Currently, netarsudil, a ROCK inhibitor, is only used in clinical patients for glaucoma or ocular hypertension, but not for cancer. In this review, we will discuss comprehensive ROCK/LIMK/cofilin signaling pathway in cancers and its inhibitors for developing cancer therapy.

In vitro inhibitory properties of sesquiterpenes from Chloranthus serratus on cell motility via down-regulation of LIMK1 activation in human breast cancer.

BACKGROUND: LIM kinase 1 plays an important role in tumor cell invasion and metastasis by regulating architecture of actin cytoskeleton, and inhibiting activity of this kinase may be a promising strategy to prevent cancer cells from distant spread. In our previous studies, we found several extracts from the medical herbs in genus Chloranthus to exhibit anti-metastatic effects. PURPOSE: The aim of this study is to find LIMK1 inhibitors from Chloranthus serratus, a medical herb from genus Chloranthus and to evaluate their effects on cell motility. METHODS: Three sesquiterpenes, chloranthalactone E (compound 1), serralactone A (compound 2, SERA is used in the further testing), and 8ß, 9α-dihydroxylindan-4(5), 7(11)-dien-8α, 12-olide (compound 3) were isolated from Chloranthus serratus, and the anti-LIMK1 activities of these compounds were investigated by kinase-Glo® luminescent kinase assay. Then, the anti-LIMK1 properties of SERA were verified by kinase-Glo® luminescent kinase assay and western blot assay. The effects of SERA on F-actin polymerization and cell migration were investigated by Phalloidin dying, AP 48 chamber system and ORIS™ cell migration assay. Furthermore, the inhibitory effects of SERA on LIMK1 were confirmed by overexpression of LIMK1 and small interfering RNA (siRNA) mediated gene silencing. RESULTS: we reported here that among the three sesquiterpenes, SERA showed significantly inhibition on LIMK1 activity, and the IC50 values on MDA-MB-231 and MDA-MB-468 cells were 3.14 µM and 4.64 µM, respectively. Furthermore, it was also found that SERA significantly suppressed LIMK1 and cofilin1 phosphorylation, F-actin polymerization and also cell migration. Data from LIMK1 overexpression and RNA interfering assay confirmed that the inhibitory effects of SERA on LIMK1 was antagonized and enhanced by the overexpression and knockdown of LIMK1. CONCLUSION: collectively, it was concluded that SERA exhibited significant inhibitory effects on breast cancer cells migration, and these effects of this sesquiterpene are due to its properties reducing the activation of LIM kinase 1.

Electroacupuncture Regulates Hippocampal Synaptic Plasticity via miR-134-Mediated LIMK1 Function in Rats with Ischemic Stroke.

MircoRNAs (miRs) have been implicated in learning and memory, by regulating LIM domain kinase (LIMK1) to induce synaptic-dendritic plasticity. The study aimed to investigate whether miRNAs/LIMK1 signaling was involved in electroacupuncture- (EA-) mediated synaptic-dendritic plasticity in a rat model of middle cerebral artery occlusion induced cognitive deficit (MICD). Compared to untreatment or non-acupoint-EA treatment, EA at DU20 and DU24 acupoints could shorten escape latency and increase the frequency of crossing platform in Morris water maze test. T2-weighted imaging showed that the MICD rat brain lesions were located in cortex, hippocampus, corpus striatum, and thalamus regions and injured volumes were reduced after EA. Furthermore, we found that the density of dendritic spine and the number of synapses in the hippocampal CA1 pyramidal cells were obviously reduced at Day 14 after MICD. However, synaptic-dendritic loss could be rescued after EA. Moreover, the synaptic-dendritic plasticity was associated with increases of the total LIMK1 and phospho-LIMK1 levels in hippocampal CA1 region, wherein EA decreased the expression of miR-134, negatively regulating LIMK1 to enhance synaptic-dendritic plasticity. Therefore, miR-134-mediated LIMK1 was involved in EA-induced hippocampal synaptic plasticity, which served as a contributor to improving learning and memory during the recovery stage of ischemic stroke.

Reduced corporal fibrosis to protect erectile function by inhibiting the Rho-kinase/LIM-kinase/cofilin pathway in the aged transgenic rat harboring human tissue kallikrein 1.

Our previous studies have demonstrated that erectile function was preserved in aged transgenic rats (TGR) harboring the human tissue kallikrein 1 (hKLK1), while the molecular level of hKLK1 on corporal fibrosis to inhibit age-related erectile dysfunction (ED) is poorly understood. Male wild-type Sprague-Dawley rats (WTR) and TGR harboring the hKLK1 gene were fed to 4- or 18-month-old and divided into three groups: young WTR (yWTR) as the control, aged WTR (aWTR), and aged TGR (aTGR). Erectile function of all rats was assessed by cavernous nerve electrostimulation method. Masson's trichrome staining was used to evaluate corporal fibrosis in the corpus cavernosum. We found that the erectile function of rats in the aWTR group was significantly lower than that of other two groups. Masson's trichrome staining revealed that compared with those of the yWTR and aTGR groups, the ratio of smooth muscle cell (SMC)/collagen (C) was significantly lower in the aWTR group. Immunohistochemistry and Western blotting analysis were performed, and results demonstrated that expression of α-SMA was lower, while expressions of transforming growth factor-ß 1 (TGF-ß1), RhoA, ROCK1, p-MYPT1, p-LIMK2, and p-cofilin were higher in the aWTR group compared with those in other two groups. However, LIMK2 and cofilin expressions did not differ among three groups. Taken together, these results indicated that the RhoA/ROCK1/LIMK/cofilin pathway may be involved in the corporal fibrosis caused by advanced age, and hKLK1 may reduce this corporal fibrosis by inhibiting the activation of this pathway to ameliorate age-related ED.

Preserved Erectile Function in the Aged Transgenic Rat Harboring Human Tissue Kallikrein 1.

INTRODUCTION: Human tissue kallikrein 1 (hKLK1) has enormous potential for the protection of vasodilation and endothelial function in the cardiovascular system. Our previous study proved the decreased expression of kallikrein 1 in the corpus cavernosum (CC) of aged rats, but the role of kallikrein 1 in age-related erectile dysfunction remains unknown. AIM: To explore the effect and underlying mechanisms of hKLK1 on age-related erectile dysfunction. METHODS: Male wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring the hKLK1 gene (TGR) were fed to 4 and 27 months of age, respectively, and divided into four groups: young WTR (yWTR) as the control, young TGR (yTGR), aged WTR (aWTR), and aged TGR (aTGR). Rats' erectile function was evaluated by the cavernous nerve electrostimulation method. Then, CCs were collected for verification of hKLK1 followed by measurement of nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) and RhoA-Rho-kinase (ROCK) signaling activities. Masson trichrome staining and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling assay were conducted to evaluate penile fibrosis and apoptosis. MAIN OUTCOME MEASURES: Erectile response, NO-cGMP and RhoA-ROCK pathway-related indices, ratio of smooth muscle to collagen, and apoptosis index. RESULTS: The hKLK1 alleviated the decrease of erectile function in the aWTR group. Endothelial NO synthase (eNOS) and phospho-eNOS(Ser1177) expressions, NO synthase activity, and NO and cGMP levels were decreased, whereas phospho-eNOS(Thr495), L-type Ca(2+) channel, RhoA, ROCK1, ROCK2, and transforming growth factor ß1 proteins were increased in the CCs of the aWTR group compared with the control yWTR group. These changes were obviously mitigated in the aTGR group. Moreover, hKLK1 prevented the sharp decrease of the ratio of smooth muscle to collagen and the increase of the apoptosis index in the CCs of the aWTR group. CONCLUSION: These results suggest that hKLK1 could play a preventive role in age-related erectile dysfunction by activation of the NO-cGMP pathway and inhibition of the RhoA-ROCK pathway and by antitissue fibrotic and apoptotic effects.

Cucurbitacin I elicits the formation of actin/phospho-myosin II co-aggregates by stimulation of the RhoA/ROCK pathway and inhibition of LIM-kinase.

Cucurbitacins are cytotoxic triterpenoid sterols isolated from plants. One of their earliest cellular effect is the aggregation of actin associated with blockage of cell migration and division that eventually lead to apoptosis. We unravel here that cucurbitacin I actually induces the co-aggregation of actin with phospho-myosin II. This co-aggregation most probably results from the stimulation of the Rho/ROCK pathway and the direct inhibition of the LIMKinase. We further provide data that suggest that the formation of these co-aggregates is independent of a putative pro-oxidant status of cucurbitacin I. The results help to understand the impact of cucurbitacins on signal transduction and actin dynamics and open novel perspectives to use it as drug candidates for cancer research.

Involvement of Rho-Kinase/LIM Kinase/Cofilin Signaling Pathway in Corporal Fibrosis after Cavernous Nerve Injury in Male Rats.

INTRODUCTION: The molecular mechanism of corporal fibrosis leading to erectile dysfunction (ED) following cavernous nerve (CN) injury is poorly understood. AIM: To determine whether the LIMK2/cofilin pathway, the downstream effectors of ROCK1, was involved in ED and corporal fibrosis following bilateral CN injury in male rats. METHODS: Forty-eight 10-week-old male Sprague-Dawley rats were equally divided into three groups: sham surgery (S); bilateral CN crush injury (I); and bilateral CN resection (R). Within each groups, two subgroups were analyzed at 1 and 4 weeks postoperatively. MAIN OUTCOME MEASURES: Electrostimulation was performed to assess erectile function by the ratio of maximal intracavernous pressure to mean arterial pressure (ICP/MAP) and areas under the ICP curve to MAP (AUC/MAP). Penile tissue was processed for Masson's trichrome staining, Western blot (ROCK1, total LIMK2, phospho-LIMK2, total cofilin, phospho-cofilin), immunohistochemistry (alpha-SM actin [α-SMA]), and double immunofluorescent staining (ROCK1, phospho-LIMK2, vimentin). RESULTS: At each time point, both I and R groups showed a significantly lower percent of ICP/MAP and AUC, and decreased SM cell/collagen ratio and expression of α-SMA than S group. Densitometry revealed a significantly higher expression of ROCK1 in I and R groups compared with S group at all time points. The LIMK2 phosphorylation in I and R groups significantly increased at 1 week, but not at 4 weeks. The cofilin phosphorylation in R group significantly increased to that in S group starting at 1 week, while that in I group was increased significantly at 4 weeks. The double immunofluorescent staining noted that coexpression of vimentin with ROCK1 or phospho-LIMK2 in I and R groups was significantly increased mainly in the subtunical area at 1 week but not at 4 weeks. CONCLUSIONS: The ROCK1/LIMK2/cofilin pathway may be involved in ED related to corporal fibrosis, and it appears to be functional particularly in the early period after CN injury.