Fangyukangsuan granules ameliorate hyperuricemia and modulate gut microbiota in rats.

Hyperuricaemia (HUA) is a metabolic disorder characterised by high blood uric acid (UA) levels; moreover, HUA severity is closely related to the gut microbiota. HUA is also a risk factor for renal damage, diabetes, hypertension, and dyslipidaemia; however, current treatments are associated with detrimental side effects. Alternatively, Fangyukangsuan granules are a natural product with UA-reducing properties. To examine their efficacy in HUA, the binding of small molecules in Fangyukangsuan granules to xanthine oxidase (XOD), a key factor in UA metabolism, was investigated via molecular simulation, and the effects of oral Fangyukangsuan granule administration on serum biochemical indices and intestinal microorganisms in HUA-model rats were examined. Overall, 24 small molecules in Fangyukangsuan granules could bind to XOD. Serum UA, creatinine, blood urea nitrogen, and XOD levels were decreased in rats treated with Fangyukangsuan granules compared to those in untreated HUA-model rats. Moreover, Fangyukangsuan granules restored the intestinal microbial structure in HUA-model rats. Functional analysis of the gut microbiota revealed decreased amino acid biosynthesis and increased fermentation of pyruvate into short-chain fatty acids in Fangyukangsuan granule-treated rats. Together, these findings demonstrate that Fangyukangsuan granules have anti-hyperuricaemic and regulatory effects on the gut microbiota and may be a therapeutic candidate for HUA.

Visceral and ectopic fat are more predictively associated with primary liver cancer than overall obesity from genetic sights: A Mendelian randomization study.

Several observational studies have reported an association between obesity and primary liver cancer (PLC), while the causality behind this association and the comparison of the risk effects of different obesity indicators on PLC remain unclear. In this study, we performed two-sample Mendelian randomization (MR) analyses to assess the associations of genetically determined liver fat, visceral adipose tissue (VAT), and body mass index (BMI) with the risk of PLC. The summary statistics of exposures were obtained from two genome-wide association studies (GWASs) based on the UK Biobank (UKB) imaging cohort and the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort. GWAS summary statistics for PLC were obtained from FinnGen consortium R7 release data, including 304 PLC cases and 218 488 controls. Inverse-variance weighted (IVW) was used as the primary analysis, and a series of sensitivity analyses were performed to further verify the robustness of these findings. IVW analysis highlighted a significant association of genetically determined liver fat (OR per SD increase: 7.14; 95% CI: 5.10-9.99; P = 2.35E-30) and VAT (OR per SD increase: 5.70; 95% CI: 1.32-24.72; P = .020) with PLC but not of BMI with PLC. The findings were further confirmed by a series of MR methods. No evidence of horizontal pleiotropy between these associations existed. Our study suggested that genetically determined liver fat and VAT rather than BMI were associated with an increased risk of PLC, which suggested that visceral fat distribution is more predictive of the clinical risk of PLC than common in vitro measures.

Standardized lymph node dissection for gallbladder cancer under laparoscopy: en-bloc resection technique.

PURPOSE: Positive lymph node (LN) is a key prognostic factor in radically resected gallbladder cancer (GBCA). However, only a few underwent an adequate lymphadenectomy, and the number and extent of lymph node dissection (LND) have not been standardized. This study aims to develop an en bloc and standardized surgical procedure of LND for GBCA under laparoscopy. METHODS: Data of patients with GBCA underwent laparoscopic radical resection using a standardized and en bloc technique for LND were collected. Perioperative and long-term outcomes were retrospectively analyzed. RESULTS: A total of 39 patients underwent laparoscopic radical resection using standardized and en bloc technique for LND except one case (open conversion rate: 2.6%). Patients with stage T1b had significantly lower LNs involved rate than patients with stage T3 (P = 0.04), whereas median LN count in stage T1b was significantly higher than that in stage T2 (P = 0.04), which was significantly higher than that in stage T3 (P = 0.02). Lymphadenectomy with ≥ 6 LNs accounted for 87.5% in stage T1b, up to 93.3% in T2 and 81.3% in T3, respectively. All the patients in stage T1b were alive without recurrence at this writing. The 2-year recurrence-free survival rate was 80% for T2 and 25% for T3, and the 3-year overall survival rate was 73.3% for T2 and 37.5% for T3. CONCLUSION: The standardized and en bloc LND permits complete and radical removal of lymph stations for patients with GBCA. This technique is safe and feasible with low complication rates and good prognosis. Further studies are required to explore its value and long-term outcomes compared to conventional approaches.

A novel cuproptosis-related subtypes and gene signature associates with immunophenotype and predicts prognosis accurately in neuroblastoma.

Background: Neuroblastoma (NB) is the most frequent solid tumor in pediatrics, which accounts for roughly 15% of cancer-related mortality in children. NB exhibited genetic, morphologic, and clinical heterogeneity, which limited the efficacy of available therapeutic approaches. Recently, a new term 'cuproptosis' has been used to denote a unique biological process triggered by the action of copper. In this instance, selectively inducing copper death is likely to successfully overcome the limitations of conventional anticancer drugs. However, there is still a gap regarding the role of cuproptosis in cancer, especially in pediatric neuroblastoma. Methods: We characterized the specific expression of cuproptosis-related genes (CRGs) in NB samples based on publicly available mRNA expression profile data. Consensus clustering and Lasso-Cox regression analysis were applied for CRGs in three independent cohorts. ESTIMATE and Xcell algorithm was utilized to visualize TME score and immune cell subpopulations' relative abundances. Tumor Immune Dysfunction and Exclusion (TIDE) score was used to predict tumor response to immune checkpoint inhibitors. To decipher the underlying mechanism, GSVA was applied to explore enriched pathways associated with cuproptosis signature and Connectivity map (CMap) analysis for drug exploration. Finally, qPCR verified the expression levels of risk-genes in NB cell lines. In addition, PDHA1 was screened and further validated by immunofluorescence in human clinical samples and loss-of-function assays. Results: We initially classified NB patients according to CRGs and identified two cuproptosis-related subtypes that were associated with prognosis and immunophenotype. After this, a cuproptosis-related prognostic model was constructed and validated by LASSO regression in three independent cohorts. This model can accurately predict prognosis, immune infiltration, and immunotherapy responses. These genes also showed differential expression in various characteristic groups of all three datasets and NB cell lines. Loss-of-function experiments indicated that PDHA1 silencing significantly suppressed the proliferation, migration, and invasion, in turn, promoted cell cycle arrest at the S phase and apoptosis of NB cells. Conclusions: Taken together, this study may shed light on new research areas for NB patients from the cuproptosis perspective.

Immune-related gene signature associates with immune landscape and predicts prognosis accurately in patients with Wilms tumour.

Wilms tumour (WT) is the most common kidney malignancy in children. Chemoresistance is the leading cause of tumour recurrence and poses a substantial therapeutic challenge. Increasing evidence has underscored the role of the tumour immune microenvironment (TIM) in cancers and the potential for immunotherapy to improve prognosis. There remain no reliable molecular markers for reflecting the immune landscape and predicting patient survival in WT. Here, we examine differences in gene expression by high-throughput RNA sequencing, focused on differentially expressed immune-related genes (IRGs) based on the ImmPort database. Via univariate Cox regression analysis and Lasso-penalized Cox regression analysis, IRGs were screened out to establish an immune signature. Kaplan-Meier curves, time-related ROC analysis, univariate and multivariate Cox regression studies, and nomograms were used to evaluate the accuracy and prognostic significance of this signature. Furthermore, we found that the immune signature could reflect the immune status and the immune cell infiltration character played in the tumour microenvironment (TME) and showed significant association with immune checkpoint molecules, suggesting that the poor outcome may be partially explained by its immunosuppressive TME. Remarkably, TIDE, a computational method to model tumour immune evasion mechanisms, showed that this signature holds great potential for predicting immunotherapy responses in the TARGET-wt cohort. To decipher the underlying mechanism, GSEA was applied to explore enriched pathways and biological processes associated with immunophenotyping and Connectivity map (CMap) along with DeSigN analysis for drug exploration. Finally, four candidate immune genes were selected, and their expression levels in WT cell lines were monitored via qRT-PCR. Meanwhile, we validated the function of a critical gene, NRP2. Taken together, we established a novel immune signature that may serve as an effective prognostic signature and predictive biomarker for immunotherapy response in WT patients. This study may give light on therapeutic strategies for WT patients from an immunological viewpoint.

κ-Carrageenan oligosaccharides induce microglia autophagy through AMPK/ULK1 pathway to regulate their immune response.

Microglia are the main effector cells of immune response in central nervous system and are important targets for disease prevention and treatment. Κ-carrageenan Oligosaccharide (KOS), obtained by enzymatic hydrolysis from carrageenan of marine red algae, can inhibit the release of inflammatory factors from the over-activated microglia. The mechanism of microglia autophagy induced by KOS and its relationship with inflammation were studied to explore the development prospect of KOS in the research and treatment of inflammatory related diseases. The effect of KOS on inducing autophagy was detected by the secretion of cytokines by lipopolysaccharide (LPS)-activated microglia, respectively. The protein expression of autophagy-related signaling pathways were detected by Western Blot. The results showed that KOS could significantly protect the microglia from over-activated inflammatory by inducing the autophagy and inhibiting the release of inflammatory cytokines. And KOS could reduce the expression of the protein that related to the AMPK/ULK1 pathways in microglia, so as to regulate the autophagy pathway, and inhibit the inflammatory response of over-activated microglia. The study on the effect of KOS on microglia autophagy and excessive inflammatory response will provide a theoretical basis for further studies on the inhibition of nerve injury by regulating microglia autophagy and inflammatory response.

κ-Carrageenan Oligosaccharides Inhibit the Inflammation of Lipopolysaccharide-Activated Microglia Via TLR4/NF-κB and p38/JNK MAPKs Pathways.

Microglial inflammation plays an essential role in neurodegenerative disease. Our previous studies had shown that κ-carrageenan oligosaccharides (KOS) could inhibit the excessive activation of microglia that induced by LPS, while the interrelated mechanisms were still indistinct. Therefore, we detected the inflammatory signaling pathway on LPS-activated microglia that pretreat by different content of KOS to reveal the mechanism on KOS's inhibition of microglia inflammatory response. ELISA was used to detect the effects of KOS on the secretion of interleukin-1 (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and prostaglandin E2 (PG-2) by LPS-activated microglia, respectively. The production of reactive oxygen species (ROS) and nitric oxide (NO) in microglia cells was detected by flow cytometry, and the protein expression of immunoinflammation-related signaling pathways were detected by Western Blot. The results showed that KOS could significantly protected the microglia from the over-activated inflammatory by inhibiting the release of inflammatory cytokines and the oxidative stress response. And KOS could reduce the expression of the protein that related to the TLR4/NF-κB and p38/JNK MAPKs pathways activated by LPS in microglia. However, there may be no specific target of KOS in cells. Therefore, KOS, a natural algal source oligosaccharide, has immunomodulatory effects and can be used as a potential intervention therapy for inflammatory related neurodegenerative diseases.

[Establishment of an Animal Model of Vesicoureteral Reflux Renal Injury through Partial Bladder Outlet Obstruction].

OBJECTIVE: To establish an animal model of reflux renal damage through bladder outlet obstruction. METHODS: Sixty male C57BL/6 mice aged 6-8 weeks were randomly assigned to a control group, a sham operation group, and a partial bladder outlet obstruction (PBOO) group, with 20 mice in each group. Laparotomy were performed on the PBOO mice under anesthesia in order to separate the bladder necks and to perform guided partial ligation of the bladder neck with a metal rod of 0.3 mm diameter. Mice in the sham operation group had laparotomy and had their bladder necks separated without ligation. The control group did not receive any treatment. 7 days after the surgery, 12 surviving mice were randomly selected from each group to observe the general changes of the bladder, ureter, renal pelvis and kidney. Retrograde urography was performed through the bladder. Kidney tissues were extracted for histopathological analysis. The expression levels of Vimentin, proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA) were examined with Western blot, immunohistochemistry and immunofluorescence staining tests, respectively. RESULTS: Compared with the control and sham operation group, the bladder, ureter, and renal pelvis of the mice in the PBOO group were significantly enlarged, vesicoureteral reflux was more obvious, the kidney volume and mass increased ( P<0.001), and renal parenchyma became thinner ( P<0.000 1). Histopathological staining showed glomerular atrophy, renal tubule expansion, tubulointerstitial inflammatory cell infiltration, glomerular basement membrane hyperplasia and obvious interstitial fibrosis. Western blot, immunofluorescence and immunohistochemistry staining showed that the expression levels of Vimentin, PCNA and α-SMA in kidney tissue were elevated ( P<0.000 1). CONCLUSION: After PBOO, the bladder, ureter, and kidney of the mice showed obvious morphological alteration and presented reflux renal fibrosis-like damage. This can be used as an animal model to study the pathological alteration mechanism and therapeutic measures of renal fibrosis caused by bladder outlet obstruction.

Systemic air embolism in a fungal pneumonia patient with lung cavities formation and review of literature.

Systemic air embolism is a rare but potentially fatal complication related to many factors. The purpose of this article is to alert clinicians once patients occurs an abnormal neurological and cardiovascular status, following minor traumatic treatment, air embolism should be considered. A 20-year-old man who presented with fungal pneumonia with lung cavities formation was admitted to an intensive care unit (ICU) and received positive airway pressure ventilation. Four days later, the fungal pneumonia was improved, but the patient's blood pressure and arterial oxygen saturation deteriorated, so computed tomography (CT) scans were preformed to reevaluate him. The scans detected air embolism in the left atrium and ventricle, ascending aorta, aortic arch and its branches (right brachiocephalic, bilateral common carotid and right subclavian arteries), descending aorta and right coronary artery. A CT scan of the abdomen revealed air in the spleen, cauda pancreatic, superior mesenteric artery and right external iliac artery. The patient died two days later from multiple organ dysfunction. We suggest that vascular air embolism should be considered under mechanical ventilation when patients' neurologic and cardiovascular status deteriorates, and hyperbaric oxygen therapy should be conducted immediately.

WITHDRAWN: Individualized and pancreatic duct diameter-based strategy for pancreaticoenteric anastomosis during pancreaticoduodenectomy.

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Gemcitabine-based combination therapy compared with gemcitabine alone for advanced pancreatic cancer: a meta-analysis of nine randomized controlled trials.

BACKGROUND: Pancreatic cancer is one of the most aggressive malignancies and chemotherapy is an effective strategy for advanced pancreatic cancer. Gemcitabine (GEM) is one of first-line agents. However, GEM-based combination therapy has shown promising efficacy in patients with advanced pancreatic cancer. This meta-analysis aimed to compare the efficacy and safety of GEM-based combination therapy versus GEM alone in the treatment of advanced pancreatic cancer. DATA SOURCES: A comprehensive search of literature was performed using PubMed, EMBASE, Web of Science and Cochrane Central Register of Controlled Trials. A quantitative meta-analysis was performed based on the inclusion criteria from all eligible randomized controlled trials. The outcome indicators included overall survival (OS), 6-month survival, 1-year survival, progression-free survival/time-to-progression (PFS/TTP), and toxicities. RESULTS: A total of nine randomized controlled trials involving 1661 patients were included in this meta-analysis. There was significant improvement in the GEM-based combination therapy with regard to the OS (HR=0.85, 95% CI: 0.76-0.95, P=0.003), PFS (HR=0.76, 95% CI: 0.65-0.90, P=0.002), 6-month survival (RR=1.09, 95% CI: 1.01-1.17, P=0.03), and the overall toxicity (RR=1.68, 95% CI: 1.52-1.86, P<0.01). However, there was no significant difference in the 1-year survival. CONCLUSIONS: GEM-based combination chemotherapy might improve the OS, 6-month survival, and PFS in advanced pancreatic cancer. However, combined therapy also added toxicity.

Ischemic preconditioning enhances hepatocyte proliferation in the early phase after ischemia under hemi-hepatectomy in rats.

BACKGROUND: Ischemia/reperfusion (I/R) injury is an important barrier to liver surgery and transplantation because it impairs remnant liver/reduced-size-graft regeneration. Ischemic preconditioning (IPC), as an effective measure to overcome I/R injury, has been shown to enhance the regenerative capacity of hepatocytes. However, investigations have always focused on regeneration in the late phase after reperfusion. This study aimed to investigate whether IPC enhances hepatocyte proliferation in the early phase after reperfusion and possible underlying mechanisms. METHODS: A total of 90 rats were divided into three groups: hemi-hepatectomy alone (PHx group), 60 minutes of ischemia plus hemi-hepatectomy (I/R group), and a cycle of 10 minutes of alternating I/R prior to 60 minutes of ischemia plus hemi-hepatectomy (IPC group). Each group was divided into five subgroups sacrificed after 0.5, 2, 6, 12 or 24 hours (n=6/subgroup). Subsequently, serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were measured; caspase-3 and proliferating cell nuclear antigen (PCNA) proteins were also determined by Western blotting. Furthermore, PCNA was detected by immunohistochemistry to identify the expression site. RESULTS: Serum ALT and AST levels after 2-24 hours of reperfusion in the PHx and IPC groups were remarkably decreased compared to the I/R group, and the serum TNF-alpha was relatively lower. A significant increase of serum IL-6 levels was found in the PHx and IPC groups compared with the I/R group at each time point. Furthermore, PCNA expression was remarkably increased in the IPC group after 6-12 hours of reperfusion, and in the earlier 0.5 and 6 hours time points after reperfusion have shown the massive PCNA-positive hepatocytes. At the same time, the expression of liver p-JNK was higher in the IPC group in the early phase after reperfusion than that of the I/R group and its expression was consistent with the PCNA. CONCLUSION: IPC can initiate hepatocyte proliferation in the early phase after ischemia under hemi-hepatectomy, and may be associated with p-JNK expression and triggered by TNF-alpha/IL-6 signals.

Glomerular expression of kidney injury molecule-1 and podocytopenia in diabetic glomerulopathy.

BACKGROUND/AIMS: Studies have shown that kidney injury molecule-1 (KIM-1) is upregulated in damaged renal proximal tubules. In this study, we examined KIM-1 expression in glomerular epithelial cells in diabetic glomerulopathy. METHODS: Renal histology, immunostaining and Western blot for protein level, and real-time PCR for mRNA expression of KIM-1 and podocyte markers were evaluated in untreated or losartan-treated Zucker lean (Fa/+) and Zucker diabetic fatty (Fa/Fa) rats. RESULTS: The diabetic rats showed an increased glomerular expression of KIM-1. KIM-1 staining was localized primarily in the hyperplastic parietal epithelium of Bowman's capsule in the early stages of diabetes with subsequent increase in KIM-1-positive cells in the glomerular tuft in the more advanced stages. The increase in glomerular KIM-1 was associated with a decrease in podocytes in Fa/Fa rats. Antiproteinuric treatment with losartan attenuated podocytopenia and decreased renal expression of KIM-1 in treated diabetic rats. In an in vitro study, albumin overload increased KIM-1 protein in the primary cultures of rat glomerular epithelial cells. CONCLUSION: These results show that glomerular KIM-1 expression was increased, in proportion to the extent of proteinuria and podocytopenia in the diabetic animals, supporting that KIM-1 could be used as a potential biomarker for glomerular injury in proteinuric kidney disease.

Outcomes and mechanisms of ischemic preconditioning in liver transplantation.

BACKGROUND: Liver transplantation is so far the most effective therapeutic modality for end-stage liver diseases, but ischemia/reperfusion (I/R) injury represents a critical barrier to liver transplantation. Primary graft dysfunction and small-for-size syndrome are closely associated with I/R injury. Ischemic preconditioning (IPC) is defined as a brief period of liver ischemia followed by reperfusion, and has demonstrated protections against a prolonged I/R injury and improved the capacity of regeneration. The article aimed to review IPC literatures for the understanding of the effects of IPC on I/R injury involving in the procurement of donor liver and protective mechanisms. DATA SOURCES: A literature search of MEDLINE and Web of Science databases using "liver transplantation", "liver regeneration", "hepatectomy", "ischemia/reperfusion" and "ischemic preconditioning" was performed, and then a large amount of related data was collected. RESULTS: The literature search provided a huge amount of evidence for the protective effects of IPC on I/R injury in liver transplantation, including reduction of blood loss in hepatectomy, intraoperative hemodynamic stability and its significant role in liver regeneration. The mechanism involves in balancing inflammatory cytokines, enhancing energy status and mitigating microcirculatory disturbance. CONCLUSION: IPC plays an essential role in hepatectomy before and after harvest of living donor liver and implantation of liver graft.

Ischemic preconditioning attenuates morphological and biochemical changes in hepatic ischemia/reperfusion in rats.

OBJECTIVE: Ischemic preconditioning (IPC) has been gradually introduced into clinical liver surgery and transplantation in recent years. However, the protective effects of IPC on hepatic warm ischemia/reperfusion (I/R) injury and the potential mechanisms involved are not fully understood. We aimed to evaluate whether the reduction of apoptotic sinusoidal endothelial cells (SECs), induced by IPC, contributes to its protective effect. METHODS: Male Wistar rats were randomized into three experimental groups: the continuous clamping group underwent 60 min of 70% hepatic ischemia; the IPC group received 10 min ischemia followed by 10 min reperfusion prior to ischemia, and the sham control (sham) underwent a sham operation without ischemia. Hepatocyte and SEC apoptosis, liver necrotic areas and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid, tumor necrosis factor, myeloperoxidase (MPO) and malondialdehyde were determined. Expression of cysteine-aspartic acid protease-3 (caspase-3) in hepatocytes and SECs was also investigated. Furthermore, the hepatic leukocyte infiltration was assessed by intravital fluorescence microscopy. RESULTS: IPC exhibited a significant alleviation of their postischemic liver function. Serum AST, ALT and tissue MPO were significantly decreased by IPC, and the degree of hepatocyte and SEC apoptosis was significantly inhibited, as shown by the decreased numbers of adherent leukocytes. CONCLUSIONS: IPC attenuates hepatic I/R injury by the reduction of leukocyte infiltration, the reduction hepatic enzymatic leakage and the depression of apoptotic cells. SECs are more sensitive to apoptosis induced by warm I/R injury compared to hepatocytes.

[Determination of metals in Ginkgo biloba leaves by atomic absorption spectrometry with microwave digestion].

Microwave digestion technique was used in the decomposition of Ginkgo biloba leaves from six different trees at the same age in the same area. HNO3-H2O2 (5 : 1 v/v) was used as microwave digestion agent at a suitable temperature and time. The contents of Ca, Mg, K, Na, Cu, Zn and Zn/Cu were determined by flame atomic absorption spectrometry to study the distribution rule of metallic elements in the trees at the same age and in the same area. The recovery ratio ranged from 95.2% to 104.6%. The results showed that there were certain differences between different trees in the distribution of metallic elements. The contents of calcium were from 39 586 to 48 320 microg x g(-1), and those of magnesium from 10 076 to 12 918 microg x g(-1), of potassium from 2 004 to 5 240 microg x g(-1), of sodium from 9.05 to 35.30 microg x g(-1), and of copper from 1.50 to 3.05 microg x g(-1), while Zn/Cu values were from 2.68 to 5.93 in the leaves of 6 different trees in the same growing area. Therefore there were abounding calcium, magnesium and potassium, while the content of sodium and Zn/Cu values were lower, and the metal contents were different in the leaves. The experimental results provided useful bases for studying the distribution rule of metallic elements in Ginkgo biloba leaves, the relationship between the contents of calcium, magnesium, potassium and sodium and the Zn/Cu value in ginkgo biloba leaves and the treatment for cardio-cerebral vascular disease.

Up-regulation of the RhoA/Rho-kinase signaling pathway in corpus cavernosum from endothelial nitric-oxide synthase (NOS), but not neuronal NOS, null mice.

We tested the hypothesis that the basal release of nitric oxide (NO) from endothelial cells modulates contractile activity in the corpus cavernosum (CC) via inhibition of the RhoA/Rho-kinase signaling pathway. Cavernosal strips from wild-type (WT), endothelial nitric-oxide synthase knockout [eNOS(-/-)], and neuronal nitric-oxide synthase knockout [nNOS(-/-)] mice were mounted in myographs, and isometric force was recorded. mRNA and protein expression of key molecules in the RhoA/Rho-kinase pathway were analyzed by real-time polymerase chain reaction and Western blot, respectively. The cGMP levels were determined. The Rho-kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl] homopiperazine (H-1152) reduced cavernosal contractions evoked by phenylephrine or electrical field stimulation (EFS) in a concentration-dependent manner, although this inhibition was less effective in tissues from eNOS(-/-) mice. Y-27632 enhanced relaxations induced by sodium nitroprusside, EFS, and NO (administered as acidified NaNO2) without affecting the cGMP content of the cavernosal strips. This enhancement was less prominent in CC from eNOS(-/-). The protein expression of RhoA, Rho-guanine dissociation inhibitor, and Rho-kinase beta did not differ among the strains. However, in eNOS(-/-) CC, the protein expression of Rho-kinase alpha and both mRNA and protein expression of p115-Rho-associated guanine exchange factor (RhoGEF), PDZ-RhoGEF, and leukemia-associated RhoGEF were up-regulated. Phosphorylation of MYPT1 at Thr696 was higher in tissues from eNOS(-/-) mice. A high concentration of Y-27632 significantly enhanced NO release in CC stimulated by EFS. These results suggest a basal release of NO from endothelial cells, which inhibits contractions mediated by the RhoA/Rho-kinase pathway and modulates the expression of proteins related to this pathway in mouse CC. It indicates that endothelial integrity is essential to the maintenance of erectile function.