The Role of OCTA in Evaluating Diabetic Retinopathy Progression: A Meta-Analysis.

Background: Diabetic retinopathy (DR) substantially threatens ocular health, necessitating the accurate and prompt assessment of its onset and progression. Optical coherence tomography angiography (OCTA) is a valuable tool for evaluating periocular microvascular indicators, offering insights crucial for diagnosing and treating DR. Objective: This meta-analysis aims to evaluate the progression of diabetic retinopathy (DR) by examining periocular microvascular indicators using optical coherence tomography angiography (OCTA). The objective is to provide substantive evidence for the future diagnosis and treatment of DR. Methods: We analyzed the relevant research retrieved from PubMed and Web of Science until January 2023. The inclusion and exclusion criteria were carefully applied to select eligible studies. Quality assessment was performed using the Newcastle-Ottawa Scale, with studies scoring 4 or less excluded. Meta-analysis was conducted using Revman 5.3 software and focused on key indicators, including peripapillary vascular length density (pVLD) and peripapillary vascular density (pVD). Heterogeneity was assessed using I2 and P values, with effect sizes determined via fixed-effect or random-effects models based on heterogeneity levels. Results: Six studies involving 839 DR-afflicted eyes and 3209 non-DR eyes were included after screening. All selected articles exhibited high reference value, with quality scores ranging from 5 to 8 points. The meta-analysis demonstrated that DR patients displayed significantly lower pVD and pVLD in the superficial (SCP) and deep capillary plexus (DCP) compared to non-DR patients (P < .05). These findings remained consistent across different effect models, reaffirming their validity. Conclusions: Patients with DR exhibit reduced levels of pVD and pVLD in the SCP and DCP compared to non-DR individuals. OCTA examination of periocular microvascular indicators emerges as an effective tool for assessing the onset and progression of DR.

Imaging-Guided Biomimetic M1 Macrophage Membrane-Camouflaged Magnetic Nanorobots for Photothermal Immunotargeting Cancer Therapy.

Biohybrid micro/nanorobots have demonstrated improved therapeutic outcomes for targeting and treating diseases in preclinical trials. However, in vivo applications remain challenging due to a lack of sufficient targeting. Based on evidence that immune cells play a role in the immune modulation in the tumor microenvironment, we developed M1 macrophage membrane-coated magnetic photothermal nanocomplexes (MPN) for photoacoustic (PA) imaging-guided tumor therapy. The MPN were able to inherit the protein from the original macrophage cells and exert a targeted immunosuppression role. Integrating black phosphorus quantum dots and DOX also greatly enhanced reactive oxygen species generation and chemo-phototherapy efficacy. The results suggest that the MPN can be employed as an excellent tumor immunotargeting nanorobotic platform for modulating the tumor microenvironment under PA imaging and magnetic guidance and, thus, exert synergistic therapeutic efficacies.

Side-Chain-Tuned Molecular Packing Allows Concurrently Boosted Photoacoustic Imaging and NIR-II Fluorescence.

It is generally considered that photoacoustic imaging (PAI) and fluorescence imaging (FLI) cannot be enhanced concurrently, as they are dependent on competitive photophysical processes at the single-molecule level. Herein, we reveal that BDTR9-OC8 and BDTR9-C8, which have identical π-conjugated backbones but are substituted by side chains of different rigidity, show distinct phototheranostic properties in the aggregated state. The NIR-II FLI and PAI brightness of BDTR9-C8 nanoparticles are enhanced by 4.6 and 1.4 times compared with BDTR9-OC8 nanoparticles. Theoretical calculations and GIWAXS analysis revealed that BDTR9-C8 with rigid side chains shows a relative amorphous condensed state, which will benefit the efficient transportation of photo-generated excitons and phonons, subsequently enhancing the FLI and PAI signals. Besides, both nanoparticles exhibit excellent photothermal conversion efficiency due to their strong light-harvesting capability and are considered effective photothermal therapy materials. This work provides an illuminating strategy for material design in the future.

Infection microenvironment-activated nanoparticles for NIR-II photoacoustic imaging-guided photothermal/chemodynamic synergistic anti-infective therapy.

Subcutaneous abscesses caused by drug-resistant bacteria pose huge challenges to human health. The design of infection microenvironment-activated biomaterials has an advantage for the diagnosis and treatment of infective diseases due to its high specificity and efficiency. Herein, a novel theranostic platform based on Cu2O nanoparticles (NPs) is successfully constructed via a simple, fast and low-cost approach. The Cu2O NPs exhibit high sensitivity to overexpressed H2S and H2O2 in the bacterial infection microenvironment. After in situ injection, the Cu2O NPs will rapidly react with the endogenous H2S to generate Cu9S8 NPs, which exhibits high absorbance in the second near-infrared (NIR-II) biowindow. The Cu9S8 NPs serving as NIR-II photoacoustic contrast agents can exactly distinguish between inflammatory and normal tissues. With the guidance of NIR-II photoacoustic imaging (PAI), H2S-activated photothermal antibacterial therapy (PTAT) can realize excellent antibacterial performance under 1060 nm laser irradiation. Meanwhile, the Cu2O NPs can effectively catalyze H2O2 at the site of inflammation to produce hydroxyl radicals with strong antibacterial property via Fenton-like reaction, resulting in the damage of bacterial cell membrane. Furthermore, the application of Cu2O NPs can enhance epidermic migration and facilitate the re-epithelialization of the infected skin. In vivo experiment shows that 97.9% methicillin-resistant Staphylococcus aureus are eliminated by the synergistic PTAT and chemodynamic antibacterial therapy.

High expression of squamous cell carcinoma antigen in poorly differentiated adenocarcinoma of the stomach: A case report.

BACKGROUND: Squamous cell carcinoma antigen (SCCA) is regarded as a specific indicator of epithelial malignancies and is widely used in the diagnosis of squamous cell carcinoma (SCC). However, the expression of SCCA in gastric adenocarcinoma has not been studied in detail. CASE SUMMARY: A 52-year-old man was admitted to our hospital for a 2.5 cm × 2.5 cm ulcer at the antrum-body junction with dull pain and fullness in the upper abdomen for 2 mo. His pre-surgery serological testing results showed 0.51 ng/mL SCCA (reference interval, < 1.5 ng/mL) and 9.9 ng/mL carcinoembryonic antigen (reference range, < 4.7 ng/mL). He underwent radical distal gastrectomy and Roux-en Y anastomosis and was diagnosed with poorly differentiated mucinous adenocarcinoma (Lauren classification: Diffuse) by pathological examination of the resected lesion. Immunohistochemistry showed that SCCA was highly expressed in the cytoplasm of cancer cells. After surgery, the patient received an S-1 adjuvant chemotherapy regimen for six cycles containing tegafur, gimeracil, and oteracil potassium. He showed no sign of recurrence or metastasis within 24-mo follow-up. CONCLUSION: This is a frontal report of SCCA overexpression in poorly differentiated adenocarcinoma of the stomach.

An All-Round Athlete on the Track of Phototheranostics: Subtly Regulating the Balance between Radiative and Nonradiative Decays for Multimodal Imaging-Guided Synergistic Therapy.

Aiming to achieve versatile phototheranostics with the integrated functionalities of multiple diagnostic imaging and synergistic therapy, the optimum use of dissipated energy through both radiative and nonradiative pathways is definitely appealing, yet a significantly challenging task. To the best of the knowledge, there have been no previous reports on a single molecular species effective at affording all phototheranostic modalities including fluorescence imaging (FLI), photoacoustic imaging (PAI), photothermal imaging (PTI), photodynamic therapy (PDT), and photothermal therapy (PTT). Herein, a simple and highly powerful one-for-all phototheranostics based on aggregation-induced emission (AIE)-active fluorophores is tactfully designed and constructed. Thanks to its strong electron donor-acceptor interaction and finely modulated intramolecular motion, the AIE fluorophore-based nanoparticles simultaneously exhibit bright near-infrared II (NIR-II) fluorescence emission, efficient reactive oxygen species generation, and high photothermal conversion efficiency upon NIR irradiation, indicating the actualization of a balance between radiative and nonradiative energy dissipations. Furthermore, the unprecedented performance on NIR-II FLI-PAI-PTI trimodal-imaging-guided PDT-PTT synergistic therapy is demonstrated by the precise tumor diagnosis and complete tumor elimination outcomes. This study thus brings a new insight into the development of superior versatile phototheranostics for practical cancer theranostics.

Mo2 C-Derived Polyoxometalate for NIR-II Photoacoustic Imaging-Guided Chemodynamic/Photothermal Synergistic Therapy.

To overcome the current limitations of chemodynamic therapy (CDT), a Mo2 C-derived polyoxometalate (POM) is readily synthesized as a new CDT agent. It permits synergistic chemodynamic and photothermal therapy operating in the second near-infrared (NIR-II) biological transparent window for deep tissue penetration. POM aggregated in an acidic tumor micro-environment (TME) whereby enables specific tumor targeting. In addition to the strong ability to produce singlet oxygen (1 O2 ) presumably via Russell mechanism, its excellent photothermal conversion enhances the CDT effect, offers additional tumor ablation modality, and permits NIR-II photoacoustic imaging. Benefitting from the reversible redox property of molybdenum, the theranostics based on POM can escape from the antioxidant defense system. Moreover, combining the specific responsiveness to TME and localized laser irradiation, side-effects shall be largely avoided.

Effects of intermittent hypoxia training on leukocyte pyruvate dehydrogenase kinase 1 (PDK-1) mRNA expression and blood insulin level in prediabetes patients.

PURPOSE: Intermittent hypoxia training/treatment (IHT) is an emerging therapeutic approach to alleviate chronic diseases, such as diabetes. The present study investigated the effects of IHT on blood leucocyte pyruvate dehydrogenase kinase 1 (PDK-1) mRNA expression and its relationship with the changes in blood insulin level. METHODS: Seven adult healthy volunteers and 11 prediabetic patients participated in this study. A 3-week course of IHT consisted of a 40-min session of 4 cycles of 5-min 12% O2 and 5-min room air breathing per day, 3 sessions per week for 3 weeks (i.e., total 9 sessions of IHT). Plasma insulin levels and leukocyte PDK-1 mRNA expression were determined at various time points either under fasting condition or following oral glucose tolerance test (OGTT). Correlation between the IHT-induced changes in PDK-1 mRNA and insulin or glucose levels in the same serological samples was analyzed. RESULTS: At pre-IHT baseline, PDK-1 mRNA expression was two times higher in prediabetes than control subjects. IHT resulted in significant augmentation in PDK-1 mRNA expression (> twofold) in prediabetes at the end of 3-week IHT and remained elevated 1 month after IHT, which was correlated with a significantly reduced insulin release and lower blood glucose after glucose loading with OGTT. CONCLUSION: IHT can trigger beneficial effects in normalizing blood insulin levels in prediabetic patients under oral glucose load, which were closely correlated with an enhanced mRNA expression of PDK-1 in leukocytes. Further clinical trials are warranted to validate the utility of IHT as a non-invasive complementary therapy against diabetes-associated pathologies.

Cluster analysis of Dendrobium officinale from different populations through agronomy characterization / 中国中药杂志

Through resources investigation and sample collection,a total number of 392 Dendrobium officinale from 38 different populations,9 provinces were processed for measuring and observing.Fourteen agronomy characterizations like stem height,stem diameter,number of node in stem were selected for further classification.The cluster analysis was performed using Ward and Euclidean method.The results showed that the threshold of genetic distance was 2.77.Thirtyeight populations were divided into 6 groups.The factor analysis showed that,the leaf shape,stem shape,pitch length and leaf color were very important factors for classification.This study establish the foundation for analyzing the genetic relationship of D.officinale from different populations.

Potential Therapeutic Strategies for Hypertension-Exacerbated Cardiotoxicity of Anticancer Drugs.

Despite their recognized cardiotoxic effects, anthracyclines remain an essential component in many anticancer regimens due to their superior antitumor efficacy. Epidemiologic data revealed that about one-third of cancer patients have hypertension, which is the most common comorbidity in cancer registries. The purpose of this review is to assess whether anthracycline chemotherapy exacerbates cardiotoxicity in patients with hypertension. A link between hypertension comorbidity and anthracycline-induced cardiotoxicity (AIC) was first suggested in 1979. Subsequent preclinical and clinical studies have supported the notion that hypertension is a major risk factor for AIC, along with the cumulative anthracycline dosage. There are several common or overlapping pathological mechanisms in AIC and hypertension, such as oxidative stress. Current evidence supports the utility of cardioprotective modalities as adjunct treatment prior to and during anthracycline chemotherapy. Several promising cardioprotective approaches against AIC pathologies include dexrazoxane, early hypertension management, and dietary supplementation of nitrate with beetroot juice or other medicinal botanical derivatives (e.g., visnagin and Danshen), which have both antihypertensive and anti-AIC properties. Future research is warranted to further elucidate the mechanisms of hypertension and AIC comorbidity and to conduct well-controlled clinical trials for identifying effective clinical strategies to improve long-term prognoses in this subgroup of cancer patients.

Intermittent hypoxia training as non-pharmacologic therapy for cardiovascular diseases: Practical analysis on methods and equipment.

The global industrialization has brought profound lifestyle changes and environmental pollutions leading to higher risks of cardiovascular diseases. Such tremendous challenges outweigh the benefits of major advances in pharmacotherapies (such as statins, antihypertensive, antithrombotic drugs) and exacerbate the public healthcare burdens. One of the promising complementary non-pharmacologic therapies is the so-called intermittent hypoxia training (IHT) via activation of the human body's own natural defense through adaptation to intermittent hypoxia. This review article primarily focuses on the practical questions concerning the utilization of IHT as a non-pharmacologic therapy against cardiovascular diseases in humans. Evidence accumulated in the past five decades of research in healthy men and patients has suggested that short-term daily sessions consisting 3-4 bouts of 5-7 min exposures to 12-10% O2 alternating with normoxic durations for 2-3 weeks can result in remarkable beneficial effects in treatment of cardiovascular diseases such as hypertension, coronary heart disease, and heart failure. Special attentions are paid to the therapeutic effects of different IHT models, along with introduction of a variety of specialized facilities and equipment available for IHT, including hypobaric chambers, hypoxia gas mixture deliver equipment (rooms, tents, face masks), and portable rebreathing devices. Further clinical trials and thorough evaluations of the risks versus benefits of IHT are much needed to develop a series of standardized and practical guidelines for IHT. Taken together, we can envisage a bright future for IHT to play a more significant role in the preventive and complementary medicine against cardiovascular diseases.

Beetroot juice reduces infarct size and improves cardiac function following ischemia-reperfusion injury: Possible involvement of endogenous H2S.

Ingestion of high dietary nitrate in the form of beetroot juice (BRJ) has been shown to exert antihypertensive effects in humans through increasing cyclic guanosine monophosphate (cGMP) levels. Since enhanced cGMP protects against myocardial ischemia-reperfusion (I/R) injury through upregulation of hydrogen sulfide (H2S), we tested the hypothesis that BRJ protects against I/R injury via H2S. Adult male CD-1 mice received either regular drinking water or those dissolved with BRJ powder (10 g/L, containing ∼ 0.7 mM nitrate). Seven days later, the hearts were explanted for molecular analyses. Subsets of mice were subjected to I/R injury by occlusion of the left coronary artery for 30 min and reperfusion for 24 h. A specific inhibitor of H2S producing enzyme--cystathionine-γ-lyase (CSE), DL-propargylglycine (PAG, 50 mg/kg) was given i.p. 30 min before ischemia. Myocardial infarct size was significantly reduced in BRJ-fed mice (15.8 ± 3.2%) versus controls (46.5 ± 3.5%, mean ± standard error [SE], n = 6/group, P < .05). PAG completely blocked the infarct-limiting effect of BRJ. Moreover, BRJ significantly preserved ventricular function following I/R. Myocardial levels of H2S and its putative protein target--vascular endothelial growth factor receptor 2 (VEGFR2) were significantly increased by BRJ intake, whereas CSE mRNA and protein content did not change. Interestingly, the BRJ-induced cardioprotection was not associated with elevated blood nitrate-nitrite levels following I/R nor induction of cardiac peroxiredoxin 5, a mitochondrial antioxidant enzyme previously linked to nitrate-induced cardioprotection. We conclude that BRJ ingestion protects against post-I/R myocardial infarction and ventricular dysfunction possibly through CSE-mediated endogenous H2S generation. BRJ could be a promising natural and inexpensive nutraceutical supplement to reduce cardiac I/R injury in patients.

Human enteroids as an ex-vivo model of host-pathogen interactions in the gastrointestinal tract.

Currently, 9 out of 10 experimental drugs fail in clinical studies. This has caused a 40% plunge in the number of drugs approved by the US Food and Drug Administration (FDA) since 2005. It has been suggested that the mechanistic differences between human diseases modeled in animals (mostly rodents) and the pathophysiology of human diseases might be one of the critical factors that contribute to drug failure in clinical trials. Rapid progress in the field of human stem cell technology has allowed the in-vitro recreation of human tissue that should complement and expand upon the limitations of cell and animal models currently used to study human diseases and drug toxicity. Recent success in the identification and isolation of human intestinal epithelial stem cells (Lgr5(+)) from the small intestine and colon has led to culture of functional intestinal epithelial units termed organoids or enteroids. Intestinal enteroids are comprised of all four types of normal epithelial cells and develop a crypt-villus differentiation axis. They demonstrate major intestinal physiologic functions, including Na(+) absorption and Cl(-) secretion. This review discusses the recent progress in establishing human enteroids as a model of infectious diarrheal diseases such as cholera, rotavirus, and enterohemorrhagic Escherichia coli, and use of the enteroids to determine ways to correct the diarrhea-induced ion transport abnormalities via drug therapy.

Biflavanones with anti-proliferative activity against eight human solid tumor cell lines from Stellera chamaejasme.

Six 3,3″-biflavanones, including a new compound isochamaejasmenin C (1), were isolated from EtOH extracts of the roots of Stellera chamaejasme L. Their structures were elucidated on the basis of spectroscopic methods, including HR-ESI-MS and 2D NMR techniques. The absolute configurations on 2, 3, 3″, and 2″ of compounds 1, 3, and 6, which represent three geometry types of these compounds, were determined by TDDFT quantum chemical calculations of their ECD spectra. All compounds were evaluated for their cytotoxicities against eight human solid tumor cell lines and compounds 2, 4, and 6 showed strong anti-proliferative effects against all these cell lines with IC50 values ranging from 1.08 to 15.97 µM, which were in the same range as the positive control paclitaxel.

Dietary nitrate supplementation protects against Doxorubicin-induced cardiomyopathy by improving mitochondrial function.

OBJECTIVES: The aim of this study was to test the hypothesis that long-term dietary nitrate supplementation protects against doxorubicin-induced cardiomyopathy by improving ventricular function and reducing mitochondrial respiratory chain damage. BACKGROUND: Doxorubicin is a powerful anthracycline antibiotic used to treat divergent human neoplasms. Its clinical use is limited because of severe cardiotoxic side effects. Dietary nitrate and nitrite are essential nutrients for maintenance of steady-state tissue levels of nitric oxide and may play a therapeutic role in diseases associated with nitric oxide insufficiency or dysregulation. Dietary nitrate and nitrite supplementation alleviates myocardial injury caused by ischemia-reperfusion and cardiac arrest-resuscitation. METHODS: Adult male CF-1 mice were given a single dose of doxorubicin (15 mg/kg intraperitoneally), and left ventricular contractile function was assessed 5 days later using both echocardiography and pressure-volume Millar catheterization. A nitrate supplementation regimen (1 g/l sodium nitrate in drinking water) was started 7 days before doxorubicin injection and continued thereafter. Cardiomyocyte necrosis and apoptosis, tissue lipid peroxidation, and plasma nitrate and nitrite levels were assessed. In addition, mitochondrial complex I activity, oxidative phosphorylation capacity, and hydrogen peroxide generation were determined in parallel experiments. RESULTS: Doxorubicin caused impairment of ventricular contractility and cell death, which were significantly reduced by nitrate supplementation (p < 0.05). These cardioprotective effects were associated with a significant decrease in tissue lipid peroxidation. Nitrate supplementation significantly preserved mitochondrial complex I activity and oxidative phosphorylation and attenuated hydrogen peroxide generation after doxorubicin treatment. CONCLUSIONS: Long-term oral intake of inorganic nitrate attenuates doxorubicin-induced ventricular dysfunction, cell death, oxidative stress, and mitochondrial respiratory chain damage. Nitrate could be a promising therapeutic agent against doxorubicin-induced cardiotoxicity.

Identification of protein targets underlying dietary nitrate-induced protection against doxorubicin cardiotoxicity.

We recently demonstrated protective effect of chronic oral nitrate supplementation against cardiomyopathy caused by doxorubicin (DOX), a highly effective anticancer drug. The present study was designed to identify novel protein targets related to nitrate-induced cardioprotection. Adult male CF-1 mice received cardioprotective regimen of nitrate (1 g NaNO(3) per litre of drinking water) for 7 days before DOX injection (15 mg/kg, i.p.) and continued for 5 days after DOX treatment. Subsequently the heart samples were collected for proteomic analysis with two-dimensional differential in-gel electrophoresis with 3 CyDye labelling. Using 1.5 cut-off ratio, we identified 36 proteins that were up-regulated by DOX in which 32 were completely reversed by nitrate supplementation (89%). Among 19 proteins down-regulated by DOX, 9 were fully normalized by nitrate (47%). The protein spots were further identified with Matrix Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF)/TOF tandem mass spectrometry. Three mitochondrial antioxidant enzymes were altered by DOX, i.e. up-regulation of manganese superoxide dismutase and peroxiredoxin 3 (Prx3), and down-regulation of Prx5, which were reversed by nitrate. These results were further confirmed by Western blots. Nitrate supplementation also significantly improved animal survival rate from 80% in DOX alone group to 93% in Nitrate + DOX group 5 days after the DOX treatment. In conclusion, the proteomic analysis has identified novel protein targets underlying nitrate-induced cardioprotection. Up-regulation of Prx5 by nitrate may explain the observed enhancement of cardiac antioxidant defence by nitrate supplementation.

Silencing heat shock factor 1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia-reperfusion injury in mice.

Induction of heat shock factor 1 (HSF1) is known to associate with cellular response to divergent pathophysiological stresses including whole body hyperthermia (WBH) and ischemia-reperfusion. However, a direct cause-effect relationship between HSF1 activation and cytoprotection induced by myocardial preconditioning has not been conclusively established, mainly due to the limitations of available experiment tools. In the present studies, we used a novel approach to block HSF1 with small interfering RNA (siRNA) technique in vivo. Male adult ICR mice were treated intraperitoneally with amine (vehicle) or siRNA specific to HSF1 (siRNA-HSF1). Three days later, WBH preconditioning protocol (rectal temperature 42 degrees C for 15 min) was applied to these mice under light anesthesia. WBH preconditioning resulted in 2.7-fold and 3.4-fold increase in cardiac HSF1 mRNA and protein expression respectively 2 hours after WBH, which was inhibited in the siRNA-treated mice. The silencing effect of siRNA on HSF1 was associated with complete loss of the infarct- limiting protection by WBH preconditioning after 48 hours. Pretreatment with siRNA-HSF1 had no effect on infarct size in the sham control animals as compared with the amine-treated group. DNA micro-array analysis revealed that siRNA-HSF1 caused a general inhibition on multiple members of HSP family, except Hsp32, Hsp47 and Hsp60. In addition, the silencing effect of siRNA on HSF1 and HSPs gene expression was transient and its inhibitory effect disappeared by 10 days after treatment. siRNA-HSF1 also impaired the thermotolerance of the heat shocked mice as indicated by higher mortality following WBH. For the first time, we have applied siRNA technique in the field of myocardial preconditioning to demonstrate HSF1 activation as an essential step in WBH preconditioning against cardiac ischemia-reperfusion injury.

Phosphodiesterase-5 inhibitor sildenafil preconditions adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide signaling.

We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 microM) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion.