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China passed a landmark health care reform in 2009, aimed at improving health care for all citizens by strengthening the primary care system, largely through improvements to infrastructure. However, research has shown that the work attitudes of primary care physicians (PCPs) can greatly affect the stability of the overall workforce and the quality and delivery of health care. The purpose of this study is to investigate the relationship between reported work attitudes of PCPs and their personal, work, and educational characteristics. A multi-stage, complex sampling design was employed to select a sample of 434 PCPs practicing in urban and rural primary care settings, and a survey questionnaire was administered by researchers with sponsorship from the Ministry of Health. Four outcome measures describing work attitudes were used, as well as a number of personal-, work-, and practice-related factors. Findings showed that although most PCPs considered their work as important, a substantial number also reported large workloads, job pressure, and turnover intentions. Findings suggest that policymakers should focus on training and educational opportunities for PCPs and consider ways to ease workload pressures and improve salaries. These policy improvements must accompany reform efforts that are already underway before positive changes in reduced disparities and improved health outcomes can be realized in China.

Objective: Levobupivacaine, an amide local anesthetic widely used in regional anesthesia, is reported in recent studies that it is a potent inhibitor of platelet functions. However, the concentrations of levobupivacaine were limitedly estimated in these reports. Additionally, the mechanisms by which it affects platelet function and blood coagulation is still not entirely known. The purpose of this study was to further investigate its effects on platelet function and the possible signaling mechanisms under various concentrations of levobupivacaine., Methods: Blood samples collected from healthy volunteers were separated into whole blood, platelet-rich-plasma and washed platelets. The effect of levobupivacaine on platelet aggregation was studied using platelet function analyzer (PFA-100) and platelet aggregometer. Agonist-induced platelet adenosine triphosphate (ATP) release, cytosolic calcium mobilization, thromboxane B2 (TxB2) secretion and platelet P-selectin translocation under various concentrations of levobupivacaine were investigated., Results: Our results indicated that levobupivacaine possessed negative effect on platelet aggregation. The closure times of (PFA-100) were lengthened and the agonist-induced platelet aggregation was significantly attenuated by levobupivacaine even at a low dose (50 μgml(-1)). Pretreatment with levobupivacaine produced significant changes in agonist-induced platelet P-selectin translocation, ATP release, thromboxane A2 (TxA2) production, and calcium mobilization in a dose-dependent manner. The p38 mitogen-activated protein kinases (MAPK), protein kinase C (PKC) δ subtype, cytosolic phospholipase A2 (cPLA2), and protein kinase B (PKB or Akt) were involved in collagen-induced platelet signaling, which would be responsible for antiplatelet effects of levobupivacaine., Conclusion: We explored possible targets of levobupivacaine on platelets aggregation signaling mechanisms. Our data revealed that p38 MAPK, PKC δ subtype, cPLA2, and Akt were pathways involved in collagen-induced platelet signaling, which might be responsible for antiplatelet effects of levobupivacaine. Our study did provide direct evidence bolstering the critical mechanisms of levobupivacaine within different contexts. Additionally, levobupivacaine imposed a negative effect on platelet aggregation through multiple signaling pathways., (Copyright © 2012. Published by Elsevier B.V.)

"It has been well known that both oxidative stress and inflammatory activity play crucial roles in the pathogenesis of type 1 diabetes mellitus (T1DM). Resveratrol (RSV), a naturally occurring polyphenol found in grapes and red wine, has recently been shown to exert potent anti-diabetic, anti-oxidative and anti-inflammatory actions. In the present study, we investigated the effect of RSV on oxidative stress and inflammatory response in the liver and spleen of streptozotocin (STZ)-induced type 1 diabetic animal models. Male Long-Evans rats were injected with 65 mg/kg STZ to induce diabetes for 2 weeks, and subsequently administrated with the dosage of 0.1 or 1 mg/kg/day RSV for 7 consecutive days. Hepatic and splenic tissues were dissected for evaluation of oxidative and inflammatory stress. Oxidative stress was assessed by quantification of oxidative indicators including superoxide anion content, lipid and protein oxidative products, as well as manganese superoxide dismutase (Mn-SOD) and nitro-tyrosine protein expression levels. Inflammatory stress was evaluated by the levels of nuclear factor κB (NF-κB) and the proinflammatory cytokine tumor necrosis factorα (TNF-α), interleukin 1 β (IL-1 β ) and IL-6. The experimental results indicated that RSV significantly decreased oxidative stress (superoxide anion content, protein carbonyl level and Mn-SOD expression) in both tissues and hepatic inflammation (NF- κB and IL-1 β ), but implicated proinflammatory potential of RSV in diabetic spleen (TNF-α and IL-6). The results of this study suggest that RSV may serve as a potent antioxidant, but RSV possesses a proinflammatory potential in certain circumstances in diabetes."

China is a country with vast regional differences and uneven economic development, which have led to widening gaps between the rich and poor in terms of access to healthcare, quality of care, and health outcomes. China's healthcare reform efforts must be tailored to the needs and resources of each region and community. Building and strengthening primary care within the Chinese health care system is one way to effectively address health challenges. This paper begins by outlining the concept of primary care, including key definitions and measurements. Next, results from a number of studies will demonstrate that primary care characteristics are associated with savings in medical costs, improvements in health outcomes and reductions in health disparities. This paper concludes with recommendations for China on successfully incorporating a primary care model into its national health policy, including bolstering the primary care workforce, addressing medical financing structures, recognizing the importance of evidence-based medicine, and looking to case studies from countries that have successfully implemented health reform.

The pathogenesis of lupus nephritis is mainly attributable to a complex interaction between the innate and adaptive immune systems, including T and B cell function abnormalities. In addition to autoantibody production and immune complex deposition, Th1 and Th17 cytokines may play key roles in the development and progression of lupus nephritis. Acute onset of severe lupus nephritis remains a challenge in terms of prevention and treatment. In the present study, we evaluated the therapeutic effects of DCB-SLE1, an extract of a mixture of four traditional Chinese medicinal herbs (Atractylodis macrocephalae Rhizoma, Eucommiae cortex, Lonicerae caulis, and Hedyotidis diffusae Herba), on an accelerated severe lupus nephritis model, characterized by acute onset of proteinuria, azotemia, autoantibody production, and development of severe nephritis, induced by twice weekly injection of New Zealand black/white F1 mice with Salmonella-type lipopolysaccharide. DCB-SLE1 was administered daily by gavage starting 2 days after the first dose of induction of lipopolysaccharide, and the mice were euthanized at week 1 or week 5. The results showed that DCB-SLE1 significantly ameliorated the hematuria, proteinuria, renal dysfunction, and severe renal lesions by 1) suppression of B cell activation and decreased autoantibody production; 2) negative regulation of T cell activation/proliferation and natural killer cell activity; 3) suppression of IL-18, IL-6, and IL-17 production and blocking of NF-κB activation in the kidney; and 4) prevention of lymphoid and renal apoptosis. These results show that DCB-SLE1 can protect the kidney from autoimmune response-mediated acute and severe damage through systemic immune modulation and anti-inflammation pathways.

Background: Diabetic nephropathy (DN) has been recognized as the leading cause of end-stage renal disease. Resveratrol (RSV), a polyphenolic compound, has been indicated to possess an insulin-like property in diabetes. In the present study, we aimed to investigate the renoprotective effects of RSV and delineate its underlying mechanism in early-stage DN., Methods: The protective effects of RSV on DN were evaluated in streptozotocin (STZ)-induced diabetic rats., Results: The plasma glucose, creatinine, and blood urea nitrogen were significantly elevated in STZ-induced diabetic rats. RSV treatment markedly ameliorated hyperglycemia and renal dysfunction in STZ-induced diabetic rats. The diabetes-induced superoxide anion and protein carbonyl levels were also significantly attenuated in RSV-treated diabetic kidney. The AMPK protein phosphorylation and expression levels were remarkably reduced in diabetic renal tissues. In contrast, RSV treatment significantly rescued the AMPK protein expression and phosphorylation compared to non-treated diabetic group. Additionally, hyperglycemia markedly enhanced renal production of proinflammatory cytokine IL-1β. RSV reduced IL-1β but increased TNF-α and IL-6 levels in the diabetic kidneys., Conclusions: Our findings suggest that RSV protects against oxidative stress, exhibits concurrent proinflammation and anti-inflammation, and up-regulates AMPK expression and activation, which may contribute to its beneficial effects on the early stage of DN.

Antrodia camphorata is used in folk medicine for the treatment of inflammation syndromes and liver-related diseases in Taiwan. The goal of this study was to evaluate the efficacy of the mycelial extract of A. camphorata (ACE) for the treatment of systemic lupus erythematosus (SLE) in SLE-prone NZB/W F1 mice. After antibodies against double-stranded DNA appeared in NZB/W mice, the mice were orally administered varying dosages of ACE (100, 200 and 400 mg kg(-1)) for 5 consecutive days per week for 12 weeks via gavage. To assess the efficacy of ACE, we measured SLE-associated biochemical and histopathological biomarkers levels of blood urine nitrogen (BUN), blood creatinine, urine protein and urine creatinine and thickness of the kidney glomerular basement membrane by staining with periodic acid-Schiff. Antroquinonol, an active component of ACE, was investigated for anti-inflammation activity in lipopolysaccharide-induced RAW 267.4 cells. ACE at 400 mg kg(-1) significantly suppressed urine protein and serum BUN levels and decreased the thickness of the kidney glomerular basement membrane. Antroquinonol significantly inhibited the production of tumor necrosis factor-α and interleukin-1β by 75 and 78%, respectively. In conclusion, ACE reduced urine protein and creatinine levels and suppressed the thickening of the kidney glomerular basement membrane, suggesting that ACE protects the kidney from immunological damage resulting from autoimmune disease.

Carthamus tinctorius (CT), also named safflower, is a traditional Chinese medicine widely used to improve blood circulation. CT also has been studied for its antitumor activity in certain cancers. To investigate the effects of CT on the dendritic cell (DC)-based vaccine in cancer treatment, cytokine secretion of mouse splenic T lymphocytes and the maturation of DCs in response to CT were analyzed. To assess the antitumor activity of CT extract on mouse CD117(+) (c-kit)-derived DCs pulsed with JC mammal tumor antigens, the JC tumor was challenged by the CT-treated DC vaccine in vivo. CT stimulated IFN-γ and IL-10 secretion of splenic T lymphocytes and enhanced the maturation of DCs by enhancing immunological molecule expression. When DC vaccine was pulsed with tumor antigens along with CT extract, the levels of TNF-α and IL-1β were dramatically increased with a dose-dependent response and more immunologic and co-stimulatory molecules were expressed on the DC surface. In addition, CT-treated tumor lysate-pulsed DC vaccine reduced the tumor weight in tumor-bearing mice by 15.3% more than tumor lysate-pulsed DC vaccine without CT treatment. CT polarized cytokine secretion toward the Th1 pathway and also increased the population of cytotoxic T lymphocytes ex vivo. In conclusion, CT activates DCs might promote the recognition of antigens and facilitate antigen presentation to Th1 immune responses.

Background: Antioxidants effectively reduce ischemia-reperfusion (IR) injury. The cardioprotective effects of luteolin, a flavonoid that exhibits antioxidant properties and is widely available in many fruits and vegetables, were examined in rats subjected to myocardial IR injury., Methods and Results: Rats were subjected to myocardial ischemia or reperfusion injury to evaluate the antiarrhythmic effects of luteolin. Myocardial infarct size was determined histochemically with triphenyltetrazolium chloride staining of the left ventricle. Luteolin was administered intravenously 15min before occlusion of the coronary artery. The incidence and duration of ventricular tachycardia and ventricular fibrillation and mortality during myocardial ischemia were significantly reduced by luteolin (10µg/kg). Similarly, luteolin (1µg/kg) reduced ventricular arrhythmias and mortality during the reperfusion phase. Pretreatment with luteolin decreased plasma lactate dehydrogenase and nitric oxide (NO) levels. Luteolin (10µg/kg) significantly reduced the myocardial infarct size, as well as malondialdehyde production in tissue samples of myocardial IR injury. Luteolin also downregulated inducible NO synthase protein and mRNA expression, but did not significantly alter neuronal NO synthase or endothelial NO synthase expression., Conclusions: Luteolin is capable of protecting the myocardium against IR injury. The actions of luteolin are at least partly mediated through downregulation of NO production and its own antioxidant properties.

Objectives: The nitric oxide (NO)-soluble guanylate cyclase (sGC) signalling pathway is attributed to the prevention of ischaemia-reperfusion (I/R)-induced leucocyte-endothelium adhesive interactions. YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole), a NO-independent sGC activator, has been shown to exert cardiovascular benefits, but its action on leucocyte-endothelium interactions remains unknown. In this study, the direct effect and the underlying mechanism of the anti-adhesive action of YC-1 have been examined in cremasteric microcirculation., Methods: Rat cremaster muscle was subjected to 4 h pudic-epigastric artery ischaemia followed by 2 h reperfusion and intravital microscopy was used to observe leucocyte-endothelium interaction and to quantify functional capillaries in rat cremaster muscle flaps., Key Findings: The values for leucocyte rolling, adhering and transmigrating were 5.5-, 6.9- and 8.8-fold greater, respectively, in I/R than in sham-control animals. YC-1 treatment rescued functional capillary density and reduced leucocyte rolling, adhering and transmigrating in I/R injured cremaster muscles to levels observed in sham-controls. Interestingly, these effects were completely blocked by the MEK (extracellular signal-regulated kinase (ERK) kinase) inhibitor (PD98059) but not by sGC or protein kinase C inhibitors. Cotreatment of PD98059 with YC-1 caused a 3.3-, 7.5- and 8.3-fold increase in the values for leucocyte rolling, adhering and transmigrating, respectively, in postcapillary venules of I/R-injured cremaster muscle., Conclusions: This study has indicated that the anti-adhesive and functional capillary density rescue properties of YC-1 were mediated predominantly by the activation of ERK but not sGC, although YC-1 was identified to be a sGC activator. A better understanding of the action of YC-1 on the microvasculature may help shed light on its therapeutic potential for cardiovascular disease., (© 2010 The Authors. JPP © 2010 Royal Pharmaceutical Society of Great Britain.)

Resveratrol (RSV), a natural phenolic compound, has been found to display cardiovascular protective and insulin-sensitizing properties. In this study, the effects of RSV and its combination with insulin on mortality, hemodynamics, insulin signaling, and nitrosative stress were compared in streptozotocin (STZ)-induced diabetic rats with or without acute myocardial ischemia/reperfusion (I/R) injury. Under normoxic conditions, cardiac systolic and diastolic functions and insulin-mediated Akt/GLUT4 (glucose transporter 4) activation were impaired in STZ-diabetic rats. The combination of RSV and insulin significantly prevented the above diabetes-associated abnormalities. Notwithstanding that, the diabetic state rendered the animals more susceptible to myocardial I/R injury, and the mortality rate and inducible nitric oxide synthase (iNOS)/nitrotyrosine protein expression and superoxide anion production were also further increased in I/R-injured diabetic hearts. In contrast, RSV treatment alone resulted in a lower mortality rate (from 62.5 to 18%) and better cardiac systolic function than its combination with insulin. RSV also inhibited iNOS/nitrotyrosine protein overexpression and superoxide anion overproduction in I/R-injured diabetic myocardium. Hyperglycemia, impairment of insulin signaling, overexpression of iNOS/nitrotyrosine, and superoxide anion overproduction were markedly rescued by the combination treatment, which did not show an improvement in mortality rate (30%) or cardiac performance over RSV treatment alone. These results indicate that insulin and RSV synergistically prevented cardiac dysfunction in diabetes and this may be in parallel with activation of the insulin-mediated Akt/GLUT4 signaling pathway. Although activation of the protective signal (Akt/GLUT4) and suppression of the adverse markers (iNOS, nitrotyrosine, and superoxide anion) were simultaneously observed in insulin and RSV combination treatment, insulin counteracted the advantage of RSV in diabetics with acute heart attack., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Boehmeria nivea extract (BNE) is widely used in southern Taiwan as a folk medicine for hepato-protection and hepatitis treatment. In previous studies, we demonstrated that BNE could reduce the supernatant hepatitis B virus (HBV) DNA in HBV-producing HepG2 2.2.15 cells. In the present study, we established an animal model of HBV viremia and used it to validate the efficacy of BNE in vivo. In this animal model, serum HBV DNA and HBsAg were elevated in accordance with tumor growth. To evaluate the anti-HBV activity of BNE, HBV-viremia mice were built up after one subcutaneous inoculation of HepG2 2.2.15 tumor cells in severe combined immunodeficiency mice over 13 days. The levels of serum HBV DNA were elevated around 10(5)-10(6) copies per milliliter. Both oral and intraperitoneal administration of BNE were effective at inhibiting the production of HBsAg and HBV DNA, whereas tumor growth was not affected by all test articles. Intraperitoneal administration of BNE appeared to have greater potential to inhibit serum HBV DNA levels compared with oral administration under the same dosage. Notably, reduced natural killer cell activity was also observed after high dosage of BNE administration, and this correlated with reduced serum HBV DNA. In conclusion, BNE exhibited potential anti-HBV activity in an animal model of HBV viremia.

Objectives: Based on a recent patient survey from Taiwan, where there is universal health insurance coverage and unrestricted physician choice, this study examined the relationship between physician specialty and the quality of primary medical care experiences., Methods: We assessed ambulatory patients' experiences with medical care using the Primary Care Assessment Tool, representing 7 primary care domains: first contact (ie, accessibility and utilization); longitudinality (ie, ongoing care); coordination (ie, referrals and information systems); comprehensiveness (ie, services available and provided); family centeredness; community orientation; and cultural competence., Results: Having a primary care physician was significantly associated with patients reporting higher quality of primary care experiences. Specifically, relative to specialty care physicians, primary care physicians enhanced accessibility, achieved better community orientation and cultural competence, and provided more comprehensive services., Conclusions: In an area with universal health insurance and unrestricted physician choice, ambulatory patients of primary care physicians rated their medical care experiences as superior to those of patients of specialists. In addition to providing health insurance coverage, promoting primary care should be included as a health policy to improve patients' quality of ambulatory medical care experiences.

Plectranthus amboinicus (P. amboinicus) is a folk herb that is used to treat inflammatory diseases or swelling symptoms in Taiwan. We investigated therapeutic efficacy of P. amboinicus in treating Rheumatoid Arthritis (RA) using collagen-induced arthritis animal model. Arthritis was induced in Lewis rats by immunization with bovine type II collagen. Serum anti-collagen IgG, IgM and C-reactive protein (CRP) were analyzed. To understand the inflammation condition of treated animals, production of TNF-α, IL-6 and IL-1β from peritoneal exudates cells (PEC) were also analyzed. P. amboinicus significantly inhibited the footpad swelling and arthritic symptoms in collagen-induced arthritic rats, while the serum anti-collagen IgM and CRP levels were consistently decreased. The production of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β were also decreased in the high dosage of P. amboinicus group. Here, we demonstrate the potential anti-arthritic effect of P. amboinicus for treating RA, which might confer its anti-rheumatic activity. This differs the pharmacological action mode of indomethacin.

The Electronic Surveillance System for the Early Notification of Community-Based Epidemics (ESSENCE) enables health care practitioners to detect and monitor health indicators of public health importance. ESSENCE is used by public health departments in the National Capital Region (NCR); a cross-jurisdictional data sharing agreement has allowed cooperative health information sharing in the region since 2004. Emergency department visits for influenza-like illness (ILI) in the NCR from 2008 are compared to those of 2009. Important differences in the rates, timing, and demographic composition of ILI visits were found. By monitoring a regional surveillance system, public health practitioners had an increased ability to understand the magnitude and character of different ILI outbreaks. This increased ability provided crucial community-level information on which to base response and control measures for the novel 2009 H1N1 influenza outbreak. This report underscores the utility of automated surveillance systems in monitoring community-based outbreaks. There are several limitations in this study that are inherent with syndrome-based surveillance, including utilizing chief complaints versus confirmed laboratory data, discerning real disease versus those healthcare-seeking behaviors driven by panic, and reliance on visit counts versus visit rates.

In this study, we established systemic in-vivo evidence from molecular to organism level to explain how diabetes can aggravate myocardial ischemia-reperfusion (I/R) injury and revealed the role of insulin signaling (with specific focus on Akt/GLUT4 signaling molecules). The myocardial I/R injury was induced by the left main coronary artery occlusion for 1 hr and then 3 hr reperfusion in control, streptozotocin (STZ)-induced insulinopenic diabetes, and insulin-treated diabetic rats. The diabetic rats showed a significant decrease in heart rate, and a prolonged isovolumic relaxation (tau) which lead to decrease in cardiac output (CO) without changing total peripheral resistance (TPR). The phosphorylated Akt and glucose transporter 4 (GLUT 4) protein levels were dramatically reduced in both I/R and non-I/R diabetic rat hearts. Insulin treatment in diabetes showed improvement of contractile function as well as partially increased Akt phosphorylation and GLUT 4 protein levels. In the animals subjected to I/R, the mortality rates were 25%, 65%, and 33% in the control, diabetic, and insulin-treated diabetic group respectively. The I/R-induced arrhythmias and myocardial infarction did not differ significantly between the control and the diabetic groups. Consistent with its anti-hyperglycemic effects, insulin significantly reduced I/R-induced arrhythmias but had no effect on I/R-induced infarctions. Diabetic rat with I/R exhibited the worse hemodynamic outcome, which included systolic and diastolic dysfunctions. Insulin treatment only partially improved diastolic functions and elevated P-Akt and GLUT 4 protein levels. Our results indicate that cardiac contractile dysfunction caused by a defect in insulin-stimulated Akt/GLUT4 may be a major reason for the high mortality rate in I/R injured diabetic rats.

Background and Purpose: Piceatannol is more potent than resveratrol in free radical scavenging in association with antiarrhythmic and cardioprotective activities in ischaemic-reperfused rat hearts. The present study aimed to investigate the antiarrhythmic efficacy and the underlying ionic mechanisms of piceatannol in rat hearts., Experimental Approach: Action potentials and membrane currents were recorded by the whole-cell patch clamp techniques. Fluo-3 fluorimetry was used to measure cellular Ca2+ transients. Antiarrhythmic activity was examined from isolated Langendorff-perfused rat hearts., Key Results: In rat ventricular cells, piceatannol (3-30 micromol.L(-1)) prolonged the action potential durations (APDs) and decreased the maximal rate of upstroke (V(max)) without altering Ca2+ transients. Piceatannol decreased peak I(Na) and slowed I(Na) inactivation, rather than induced a persistent non-inactivating current, which could be reverted by lidocaine. Resveratrol (100 micromol.L(-1)) decreased peak I(Na) without slowing I(Na) inactivation. The inhibition of peak I(Na) or V(max) was associated with a negative shift of the voltage-dependent steady-state I(Na) inactivation curve without altering the activation threshold. At the concentrations more than 30 micromol.L(-1), piceatannol could inhibit I(Ca,L), I(to), I(Kr), Ca2+ transients and Na+-Ca2+ exchange except I(K1). Piceatannol (1-10 micromol.L(-1)) exerted antiarrhythmic activity in isolated rat hearts subjected to ischaemia-reperfusion injury., Conclusions and Implications: The additional hydroxyl group on resveratrol makes piceatannol possessing more potent in I(Na) inhibition and uniquely slowing I(Na) inactivation, which may contribute to its antiarrhythmic actions at low concentrations less than 10 micromol.L(-1).

Introduction: Public health surveillance systems need to be refined. We intend to use a generic approach for early identification of patients with severe influenza-like illness (ILI) by calculating a score that estimates a patients disease-severity. Accordingly, we built the Intelligent Severity Score Estimation Model (ISSEM), structured so that the inference process would reflect experts decision-making logic. Each patients disease-severity score is calculated from numbers of respiratory ICD9 encounters, and laboratory, radiologic, and prescription-therapeutic orders in the EMR. Other ISSEM components include chronic disease evidence, probability of immunodeficiency, and the providers general practice-behavior patterns., Results: Sensitivity was determined from 200 randomly selected patients with upper- and lower-respiratory tract ILI; specificity, from 300 randomly selected patients with URI only. For different age groups, ISSEM sensitivity ranged between 90% and 95%; specificity was 72% to 84%., Conclusion: Our preliminary assessment of ISSEM performance demonstrated 93.5% sensitivity and 77.3% specificity across all age groups.

Background: Previous research has demonstrated that pretreatment with kappa opioid receptor (KOR) agonist protects against ischemia reperfusion (I/R) injury in cardiomyocytes and neuron cells through activation of protein kinase C. The purpose of this study is to investigate the KOR agonist's effect on I/R-injured cremaster muscle and its underlying mechanism., Method and Material: Male Sprague Dawley rats were randomized into 3 groups (n = 6 each group). Group I was the ischemia reperfusion (I/R) injury group (4 hours of ischemia followed by 90 minutes of reperfusion). Group II was the U-50488H (selective KOR agonist)-pretreated group (KOR agonist + I/R injury). Group III was pretreated with U-50488H + nor-binaltorphimine (NBI, a selective KOR antagonist) (KOR agonist + antagonist + I/R injury). The numbers of leukocyte rolling, adhering, and transmigrating, functional capillary, and swelling index of the vessel wall of the postcapillary venule were observed under intravital videomicroscopy. Biochemically, the lactate dehydrogenase, creatine phosphokinase isoenzyme, expression of E-selectin, and intercellular adhesion molecule-1 (ICAM-1) were analyzed., Results: The U-50488H-pretreated group had significantly decreased the number of leukocyte sticking (P < 0.001) and transmigrating (P < 0.001) as compared with the I/R-injury group and the U-50488H + NBI-pretreated group. The numbers of functional capillary in the U-50488H-pretreated group were significantly less attenuated compared with the I/R-injury group and U-50488H + NBI-pretreated group (P < 0.001). The expression of the ICAM-1 in the cremaster muscle was evidently reduced in the U-50488H-pretreated group than in the I/R-injury group or the U-50488H + NBI-pretreated group., Conclusion: Administration of KOR agonist protects the muscle flap microcirculation from I/R injury, which can be abolished by concomitant KOR antagonist administration. The KOR agonist-induced protection from ischemia reperfusion injury may be related to decreased expression of adhesion molecule ICAM-1.

Background: Our prior study showed that resveratrol could suppress infarct volume and exert neuroprotective effect on rats subjected to focal cerebral ischemia (FCI) injury. Recently, it has been reported in some literature that resveratrol protects the spinal cord, kidney, and heart from ischemia-reperfusion injury through upregulation of nitric oxide (NO). Therefore, this study was designed to investigate the role of nitric oxide on the neuroprotective mechanisms of resveratrol on rats after FCI injury., Methods: The FCI injury was induced by the middle cerebral artery (MCA) occlusion for 1 hour and then a 24-hour reperfusion followed in the anesthetized Long-Evans rats. Resveratrol was intravenously injected after 1 hour MCA occlusion., Results: Treatment of resveratrol (0.1 and 1 microg/kg) decreased the lactate dehydrogenase (LDH) in plasma and malondialdehyde (MDA) in FCI injury brain tissue, whereas the level of NO in plasma was increased. In addition, resveratrol downregulated protein and mRNA expression of inducible nitric oxide synthase (iNOS), and upregulated protein and mRNA expression of endothelial nitric oxide synthase (eNOS), while the expression of protein and mRNA of neuronal nitric oxide synthase (nNOS) was unchanged. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME, the nonselective NOS inhibitor) or L-N(5)-(1-iminoethyl)-ornithine (L-NIO, the eNOS selective inhibitor) completely blocked the effect of resveratrol in decreasing infarction volumes., Conclusions: This study demonstrated the important role of NO in the neuroprotective effect of resveratrol in FCI injury.

Although insulin resistance is recognized as a potent and prevalent risk factor for coronary heart disease, less is known as to whether insulin resistance causes an altered cardiac phenotype independent of coronary atherosclerosis. In this study, we investigated the relationship between insulin resistance and cardiac contractile dysfunctions by generating a new insulin resistance animal model with rats on high cholesterol-fructose diet. Male Sprague-Dawley rats were given high cholesterol-fructose (HCF) diet for 15 wk; the rats developed insulin resistance syndrome characterized by elevated blood pressure, hyperlipidemia, hyperinsulinemia, impaired glucose tolerance, and insulin resistance. The results show that HCF induced insulin resistance not only in metabolic-response tissues (i.e., liver and muscle) but also in the heart as well. Insulin-stimulated cardiac glucose uptake was significantly reduced after 15 wk of HCF feeding, and cardiac insulin resistance was associated with blunted Akt-mediated insulin signaling along with glucose transporter GLUT4 translocation. Basal fatty acid transporter FATP1 levels were increased in HCF rat hearts. The cardiac performance of the HCF rats exhibited a marked reduction in cardiac output, ejection fraction, stroke volume, and end-diastolic volume. It also showed decreases in left ventricular end-systolic elasticity, whereas the effective arterial elasticity was increased. In addition, the relaxation time constant of left ventricular pressure was prolonged in the HCF group. Overall, these results indicate that insulin resistance reduction of cardiac glucose uptake is associated with defects in insulin signaling. The cardiac metabolic alterations that impair contractile functions may lead to the development of cardiomyopathy.

Low-density lipoprotein (LDL) has been implicated in the initiation and progression of atherosclerotic vascular disease. But whether LDL can elicit similar effects in the microcirculation remain unexplored. To approach this issue, the hypothesis that LDL promotes oxidative stress in monocytes and results in microvascular inflammation was tested. Native LDL was capable of stimulating respiratory burst in rat monocytes, and this was blocked by BAPTA, cytochalasin B, apocynin, and diphenyliodonium. In monocytes from rats on a high-cholesterol (4%) diet, increased intracellular calcium, actin polymerization, respiratory burst, and surface CD18 expression were found. Concurrently, leukocyte-endothelial interaction was enhanced in the cremaster microcirculation. Rosiglitazone, an insulin-sensitizing agent with antiinflammatory properties, was found to suppress native-LDL-induced actin polymerization and respiratory burst in monocytes. It also improved leukocytes activation and leukocyte-endothelial interaction due to the high cholesterol intake. Hence, native LDL stimulation of monocytes contributed to hypercholesterolemia-associated microvascular inflammation, which could be treated by rosiglitazone.

Background: This study addressed the effects of apocynin, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, on hepatic oxidative burden and liver injury during diet-induced hypercholesterolaemia., Methods and Results: Male Wistar rats were fed a 4% cholesterol-enriched diet for 3 weeks. Apocynin was administered in drinking water concurrently. The high-cholesterol diet (HC) significantly increased the serum level of cholesterol and hepatic cholesterol ester deposition, and these parameters were similar between the HC and high-cholesterol diet plus apocynin (HCA) groups. The HC group showed abnormal liver function tests [alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (Alk-P)] as well as increased Evans blue extravasation and macrophages infiltration. Apocynin treatment could suppress these inflammation-related parameters. In vivo measurement of NADPH-derived cellular autofluorescence suggested that HC increased oxidative stress in hepatocytes. Biochemical analysis of redox status including thiobarbituric acid reactive substances, reduced glutathione, and oxidized glutathione also confirmed the phenomenon. Apocynin treatment was able to alleviate these indices of oxidative burden owing to HC. Furthermore, apocynin-abrogated HC induced gp91(phox) expression, suggesting the involvement of NADPH oxidase in the pathogenesis., Conclusions: We concluded that apocynin suppressed NADPH oxidase activation and subsequent liver injuries owing to high-cholesterol intake in rats. The impacts of cholesterol metabolism disorders on pathogenesis and progression of steatohepatitis warrant further clinical investigation.

Background: Ischemic - reperfusion (IR) injury is a multifactorial process that leads to tissue damage and rejection in composite tissue allotransplantation (CTA). Antioxidant or free radical scavenger may reduce IR injury, so the effects of resveratrol, a natural antioxidant, on amelioration of leukocyte - endothelial cell adhesive interaction and prevention of transplant rejection in CTA were investigated., Methods and Results: In a microcirculatory study, resveratrol significantly reduced the number of IR-induced leukocytes rolling, adhering, and transmigrating in the postcapillary venules of the cremaster muscle. In the CTA study using groin skin flap allotransplantation across the MHC barrier 8-11-week-old Brown Norway donors (RT1(n)) and 10-11-week-old Lewis recipients (RT1(l)) rats were randomized into 4 groups: isograft control, allograft control, and 2 groups that received different doses of resveratrol (0.1 or 0.5 mg/kg) for 7 days. Allograft control animals rejected their allograft between 5 and 7 days postoperatively, whereas resveratrol-treated recipients had a moderate survival prolongation compared with the allograft control group. Consistent with these observations, histology results also showed reduction of lymphocytic infiltration and necrosis in resveratrol-treated subjects., Conclusion: Resveratrol treatment prolonged groin skin flap allotransplant survival in the recipient and ameliorated the leukocyte - endothelial cell adhesive interactions that may lead to attenuated and delayed rejection in CTA.

Resveratrol (trans-3, 4', 5-trihydroxystilbene), a natural antioxidant derived from grapes, has beneficial effects against coronary heart disease. Its electrophysiological characteristics for antiarrhythmic efficacy are largely unknown; thus, this study aims to explore the resveratrol's antiarrhythmic effects and conduction system in isolated hearts as well as its electrophysiological effects on cardiac myocytes. In the experiment, resveratrol suppressed the ischemia/reperfusion-induced ventricular arrhythmias in Langendorff-perfused rat hearts. In the current clamp study of the experiment, resveratrol prolonged the action potential duration (APD(50) and APD(90)) and suppressed the upstroke velocity of the action potential (V(max)). In the voltage clamp study, resveratrol inhibited sodium inward current (I(Na)) in a concentration-dependent manner and negative-shifted the voltage-dependent inactivation curve. Resveratrol also reduced the calcium inward current (I(Ca), 51.2+/-13.3% at 100 microM). Furthermore, the transient (I(to)) and sustained (I(ss)) outward potassium currents were decreased 60.2+/-5.7% and 42.3+/-5.2% after exposure to resveratrol (100 microM), respectively. The inward rectifier potassium current (I(K1)) was also reduced 24.2+/-7.0% in the presence of resveratrol (100 microM). In the isolated heart perfusion model, resveratrol (100 microM) prolonged AV nodal refractory period, the Wenckebach cycle length and the conduction through AV node and His-Purkinje system. In conclusion, resveratrol increased the cardiac effective refractory period mainly through inhibiting the ionic channels including I(Na), I(to) and I(ss) which could contribute to the conversion of ischemia/reperfusion-induced lethal arrhythmias.

Background: Ischemic preconditioning (IPC) produces immediate tolerance to subsequent prolonged ischemia/reperfusion (I/R), although the underlying mechanism remains unknown. The purpose of this study was to examine the role of nitric oxide (NO) and protein kinase C (PKC) in IPC-attenuated post ischemic leukocyte-endothelium interactions., Methods and Results: Male Sprague-Dawley rats were randomized (n=8 per group) into 5 groups: sham-operated control group, IPC group, I/R group (4 h of pubic epigastric artery ischemia followed by 2 h of reperfusion), IPC+I/R group (30 min of ischemia followed by 30 min of reperfusion before I/R), and chelerythrine (PKC inhibitor)+IPC+I/R group. Intravital microscopy was used to observe leukocyte-endothelium interaction and to quantify functional capillaries in rat cremaster muscle flaps. The mRNA expressions of neuronal (n) NO synthase (NOS), inducible (i) NOS, and endothelial (e) NOS were determined by reverse transcription-polymerase chain reaction. The results showed that besides increasing functional capillary density, IPC also prevents I/R-induced increases in leukocyte rolling, adhesion, and migration. In the chelerythrine+IPC+I/R group, the IPC protective action was inhibited by the addition of chelerythrine. It was also observed that IPC upregulated nNOS, iNOS, and eNOS mRNA in I/R injured tissue, but this effect was not blocked by chelerythrine. Furthermore, specifically pretreated nNOS and iNOS inhibitors, along with a nonselective NOS inhibitor, were used in the IPC+I/R group to examine their possible antagonistic effects on leukocyte-endothelium interactions. Inhibition of the nNOS and iNOS activities did not block the beneficial effects of IPC. In contrast, pretreatment with the nonselective NOS inhibitor (NG-nitro-L-arginine methylester) in the IPC+I/R group almost completely blocked the protective effect of IPC., Conclusions: Both NOS and PKC play a protective role during IPC, but probably in distinct ways. Furthermore, the results also indicate that eNOS, but not nNOS nor iNOS, is the key mediator of IPC-attenuated I/R-induced microcirculatory disturbance.

Aberrant energy metabolism is one characteristic of diabetes mellitus (DM). Two types of DM have been identified, type 1 and type 2. Most of type 2 DM patients eventually become insulin dependent because insulin secretion by the islets of Langerhans becomes exhausted. In the present study, we show that resveratrol (3,5,4'-trihydroxylstilbene) possesses hypoglycemic and hypolipidemic effects in streptozotocin-induced DM (STZ-DM) rats. In resveratrol-treated STZ-DM rats, the plasma glucose concentration on day 14 was reduced by 25.3 +/- 4.2%, and the triglyceride concentration was reduced by 50.2 +/- 3.2% compared with the vehicle-treated rats. In STZ-nicotinamide DM rats, the plasma glucose concentration on day 14 was reduced by 20.3 +/- 4.2%, and the triglyceride concentration was reduced by 33.3 +/- 2.2% compared with the vehicle-treated rats. Resveratrol administration ameliorates common DM symptoms, such as body weight loss, polyphagia, and polydipsia. In STZ-nicotinamide DM rats, resveratrol administration significantly decreased insulin secretion and delayed the onset of insulin resistance. Further studies showed that glucose uptake by hepatocytes, adipocytes, and skeletal muscle and hepatic glycogen synthesis were all stimulated by resveratrol treatment. Because the stimulation of glucose uptake was not attenuated in the presence of an optimal amount of insulin in insulin-responsive cells, the antihyperglycemic effect of resveratrol appeared to act through a mechanism(s) different from that of insulin.

Background: In vitro experimental studies showed that A2a receptor agonists reduce allostimulatory functions of the dendritic cells through modulation of surface expression of the costimulatory molecules and down-regulation of cytokines. Similar suppressive effects on dendritic cells have also been observed with Cyclosporine A (CsA). In this study we sought to explore if combined use of these two drugs in vivo could prolong the survival of composite tissue allografts across MHC barriers., Materials and Methods: A total of 24 hindlimb transplantations were performed across a major MHC barrier from Brown Norway rats (BN; RT1n) to Lewis rats (Lew; RT1l) In the control group, either isografts (group 1, n = 2) or allografts (group 2, n = 3) were performed and no treatment was given. In the experimental group three kinds of treatment protocols were used: (1) A2a adenosine receptor agonist alone (group 3, n = 6); (2) CsA alone (group 4, n = 7); and (3) A2a adenosine receptor agonist + CsA combined treatment (group 5, n = 6). Mean survival times of each group as well as cytokine levels including IL-2, IL-4, IL-10, INF-gamma, and TNF-alpha were analyzed by ELISA., Results: Isografts survived indefinitely. The mean survival times for allograft groups (group 2 to 5) were 9.8 +/- 1.3, 10.5 +/- 1.0, 29.8 +/- 1.7, and 22 +/- 1.4 days, respectively. Statistically, there was no difference between the allograft control group and the A2a adenosine receptor agonist treated group (P = 0.35). However, survival of the allografts in the CsA-treated group was significantly higher than the A2a adenosine receptor agonist treated (P < 00001) and combined CsA + A2a adenosine receptor agonist treated group (P < 0.0001). In vivo A2a adenosine receptor agonist treatment alone increased the levels of IL-2, INF-gamma, and TNF-alpha, which are important cytokines for induction of allotransplant rejection. However A2a adenosine receptor agonist in combination with CsA significantly reduced the levels of suppressor cytokines IL-4 and IL-10., Conclusion: As opposed to the previous in vitro studies, the results from this in vivo study showed that A2a adenosine receptor agonist treatment does not prolong composite tissue allograft survival. Compared to CsA treatment alone, the allotransplant survival was even shorter with the combined treatment. Treatment with A2a adenosine receptor agonist possibly promotes the differentiation of alloreactive CD4 cells predominantly into T-helper 1 phenotype. As a result, the levels of stimulatory cytokines contributing to allograft rejection are increased and suppressor cytokines are reduced, leading to accelerated allograft rejection.

Dissection and microsurgical anastomosis in small and thin-walled vessels is challenging. Temporary assisting suspension suture technique was developed to overcome those difficulties in establishing successful composite tissue allotransplantation in mice. The operations were performed in 12- to 16-week-old Balb/c mice weighing 25 to 30 grams as both donor and recipient animals. Extended vascularized groin cutaneous flaps based on the superficial epigastric vessels were used. A total of 10 groin cutaneous flaps were transplanted. Three temporary assisting suspension sutures of 11-0 nylon were placed at the 12-, 4-, and 8-o'clock positions to donor and recipient artery and vein before the anastomosis. This technique allowed atraumatic dissection of delicate and thin vessels, prevented vessel wall collapse, and facilitated adequate exposure of the lumen during placement of the permanent microvascular sutures. Thus, the microvascular anastomosis was performed in an unusual manner. The temporary assisting suspension sutures were removed just before the permanent suture was tied down. The mean operation time was 1 hour and 45 minutes with an ischemia time of 1 hour. Ninety-percent success in immediate and late-term patency rates was achieved, which was confirmed by transplant survival. This technique was proven to be useful for microvascular anastomosis in thin-walled vessels and is recommended.

Background: Allografts from cadaveric sources may be an alternative for replacing the missing auricle. In this report, the technical aspects of orthotopic composite auricle allotransplantation and an effective short-term immunosuppression protocol in a rat model are described., Methods: A total of seven transplantations were performed in the experimental group. The donors were Brown Norway (RT1(n)) rats and the recipients were Lewis (RT1(1)) rats. In the pilot study, 11 isotransplantations (Lewis to Lewis) were performed in either heterotopic (n = 4) or orthotopic (n = 7) locations to establish the surgical technique. Composite auricle allografts were harvested and transplanted based on the posterior facial vein, the external carotid artery, and the great auricular nerve. A plastic square mold sutured over the transplants was used to prevent mechanical trauma to the transplants. Cyclosporine A initiated as 16 mg/kg/day for 2 weeks and tapered to a dosage of 8 mg/kg/day for another 2 weeks was the only immunosuppression regimen., Results: All allografts survived with perfect viability for the follow-up period of 30 days. There were no signs of rejection, infection, or graft-versus-host disease, although significant weight loss was observed resulting from the immunosuppressive treatment. However, signs of rejection started 4 to 6 days after cessation of the cyclosporine A treatment, including edema, localized epidermal desquamation, and erythema formation that eventually progressed to necrosis within 11 to 14 days. The histologic outcomes were well correlated with the macroscopic appearance., Conclusions: It is feasible to elevate and transplant the composite auricle in rats as a single neurosensorial facial subunit. A tapered dose of cyclosporine A from 16 mg/kg to 8 mg/kg allows maintaining allograft survival for 30 days across a strong major histocompatibility complex barrier. This model is reliable and reproducible and has the potential to be used for future immunologic studies to prevent or to induce transplantation tolerance.

In this study, the authors explored the feasibility of transplanting a "pectoral skin flap" as an alternative model for composite skin allotransplantation research. Genetically inbred male Lewis rats (LEW; RT1l) (n = 13), weighing 250 to 300 gr, were used as recipients and Brown Norway rats (BN; RT1n) (n = 8) rats were used as donors. Five Brown Norway rats had bilateral and three had unilateral flap harvest with a total of 13 flap transplantations to Lewis rats. All flaps were transplanted to the groin regions of the recipients and microsurgical anastomoses with 10-0 nylon were performed between the axillary vessels of the flaps and the femoral vessels of the recipient animals. The mean operation time was 55 min, with an ischemia time of 25 min. All transplantations were successful immediately postoperatively. The recipient animals were treated with Cyclosporine A (16 mg/kg/day) from the day of the surgery for 1 week, and then discontinued. One flap developed infection at postoperative day 11 and one flap had partial (50 percent) necrosis. The early signs of rejection started 8 to 9 days after the cessation of the immunosuppressive treatment, and all flaps were acutely and uniformly rejected within 3 to 4 days. The feasibility of harvesting two flaps from one donor animal allowed saving donor animal lives and reducing donor-specific variables.

According to our experience of over 100 hindlimb transplantations, in rats, catching the posterior wall with an anterior wall suture is the most common cause of thrombosis and acute transplant failure. It is generally due to inadequate visibility of the lumen and consequent blind suture placement. With an intention to find a superior suture technique, a comparative study between standard end-to-end interrupted and combined continuous sutures and interrupted tie techniques was performed. Twenty hindlimb transplantations to rats' femoral vessels were performed in each group. Anastomosis time, patency, complications, and animal and transplant survivals were compared. In the interrupted suture group (group I), anastomosis failure was encountered in one vein, with 95% of immediate patency rate. A redo of the venous anastomosis was performed and successful. The combined suture technique (group II) had 100% patency rates without complications. The average time for arterial anastomosis in groups I and II was 8.5 and 8.9 min, and for venous anastomosis, 10.7 and 9.6 min, respectively. At 2 weeks, no differences were found between groups as related to transplant and animal survival.

A nonfunctional heterotopic hindlimb allotransplantation model is presented, and a study was performed between this model and standard orthotopic hindlimb transplantation to compare operation and ischemia time, overall morbidity, and mortality rates. In this model, the skin component of the hindlimb was stripped away to the ankle level, and the remaining part along with the vascularized epigastric skin was transplanted heterotopically to the inguinal space of the recipient. No osteotomy and intramedullary fixation were performed. Thirty heterotopic and orthotopic hindlimb transplantations were performed in two groups. Each group involved 15 transplantations from inbred, male Brown Norway (BN; RT1n) weighing 150-200 g to Lewis rats (LEW; RT1(1)) weighing 250-300 g. The animals were followed up for 2 weeks under immunosuppression with cyclosporine A (16 mg/kg/day). Mean operation times for heterotopic and orthotopic hindlimb transplantations were 60 and 105 min, with ischemia times of 35 and 85 min, respectively. No animal deaths or major complications were encountered in heterotopic hindlimb transplantation during the follow-up period. Seroma formation was observed in one animal as the only minor complication. The mortality rate for orthotopic hindlimb transplantation was 26.7%, and there were minor complications in 35%, including infection, ulceration, and loss of rigid fixation. This model circumvents the disadvantages of osteotomy and intramedullary fixation, which may increase the risk of blood loss, embolus, and infection in immunosuppressed animals. It also helps avoid tension or kinking on the anastomosis due to inadequate judgment of the osteotomy level, distortion associated with loss of rigid fixation, and weight mismatch between donor and recipient. From the immunological point of view, insult to bone marrow is avoided, and a relatively constant amount of bone marrow is introduced to be used in chimerism-based tolerance studies. We recommend this model for composite-tissue allotransplantation studies when functional recovery is not of primary importance., (Copyright 2005 Wiley-Liss, Inc.)

Diazoxide is a selective mitochondria ATP-sensitive potassium (K(ATP)) channel opener, which has been reported to preserve the microvascular integrity of ischemia-reperfusion (I/R)-injured tissues. Our study aimed to assess diazoxide's effects on I/R-injured cremaster muscles and to further elucidate its underlying mechanisms. Male Sprague Dawley (SD) rats were randomized (n = 8 per group) into four groups: sham-operated control group, I/R group (4 h of pudic epigastic artery ischemia followed by 2 h of reperfusion), diazoxide + I/R group, and chelerythrine (PKC inhibitor)+diazoxide+I/R group. Microscopically, we observed that I/R markedly increased the number of rolling, adhering, and transmigrating leukocytes. I/R also markedly decreased the number of functional capillaries. Biochemically, we found that I/R significantly increased TNF-alpha, E-selectin,L-selectin and P-selectin expressions. However, I/R did not cause significant changes in ICAM-1 and PECAM-1 expressions. On the other hand, in I/R + diazoxide group, we found that diazoxide reduced the number of rolling, adhering, and transmigrating leukocytes. Furthermore, biochemical study revealed that diazoxide caused only a decrease in L-selectin expression but had no effect on TNF-alpha, E-selectin, P-selectin, ICAM-1, and PECAM-1 expressions. Finally, in chelerythrine + diazoxide + I/R group, we observed that diazoxide's protective effects were blocked by the addition of chelerythrine. Diazoxide's ability to protect against I/R injury was confirmed by the observation that it reduced the number of rolling, adhering, and transmigrating leukocytes, and increased the number of functional capillaries. Our results indicated that diazoxide operated via a PKC-dependent pathway to achieve protection against I/R injury.

Ischemia-reperfusion (I/R) injury is a complex process involving the generation and release of inflammatory cytokines, and the accumulation and infiltration of neutrophils and macrophages, which disturbs the microcirculatory hemodynamics. Nonetheless, ischemic preconditioning (IPC) is known to produce immediate tolerance to subsequent prolonged I/R insults, although its underlying mechanism largely remains unknown. Our study investigated the role of the IkappaB-alpha-NF-kappaB-TNF-alpha (tumor necrosis factor-alpha) pathway in IPC's ability to ameliorate I/R-induced microcirculatory disturbances in rat cremaster muscle flaps. Male Sprague-Dawley rats were randomized (n = 8 per group) into 3 groups: a sham-operated control group, an I/R group (4 h of pudic epigastric artery ischemia followed by 2 h of reperfusion), and an IPC+I/R group (3 cycles of 10 min of ischemia followed by 10 min reperfusion before I/R). Intravital microscopy was used to observe leukocyte/endothelial cell interactions and quantify functional capillaries in cremaster muscles. I/R markedly increased the number of rolling, adhering, and migrating leukocytes. It was also observed that I/R significantly increased TNF-alpha expression in these injured tissues. On the other hand, IPC prevented I/R-induced increases in leukocyte rolling, adhesion, and transmigration. Moreover, TNF-alpha protein production and its mRNA expression were downregulated in the IPC group. Finally, I/R-induced IkappaB-alpha phosphorylation and NF-kappaB (p65) nuclear translocation were both suppressed by IPC. These results indicated that IPC attenuated NF-kappaB activation and subsequently reduced TNF-alpha expression, which resulted in the amelioration of microcirculatory disturbances in I/R-injured cremaster muscles., (2004 National Science Council, ROC and S. Karger AG, Basel)

We previously showed that resveratrol (3,4',5-trihydroxystilbene) stimulates NO production and is cardioprotective in rat heart subjected to ischemia-reperfusion (I/R rat heart). We now show that in I/R rat heart, inducible nitric oxide synthase (iNOS) expression is markedly induced, while expression of endothelial nitric oxide synthase (eNOS) and nueronal nitric oxide synthase (nNOS) is unchanged. In animals preconditioned with resveratrol (0.5 to 1 mg/kg body wt), I/R-induced iNOS induction is abrogated; however, expression of eNOS and nNOS is greatly upregulated. The protective effects of resveratrol on I/R rat heart include reduced rhythm disturbances, reduced cardiac infarct size, and decreased plasma levels of lactate dehydrogenase (LDH) and creatine kinase (CK). Among these, the reductions in LDH/CK levels and infarct size are NO-dependent as the coadministration of N(omega)-nitro-L-arginine methyl ester (L-NAME, 1 mg/kg body wt) with resveratrol abolishes the resveratrol effect. In contrast, the reductions in the severity of ventricular arrhythmia and mortality rate are not affected by L-NAME coadministration, suggesting that a NO-independent mechanism is involved.

1. O-methyl-neocaryachine (OMNC) suppressed the ischaemia/reperfusion-induced ventricular arrhythmias in Langendorff-perfused rat hearts (EC50=4.3 microM). Its electrophysiological effects on cardiac myocytes and the conduction system in isolated hearts as well as the electromechanical effects on the papillary muscles were examined. 2. In rat papillary muscles, OMNC prolonged the action potential duration (APD) and decreased the maximal rate of depolarization (V(max)). As compared to quinidine, OMNC exerted less effects on both the V(max) and APD but a positive inotropic effect. 3. In the voltage clamp study, OMNC decreased Na+ current (I(Na)) (IC50=0.9 microM) with a negative-shift of the voltage-dependent inactivation and a slowed rate of recovery from inactivation. The voltage dependence of I(Na) activation was, however, unaffected. With repetitive depolarizations, OMNC blocked I(Na) frequency-dependently. OMNC blocked I(Ca) with an IC(50) of 6.6 microM and a maximum inhibition of 40.7%. 4. OMNC inhibited the transient outward K+ current (I(to)) (IC50=9.5 microM) with an acceleration of its rate of inactivation and a slowed rate of recovery from inactivation. However, it produced little change in the steady-state inactivation curve. The steady-state outward K+ current (I(SS)) was inhibited with an IC50 of 8.7 microM. The inward rectifier K+ current (I(K1)) was also reduced by OMNC. 5. In the perfused heart model, OMNC (3 to 30 microM) prolonged the ventricular repolarization time, the spontaneous cycle length and the atrial and ventricular refractory period. The conduction through the AV node and His-Purkinje system, as well as the AV nodal refractory period and Wenckebach cycle length were also prolonged (30 microM). 6. In conclusion, OMNC blocks Na+, I(to) and I(SS) channels and in similar concentrations partly blocks Ca2+ channels. These effects lead to a modification of the electromechanical function and may likely contribute to the termination of ventricular arrhythmias. These results provide an opportunity to develop an effective antiarrhythmic agent with modest positive inotropy as well as low proarrhythmic potential.

1. Dietary antioxidants are thought to be beneficial in reducing the incidence of coronary heart disease. In this study, we compared resveratrol and analogues on their antioxidation and free radical scavenging activities to their protective effects on ischaemia-reperfusion induced injuries of rat hearts. 2. Astringinin (3,3',4',5-tetrahydroxystilbene) was shown to be a more potent inhibitor than other analogues against Cu(2+)-induced LDL (low-density lipoprotein) oxidation, as measured by the formation of conjugated diene and TBARS (thiobarbituric acid-reactive substance) and by the electrophoretic mobility of the oxidized LDL. 3. Resveratrol (trans-3,4',5-trihydroxystilbene) and astringinin scavenged the stable free radical DPPH (1,1-diphenyl-2-picryl-hydrazyl) with an IC(0.200) of 7.1 and 4.3 microM, respectively. 4. Astringinin has a superoxide anion scavenging activity about 160 fold more potent than resveratrol. 5. After a 30 min global ischemia followed by 2 h reperfusion, astringinin (10 microM) significantly reduced infarct size, superoxide anion production and increased functional recovery of the coronary flow in Langendorff-perfused rat hearts. 6. The result showed there is a positive correlation between the anti-oxidation and cardioprotective activities among these phenolic compounds. Our finding together with the fact that astringinin is more water-soluble than resveratrol suggest that astringinin could potentially be used as an anti-oxidant and cardioprotective agent in biological systems.

Resveratrol is found in a wide variety of plant species. It is present in the seeds and skin of grapes and constitutes one of the major components of red wine. This study was undertaken to evaluate whether resveratrol could effectively suppress infarct size from the damaging effects of focal cerebral ischemia. The middle cerebral artery was occluded for 1 hr and 24 hr reperfusion in anesthetized Long-Evans rats. In pretreatment or treatment groups, resveratrol, at dosages of 10(-6), 10(-7), 10(-8) and 10(-9) g/kg, was intravenous injected 15 minutes before middle cerebral artery (MCA) occlusion or when the common carotid arteries clips were removed respectively. Pretreatment or treatment of resveratrol (10(-6), 10(-7), 10(-8) and 10(-9) g/kg) did not produce any changes in pH, blood gases, heart rate or mean arterial blood pressure, but it significantly reduced the total volume of infarction at the doses 10(-6) and 10(-7) g/kg. Our study suggests resveratrol is a potent neuroprotective agent in focal cerebral ischemia. Its beneficial effects may be related to its anti-platelet aggregation activity, vasodilating effect, antioxidant property or by all mechanisms together.

Oxidative stress plays an important role in the pathogenesis of myocardial ischemia and infarction. Antioxidants might then be beneficial in the prevention of these diseases. Astringinin (3,3',4',5-tetrahydroxystilbene), a resveratrol (3,4',5-trihydroxystilbene) analogue with considerably higher antioxidative activity and free radical scavenging capacity, was introduced to examine its cardioprotective effects in ischemia or ischemia-reperfusion (I/R) rats. In the present study, the left main coronary artery was occluded by the following procedures: (i) 30 min occlusion, (ii) 5 min occlusion followed by 30 min reperfusion, and (iii) 4 h occlusion. Animals were infused with and without astringinin before coronary artery occlusion. Mortality, and the severity of ischemia- and I/R-induced arrhythmias were compared. Pretreatment of astringinin dramatically reduced the incidence and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) during either ischemia or I/R period. Astringinin at 2.5 x 10(-5) and 2.5 x 10(-4) g/kg completely prevented the mortality of animals during ischemia or I/R. During the same period, astringinin pretreatment also increased nitric oxide (NO) and decreased lactate dehydrogenase (LDH) levels in the carotid blood. In animals subjected to 4 h coronary occlusion, the cardiac infarct size (expressed as a percentage of occluded zone) was reduced from 44.4 + or - 4.1% to 19.1 + or - 2.4% by astringinin (2.5 x 10(-4) g/kg). We conclude that, astringinin is a potent antiarrhythmic agent with cardioprotective activity in ischemic and ischemic-reperfused rat heart. The beneficial effects of astringinin in the ischemic and ischemic-reperfused hearts may be correlated with its antioxidant activity and upregulation of NO production.

Background: The major objective of the present study was to examine the cardioprotective effect of resveratrol, an antioxidant presents in red wines, in the rat after ischemia and ischemia-reperfusion (I-R)., Methods: The left main coronary artery was occluded for 30 or 5 min followed by a 30-min reperfusion in anesthetized rats. Animals were preinfused with and without resveratrol before occlusion and the severity of ischemia- and I-R-induced arrhythmias and mortality were compared., Results: Resveratrol pretreatment had no effect on ischemia-induced arrhythmias nor on mortality. In contrast, a dramatic protective effects were observed against I-R-induced arrhythmias and mortality. Resveratrol pretreatment both reduced the incidence and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF). During the same period, resveratrol pretreatment also increased nitric oxide (NO) and decreased lactate dehydrogenase levels in the carotid blood., Conclusions: Resveratrol is a potent antiarrhythmic agent with cardioprotective properties in I-R rats. The cardioprotective effects of resveratrol in the I-R rats may be correlated with its antioxidant activity and upregulation of NO production.

This study was designed to evaluate the in vivo effect of magnolol and honokiol on the acute phase of coronary ligation in the presence of nitric oxide inhibitor (L-NAME) or cyclooxygenase inhibitor (aspirin). After Sprague-Dawley rats were anesthetized with urethane, the changes of ventricular arrhythmia induced by coronary ligation for 30 min were determined with or without pretreatment with study medications. The incidence and duration of ventricular arrhythmia were significantly reduced after intravenous pretreatment (15 min before coronary ligation) with 10(-7) g/kg magnolol or 10(-7) g/kg honokiol. However, the antiarrhythmic effect of magnolol or honokiol could be abolished with the pretreatment of 1 mg/kg L-NAME, but not with pretreatment of 100 mg/kg aspirin. The abolishment of the myocardial beneficial effect of magnolol and honokiol by L-NAME, instead of aspirin, suggests an involvement of an increased nitric oxide synthesis in the protection offered by magnolol and honokiol against arrhythmia during myocardial ischemia.

Background: Abnormal proliferation of vascular smooth muscle cells (VSMC) is a key event in the pathogenesis of atherosclerosis and many vascular diseases. It is known that nitric oxide released from the endothelium participates in the regulation of VSMC proliferation via a cyclic 3',5'-guanosine monophosphate (cGMP)-mediated mechanism. In a series of experiments, sodium nitroprusside (SNP) and A02131-1 were evaluated for their antiproliferative effect and the mechanism of their cGMP-elevating action., Methods and Results: The effect of SNP and A02131-1 on epidermal growth factor (EGF)-stimulated proliferation of rat aortic smooth muscle cells (VSMC) was examined. Cell proliferation was measured in terms of [3H]thymidine incorporation, flow cytometry, and the cell number. Further, their effect on the EGF-activated signal transduction pathway was assessed by measuring mitogen-activated protein kinases (MAPK), MAPK kinase (MEK). Raf-1 activity, and the formation of active form of Ras. SNP and A02131-1 inhibited EGF-induced DNA synthesis and subsequent proliferation of VSMC. These two increased cGMP but only a little cAMP in VSMC. A similar antiproliferative effect was observed with 8-bromo-cGMP. The antiproliferative effect of the two was reversed by KT5823 but not by dideoxyadenosine nor Rp-cAMPS. SNP and A02131-1 blocked the EGF-inducible cell cycle progression at the G1/S phase. Further experiments indicated that the two cGMP-elevating agents primarily blocked the activation of Raf-1 by EGF-activated Ras., Conclusions: These results demonstrate that cGMP-elevating agents inhibit [3H]thymidine incorporation and thus the growth of VSMC, and this inhibition appears to attenuate EGF-activated signal transduction pathway by preventing Ras-dependent activation of Raf-1.

We previously found that TGF-beta1 inhibits PDGF mitogenicity in MG-63 cells and the inhibition is correlated with a suppression of the PDGF-induced receptor autophosphorylation. In this study, we analyze if all the PDGF receptor signaling pathways are similarly affected by the TGF-beta1 pretreatment. We show that TGF-beta1 suppresses PDGF-stimulated tyrosine phosphorylation of PLC-gamma1, and the phosphorylation of Erk. In contrast, the tyrosine phosphorylation of PI3-kinase is not affected. Thus, TGF-beta1 selectively suppresses two out of three PDGF receptor signaling pathways despite of its prominent inhibition of the PDGF-induced receptor autophosphorylation. The results also indicate that activation of the PI3-kinase pathway alone by PDGF is not sufficient in supporting the optimum growth of MG-63 cells under the culture conditions employed.

It has been shown that cAMP may perturb the polypeptide growth factor-induced nuclear events. However, the possible interactions of the cAMP-protein kinase A (cAMP-PKA) and receptor tyrosine kinase pathways in the cytosol have not been fully elucidated. In this study, we use human astrocytoma cells as a model to investigate this issue. The results show that platelet-derived growth factor (PDGF)-induced receptor autophosphorylation in human astrocytoma cells is suppressed by dibutyryl-cAMP pretreatment and such suppression is not due to changes in the ligand-receptor binding properties. Further studies show that PDGF-induced tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and phosphatidylinositol 3-kinase (PI 3-kinase) are also suppressed in dibutyryl-cAMP-pretreated cells. The suppression of PLC-gamma 1 tyrosine phosphorylation was accompanied by a decreased production of water soluble inositol phosphates. In contrast, similar treatment with normal human astrocytes potentiates the tyrosine phosphorylation of PLC-gamma 1 and PI 3-kinase. The results indicate that cAMP can either negatively or positively modulate the PDGF receptor tyrosine kinase activity depending on the cell types examined.

It has been shown that the intracellular cAMP levels were decreased in human malignant astrocytomas. On the other hand, various growth factors and their receptors were found to be overexpressed in these tumors. It is therefore intriguing as to whether there is interplay between the two phenomena in the modulation of the astrocytoma cell growth. In a basal medium consisting of 75% DMEM, 25% Ham's F-12 supplemented with 2% FBS, we show that the mitogenic effects of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) on human astrocytoma cells were suppressed by dibutyryl-cAMP. Dibutyryl-cAMP alone neither potentiated nor inhibited the tumor cell growth. Further studies show that PDGF-induced receptor autophosphorylation in human astrocytoma cells is suppressed by increased intracellular cAMP levels as measured by immunoprecipitation with anti-PDGF receptor and antiphosphotyrosine antibodies. Our results indicate that there is antagonistic interplay between the receptor tyrosine kinase pathway and cAMP-dependent protein kinase pathway in the control of the malignantly transformed glial cells. A reduced cAMP level seen in many human astrocytoma cells may favor their response to growth factor mitogenesis.

The sequence of the 3' 1184 nucleotides of tobacco vein-banding mosaic virus (TVBMV) genome has been determined. It contains a single open reading frame which encompasses the whole of the coat protein of TVBMV. The sequence of the first 20 amino acids at the N-terminal region of the coat protein has also been determined chemically to be GDDQTVDAGKNVQSNQKQRN. The sequence matches the translation product of the open reading frame starting with amino acid-271; a glycine residue. Thus the coat protein of TVBMV has a calculated M(r) of 30,210. The 3' non-coding region of TVBMV is 185 nucleotides in length. Sequence alignment of the coat proteins or the 3' non-coding regions from TVBMV and other reported potyviruses indicated that TVBMV is a separate species of the potyvirus genus.

In the prokaryote Synechococcus RF-1, circadian changes in the uptake of l-leucine and 2-amino isobutyric acid were observed. Uptake rates in the light period were higher than in the dark period for cultures entrained by 12/12 hour light/dark cycles. The periodic changes in l-leucine uptake persisted for at least 72 hours into continuous light (L/L). The rhythm had a free-running period of about 24 hours in L/L at 29 degrees C. A single dark treatment of 12 hours could initiate rhythmic leucine uptake in an L/L culture. The phase of rhythm could be shifted by a pulse of low temperature (0 degrees C). The free-running periodicity was "temperature-compensated" from 21 to 37 degrees C. A 24 hour depletion of extracellular Ca(2+) before the free-running L/L condition reduced the variation in uptake rate but had little effect on the periodicity of the rhythm. The periodicity was also not affected by the introduction of 25 mm NaNO(3). The uptake rates for 20 natural amino acids were studied at 12 hour intervals in cultures exposed to 12/12 hour light/dark cycles. For eight of these amino acids (l-Val, l-Leu, l-Ile, l-Pro, l-Phe, l-Trp, l-Met, and l-Tyr), the light/dark uptake rate ratios had values greater than 3 and the rhythm persisted in L/L.