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The study aims to discover risk factors significantly correlated with insulin resistance among adolescents in Taiwan. A total of 339 study subjects were recruited in this cross-sectional study. A self-administered questionnaire and physical examinations including anthropometrics and biochemistry profiles were collected. Insulin resistance was assessed using homeostasis model assessment for insulin resistance (HOMA-IR). Study subjects had a significantly increased risk of IR for those with abnormal level of body mass index (odds ratio [OR] = 3.54; 95% confidence interval [CI] = 1.81-6.91), body fat (OR = 2.71; 95% CI = 1.25-5.88), and waist circumference (OR = 25.04; 95% CI = 2.93-214.14) when compared with those who have normal values. Furthermore, a significantly joint effect of 10.86-fold risk for HOMA-IR abnormality among body fat, body mass index, and systolic blood pressure was observed. The identification of risk factors significantly correlated with IR will be important to prevent metabolic syndrome-related diseases and complications for adolescents in their future life., (© 2012 APJPH.)

Background: In the past several decades, Polygonum viviparum L. (PV) was reported to have antibacterial, antiulcer, antioxidant, antitumor, anti-inflammatory, and antiarthritic properties. The anti-inflammatory pathway was recently elucidated through cytosolic nuclear factor E2-related factor 2 (Nrf2) activation and heme oxygenase (HO)-1 protein expression. PV is a perennial herb and widely distributed in high-elevation mountain regions, such as the Tibetan Plateau. In Tibetan traditional medicine, PV is usually used to boost the blood circulation to dissipate blood stasis. Therefore, this study focused on how PV improves the vascular circulation and acts on vascular tissues., Methods: In this study, we isolated aortas from Sprague-Dawley rats (male, weight about 250~350 g), and detected the effects of PV on phenylephrine (PE)-induced contraction and cyclic guanosine 3',5'-monophosphate (cGMP) formation using aortic rings. In addition, human umbilical vein endothelial cells (HUVECs) were used to exam nitric oxygen (NO) synthase (NOS) activity by directly measuring NO production in the culture medium. Endothelial (e) NOS phosphorylation, and cytosolic Nrf2 and HO-1 expressions were measured using a Western blot analysis., Results: PV dose-dependently relaxed PE-induced contractions in endothelial-intact but not -denuded aorta. The concentration to produce 50% relaxation was 22.04±1.77 μg/ml. PV-induced vasorelaxation was markedly blocked by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME), an NOS inhibitor, methylene blue (MB), a guanylyl cyclase inhibitor, and hemoglobin, an NO scavenger. PV increased cGMP formation; however, this effect was also suppressed by co-pretreatment with l-NAME, MB, hemoglobin, and Ca2+-free medium. In HUVECs, PV increased NO formation, which was greatly attenuated by NOS inhibitors (L-NAME and L-NMMA) and by removing extracellular Ca2+ and chelating intracellular Ca2+ with BAPTA-AM. In addition, PV promoted eNOS phosphorylation, Nrf2 degradation, and HO-1 protein expression according to a Western blot analysis., Conclusions: The results suggest that PV possesses vasorelaxing action in an endothelium-dependent manner and works through activating Ca2+/calmodulin- dependent NO synthesis; when NO is released and then transferred to smooth muscle cells, NO activates guanylyl cyclase and increases cGMP formation, ultimately resulting in vasorelaxation. Thus, PV can be considered for application as a potential therapeutic approach for vascular-associated disorders.

Chronic liver diseases, including cancer, are characterized by inflammation and elevated serum ferritin (SF). However, the causal-relationship remains unclear. This study used primary rat hepatic stellate cells (HSC) as a model to investigate effects of physiological SF concentrations (10, 100 and 1000 pM) because HSCs play a central role in the development and progression of liver fibrosis. Physiological concentrations of SF, either horse SF or human serum, induced pro-inflammatory cytokine IL1β, IL6 and TNFα secretion in rat activated HSCs (all p<0.05). By contrast, treatment did not alter activation marker αSMA expression. The presence of SF markedly enhanced expression of Grp78 mRNA (p<0.01). Furthermore, transient knock down of Grp78 by endotoxin EGF- SubA abolished SF-induced IL1β and TNFα secretion in activated HSCs (all p<0.05). In conclusion, our results showed that at physiological concentrations SF functions as a pro-inflammatory mediator in primary rat HSCs. We also provide a molecular basis for the action of SF and identified Grp78-associated ER stress pathways as a novel potential therapeutic target for resolution of fibrosis and possible prevention of liver cancer.

Vascular inflammatory process has been suggested to play a key role in the initiation and progression of atherosclerosis, a major complication of diabetes mellitus. Recent studies have shown that brazilin exhibits antihepatotoxic, antiplatelet, cancer preventive, or anti-inflammatory properties. Thus, we investigated whether brazilin suppresses vascular inflammatory process induced by high glucose (HG) in cultured human umbilical vein endothelial cells (HUVEC). HG induced nitrite production, lipid peroxidation, and intracellular reactive oxygen species formation in HUVEC cells, which was reversed by brazilin. Western blot analysis revealed that brazilin markedly inhibited HG-induced phosphorylation of endothelial nitric oxide synthase. Besides, we investigated the effects of brazilin on the MAPK signal transduction pathway because MAPK families are associated with vascular inflammation under stress. Brazilin blocked HG-induced phosphorylation of extracellular signal-regulated kinase and transcription factor NF-κB. Furthermore, brazilin concentration-dependently attenuated cell adhesion molecules (ICAM-1 and VCAM-1) expression induced by various concentrations of HG in HUVEC. Taken together, the present data suggested that brazilin could suppress high glucose-induced vascular inflammatory process, which may be closely related with the inhibition of oxidative stress, CAMs expression, and NF-κB activation in HUVEC. Our findings may highlight a new therapeutic intervention for the prevention of vascular diseases.

Ageing dynamically disrupts the multilayered supporting components of the skin that are held together by cell adhesion molecules (CAMs). Skin specimens from 33 female Chinese patients undergoing lower blepharoplasty were divided into three age groups and examined by haematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and Elastica-van Gieson (EVG) stains, western blotting, surface electron microscopy (SEM) and biomechanical tension analysis. The SEM density (skin surface topology) showed a negative linear relationship with age. The triangular pattern of the skin surface in the younger group gradually broke down into quadrangular and irregular patterns in the older group. Collagens and elastic fibres in the dermis showed anisotropy and decreased density in the older groups compared with the younger group, especially in the papillary dermis. Anisotropy means that physical properties differ according to the direction of measurement. E-cadherin and integrin αv (whose functions are to bind epidermal and dermal elements respectively) increased and decreased, respectively, in the oldest group. Skin resilience decreased significantly in this group under repetitive stress. In conclusion, a loss of skin surface textures, integrin αv expressions, epidermal-dermal connections and dermal compactness led to the multilayered structure of the skin becoming separated. This in turn decreased resilience during ageing. These findings may therefore explain why aged skins cannot tolerate repetitive facial expressions, and why this action produces further dynamic wrinkles., (© 2013 The Authors. International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology.)

Background: Polygonum viviparum L. (PV) is a member of the family Polygonaceae and is widely distributed in high-elevation areas. It is used as a folk remedy to treat inflammation-related diseases. This study was focused on the anti-inflammatory response of PV against lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophages., Results: Treatment with PV did not cause cytotoxicity at 0-50 µg mL(-1) in RAW264.7 macrophages, and the IC(50) value was 270 µg mL(-1). PV inhibited LPS-stimulated nitric oxide (NO), prostaglandin (PG)E(2) , interleukin (IL)-1β and tumour necrosis factor (TNF)-α release and inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 protein expression. In addition, PV suppressed the LPS-induced p65 expression of nuclear factor (NF)-κB, which is associated with the inhibition of IκB-α degradation. These results suggest that, among mechanisms of the anti-inflammatory response, PV inhibits the production of NO and these cytokines by down-regulating iNOS and COX-2 gene expression. Furthermore, PV can induce haem oxygenase (HO)-1 protein expression through nuclear factor E2-related factor 2 (Nrf2) activation. A specific inhibitor of HO-1, zinc(II) protoporphyrin IX, inhibited the suppression of iNOS and COX-2 expression by PV., Conclusion: These results suggest that PV possesses anti-inflammatory actions in macrophages and works through a novel mechanism involving Nrf2 actions and HO-1. Thus PV could be considered for application as a potential therapeutic approach for inflammation-associated disorders., (Copyright © 2012 Society of Chemical Industry.)

This study aimed to determine whether brazilin exhibits anti-inflammatory effects that inhibit T helper cell type II (T(H)2) responses and whether it suppresses allergic inflammation reactions in a murine model of asthma. We found that brazilin inhibited the mRNA and protein expression of interleukin (IL)-4 and IL-5 induced by phorbol myristate acetate (PMA) and cAMP in EL-4 T cells in a dose-dependent manner. Following the intratracheal instillation of brazilin in ovalbumin (OVA)-immunized mice, we found that brazilin-treated mice exhibited decreases in the release of IL-4, IL-5, IL-13, eotaxin-1, and tumor necrosis factor-α in bronchoalveolar lavage fluid (BALF); inhibited T(H)2 functioning via a decrease in IL-4 production; and exhibited attenuation of OVA-induced lung eosinophilia, airway hyperresponsiveness, and airway remodeling. These results suggest that brazilin exhibits anti-T(H)2 effects both in vitro and in vivo and may possess therapeutic potential for allergic diseases.

Background: Doxorubicin (DOX) is an effective antineoplastic drug; however, clinical use of DOX is limited by its dose-dependent cardiotoxicity. It is well known that reactive oxygen species (ROS) play a vital role in the pathological process of DOX-induced cardiotoxicity. For this study, we evaluated the protective effects of guggulsterone (GS), a steroid obtained from myrrh, to determine its preliminary mechanisms in defending against DOX-induced cytotoxicity in H9C2 cells., Methods: In this study, we used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release measurements, and Hoechst 33258 staining to evaluate the protective effect of GS against DOX-induced cytotoxicity in H9C2 cells. In addition, we observed the immunofluorescence of intracellular ROS and measured lipid peroxidation, caspase-3 activity, and apoptosis-related proteins by using Western blotting., Results: The MTT assay and LDH release showed that treatment using GS (1-30 μM) did not cause cytotoxicity. Furthermore, GS inhibited DOX (1 μM)-induced cytotoxicity in a concentration-dependent manner. Hoechst 33258 staining showed that GS significantly reduced DOX-induced apoptosis and cell death. Using GS at a dose of 10-30 μM significantly reduced intracellular ROS and the formation of MDA in the supernatant of DOX-treated H9C2 cells and suppressed caspase-3 activity to reference levels. In immunoblot analysis, pretreatment using GS significantly reversed DOX-induced decrease of PARP, caspase-3 and bcl-2, and increase of bax, cytochrome C release, cleaved-PARP and cleaved-caspase-3. In addition, the properties of DOX-induced cancer cell (DLD-1 cells) death did not interfere when combined GS and DOX., Conclusion: These data provide considerable evidence that GS could serve as a novel cardioprotective agent against DOX-induced cardiotoxicity.

The study was to examine the effects of Sanguis draconis ethanol extract (SDEE) on streptozotocin (STZ)- and cytokine-induced β-cell damage. In vitro, SDEE did not cause cytotoxicity below 200 μg/ml, and can prevent STZ (5mM)-induced cell death and apoptosis below 100 μg/ml on RIN-m5F cells. SDEE inhibits IL-1β/IFN-γ-stimulated NO, TNF-α release, and iNOS expression. Furthermore, SDEE suppressed the IL-1β/IFN-γ- or STZ-induced p65 expression of NF-κB, which is associated with inhibition of IκB-α degradation. In vivo, treatment of ICR mice with STZ (100 mg/kg, i.p. single injection) resulted in hyperglycemia and hypoinsulinemia, which was further evidenced by blood glucose and plasma insulin. The diabetogenic effects of STZ were completely prevented when mice were orally administered with SDEE for 3 weeks, however, the blood glucose and plasma insulin showed no significant change after SDEE administration alone. In addition, SDEE also can inhibit STZ-induced iNOS protein expression, pancreatic injury and lipid peroxidation. In conclusions, the molecular mechanism by which SDEE inhibits iNOS gene expression appears to involve the inhibition of NF-κB activation. These results suggest the possible therapeutic value of S. draconis and could be potentially developed into a novel drug for preventing the progression of diabetes mellitus., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objectives: To elucidate a novel anti-inflammatory mechanism of myrrh against lipopolysaccharide (LPS)-induced inflammation., Methods: RAW264.7 macrophages were cultured in DMEM and then cells were treated with LPS or LPS plus a myrrh methanol extract (MME) for 24h. The culture medium was collected for determination of nitric oxide (NO), prostaglandin (PG)E(2) , interleukin (IL)-1β, and tumour necrosis factor (TNF)-α, and cells were harvested by lysis buffer for Western blot analysis., Key Findings: Our data showed that treatment with the MME (1∼100µg/ml) did not cause cytotoxicity or activate haem oxygenase-1 (HO-1) protein synthesis in RAW264.7 macrophages. Furthermore, the MME inhibited LPS-stimulated NO, PGE(2) , IL-1β and TNF-α release and inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 protein expression. Zn(II) protoporphyrin IX, a specific inhibitor of HO-1, blocked the inhibition of iNOS and COX-2 expression by the MME., Conclusions: These results suggest that among mechanisms of the anti-inflammatory response, the MME inhibited the production of NO, PGE(2) , IL-1β and TNF-α by downregulating iNOS and COX-2 gene expression in macrophages and worked through the action of HO-1., (© 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.)

Background: Safflower, whose botanic name is Carthamus tinctorius L., is a member of the family Compositae or Asteraceae. Carthamus yellow (CY) is the main constituent of safflower and is composed of safflomin A and safflomin B. Dried safflower petals are used in folk medicine and have been shown to invigorate blood circulation, break up blood stasis, and promote menstruation. In addition, dried safflower petals contain yellow dyes that are used to color food and cosmetics. In this study, we investigated the effects of dried safflower petals aqueous extracts (SFA) and CY on lipopolysaccharide (LPS)-induced inflammation using RAW264.7 macrophages., Results: Our data showed that treatment with SFA (1-1000 microg mL(-1)) and CY (1-2000 microg mL(-1)) does not cause cytotoxicity in cells. SFA and CY inhibited LPS-stimulated nitric oxide (NO), prostaglandin E(2) (PGE(2)), and interleukin 1β (IL-1β) release, through attenuation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. Further, SFA and CY suppressed the LPS-induced phosphorylation of nuclear factor-κB, which was associated with the inhibition of IκB-α degradation., Conclusion: These results suggest that SFA and CY provide an anti-inflammatory response through inhibiting the production of NO and PGE(2) by the downregulation of iNOS and COX-2 gene expression. Thus safflower petals have the potential to provide a therapeutic approach to inflammation-associated disorders., (2010 Society of Chemical Industry.)

Brazilin, the main constituent of Caesalpinia sappan L., is a natural red pigment that has been reported to possess anti-inflammatory properties. This study aimed to identify a novel anti-inflammatory mechanism of brazilin. We found that brazilin did not cause cytotoxicity below 300 microM, and activated heme oxygenase-1 (HO-1) protein synthesis in a concentration-dependent manner at 10-300 microM in RAW264.7 macrophages without affecting mRNA transcription of HO-1. Additionally, brazilin increased bilirubin production and HO-1 activity in RAW264.7 macrophages. In lipopolysaccharide (LPS)-stimulated macrophages, brazilin suppressed the release of nitric oxide (NO), prostaglandin E(2) (PGE(2)), interleukin (IL)-1beta and tumor necrosis factor-alpha (TNF-alpha), and reduced the expression of inducible nitric oxide synthase (iNOS). A specific inhibitor of HO-1, Zn(II) protoporphyrin IX, blocked the suppression of NO production, cytokines release and iNOS expression by brazilin. These results suggest that brazilin possesses anti-inflammatory actions in macrophages and works through a novel mechanism involving the action of HO-1.

Aim of the Study: Shikonin/alkannin (SA) derivatives, analogs of naphthoquinone pigments, are the major components of root extracts of the Chinese medicinal herb (Lithospermum erythrorhizon; LE) and widely distributed in several folk medicines. In the present study, the effect and the underline molecular mechanism of shikonin derivatives isolated from root extracts of Lithospermum euchroma on lipopolysaccharide (LPS)-induced inflammatory response were investigated., Materials and Methods: Effects of five SA derivatives, including SA, acetylshikonin, beta,beta-dimethylacrylshikonin, 5,8-dihydroxy-1.4-naphthoquinone, and 1,4-naphthoquinone on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in mouse macrophage RAW264.7 cells were examined., Results: Data suggested that SA derivatives inhibited LPS-induced NO and PGE(2) production, and iNOS protein expression. RT-PCR analysis showed that SA derivatives diminished LPS-induced iNOS mRNA expression. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in LPS-stimulated RAW 264.7 cells was concentration-dependently suppressed by SA derivatives. SA inhibited NF-kappaB activation by prevention of the degradation of inhibitory factor-kappaB and p65 level in nuclear fractions induced by LPS., Conclusions: Taken together, these results suggest that the anti-inflammatory properties of SA derivatives might result from inhibition of iNOS protein expression through the downregulation of NF-kappaB activation via suppression of phosphorylation of ERK, in LPS-stimulated RAW 264.7 cells.

Sanguinarine (SANG) has been suggested to be one of the principle constituents responsible for the toxicity of Argemone mexicana seed oil. In this study, we focused on the possible mechanism of SANG-induced hepatotoxicity. The serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities, hepatic vacuolization, lipid accumulation and lipid peroxidation of the liver were increased, and triglyceride (TG) was decreased in SANG-treated mice (10 mg kg(-1) i.p.), indicating damage to the liver. SANG induced cell death and DNA fragmentation, in a concentration- (0-30 microm) and time-dependent (0-24 h) manner, and the cytotoxicity of SANG (15 microm) was accompanied by an increase in reactive oxygen species and a lessening in protein thiol content; these outcomes were reversed by glutathione, N-acetyl-l-cysteine and 1,4-dithiothretol, and slightly improved by other antioxidants in hepatocytes. SANG can affect the function of mitochondria, leading to the depletion of the mitochondrial membrane potential and adenosine 5'-triphosphate content of hepatocytes. SANG caused an uncoupling effect of the respiratory chain at lower concentrations, but inhibited the respiratory chain at higher concentrations in mitochondria isolated from rat liver. In conclusion, the data suggest that SANG is a liver toxin that induces cytotoxicity in liver cells, possibly through oxidation of protein thiols, resulting in oxidative stress on the cells and disturbance of mitochondrial function.

Sanguis Draconis (SD) is a kind of dragon's blood resin that is obtained from Daemomorops draco (Palmae). It is used in traditional medicine and has shown anti-inflammatory activity in some diseases. In this study, we examined the effects of Sanguis Dranonis ethanol extract (SDEE) on LPS-induced inflammation using RAW 264.7 cells. Our data indicated that SDEE inhibits LPS-stimulated NO, PGE2, IL-1 beta and TNF-alpha release, and iNOS and COX-2 expression. Furthermore, SDEE suppressed the LPS-induced p65 expression of NF-kappa B, which was associated with the inhibition of I kappa B-alpha degradation. We also found that the expression of HO-1 was significantly increased in RAW 264.7 cells by SDEE. These results suggest among possibilities of anti-inflammation that SDEE inhibits the production of NO and PGE2 by the down-regulation of iNOS and COX-2 gene expression via the suppression of NF-kappaB (p65) activation. SDEE can induce HO-1 over-expression in macrophage cells, which indicates that it may possess antioxidant properties. This result means that SEDD its anti-inflammatory effects in macrophages may be through a novel mechanism that involves the action of HO-1. Thus, SD could provide a potential therapeutic approach for inflammation-associated disorders.

Heme oxygenase-1 (HO-1) is an enzyme with potent immunoregulatory capacity. To evaluate the effect of HO-1 on autoimmune diabetes, female NOD mice at 9 weeks of age received a single intravenous injection of a recombinant adeno-associated virus bearing HO-1 gene (AAV-HO-1; 0.5 x 10(10)-2.5 x 10(10) viruses/mouse). In a dose-dependent manner, HO-1 transduction reduced destructive insulitis and the incidence of overt diabetes examined over a 15-week period. HO-1-mediated protection was associated with a lower type 1 T-helper cell (Th1)-mediated response. Adaptive transfer experiments in NOD.scid mice demonstrated that splenocytes isolated from AAV-HO-1-treated mice were less diabetogenic. Flow cytometry analysis revealed no significant difference in the percentages of CD4(+)CD25(+) regulatory T-cells between saline-treated and AAV-HO-1-treated groups. However, the CD11c(+) major histocompatibility complex II(+) dendritic cell population was much lower in the AAV-HO-1-treated group. A similar protective effect against diabetes was observed in NOD mice subjected to carbon monoxide (CO) gas (250 ppm CO for 2 h, twice per week). These data suggest that HO-1 slows the progression to overt diabetes in pre-diabetic NOD mice by downregulating the phenotypic maturity of dendritic cells and Th1 effector function. CO appears to mediate at least partly the beneficial effect of HO-1 in this disease setting.

In researches of ketone bodies, D-3-hydroxybutyrate (D-3HB) is usually the major one which has been investigated; in contrast, little attention has been paid to L-3-hydroxybutyrate (L-3HB), because of its presence in trace amounts, its dubious metabolism, and a lack of knowledge about its sources. In the present study we determined the distributions of enantiomers of 3-hydroxybutyrate (3HB) in rat brain, liver, heart, and kidney homogenates, and we found the heart homogenate contained an enriched amount of L-3HB (37.67 microM/mg protein) which generated a significant ratio of 66/34 (D/L). The ratio was altered to be 87/13 in the diabetic rat heart homogenate. We subsequently found this changed ratio of D/L-3HB may contribute to reduce glucose utilization in cardiomyocytes. Glucose utilization by cardiomyocytes with 5 mM of D-3HB was decreased to 61% of the control, but no interference was observed when D-3HB was replaced with L-3HB, suggesting L-3HB is not utilized for the energy fuel as other ketone bodies are. In addition, the reduced glucose utilization caused by D-3HB gradually recovered in a dose-dependent manner with administration of additional L-3HB. The results gave the necessity of taking L-3HB together with D-3HB into account with regard to glucose utilization, and L-3HB may be a helpful substrate for improving inhibited cardiac pyruvate oxidation caused by hyperketonemia.

In this study, the toxic effect of sanguinarine (SANG) on heart was studied with isolated cardiac muscle strip isolated from Wistar rat. SANG induced positive inotropic action followed by contracture on the left ventricle and both atria strips. In addition, SANG dose-dependently inhibited spontaneous beat of the right atrium. SANG-induced contracture was completely suppressed by pretreatment with La3+ or in a Ca2+ free Tyrode solution containing 2.5 mM EGTA. Incubating isolated cardiomyocytes with SANG enhanced the 45Ca2+ influx, which could be inhibited by pretreatment with La3+. However, the SANG-induced 45Ca2+ influx could not be inhibited by pretreatment with other Ca2+ channel blockers, such as nifedipine, verapamil, diltiazem, nickel and manganese, and amiloride. Although antioxidants can inhibit the SANG-induced lipid peroxidation, they could not prevent the SANG-induced contracture. N-acetylcysteine and dithiothreitol, the sulfhydryl reducing agents, were shown to be effective in preventing the SANG-induced contracture. These data suggested that the SANG-induced contracture is caused by the influx of extracellular Ca2+ through a La3+-sensitive Ca2+ channel.

Background: Heme oxygenase-1 (HO-1) is a stress-response enzyme implicated in cardioprotection. To explore whether HO-1 has a role in cardiac remodeling response, the effect of its overexpression on angiotensin II (Ang II)-induced cardiac hypertrophy was examined., Methods and Results: HO-1 was induced in cultured rat neonatal cardiomyocytes by treatment with cobalt protoporphyrin IX (CoPPIX) or a recombinant adenovirus carrying the human HO-1 gene. Ang II-induced myocyte hypertrophy assessed by increments in cell size, [3H]leucine uptake, and protein content was suppressed by HO-1 overexpression. Cotreatment of cells with tin protoporphyrin IX, a HO inhibitor, significantly reversed the suppressive effect of HO-1. Bilirubin, one of the byproducts of heme degradation by HO-1, mediated the suppressive effect through the inhibition of Ang II-induced production of reactive oxygen species, as detected by a 2',7'-dichlorofluorescein probe. The antihypertrophic effect of HO-1 was also demonstrated in rats receiving chronic Ang II infusions. Cotreatment of animals with CoPPIX significantly attenuated Ang II-induced left ventricular hypertrophy and hyperdynamic contractions, whereas concomitant treatment with tin protoporphyrin IX abolished CoPPIX-mediated cardioprotection in vivo., Conclusions: HO-1 attenuates Ang II-induced cardiac hypertrophy both in vitro and in vivo, and bilirubin mediates, at least in part, the antihypertrophic effect of HO-1 via inhibition of reactive oxygen species production after Ang II stimulation.

The effects of a naphthoquinone analogue, shikonin/alkannin (SA) and derivatives (acetylshikonin and beta,beta-dimethylacrylshikonin), on vascular reactivity were studied with isolated rat aortic rings. At lower concentrations, SA and its derivatives concentration-dependently inhibit the agonist-induced (acetylcholine and histamine) relaxation in PE precontracted aorta in an endothelium-dependent manner with IC (50) values ranging from 0.2 to 1.5 microM. In addition to the effect on agonist-induced vasorelaxation, the Ca (2+) ionophore A23187-induced vasorelaxation was also inhibited or reversed by SA. However, SA had no effect on sodium nitroprusside-induced (guanylate cyclase activator) vasorelaxation. These data suggested that SA and its derivatives might be acting as inhibitors of nitric oxide synthesis in endothelium. At a concentration greater than 10 microM, SA induced contraction of intact but not denuded aorta which could be inhibited by prior treatment with indomethacin, a cyclooxygenase inhibitor. In summary, the results from this study showed that SA and its derivatives inhibited agonist-induced relaxation at lower concentrations and induced vasocontraction at higher concentrations. All the effects seen with SA were endothelium-dependent, however, through different mechanisms. Abbreviations. SA:shikonin/alkannin PE:phenylephrine Ach:acetylcholine SNP:sodium nitroprusside eNOS:endothelial nitric oxide synthase L-NAME: Nw-nitro- L-arginine methyl ester

The vasorelaxant activity of Caesalpinia sappan L., a traditional Chinese medicine, and its major component brazilin were investigated in isolated rat aorta and human umbilical vein endothelial cells. In isolated rat aorta, C. sappan L. extract and brazilin relaxed phenylephrine-induced vasocontraction and increased cyclic guanosine 3',5'-monophosphate (cGMP) content. Induction of vasorelaxation of brazilin was endothelium-dependent and could be markedly blocked by pretreatment with nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME); N(G)-monomethyl-L-arginine acetate (L-NMMA) and guanylyl cyclase inhibitor, methylene blue; 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and nitric oxide (NO) scavenger, hemoglobin. The increasing cGMP content induced by brazilin was also blocked by pretreatment with L-NAME, methylene blue, and the removal of extracellular Ca(2+). In human umbilical vein endothelial cells, brazilin dose-dependently induced an increase in NO formation and NOS activity, which were greatly attenuated by either the removal of extracellular Ca(2+) or the chelating of intracellular Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM). Moreover, brazilin dose-dependently induced the influx of extracellular Ca(2+) in human umbilical vein endothelial cells. Collectively, these results suggest that brazilin induces vasorelaxation by the increasing intracellular Ca(2+) concentration in endothelial cells of blood vessels and hence activating Ca(2+)/calmodulin-dependent NO synthesis. The NO is released and then transferred into smooth muscle cells to activate guanylyl cyclase and increase cGMP content, resulting in vasorelaxation.