Virtual Screening and Molecular Docking to Study the Mechanism of Chinese Medicines in the Treatment of Coronavirus Infection.

BACKGROUND Heat-clearing and detoxifying herbs (HDHs) play an important role in the prevention and treatment of coronavirus infection. However, their mechanism of action needs further study. This study aimed to explore the anti-coronavirus basis and mechanism of HDHs. MATERIAL AND METHODS Database mining was performed on 7 HDHs. Core ingredients and targets were screened according to ADME rules combined with Neighborhood, Co-occurrence, Co-expression, and other algorithms. GO enrichment and KEGG pathway analyses were performed using the R language. Finally, high-throughput molecular docking was used for verification. RESULTS HDHs mainly acts on NOS3, EGFR, IL-6, MAPK8, PTGS2, MAPK14, NFKB1, and CASP3 through quercetin, luteolin, wogonin, indirubin alkaloids, ß-sitosterol, and isolariciresinol. These targets are mainly involved in the regulation of biological processes such as inflammation, activation of MAPK activity, and positive regulation of NF-kappaB transcription factor activity. Pathway analysis further revealed that the pathways regulated by these targets mainly include: signaling pathways related to viral and bacterial infections such as tuberculosis, influenza A, Ras signaling pathways; inflammation-related pathways such as the TLR, TNF, MAPK, and HIF-1 signaling pathways; and immune-related pathways such as NOD receptor signaling pathways. These pathways play a synergistic role in inhibiting lung inflammation and regulating immunity and antiviral activity. CONCLUSIONS HDHs play a role in the treatment of coronavirus infection by regulating the body's immunity, fighting inflammation, and antiviral activities, suggesting a molecular basis and new strategies for the treatment of COVID-19 and a foundation for the screening of new antiviral drugs.

Arginase inhibition by rhaponticin increases L-arginine concentration that contributes to Ca2+-dependent eNOS activation.

Although arginase primarily participates in the last reaction of the urea cycle, we have previously demonstrated that arginase II is an important cytosolic calcium regulator through spermine production in a p32-dependent manner. Here, we demonstrated that rhaponticin (RPT) is a novel medicinal-plant arginase inhibitor and investigated its mechanism of action on Ca2+-dependent endothelial nitric oxide synthase (eNOS) activation. RPT was uncompetitively inhibited for both arginases I and II prepared from mouse liver and kidney. It also inhibited arginase activity in both aorta and human umbilical vein endothelial cells (HUVECs). Using both microscope and FACS analyses, RPT treatments induced increases in cytosolic Ca2+ levels using Fluo-4 AM as a calcium indicator. Increased cytosolic Ca2+ elicited the phosphorylations of both CaMKII and eNOS Ser1177 in a time-dependent manner. RPT incubations also increased intracellular L-arginine (L-Arg) levels and activated the CaMKII/AMPK/Akt/eNOS signaling cascade in HUVECs. Treatment of L-Arg and ABH, arginase inhibitor, increased intracellular Ca2+ concentrations and activated CaMKII-dependent eNOS activation in ECs of WT mice, but, the effects were not observed in ECs of inositol triphosphate receptor type 1 knockout (IP3R1-/-) mice. In the aortic endothelium of WT mice, RPT also augmented nitric oxide (NO) production and attenuated reactive oxygen species (ROS) generation. In a vascular tension assay using RPT-treated aortic tissue, cumulative vasorelaxant responses to acetylcholine (Ach) were enhanced, and phenylephrine (PE)-dependent vasoconstrictive responses were retarded, although sodium nitroprusside and KCl responses were not different. In this study, we present a novel mechanism for RPT, as an arginase inhibitor, to increase cytosolic Ca2+ concentration in a L-Arg-dependent manner and enhance endothelial function through eNOS activation. [BMB Reports 2021; 54(10): 516-521].

High dose folic acid is a potential treatment for pulmonary hypertension, including when associated with COVID-19 pneumonia.

BACKGROUND: Pulmonary hypertension is a significant complication for some patients with COVID-19 pneumonia, especially those requiring intensive care. Tachyphylaxis to the current therapy, inhaled nitric oxide (iNO), is also common. In vitro, folic acid directly increases nitric oxide (NO) production and extends its duration of action; effects which could be of benefit in reversing pulmonary hypertension and severe hypoxaemia. Our work has shown that, in the systemic circulation, folic acid in high dose rapidly improves nitric oxide mediated vasodilation, by activating endothelial nitric oxide synthase (eNOS). HYPOTHESIS: A similar effect of high dose folic acid on pulmonary endothelial function would be expected from the same mechanism and would lead to improvement in pulmonary perfusion. We therefore hypothesise that folic acid, 5 mg or greater, is a useful therapeutic option for pulmonary hypertension and/or refractory severe hypoxaemia, in patients with severe COVID-19 associated pneumonia in whom NO therapy is considered, with a very low risk of adverse effects.

Mid-dose losartan mitigates diabetes-induced hepatic damage by regulating iNOS, eNOS, VEGF, and NF-κB expressions

Background/aim: Losartan, an antihypertensive drug, is highly preferred in patients with diabetes mellitus (DM) and hypertension because of its retarding effect on diabetic nephropathy. In this study, we investigated the potential therapeutic effect of different doses of losartan on hepatic damage in a streptozotocin (STZ, 50 mg/kg)-induced DM model in rats. Materials and methods: In this study, five different groups were formed: control, DM, low-dose losartan (5 mg/kg), mid-dose losartan (20 mg/kg), and high-dose losartan (80 mg/kg). Liver tissues of experimental groups were evaluated immunohistochemically for TUNEL, iNOS, eNOS, VEGF, and NF-κB pathways. In addition to immunohistochemical analysis, analyses of SOD and MDA, which are oxidative stress markers, were also performed and the results were evaluated together. Results: When biochemical and immunohistochemical findings were evaluated together, it was found that the results obtained from the mid-dose losartan group were closer to those of the control than the other groups. Conclusion: This study indicated that mid-dose losartan administration may have a therapeutic effect by inhibiting apoptosis and regulating iNOS, eNOS, VEGF, and NF-κB protein expressions in DM-induced hepatic damage.

Acanthopanax senticosus Induces Vasorelaxation via Endothelial Nitric Oxide-Dependent and -Independent Pathways.

Although Acanthopanax senticosus root extract (ASRE), a functional food used in Japan, improves peripheral blood circulation and exerts vasorelaxant effects in rats under healthy conditions, the underlying mechanisms currently remain unclear. Therefore, we investigated the mechanisms responsible for ASRE-induced relaxation in isolated thoracic aortas using organ bath techniques and examined whether ASRE affects systemic and peripheral circulation using a photoplethysmographic tail-cuff system and noncontact laser tissue blood flow meter in Wistar rats. Similar to acetylcholine (ACh), ASRE induced dose-dependent relaxation in aortas pre-contracted with phenylephrine; however, in contrast to ACh, ASRE-induced relaxation was partially inhibited by treatments with antagonists of nitric oxide (NO) synthase and soluble guanylyl cyclase as well as by endothelium removal. Contractile responses to phenylephrine or potassium chloride were observed in the presence of ASRE. The oral administration of ASRE (900 mg/kg/d for 1 wk) decreased systolic blood pressure in rats 3 h after the treatment and did not affect heart rate, tail blood flow, mass, or velocity; this decreasing effect was not observed on day 2. A 1-wk treatment with ASRE did not affect vasorelaxation in response to ASRE. These results demonstrate that ASRE induces vasorelaxation via endothelial NO production and an NO-independent pathway in rats. Based on these findings, positive impacts of ASRE on blood pressure and peripheral blood circulation cannot be expected under healthy conditions as the systemic effects of ASRE are temporary. Instead, caution is needed to prevent the occurrence of side effects (i.e., orthostatic dizziness) at the beginning of ASRE dosing.

Folic acid ameliorates homocysteine-induced angiogenesis and portosystemic collaterals in cirrhotic rats.

INTRODUCTION AND OBJECTIVES: Liver cirrhosis is characterized by increased intrahepatic resistance, splanchnic vasodilation/angiogenesis, and formation of portosystemic collateral vessels. Collaterals can cause lethal complications such as gastroesophageal variceal hemorrhage. Homocysteine is linked to vascular dysfunction and angiogenesis and higher levels have been reported in cirrhotic patients. It is also known that folic acid supplementation reverses the effects of homocysteine. However, the treatment effect in cirrhosis has yet to be investigated. MATERIAL AND METHODS: Liver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (CBDL). The CBDL rats randomly received (1) vehicle; (2) dl-homocysteine thiolactone (1g/kg/day); (3) dl-homocysteine thiolactone plus folic acid (100mg/kg/day); or (4) folic acid. On the 29th day, hemodynamic parameters, liver and renal biochemistry, protein expressions of proangiogenic factors, mesenteric vascular density and portosystemic shunting were evaluated. RESULTS: In the cirrhotic rats, homocysteine increased mesenteric vascular density and the severity of shunting. It also up-regulated the protein expressions of mesenteric vascular endothelial growth factor (VEGF) and phosphorylated-endothelial nitric oxide synthase (p-eNOS). These effects were reversed by folic acid treatment (P<0.05). CONCLUSION: Folic acid ameliorated the adverse effects of homocysteine in the cirrhotic rats, which may be related to down-regulation of the VEGF-NO signaling pathway.

Icariin improves SHR erectile function via inhibiting eNOS uncoupling.

The aim of the study was to investigate whether or not the effect of icariin on erectile function of SHR is associated with inhibition of eNOS uncoupling. Ten 12-week-old male WKY rats and 10 age-matched male SHR were evenly randomised into SHR control group, SHR+ icariin treatment group, WKY control group and WKY+ icariin group. After being treated for 4 weeks, ICPmax/MAP, the expression of NT, monomer and dimer of eNOS and the level of BH4, BH2, DHFR, NADPH oxidase and GTPCH1 in the corpus cavernosum were determined. The ICPmax/MAP and the value of BH4, DHFR and GTPCH1 in the SHR icariin treatment group were significantly higher than that of the SHR group and less than that of the WYK group and icariin-treated WKY group (p < 0.05). The value of BH2, NADPH oxidase, the ratio of eNOS monomers/dimmers and NT in the SHR icariin treatment group was significantly less than that of in the SHR group and higher than that of the WYK control group and icariin-treated WKY group (p < 0.05). Hypertension increases eNOS uncoupling in the corpus cavernosum of SHR. Inhibiting uncoupling of eNOS may be an important mechanism of icariin to improve SHR erectile function.

Crataegus Special Extract WS 1442 Effects on eNOS and microRNA 155.

Increased expression of microRNA 155 (miR-155) results in a decrease in endothelial nitric oxide synthase (eNOS) expression and impaired endothelial function. Factors that have been shown to increase expression of miR-155 may be mitigated by WS 1442, an extract of hawthorn leaves and flowers (Crataegus special extract) that contains a range of pharmacologically active substances including oligomeric proanthocyanidins and flavonoids. The purpose of this study is to determine the effect of WS 1442 on the expression of miR-155 and eNOS in the presence of tumor necrosis factor (TNF-α). Human umbilical vein endothelial cells (HUVECs) were studied after the exposure to TNF-α, with or without simvastatin (positive control) and WS 1442. The expression levels of eNOS, phosphorylated eNOS, and miR-155 in the different HUVEC treatment groups were determined by western blot and quantitative real-time polymerase chain reaction, respectively. To evaluate the effect of WS 1442 on the eNOS activity, the medium and intracellular nitrate/nitrite (NO) concentrations were also analyzed using a colorimetric Griess assay kit. The results demonstrated that TNF-α upregulated miR-155 expression and decreased eNOS expression and NO concentrations. WS 1442 also increased miR-155 expression and decreased eNOS expression but, unlike TNF-α, increased phosphorylated eNOS expression and NO concentrations. Surprisingly, WS 1442 increased miR-155 expression; however, WS 1442 mitigated the overall negative effect of miR-155 on decreasing eNOS expression by increasing expression of phosphorylated eNOS and resulting in an increase in NO concentrations. In the setting where miR-155 may be expressed, WS 1442 may offer vascular protection by increasing the expression of phosphorylated eNOS.

Phenolic Compounds as Arginase Inhibitors: New Insights Regarding Endothelial Dysfunction Treatment.

Endothelial dysfunction is characterised by the low bioavailability of nitric oxide with a relevant negative impact on the nitric oxide/cGMP pathway. The loss of nitric oxide/cGMP signaling may be caused by an increased arginase activity. Plant-derived substances, especially polyphenols, are compounds that have the potential to inhibit arginase activity and they may represent an attractive therapeutic option to combat clinical outcomes related to endothelial dysfunction. An extensive review was carried out using all available data published in English in the Pubmed database, and without restriction regarding the year of publication. Despite the increased number of new substances that have been tested as arginase inhibitors, it is rare to find a compound that satisfies all the toxicological criteria to be used in the development of a new drug. On the other hand, recent data have shown that substances from plants have great potential to be applied as arginase inhibitors, most of which are polyphenols. Of the relevant mechanisms in this process, the inhibition of arginase by natural products seems to act against endothelial dysfunction by reestablishing the vascular function and elevating nitric oxide levels (by increasing the amounts of substrate (L-arginine, and endothelial nitric oxide synthase activation and stabilisation) as well as decreasing the generation of reactive species (formed by uncoupledendothelial nitric oxide synthase). This review summarises several topics regarding arginase inhibition by natural substances as well as indicating this pathway as an emergent strategy to elevate nitric oxide levels in disorders involving endothelial dysfunction. In addition, some aspects regarding structural activity and future perspectives are discussed.

Silibinin from Silybum marianum Stimulates Embryonic Stem Cell Vascular Differentiation via the STAT3/PI3-K/AKT Axis and Nitric Oxide.

Silibinin, the bioactive compound of milk thistle (Silybum marianum), exerts tissue protective and regenerative effects that may include stem cell differentiation toward vascular cells. The purpose of the present study was to investigate whether silibinin stimulates blood vessel formation from mouse embryonic stem (ES) cells and to unravel the underlying signaling cascade. Vascular branching points were assessed by confocal laser scanning microscopy and computer-assisted image analysis of CD31-positive cell structures. Protein expression of vascular markers and activation of protein kinases were determined by western blot. Nitric oxide (NO) generation was investigated by use of the fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. Silibinin dose-dependently increased CD31-positive vascular branching points in embryoid bodies cultivated from ES cells. This was paralleled by increase of protein expression levels for the endothelial-specific markers vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor 2, and hypoxia-inducible factor-1α. Moreover, silibinin increased activation of endothelial nitric oxide synthase (eNOS), which boosted generation of NO in embryoid bodies and enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphoinositide 3-kinase (PI3-K) and AKT. Vasculogenesis, VE-cadherin expression, STAT3 and AKT phosphorylation, NO generation, and eNOS phosphorylation were inhibited by the small molecule STAT3 inhibitor Stattic, AKT inhibitor VIII, the PI3-K inhibitor LY294002, or the NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride. In conclusion, our findings indicate that silibinin induces vasculogenesis of ES cells via activation of STAT3, PI3-K, and AKT, which regulate NO generation by eNOS.

Low molecular weight fucoidan ameliorates hindlimb ischemic injury in type 2 diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Low molecular weight fucoidan (LMWF), extracted from Laminaria japonica Areschoug, is a traditional Chinese medicine, commonly used to alleviate edema, particularly for feet with numbness and pain. AIM OF THE STUDY: Diabetic mellitus (DM) patients are at high risk of developing peripheral arterial disease (PAD). Individuals with DM and PAD co-morbidity have a much higher risk of critical limb ischemia. LMWF showed several beneficial effects, such as anti-inflammation, anti-thrombosis, and enhancing revascularization. Therefore, we hypothesized that LMWF might be beneficial to diabetes-induced PAD, and investigated the therapeutic potential of LMWF on diabetic PAD rats. MATERIALS AND METHODS: Type 2 diabetic Goto-Kakizaki (GK) rats were made PAD by injection of sodium laurate into femoral artery. LMWF (20, 40 or 80mg/kg/day) or cilostazol (100mg/kg/day) were given to diabetic PAD rats for 4 weeks, respectively. The effects of LMWF on foot ulceration and claudication, plantar blood flow, collateral vessel formation, endothelium morphology, gastrocnemius injury, platelet aggregation, vessel vasodilation, and the expressions of inflammation factors, VEGF, eNOS, and nitric oxide were measured. RESULTS: We found that LMWF markedly ameliorated foot ulceration and claudication, and improved the plantar perfusion by reversing hyperreactive platelet aggregation, ameliorating endothelium-dependent vasodilation and revascularization on diabetic PAD rats. In addition, upregulation of several inflammatory factors, such as ICAM-1 and IL-1ß in the gastrocnemius muscles of ischemic hindlimb were suppressed by LMWF administration. And eNOS phosphorylation at Ser1177 and NO production were significantly enhanced in LMWF-treated diabetic PAD rats. CONCLUSIONS: Taken together, our findings demonstrated that LMWF exhibits therapeutic effect on hindlimb ischemia in type 2 diabetic rats likely through ameliorating endothelium eNOS dysfunction and enhancing revascularization, thus, providing a potential supplementary non-invasive treatment for diabetes-induced PAD.

Scutellarin attenuates vasospasm through the Erk5-KLF2-eNOS pathway after subarachnoid hemorrhage in rats.

Angiographic vasospasm, especially in the early phases (<72h) of subarachnoid hemorrhage (SAH), is one of the major complications after an aneurysm rupture and is often the cause of delayed neurological deterioration. Scutellarin (SCU), a flavonoid extracted from the traditional Chinese herb Erigeron breviscapus, has been widely accepted as an antioxidant, but the effect of SCU on vasospasm after SAH remains elusive. Endovascular perforation was conducted to induce SAH in Sprague-Dawley rats. Then, the underlying mechanism of the anti-vasospasm effect of SCU was investigated using a modified Garcia scale, India ink angiography, cross-sectional area analysis, immunohistochemistry staining and western blot. SCU (50µM, 100mg/kg) alleviated angiographic vasospasm and improved neurological function 48h after SAH and enhanced the expression of endothelial nitric oxide synthase (eNOS) at the intima of cerebral arteries. In addition, SCU upregulated the expression of phosphorylated extracellular-regulated kinase 5 (p-Erk5) and Kruppel-like factor 2 (KLF2) at 48h after SAH. However, the effects of SCU were reversed by the Erk5 inhibitor XMD8-92. Our results indicate that SCU could attenuate vasospasm and neurological deficits via modulating the Erk5-KLF2-eNOS pathway after SAH, which may provide an experimental basis for the clinical use of SCU treatment in SAH patients.

Arginase Inhibition Restores Peroxynitrite-Induced Endothelial Dysfunction via L-Arginine-Dependent Endothelial Nitric Oxide Synthase Phosphorylation.

PURPOSE: Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay. RESULTS: SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. N(G)-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition. CONCLUSION: These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions.

Ligusticum chuanxiong prevents ovariectomy-induced liver and vascular damage in rats.

Post-menopause, there is an increase in body weight, visceral adiposity, and risk of developing non-alcoholic fatty liver disease (NAFLD), which leads to various cardiovascular diseases (CVDs). Some natural products have proven useful for counteracting the detrimental effects of menopause. The rhizome of Ligusticum chuanxiong Hort. (LC) is a well-known medicinal herb widely used in Chinese communities for the treatment of CVDs. The hepatic and vascular protective effects of LC ethanolic extract under postmenopausal conditions were investigated on ovariectomized (OVX) rats supplemented with or without LC ethanolic extract (600 mg/kg body weight/day, p.o.) or 17ß-estradiol (1 mg/kg body weight/day, p.o.) for 12 weeks. The current findings demonstrated that consumption of LC ethanolic extract could reduce the body weight gain, improve serum lipid profile (lowering low density lipoprotein cholesterol but raising high density lipoprotein cholesterol), combat NAFLD, and protect vascular endothelium in the OVX rats. The beneficial effects of LC may be associated with its antioxidant or vasorelaxant compounds, which enhance the levels of hepatic antioxidant enzymes and up-regulate endothelial nitric oxide synthase mRNA expression, respectively. Taken together, LC may be a promising natural supplement for postmenopausal women to prevent NAFLD and CVDs.

Pien Tze Huang inhibits tumor angiogenesis in a mouse model of colorectal cancer via suppression of multiple cellular pathways.

Angiogenesis plays an essential role in cancer progression, which therefore has become an attractive target for anticancer treatment. Tumor angiogenesis is tightly regulated by multiple signaling pathways that usually function redundantly; in addition, crosstalk between these pathways forms a complicated network that is regulated by compensatory mechanisms. Given the complexity of pathogenic mechanisms underlying tumor angiogenesis, most currently used angiogenesis inhibitors that only target single pathways may be insufficient and probably generate drug resistance, thus, increasing the necessity for development of novel anticancer agents. Traditional Chinese medicines (TCM) are receiving great interest since they have relatively fewer side-effects and have been used for thousands of years to clinically treat various types of diseases including cancer. Pien Tze Huang (PZH), a well-known traditional Chinese formulation that was first prescribed 450 years ago, has long been used as an alternative remedy for cancers. However, the precise mechanism of PZH's anticancer activity remains to be further elucidated. Using a colorectal cancer mouse xenograft model, in the present study, we evaluated the effect of PZH on tumor angiogenesis and investigated the underlying molecular mechanisms. We found that PZH inhibited tumor growth since PZH treatment resulted in decrease in both tumor volume and tumor weight in CRC mice. In addition, PZH suppressed the activation of several signaling pathways such as STAT3, Akt and MAPKs. Consequently, the inhibitory effect of PZH on these pathways resulted in the inhibition of tumor angiogenesis as demonstrated by the decrease of microvessel density in tumor tissues. Moreover, PZH treatment reduced the expression of angiogenic factors including iNOS, eNOS, VEGF-A, bFGF as well as their specific receptors VEGFR2 and bFGFR. Altogether, our findings suggest that inhibition of tumor angiogenesis via suppression of multiple signaling pathways might be one of the mechanisms whereby PZH affects cancers.

Daily low-dose folic acid supplementation does not prevent nitroglycerin-induced nitric oxide synthase dysfunction and tolerance: a human in vivo study.

INTRODUCTION: Continuous treatment with nitroglycerin (GTN) causes tolerance and endothelial dysfunction, both of which may involve endothelial nitric oxide synthase (eNOS) dysfunction. eNOS dysfunction may be linked to depletion of tetrahydrobiopterin, and folic acid may be involved in the regeneration of this cofactor. It has been demonstrated that 10 mg/day folic acid supplementation prevents the development of GTN tolerance and GTN-induced endothelial dysfunction. However, the efficacy of daily lower-dose folic acid supplementation for preventing these phenomena has not been investigated. OBJECTIVE: To determine the effect of 1 mg/day folic acid supplementation on responses to sustained GTN therapy. METHODS AND RESULTS: On visit 1, 20 healthy male volunteers were randomly assigned to receive either oral folic acid (1 mg/day) or placebo for one week in a double- blind study. All subjects also received continuous transdermal GTN (0.6 mg/h). On visit 2, forearm blood flow was measured using venous occlusion strain-gauge plethysmography in response to incremental intra-arterial infusions of acetylcholine, N-monomethyl-L-arginine and GTN. Subjects in both groups displayed significantly decreased responses to acetylcholine and N-monomethyl-L-arginine infusions compared with a control group that received no treatment. Responses to GTN were also significantly diminished in both groups (P<0.05 for all). DISCUSSION: The present data demonstrate that daily supplementation with 1 mg folic acid does not prevent the development of GTN-induced eNOS dysfunction or tolerance.

Diallyl disulfide and diallyl trisulfide protect endothelial nitric oxide synthase against damage by oxidized low-density lipoprotein.

Garlic is viewed as an effective health food against atherosclerosis. In this study, we examined whether diallyl disulfide (DADS) and diallyl trisulfide (DATS) protect endothelial nitric oxide synthase (eNOS) activation against oxidized LDL (ox-LDL) insult and through what mechanism. We found that DADS and DATS reversed the suppression of eNOS Ser1177 phosphorylation by ox-LDL, and wortmannin abolished the reversal by DADS and DATS. Similarly, the inhibition of cellular cGMP and nitric oxide production by ox-LDL was reversed by DADS and DATS (p<0.05). This increase in nitric oxide bioavailability by the allyl sulfides was attenuated by wortmannin. Immunoprecipitation assay revealed that DADS and DATS preserved the interaction of eNOS with caveolin-1 in the membrane. In addition, DADS and DATS suppressed the reduction of the cellular eNOS protein content by ox-LDL. When cycloheximide was added to block protein synthesis, DADS and DATS suppressed eNOS protein degradation similarly to that noted by MG132. Ox-LDL increased chymotrypsin-like proteasome activity, and this increase was inhibited by the allyl sulfides and MG132 (p<0.05). These results suggest that DADS and DATS protect eNOS activity against ox-LDL insult. This protection can be attributed partly to their mediation of phosphatidylinositol 3-kinase/protein kinase B signaling and prevention of eNOS degradation.

Beneficial effects of high dose of L-arginine on airway hyperresponsiveness and airway inflammation in a murine model of asthma.

BACKGROUND: Disturbance in the delicate balance between L-arginine-metabolizing enzymes such as nitric oxide synthase (NOS) and arginase may lead to decreased L-arginine availability to constitutive forms of NOS (endothelial NOS), thereby increasing the nitro-oxidative stress and airway hyperresponsiveness (AHR). OBJECTIVE: In this study, we investigated the effects of high doses of L-arginine on L-arginine-metabolizing enzymes and subsequent biological effects such as cyclic guanosine monophosphate production, lipid peroxidation, peroxynitrite, AHR, and airway inflammation in a murine model of asthma. METHODS: Different doses of L-arginine were administered to ovalbumin-sensitized and challenged mice. Exhaled nitric oxide, AHR, airway inflammation, T(H)2 cytokines, goblet cell metaplasia, nitro-oxidative stress, and expressions of arginase 1, endothelial NOS, and inducible NOS in lung were determined. RESULTS: L-arginine significantly reduced AHR and airway inflammation including bronchoalveolar lavage fluid eosinophilia, T(H)2 cytokines, TGF-beta1, goblet cell metaplasia, and subepithelial fibrosis. Further, L-arginine increased ENO levels and cyclic guanosine monophosphate in lung and reduced the markers of nitro-oxidative stress such as nitrotyrosine, 8-isoprostane, and 8-hydroxy-2'-deoxyguanosine. This was associated with reduced activity and expression of arginase 1, increased expression of endothelial NOS, and reduction of inducible NOS in bronchial epithelia. CONCLUSION: We conclude that L-arginine administration may improve disordered nitric oxide metabolism associated with allergic airway inflammation, and alleviates some features of asthma.

Green and black tea are equally potent stimuli of NO production and vasodilation: new insights into tea ingredients involved.

Epidemiological studies suggest that consumption of tea is associated with beneficial cardiovascular effects. Since different types of tea are consumed throughout the world, a question of much interest is whether green tea is superior to black tea in terms of cardiovascular protection. We therefore compared the effects of green and black tea on nitric oxide (NO) production and vasodilation and elucidated the tea compounds involved. We chose a highly fermented black tea and determined concentrations of individual tea compounds in both green and black tea of the same type (Assam). The fermented black tea was almost devoid of catechins. However, both teas stimulated eNOS activity and phosphorylation in bovine aortic endothelial cells (BAEC) as well as vasorelaxation in rat aortic rings to a similar extent. In green tea, only epigallocatechin-3-gallate (EGCG) resulted in pronounced NO production and NO-dependent vasorelaxation in aortic rings. During tea processing to produce black tea, the catechins are converted to theaflavins and thearubigins. Individual black tea theaflavins showed a higher potency than EGCG in NO production and vasorelaxation. The thearubigins in black tea are highly efficient stimulators of vasodilation and NO production. Green and black tea compounds induced comparable phosphorylation of eNOS and upstream signalling kinases. Whereas stimulation of eNOS activity by EGCG was only slightly affected by pretreatment of cells with various ROS scavengers, TF3(theaflavin-3',3-digallate)-induced eNOS activity was partially inhibited by PEG-catalase. These results implicate that highly fermented black tea is equally potent as green tea in promoting beneficial endothelial effects. Theaflavins and thearubigins predominantly counterbalance the lack of catechins in black tea. The findings may underline the contribution of black tea consumption in prevention of cardiovascular diseases.

Elevation of intracavernous pressure and NO-cGMP activity by a new herbal formula in penile tissues of spontaneous hypertensive male rats.

AIM OF THE STUDY: We created a new herbal formulation that mainly consists of the seeds of Lycium chinense, Cornus officinalis, Rubus coreanus, Cuscuta chinensis and Schizandra chinensis. These materials have been long used by Korean people as they are known to be good for health and sexual function; hence we could say that their safety have been proven in a certain sense. We investigated the effects of this herbal formulation on the penile erection and corpus cavernosum of spontaneous hypertensive male Rats (SHRs). MATERIALS AND METHODS: We used male SHRs aged 16 weeks as a model of hypertension. The treatment groups received once a day oral doses of KH-204 at either 100 or 300mg/kg per day for 4 weeks. Distilled water was administered to the control group. To investigate the penile erection, the intracavernosal pressure (ICP) and mean arterial pressure (MAP) were recorded in all groups. We analyzed the distribution of NOS by immunohistochemical staining and the expressions of nNOS and eNOS in the isolated corpus cavernosum were measured by Western blotting. RESULTS: In the control group, the ICP/MAP ratio was 14.9+/-1.4% after pelvic nerve stimulation. The ICP/MAP ratio was markedly increased in the treatment group with KH-204 100 or 300mg/kg, compared with the control group. Immunohistochemical staining for NOS showed that eNOS and nNOS were stained as a brown color. Compared with the control group, the NOS activities of KH-204 100 or 300mg/kg were significantly increased. Also, the penile expression levels of nNOS and eNOS in the KH-204 100 and 300mg/kg treatment groups were more increased, and this was significant, than those of the control group, as was determined by Western blotting. CONCLUSIONS: This study showed that the KH-204 herbal formulation enhances intracavernous pressure and NO-cGMP activity in penile tissues of SHR male rats.