In silico analysis of single nucleotide polymorphism (rs34377097) of TBXA2R gene and pollen induced bronchial asthma susceptibility in West Bengal population, India.

Introduction: Prevalence of asthma is increasing steadily among general population in developing countries over past two decades. One of the causative agents of broncho-constriction in asthma is thromboxane A2 receptor (TBXA2R). However few studies of TBXA2R polymorphism were performed so far. The present study aimed to assess potential association of TBXA2R rs34377097 polymorphism causing missense substitution of Arginine to Leucine (R60L) among 482 patients diagnosed with pollen-induced asthma and 122 control participants from West Bengal, India. Also we performed in-silico analysis of mutated TBXA2R protein (R60L) using homology modeling. Methods: Clinical parameters like Forced expiratory volume in 1 second (FEV1), FEV1/Forced vital capacity (FVC) and Peak expiratory flow rate (PEFR) were assessed using spirometry. Patients' sensitivity was measured by skin prick test (SPT) against 16 pollen allergens. Polymerase chain reaction-based Restriction fragment length polymorphism was done for genotyping. Structural model of wild type and homology model of polymorphic TBXA2R was generated using AlphaFold2 and MODELLER respectively. Electrostatic surface potential was calculated using APBS plugin in PyMol. Results: Genotype frequencies differed significantly between the study groups (P=0.03). There was no significant deviation from Hardy-Weinberg equilibrium in control population (χ2=1.56). Asthmatic patients have significantly higher frequency of rs34377097TT genotype than control subjects (P=0.03). SPT of patients showed maximum sensitivity in A. indica (87.68%) followed by C. nusifera (83.29%) and C. pulcherima (74.94%). Significant difference existed for pollen sensitivity in adolescent and young adult (P=0.01) and between young and old adult (P=0.0003). Significant negative correlation was found between FEV1/FVC ratio and intensity of SPT reactions (P<0.0001). Significant association of FEV1, FEV1/FVC and PEFR was observed with pollen-induced asthma. Furthermore, risk allele T was found to be clinically correlated with lower FEV1/FVC ratio (P=0.015) in patients. Our data showed R60L polymorphism, which was conserved across mammals, significantly reduced positive electrostatic charge of polymorphic protein in cytoplasmic domain thus altered downstream pathway and induced asthma response. Discussion: The present in-silico study is the first one to report association of TBXA2R rs34377097 polymorphism in an Indian population. It may be used as prognostic marker of clinical response to asthma in West Bengal and possible target of therapeutics in future.

XJ-8, a natural compound isolated from Sanguis draxonis, inhibits platelet function and thrombosis by targeting MAP3K3.

BACKGROUND: Vascular injury initiates rapid platelet activation, which is critical for haemostasis, while it also causes fatal thrombotic diseases, such as myocardial infarction or ischemic stroke. OBJECTIVES: To study the inhibitory effects and underlying mechanisms of XJ-8, a natural compound isolated from Sanguis draxonis, on platelet activation and thrombosis. METHODS: The regulatory effects of XJ-8 on the dense granule release, thromboxane A2 (TxA2 ) synthesis, α-granule release, activation of integrin αIIbß3, and aggregation of platelets induced by multiple agonists were investigated in in vitro experiments. The effects of XJ-8 on bleeding time and FeCl3 -induced carotid artery thrombosis were also evaluated in in vivo experiments. Furthermore, we investigated the underlying mechanisms by which XJ-8 exerted its pharmacological effects. RESULTS: XJ-8 not only significantly inhibited the dense granule release, TxA2 synthesis, and aggregation of platelets induced by multiple agonists, but also exerted extending effects on bleeding time and therapeutic effects on thrombotic disease. In addition, XJ-8 selectively and moderately inhibited the activity of mitogen-activated protein kinase kinase kinase 3 (MAP3K3) and the activation of signalling pathways downstream MAP3K3, which play important roles in platelet activation. CONCLUSION: XJ-8 can inhibit platelet function and thrombosis by targeting MAP3K3 and has potential to be developed into a novel therapeutic agent for the treatment of thrombotic diseases.

The Role of 20-HETE, COX, Thromboxane Receptors, and Blood Plasma Antioxidant Status in Vascular Relaxation of Copper-Nanoparticle-Fed WKY Rats.

Recently, the addition of copper nanoparticles (NPs) in a daily diet (6.5 mg/kg) was studied in different animal models as a possible alternative to ionic forms. Male Wistar-Kyoto rats (24-week-old, n = 11) were fed with copper, either in the form of carbonate salt (Cu6.5) or metal-based copper NPs (NP6.5), for 8 weeks. The third group was fed with a half dose of each (NP3.25 + Cu3.25). The thoracic aorta and blood plasma was studied. Supplementation with NP6.5 decreased the Cu (×0.7), Cu/Zn-ratio (×0.6) and catalase (CAT, ×0.7), and increased Zn (×1.2) and superoxide dismutase (SOD, ×1.4). Meanwhile, NP3.25 + Cu3.25 decreased the Cu/Zn-ratio (×0.7), and CAT (×0.7), and increased the daily feed intake (×1.06). Preincubation with either the selective cyclooxygenase (COX)-2 inhibitor, or the non-selective COX-1/2 inhibitor attenuated vasodilation of rat thoracic aorta in the NP6.5 group exclusively. However, an increased vasodilator response was observed in the NP6.5 and NP3.25 + Cu3.25 group of rats after preincubation with an inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) formation, and the thromboxane receptor (TP) antagonist. Significant differences were observed between the NP6.5 and NP3.25 + Cu3.25 groups of rats in: dietary intake, acetylcholine-induced vasodilation, and response to COX-inhibitors. Copper NPs in a standard daily dose had more significant effects on the mechanism(s) responsible for the utilization of reactive oxygen species in the blood plasma with the participation of prostanoids derived from COX-2 in the vascular relaxation. Dietary copper NPs in both doses modified vasodilation through the vasoconstrictor 20-HETE and the TP receptors.

The Effect of Hemp (Cannabis sativa L.) Seeds and Hemp Seed Oil on Vascular Dysfunction in Obese Male Zucker Rats.

Seeds of industrial hemp (Cannabis sativa L.) contain a large amount of protein (26.3%), dietary fiber (27.5%), and fatty acids (33.2%), including linoleic, α-linolenic, and some amount of γ-linolenic acid. In our study, obese male Zucker rats (n = 6) at 8 weeks of age were supplemented for a further 4 weeks with either ground hemp seeds (12% diet) or lipid fractions in the form of hemp seed oil (4% diet). Hemp oil decreased blood plasma HDL-cholesterol (x0.76, p ≤ 0.0001), triglycerides (x0.55, p = 0.01), and calculated atherogenic parameters. Meanwhile, hemp seeds decreased HDL-cholesterol (x0.71, p ≤ 0.0001) and total cholesterol (x0.81, p = 0.006) but not the atherogenic index. The plasma antioxidant capacity of water-soluble compounds was decreased by the seeds (x0.30, p = 0.0015), which in turn was associated with a decrease in plasma uric acid (x0.18, p = 0.03). Dietary hemp seeds also decreased plasma urea (x0.80, p = 0.02), while the oil decreased the plasma total protein (x0.90, p = 0.05). Hemp seeds and the oil decreased lipid peroxidation in the blood plasma and in the heart (reflected as malondialdehyde content), improved contraction to noradrenaline, and up-regulated the sensitivity of potassium channels dependent on ATP and Ca2+. Meanwhile, acetylcholine-induced vasodilation was improved by hemp seeds exclusively. Dietary supplementation with ground hemp seeds was much more beneficial than the oil, which suggests that the lipid fractions are only partially responsible for this effect.

Activation of Nrf2/HO-1 signaling: An important molecular mechanism of herbal medicine in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress.

Introduction: Recently, Nrf2/HO-1 has received extensive attention as the main regulatory pathway of intracellular defense against oxidative stress and is considered an ideal target for alleviating endothelial cell (EC) injury. Objectives: This paper aimed to summarized the natural monomers/extracts that potentially exert protective effects against oxidative stress in ECs. Methods: A literature search was carried out regarding our topic with the keywords of "atherosclerosis" or "Nrf2/HO-1" or "vascular endothelial cells" or "oxidative stress" or "Herbal medicine" or "natural products" or "natural extracts" or "natural compounds" or "traditional Chinese medicines" based on classic books of herbal medicine and scientific databases including Pubmed, SciFinder, Scopus, the Web of Science, GoogleScholar, BaiduScholar, and others. Then, we analyzed the possible molecular mechanisms for different types of natural compounds in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress. In addition, perspectives for possible future studies are discussed. Results: These agents with protective effects against oxidative stress in ECs mainly include phenylpropanoids, flavonoids, terpenoids, and alkaloids. Most of these agents alleviate cell apoptosis in ECs due to oxidative stress, and the mechanisms are related to Nrf2/HO-1 signaling activation. However, despite continued progress in research on various aspects of natural agents exerting protective effects against EC injury by activating Nrf2/HO-1 signaling, the development of new drugs for the treatment of atherosclerosis (AS) and other CVDs based on these agents will require more detailed preclinical and clinical studies. Conclusion: Our present paper provides updated information of natural agents with protective activities on ECs against oxidative stress by activating Nrf2/HO-1. We hope this review will provide some directions for the further development of novel candidate drugs from natural agents for the treatment of AS and other CVDs.

The combination of bromelain and curcumin as an immune-boosting nutraceutical in the prevention of severe COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is still ongoing, while no treatment has been proven effective. COVID-19 pathophysiology involves the activation of three main pathways: the inflammatory, the coagulation and the bradykinin cascades. Here, we highlight for the first time the joint potential therapeutic role of bromelain and curcumin, two well-known nutraceuticals, in the prevention of severe COVID-19. Bromelain (a cysteine protease isolated from the pineapple stem) and curcumin (a natural phenol found in turmeric) exert important immunomodulatory actions interfering in the crucial steps of COVID-19 pathophysiology. Their anti-inflammatory properties include inhibition of transcription factors and subsequent downregulation of proinflammatory mediators. They also present fibrinolytic and anticoagulant properties. Additionally, bromelain inhibits cyclooxygenase and modulates prostaglandins and thromboxane, affecting both inflammation and coagulation, and also hydrolyzes bradykinin. Interestingly, curcumin has been shown in silico studies to prevent entry of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into cells as well as viral replication, while a recent experimental study has demonstrated that bromelain may also inhibit viral entry into cells. Notably, bromelain substantially increases the absorption of curcumin after oral administration. To the best of our knowledge, this is the first report highlighting the significance of bromelain and, most importantly, the potential preventive value of the synergistic effects of bromelain and curcumin against severe COVID-19.

Plantain (Plantago L.) species as modulators of prostaglandin E2 and thromboxane A2 production in inflammation.

ETHNOPHARMACOLOGICAL RELEVANCE: Worldwide distributed plantains (genus Plantago L.) are extensively used in the traditional, but some of them are also accepted in the modern medicine. Wide range of usages is mainly connected to the inflammation processes. AIM OF THE STUDY: To support usage of renowned P. lanceolata L. and P. major L., underinvestigated P. altissima L., P. argentea Chaix, P. holosteum Scop. and P. media L. methanol extracts, and their typical constituents (aucubin, apigenin, apigenin-7-O-glucoside, luteolin, luteolin-7-O-glucoside, chlorogenic and ursolic acid) in treatment of inflammation disorders, we conducted study on plantain potential to inhibit production of inflammatory mediators, prostaglandin E2 (PGE2) and thromboxane A2 (TXA2). MATERIALS AND METHODS: LPS-stimulated monocytes (U937 cell line) were used as a model-system to examine anti-inflammatory potential of plantains and their constituents. Produced PGE2 and TXA2 were quantified by LC-MS/MS; qPCR was applied to examine related gene (PLA2, COX-1, COX-2, mPGES-1, mPGES-2, cPGES, TXAS) expression; LC-MS/MS and LC-UV/VIS techniques to analyze extracts composition. RESULTS: In general, examined plantain extracts showed comparable inhibition activity of PGE2 and TXA2 production as aspirin at low-dose concentration. Underinvestigated P. altissima can be pointed out, since it exerted the strongest effect on both PGE2 production and related gene expression. Notable suppression of TXA2 production by P. lanceolata and P. major was observed. But, PCA analysis showed no obvious grouping, implicating that different mechanisms of action are responsible for each sample activity. In most cases, positive correlation was found between content of apigenin and ursolic acid and extracts suppression of PGE2 and TXA2 production and related genes expression. CONCLUSIONS: P. altissima can be regarded as promising anti-inflammatory agent, while novel aspect of P. lanceolata and P. major application (anti-aggregation) can be suggested. P. argentea and P. media could be considered as a good source of ursolic acid.

Therapeutic effects of JLX001 on cerebral ischemia through inhibiting platelet activation and thrombus formation in rats.

(3ß,5α,16α,20S)-4,4,14-trimethyl-3,20-bis(methylamino)-9,19-cyclopregnan-16-ol-dihydrochloride (JLX001), a derivative of cyclovirobuxine D (CVB-D), is a novel compound from synthesis. This study aims to confirm the therapeutic effect of JLX001 on cerebral ischemia and researchits antiplatelet and antithrombosis activities via thromboxane (TXA2)/phospholipase C-ß-3(PLCß3)/protein kinase C (PKC) pathway suppression. The therapeutic effects of JLX001 was evaluated by infarct sizes, brain edema and neurological scores in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Brain TXA2 and prostacyclin (PGI2) were measured by enzyme-linked immunosorbentassay (ELISA). P-PLCß3and activated PKC were detected by immunohistochemical method. Adenosine diphosphate (ADP) or 9, 11-dieoxy-11α, 9α-epoxymethanoeprostaglandin F2α (U46619) was used as platelet agonist in the in vivo and in vitro platelet aggregation experiments. Clotting time and bleeding time were determined. Besides, two whole-animal experiments including arteriovenous shunt thrombosis and pulmonary thromboembolism model were conducted. Results showed that JLX001 treatment markedly alleviated cerebral infarcts, edema, and neurological scores in permanent middle cerebral artery occlusion (pMCAO) rats. Brain TXA2 level, p-PLCß3and activated PKC were decreased, while PGI2level had no significant change. Besides, JLX001 inhibited platelet aggregation induced by ADP or U46619 and exhibited anti-coagulation effects with a minor bleeding risk. In the two whole-animal experiments, JLX001 inhibited thrombus formation. In summary, JLX001 attenuates cerebral ischemia injury and the underlying mechanisms relate to inhibiting platelet activation and thrombus formation via TXA2/PLCß3/PKC pathway suppression.

Role of thromboxane A2 signaling in endothelium-dependent contractions of arteries.

Thromboxane A2 (TxA2) plays a very important role in various cardiovascular diseases through its action on platelet aggregation, vasoconstriction, and proliferation. The present article focuses on the role of TxA2 signaling in endothelium-dependent contractions of arteries. Arachidonic acid (AA) is metabolized by cyclooxygenase (COX) to form the unstable prostaglandin H2 which is further converted into TxA2. After being produced by thromboxane synthase (TxAS), TxA2 ultimately stimulates TxA2/prostanoid (TP) receptor to induce vasoconstriction. The calcium ionophore A23187, the prostanoid precursor AA, or the muscarinic receptor agonist acetylcholine (ACh) can evoke endothelium-dependent contractions associated with TxA2. The endothelium-dependent contractions shown in hypertension, diabetes, atherogenesis, and other cardiovascular diseases have been significantly reduced by antagonism of COX, TxAS, or TP receptor. So inhibition of the bioavailability and/or effect of TxA2 may be promising therapeutic targets to prevent these diseases. Especially some bioactive compounds isolated from medicinal plants will provide new pharmacological approaches to promote vascular health.

Study Progress in Signaling through Thromboxane A2 Receptors in Platelet and Anti-platelet Drugs / 中国药师

Thromboxane A2 receptors (TPs) widely distribute in different organ systems and localize both on cell membranes and in intracellular structures.TPs are the members of seven-transmembrane G-protein-coupled receptor (GPCR) super family.Historical-ly, the involvement of TPs in platelet functions has received the greatest attention .TPs have the capacity to activate different signaling cascades which regulate platelet shape change , aggregation and secretion response .Currently , anti-platelet drugs primarily act on re-ceptors and /or signaling molecules in activation pathways .The signaling transduction of TPs in platelet contributes to the investigation of the effects of extracts of traditional Chinese medicine on antiplatelet aggregation and the exploration of the action mechanisms .

Effects of traditional Chinese medicine syndrome differentiation quadruple therapy on serum vascular active substance in patients with acute pancreatitis / 中国中西医结合急救杂志

Objective To observe the effects of traditional Chinese medicine (TCM) syndrome differentiation quadruple therapy on serum thromboxane A2 (TXA2), prostacyclin (PGI2) and platelet activating factor (PAF) levels in patients with acute pancreatitis (AP). Methods Ninety patients with AP admitted to the First Affiliated Hospital of Henan University of TCM from January 2016 to March 2017, and they were divided into an observation group and a control group according to the random numbers generated by computer inpatients, 45 cases in each group. The control group was given routine treatment of western medicine, and the observation group was given TCM syndrome differentiation quadruple therapy according to the patient's disease individual situation and on the basis of western medicine treatment. The TCM syndrome differentiation quadruple therapy included the following methods: intragastric administration of TCM decoction [gastrointestinal excess heat syndrome (rhubarb, sodium sulfate, aurantii fructus immaturus, magnolia bark, etc.), damp heat syndrome of liver and gallbladder (radix bupleuri, aurantii fructus immaturus, baical skullcap root, rhubarb, etc.), each group of above agents immersed in water and decocted to make juice 400 mL, once 100 mL taken orally, every 4 hours]; retention enema with TCM decoction [rhubarb, magnolia bark, aurantii fructus immaturus, sodium sulfate (dissolved) etc, each dose of agents forming decoction 400 mL, 200 mL taken for proctoclysis, once every 6 hours]; Chinese medicine package (boswellin, myrrha, dandelion, coptidis rhizoma and so on crushed and mixed with honey, then applied to the body surface of the pancreas and its periphery, 1 dose each time for 4 hours, once a day ); intravenous drip of blood-activating and stasis-resolving TCM (Dengzhanhuasu injection 100 mg added to 5% glucose solution 250 mL for intravenous drip). The times of disappearance of abdominal distension, abdominal pain, and the recovery times of bowel sound, blood amylase, lipase, C-reactive protein (CRP), white blood cell count (WBC) levels to normal were compared between the two groups; the modified CT severity index (MCTSI) score and the changes of serum TXA2, PAF and PGI2 levels were observed before and after treatment in the two groups. Results The abdominal pain and abdominal distension disappearance times in observation group were shorter than those in control group [abdominal pain (days): 5.07±1.88 vs. 6.02±1.89, abdominal distension (days): 3.50±1.49 vs. 4.40±1.53, both P < 0.05]; the recovery times of bowel sounds, WBC, CRP, amylase and lipase to normal were shorter than those of the control group [bowel sounds (days): 4.05±1.79 vs. 5.00±1.55, WBC (days): 3.93±1.49 vs. 5.98±2.90, CRP (days): 6.17±2.46 vs. 7.92±2.84, blood amylase (days): 3.5 (3.0, 5.0) vs. 5.0 (3.0, 5.5), lipase (days): 5.0 (3.0, 7.0) vs. 6.5 (5.0, 9.0), all P <0.05]; the scores of MCTSI in the two groups were lower than those before treatment and the degree of decrease in the observation group was more significant than that in the control group [2 (0, 4) vs. 4 (0, 6), P < 0.05]. The TXA2 and PAF levels of the two groups were significantly lower than those before treatment and the level of PGI2 was significantly higher than that before treatment; after treatment for 3 days, the differences between the two groups showed statistical significance and on the 7th day after treatment, the degrees of improvement in observation group were more obvious than those of the control group [TXA2 (ng/L): 276.81±31.48 vs. 345.42±47.27, PAF (ng/L): 72.65±17.61 vs. 89.77±15.59, PGI2 (ng/L): 104.43±18.67 vs. 94.37±17.91, all P < 0.05]; on the 14th day after treatment, the values of the two groups were very close and there were no statistically significant differences (all P >0.05). Conclusions The TCM differentiation syndrome quadruple therapy for treatment of AP is beneficial to the disappearance of clinical symptoms of patients with different syndromes, recovery of abnormal signs and improvement of laboratory indexes, and its early use can significantly reduce the serum levels of TXA2, PAF and increase the level of PGI2 in patients with AP.

Effects of traditional Chinese medicine syndrome differentiation quadruple therapy on serum vascular active substance in patients with acute pancreatitis / 中国中西医结合急救杂志

Objective To observe the effects of traditional Chinese medicine (TCM) syndrome differentiation quadruple therapy on serum thromboxane A2 (TXA2), prostacyclin (PGI2) and platelet activating factor (PAF) levels in patients with acute pancreatitis (AP). Methods Ninety patients with AP admitted to the First Affiliated Hospital of Henan University of TCM from January 2016 to March 2017, and they were divided into an observation group and a control group according to the random numbers generated by computer inpatients, 45 cases in each group. The control group was given routine treatment of western medicine, and the observation group was given TCM syndrome differentiation quadruple therapy according to the patient's disease individual situation and on the basis of western medicine treatment. The TCM syndrome differentiation quadruple therapy included the following methods: intragastric administration of TCM decoction [gastrointestinal excess heat syndrome (rhubarb, sodium sulfate, aurantii fructus immaturus, magnolia bark, etc.), damp heat syndrome of liver and gallbladder (radix bupleuri, aurantii fructus immaturus, baical skullcap root, rhubarb, etc.), each group of above agents immersed in water and decocted to make juice 400 mL, once 100 mL taken orally, every 4 hours]; retention enema with TCM decoction [rhubarb, magnolia bark, aurantii fructus immaturus, sodium sulfate (dissolved) etc, each dose of agents forming decoction 400 mL, 200 mL taken for proctoclysis, once every 6 hours]; Chinese medicine package (boswellin, myrrha, dandelion, coptidis rhizoma and so on crushed and mixed with honey, then applied to the body surface of the pancreas and its periphery, 1 dose each time for 4 hours, once a day ); intravenous drip of blood-activating and stasis-resolving TCM (Dengzhanhuasu injection 100 mg added to 5% glucose solution 250 mL for intravenous drip). The times of disappearance of abdominal distension, abdominal pain, and the recovery times of bowel sound, blood amylase, lipase, C-reactive protein (CRP), white blood cell count (WBC) levels to normal were compared between the two groups; the modified CT severity index (MCTSI) score and the changes of serum TXA2, PAF and PGI2 levels were observed before and after treatment in the two groups. Results The abdominal pain and abdominal distension disappearance times in observation group were shorter than those in control group [abdominal pain (days): 5.07±1.88 vs. 6.02±1.89, abdominal distension (days): 3.50±1.49 vs. 4.40±1.53, both P < 0.05]; the recovery times of bowel sounds, WBC, CRP, amylase and lipase to normal were shorter than those of the control group [bowel sounds (days): 4.05±1.79 vs. 5.00±1.55, WBC (days): 3.93±1.49 vs. 5.98±2.90, CRP (days): 6.17±2.46 vs. 7.92±2.84, blood amylase (days): 3.5 (3.0, 5.0) vs. 5.0 (3.0, 5.5), lipase (days): 5.0 (3.0, 7.0) vs. 6.5 (5.0, 9.0), all P <0.05]; the scores of MCTSI in the two groups were lower than those before treatment and the degree of decrease in the observation group was more significant than that in the control group [2 (0, 4) vs. 4 (0, 6), P < 0.05]. The TXA2 and PAF levels of the two groups were significantly lower than those before treatment and the level of PGI2 was significantly higher than that before treatment; after treatment for 3 days, the differences between the two groups showed statistical significance and on the 7th day after treatment, the degrees of improvement in observation group were more obvious than those of the control group [TXA2 (ng/L): 276.81±31.48 vs. 345.42±47.27, PAF (ng/L): 72.65±17.61 vs. 89.77±15.59, PGI2 (ng/L): 104.43±18.67 vs. 94.37±17.91, all P < 0.05]; on the 14th day after treatment, the values of the two groups were very close and there were no statistically significant differences (all P >0.05). Conclusions The TCM differentiation syndrome quadruple therapy for treatment of AP is beneficial to the disappearance of clinical symptoms of patients with different syndromes, recovery of abnormal signs and improvement of laboratory indexes, and its early use can significantly reduce the serum levels of TXA2, PAF and increase the level of PGI2 in patients with AP.

Antiplatelet and antithrombotic effects of cordycepin-enriched WIB-801CE from Cordyceps militaris ex vivo, in vivo, and in vitro.

BACKGROUND: A species of the fungal genus Cordyceps has been used as a complementary and alternative medicine of traditional Chinese medicine, and its major component cordycepin and cordycepin-enriched WIB-801CE are known to have antiplatelet effects in vitro. However, it is unknown whether they have also endogenous antiplatelet and antithrombotic effects. In this study, to resolve these doubts, we prepared cordycepin-enriched WIB-801CE, an ethanol extract from Cordyceps militaris-hypha, then evaluated its ex vivo, in vivo, and in vitro antiplatelet and antithrombotic effects. METHODS: Ex vivo effects of WIB-801CE on collagen- and ADP-induced platelet aggregation, serotonin release, thromboxane A2 (TXA2) production and its associated activities of enzymes [cyclooxygenase-1 (COX-1), TXA2 synthase (TXAS)], arachidonic acid (AA) release and its associated phosphorylation of phospholipase Cß3, phospholipase Cγ2 or cytosolic phospholipase A2, mitogen-activated protein kinase (MAPK) [p38 MAPK, extracellular signal-regulated kinase (ERK)], and blood coagulation time in rats were investigated. In vivo effects of WIB-801CE on collagen plus epinephrine-induced acute pulmonary thromboembolism, and tail bleeding time in mice were also inquired. In vitro effects of WIB-801CE on cytotoxicity, and fibrin clot retraction in human platelets, and nitric oxide (NO) production in RAW264.7 cells or free radical scavenging activity were studied. RESULTS: Cordycepin-enriched WIB-801CE inhibited ex vivo platelet aggregation, TXA2 production, AA release, TXAS activity, serotonin release, and p38 MAPK and ERK2 phosphorylation in collagen- and ADP-activated rat platelets without affecting blood coagulation. Furthermore, WIB-801CE manifested in vivo inhibitory effect on collagen plus epinephrine-induced pulmonary thromboembolism mice model. WIB-801CE inhibited in vitro NO production and fibrin clot retraction, but elevated free radical scavenging activity without affecting cytotoxicity against human platelets. CONCLUSION: WIB-801CE inhibited collagen- and ADP-induced platelet activation and its associated thrombus formation ex vivo and in vivo. These were resulted from down-regulation of TXA2 production and its related AA release and TXAS activity, and p38MAPK and ERK2 activation. These results suggest that WIB-801CE has therapeutic potential to treat platelet activation-mediated thrombotic diseases in vivo.

In vitro inhibition of platelet aggregation by peptides derived from oat (Avena sativa L.), highland barley (Hordeum vulgare Linn. var. nudum Hook. f.), and buckwheat (Fagopyrum esculentum Moench) proteins.

Bioactive compounds present in foods could have beneficial effects on human health. In this study, we report the capacity of peptides released from oat, highland barley, and buckwheat proteins after enzymatic digestion to inhibit platelet aggregation in vitro. All hydrolysates showed high antiplatelet activity, with IC50 values of 0.282mg/ml (oat flour gastrointestinal hydrolysate, 6h) to 2.496mg/ml (highland barley glutelin tryptic hydrolysate, 14h) in a dose-dependent manner. Thirty-eight peptides with more than seven residues were identified in the tryptic hydrolysates of oat globulin. Results of computational modeling revealed that nine peptides, including ALPIDVLANAYR, EFLLAGNNKR, GEEFGAFTPK, QLAQIPR, LQAFEPLR, ALPVDVLANAYR, GEEFDAFTPK, QKEFLLAGNNK, and TNPNSMVSHIAGK bound the cyclooxygenase-1 active centers with low binding energy (-6.5 to -7.5kcal/mol). This is the first report to identify antiplatelet peptides from grain hydrolysates and the binding modes at the molecular level, leading to their possible use as functional food ingredients to prevent thrombosis.

Decichine enhances hemostasis of activated platelets via AMPA receptors.

INTRODUCTION: Dencichine, one of the non-protein amino acids present in the roots of Panax notoginseng, has been found to shorten bleeding time of mice and increase the number of platelets. However, the exact underlying mechanisms have not been elucidated yet. This study was aimed to identify the hemostatic effect of dencichine and uncover its mechanisms. MATERIALS AND METHODS: Hemostatic effect was assessed by measuring tail bleeding time and coagulation indices of rats. PT, APTT, TT and FIB concentration were measured using a Sysmex CA-1500 plasma coagulation analyzer. Platelet aggregation rate was determined by using a platelet aggregometer. Concentration of cyotosolic calcium was evaluated by Fluo-3 and levels of cyclic adenosine monophosphate (cAMP) and thromboxane A2 (TXA2) were measured by ELISA method. RESULTS AND CONCLUSION: Dencichine administered orally shortened tail bleeding time, reduced APTT and TT but increased the concentration of FIB in plasma in a dose-dependent manner. When induced with trap, dencichine could elevate the cytoplasmic concentration of calcium, and secretion of TXA2 as well as the ratio of TXA2 to PGI2 from platelets. Meanwhile, it decreased the level of intracellular cAMP. However, CNQX could block the enhanced hemostatic effect of dencichine. These results suggested that dencichine exerted hemostatic function via AMPA receptors on platelets, therefore, facilitated coagulation cascade in a paracrine fashion by control of platelet cytosolic calcium influx, cAMP production and TXA2 release. Current study may contribute to its clinical use in therapy of hemorrhage.

Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis.

INTRODUCTION: Derived from the root of Panax ginseng C.A.Mey, Panax notoginsenosides (PNS) is a widely used herbal medicine to treat atherothrombotic diseases in Asian medicine. Ginsenoside Rg1 is one of the main compounds responsible for the pharmaceutical actions of PNS. As platelets play pivotal roles in atherothrombogenesis, we therefore studied the effect of Rg1 on platelet activation and its underlying mechanisms. MATERIALS AND METHODS: Human platelets are obtained from healthy subjects. Platelet activation and the inhibition of Rg1 were assessed by Born aggregometer, flow cytmetry, flow chamber and western blot. The in vivo thrombosis model was induced by 10% FeCl3 on mesenteric arterioles of wild type B57/b6 mice. RESULTS: Rg1 significantly inhibited platelet aggregation induced by thrombin, ADP, collagen and U46619, e.g., aggregation rate stimulated by 0.1UmL(-1) thrombin was decreased 46% by Rg1. Rg1 also reduced thrombin (0.1UmL(-1))-enhanced fibrinogen binding and P-selectin expression of single platelet by 81% and 66%, respectively. Rg1 affected αIIbß3-mediated outside-in signaling as demonstrated by diminished platelet spreading on immobilized fibrinogen. Rg1 also decreased the rate of clot retraction in platelet rich plasma. Furthermore, Rg1 decreased platelet adhesion on collagen surface under a shear rate correlated to the arterial flow (1000s(-1)) by approximately 70%. Western blot showed that Rg1 potently inhibited ERK phosphrylation. The in vitro findings were further evaluated in the mouse model of in vivo arterial thrombosis, and Rg1 was found to prolong the mesenteric arterial occlusion time (34.9±4.1min without and 64.3±4.9min with Rg1; p<0.01). CONCLUSIONS: Rg1 inhibits platelet activation via the inhibition of ERK pathway, and attenuates arterial thrombus formation in vivo.

Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay.

BACKGROUND: Microfluidic devices recreate the hemodynamic conditions of thrombosis. METHODS: Whole blood inhibited with PPACK was treated ex vivo with inhibitors and perfused over collagen for 300 s (wall shear rate=200 s(-1)) using a microfluidic flow assay. Platelet accumulation was measured in the presence of COX-1 inhibitor (aspirin, ASA), P2Y1 inhibitor (MRS 2179), P2Y12 inhibitor (2MeSAMP) or combined P2Y1 and P2Y12 inhibitors. RESULTS: High dose ASA (500 µM), 2MeSAMP (100 µM), MRS 2179 (10 µM), or combined 2MeSAMP and MRS 2179 decreased total platelet accumulation by 27.5%, 75.6%, 77.7%, and 87.9% (p<0.01), respectively. ASA reduced secondary aggregation rate between 150 and 300 s without effect on primary deposition rate on collagen from 60 to 150 s. In contrast, 2MeSAMP and MRS 2179 acted earlier and reduced primary deposition to collagen between 60 and 105 s and secondary aggregation between 105 and 300 s. R(COX) and R(P2Y) (defined as a ratio of secondary aggregation rate to primary deposition rate) demonstrated 9 of 10 subjects had R(COX)<1 or R(P2Y)<1 following ASA or 2MeSAMP addition, while 6 of 10 subjects had R(P2Y)<1 following MRS 2179 addition. Combined MRS 2179 and 2MeSAMP inhibited primary platelet deposition rate and platelet secondary aggregation beyond that of each individual inhibitor. Receiver-Operator Characteristic area under the curve (AUC) indicated the robustness of R(COX) and R(P2Y) to detect inhibition of secondary platelet aggregation by ASA, 2MeSAMP, and MRS 2179 (AUC of 0.874 0.966, and 0.889, respectively). CONCLUSIONS: Microfluidic devices can detect platelet sensitivity to antiplatelet agents. The R-value can serve as a self-normalized metric of platelet function for a single blood sample.

Estrogen, vascular estrogen receptor and hormone therapy in postmenopausal vascular disease.

Cardiovascular disease (CVD) is less common in premenopausal women than men of the same age or postmenopausal women, suggesting vascular benefits of estrogen. Estrogen activates estrogen receptors ERα, ERß and GPR30 in endothelium and vascular smooth muscle (VSM), which trigger downstream signaling pathways and lead to genomic and non-genomic vascular effects such as vasodilation, decreased VSM contraction and growth and reduced vascular remodeling. However, randomized clinical trials (RCTs), such as the Women's Health Initiative (WHI) and Heart and Estrogen/progestin Replacement Study (HERS), have shown little vascular benefits and even adverse events with menopausal hormone therapy (MHT), likely due to factors related to the MHT used, ER profile, and RCT design. Some MHT forms, dose, combinations or route of administration may have inadequate vascular effects. Age-related changes in ER amount, distribution, integrity and post-ER signaling could alter the vascular response to MHT. The subject's age, preexisting CVD, and hormone environment could also reduce the effects of MHT. Further evaluation of natural and synthetic estrogens, phytoestrogens, and selective estrogen-receptor modulators (SERMs), and the design of appropriate MHT combinations, dose, route and 'timing' could improve the effectiveness of conventional MHT and provide alternative therapies in the peri-menopausal period. Targeting ER using specific ER agonists, localized MHT delivery, and activation of specific post-ER signaling pathways could counter age-related changes in ER. Examination of the hormone environment and conditions associated with hormone imbalance such as polycystic ovary syndrome may reveal the causes of abnormal hormone-receptor interactions. Consideration of these factors in new RCTs such as the Kronos Early Estrogen Prevention Study (KEEPS) could enhance the vascular benefits of estrogen in postmenopausal CVD.

A prospective, randomized, double-blind, multicenter comparative study on the safety and efficacy of Celecoxib and GCSB-5, dried extracts of six herbs, for the treatment of osteoarthritis of knee joint.

ETHNOPHARMACOLOGICAL RELEVANCE: This prospective, randomized, double-blind, multicenter study compared the efficacy and safety of Celecoxib and GCSB-5, a new product from extracts of six herbs, for the treatment of knee osteoarthritis. MATERIALS AND METHODS: A total of 198 eligible patients were randomly assigned to the Celecoxib group (n=99 patients) or the GCSB-5 group (n=99 patients) for the 12-week study. The amount of change and percentage of the change in Western Ontario and McMaster Universities (WOMAC) Arthritis Index from the baseline, the change in pain on walking by visual analogue scale (VAS), physician's global assessment on response to therapy (PGART) by five point Likert scale, and the amount of rescue medicine taken were used as parameters for efficacy. Adverse drug reactions (ADRs) were carefully investigated. RESULTS: The WOMAC score improved in both the Celecoxib group and GCSB-5 group by 20.5 and 21.3 (P=0.79). The percentage of the change in WOMAC score were -42.0% and -38.9% (P=0.54). The pain VAS score decreased by 29.9 and 27.9 (P=0.58). The responders by PGART were 95.3% and 93.8% (P= 0.66), and the median amount of rescue medicine taken were 2.0 and 6.5 tablets (P=0.06). The incidence of ADRs were 31.3% and 21.2% (P=0.11). The most common ADRs were gastrointestinal system related; 17.2% in GCSB-5 group and 22.2% in Celecoxib group. Any severe ADR was not observed in either group. CONCLUSIONS: The result of this study supports that GCSB-5 is comparable to Celecoxib in terms of the efficacy and safety for the treatment of osteoarthritis of knee joint.

Neferine exerts its antithrombotic effect by inhibiting platelet aggregation and promoting dissociation of platelet aggregates.

INTRODUCTION: Neferine, a kind of isoquinoline alkaloid, extracted from the seed embryo of Nelumbo nucifera Gaertn, has long been recognized in traditional medicine as a medicinal plant with various usages. Neferine has many biological activities, including anti-hypertensive, anti-arrhythmic, negative inotropic effect and relaxation on vascular smooth muscle. Although previous studies have reported its antithrombotic effect, the mechanisms by which it exerts antithrombotic effect have not been thoroughly studied. METHOD: Washed mice platelets and mice platelet-rich-plasma (PRP) were used to investigate the effects of neferine on platelet aggregation, secretion induced by various agonists and dissociation of agonist-formed platelet aggregates. Bioflux plates coated with collagen were used to investigate the effect of neferine on platelet adhesion and thrombosis in vitro. With collagen-epinephrine-induced acute pulmonary thrombus formation mouse model, the effect of neferine on thrombosis in vivo was also examined. RESULTS: Neferine, significantly and dose-dependently, inhibited collagen-, thrombin-, U46619-induced platelet aggregation in mice washed platelets, or ADP-induced platelet aggregation in PRP. Neferine treatment decreased platelet dense granule secretion initiated by collagen, thrombin and U46619. Also, Neferine dramatically and dose-dependently promoted the dissociation of platelet aggregates pre-formed by various agonists including collagen, thrombin, U46619 or ADP. Neferine can significantly reduce the area of mice platelets adhesion to the collagen and inhibit thrombosis in vitro. In collagen-epinephrine-induced acute pulmonary thrombus mouse model, neferine, at 6 mg/kg, significantly attenuated thrombus formation. CONCLUSIONS: Neferine remarkably prevents thrombus formation by inhibiting platelet activation, adhesion and aggregation, as well as promoting disassembly of pre-formed platelet aggregates. The inhibitory effects of neferine on platelet activation might be relevant in cases involving aberrant platelet activation where neferine could be used as an anti-platelet and antithrombotic agent.