TXB2 Levels Research Articles

Stability of the thromboxane B2 biomarker of aspirin pharmacodynamics in human whole blood and in long-term stored serum samples

BackgroundSerum thromboxane B2 (sTXB2) is a validated biomarker of low-dose aspirin pharmacodynamics. In the original method, non-anticoagulated blood samples must incubate at 37°C immediately after withdrawal, be centrifuged, serum should be frozen until assayed. Timely completion of all preanalytical steps may affect the feasibility and quality of sTXB2 measurements. The storage duration of frozen serum can also affect sTXB2 stability. AimsWe assessed the stability of sTXB2: in clotted blood samples stored at 4°C before further processing; in sera stored at -40°C for over a decade. MethodVenous whole blood withdrawn from individuals on chronic low-dose aspirin was dispensed in different tubes, immediately incubated at 37°C for 1 hour. The reference tube was promptly processed following the original protocol, the remaining tubes were stored at 4°C from 12 to 72 hours before further processing. Sera stored at controlled -40°C temperature for <1 to 15 years were re-assayed. Values within the inter-assay variation limits (±9%) versus baseline were considered acceptable. ResultsBaseline sTXB2 values (5.4[2.4-13.4] ng/ml, median [IQR], n=40) were comparable with those in samples at 4°C up to 48 hours (97[86-104]% of reference, n=26), but at 72 hours the variability exceeded the inter-assay variation. TXB2 levels were stable in frozen sera for up to 10 years (101[87-108]% of reference, n=32), but decreased significantly afterwards (87[74-109]% at 15 years, p=0.005, n=32). ConclusionsTXB2 is stable in clotted blood samples stored at 4°C for up to 48 hours before further processing and in serum samples stored at -40°C over 10 years.

Prostacyclin Synthase Deficiency Leads to Exacerbation or Occurrence of Endothelium-Dependent Contraction and Causes Cardiovascular Disorders Mainly via the Non-TxA2 Prostanoids/TP Axis.

Prostaglandin I2 synthesized by endothelial COX (cyclooxygenase) evokes potent vasodilation in some blood vessels but is paradoxically responsible for endothelium-dependent constriction (EDC) in others. Prostaglandin I2 production and EDC may be enhanced in diseases such as hypertension. However, how PGIS (prostaglandin I2 synthase) deficiency affects EDC and how this is implicated in the consequent cardiovascular pathologies remain largely unknown. Experiments were performed with wild-type, Pgis knockout (Pgis-/-) and Pgis/thromboxane-prostanoid receptor gene (Tp) double knockout (Pgis-/-Tp-/-) mice and Pgis-/- mice transplanted with unfractionated wild-type or Cox-1-/- bone marrow cells, as well as human umbilical arteries. COX-derived prostanoids were measured by high-performance liquid chromatography-mass spectrometry. Vasomotor responses of distinct types of arteries were assessed by isometric force measurement. Parameters of hypertension, vascular remodeling, and cardiac hypertrophy in mice at different ages were monitored. PGF2α, PGE2, and a trace amount of PGD2, but not thromboxane A2 (TxA2), were produced in response to acetylcholine in Pgis-/- or PGIS-inhibited arteries. PGIS deficiency resulted in exacerbation or occurrence of EDC ex vivo and in vivo. Endothelium-dependent hyperpolarization was unchanged, but phosphorylation levels of eNOS (endothelial nitric oxide synthase) at Ser1177 and Thr495 were altered and NO production and the NO-dependent relaxation evoked by acetylcholine were remarkably reduced in Pgis-/- aortas. Pgis-/- mice developed high blood pressure and vascular remodeling at 16 to 17 weeks and subsequently cardiac hypertrophy at 24 to 26 weeks. Meanwhile, blood pressure and cardiac parameters remained normal at 8 to 10 weeks. Additional ablation of TP (TxA2 receptor) not only restrained EDC and the downregulation of NO signaling in Pgis-/- mice but also ameliorated the cardiovascular abnormalities. Stimulation of Pgis-/- vessels with acetylcholine in the presence of platelets led to increased TxA2 generation. COX-1 disruption in bone marrow-derived cells failed to affect the development of high blood pressure and vascular remodeling in Pgis-/- mice though it largely suppressed the increase of plasma TxB2 (TxA2 metabolite) level. Our study demonstrates that the non-TxA2 prostanoids/TP axis plays an essential role in mediating the augmentation of EDC and cardiovascular disorders when PGIS is deficient, suggesting TP as a promising therapeutic target in diseases associated with PGIS insufficiency.

Maternal nano-titanium dioxide inhalation exposure alters placental cyclooxygenase and oxidant balance in a sexually dimorphic manner

The placenta plays a critical role in nutrient-waste exchange between the maternal and fetal circulation, and thus impacts fetal growth and development. We have previously shown that nano-titanium dioxide (nano-TiO2) inhalation exposure during gestation decreased fetal female pup and placenta mass [1], which persists in the following generation [2]. In utero exposed females, once mated, their offspring's placentas had increased capacity for H2O2 production. Generation of oxidants such as hydrogen peroxide (H2O2), have been shown to impact cyclooxygenase activity, specifically metabolites such as prostacyclin (PGI2) or thromboxane (TXA2). Therefore, we hypothesized that maternal nano-TiO2 inhalation exposure during gestation results in alterations in placental production of prostacyclin and thromboxane mediated by enhanced H2O2 production in a sexually dimorphic manner. Pregnant Sprague-Dawley rats were exposed to nano-TiO2 aerosols or filtered air (sham-control) from gestational day (GD) 10–19. Dams were euthanized on GD 20, and fetal serum and placental tissue were collected based on fetal sex. Fetal placental zones (junctional zone (JZ) and labyrinth zone (LZ)) were assessed for xanthine oxidoreductase (XOR) activity, H2O2, and catalase activity, as well as 6-keto-PGF1α and TXB2 levels. Nano-TiO2 exposed fetal female LZ demonstrated significantly greater XOR activity compared to exposed males. Exposed fetal female LZ also demonstrated significantly diminished catalase activity compared to sham-control females. Exposed fetal female LZ had significantly increased abundance of 6-keto-PGF1α compared to sham-control females and increased TXB2 compared to exposed males. In the aggregate these data indicate that maternal nano-TiO2 inhalation exposure has a greater impact on redox homeostasis and PGI2/TXA2 balance in the fetal female LZ. Future studies need to address if treatment with an XO inhibitor during gestation can prevent diminished fetal female growth during maternal nano-TiO2 inhalation exposure.

Assessing Mailuoning injection in wound healing and thrombophlebitis management: A rat model study.

Thrombophlebitis is the inflammatory condition characterized by obstruction of one or more vessels, commonly in the legs, due to the formation of blood clots. It has been reported that traditional Chinese medicine, including Mailuoning injection, is advantageous for treating inflammatory and blood disorders. This research assessed the therapeutic efficacy of Mailuoning injection in the treatment of thrombophlebitis in rodents, as well as investigated its impact on fibrinolysis, inflammation, and coagulation. An experimental setup for thrombophlebitis was established in rodents via modified ligation technique. Five groups comprised the animals: sham operation group, model group, and three Mailuoning treatment groups (low, medium, and high dosages). The pain response, edema, coagulation parameters (PT, APTT, TT, FIB), serum inflammatory markers (IL-6, TNF-α, CRP), and expression levels of endothelial markers (ICAM-1, VCAM-1, NF-κB) were evaluated. Blood flow and vascular function were further assessed by measuring hemorheological parameters and the concentrations of TXB2, ET, and 6-k-PGF1α. In contrast to the sham group, model group demonstrated statistically significant increases in endothelial expression levels, coagulation latencies, and inflammatory markers (p < 0.05). The administration of mailing, specifically at high and medium dosages, resulted in a substantial reduction in inflammatory markers, enhancement of coagulation parameters, suppression of ICAM-1 and VCAM-1 expression, and restoration of hemorheological measurements to baseline (p < 0.05). Significantly higher concentrations of 6-k-PGF1α and lower levels of TXB2 and ET were observed in high-dose group, suggesting that pro- and anti-thrombotic factors were restored to equilibrium. Utilization of Mailuoning injection in rat model of thrombophlebitis exhibited significant therapeutic impact. This effect was manifested through pain alleviation, diminished inflammation, enhanced blood viscosity and facilitation of fibrinolysis. The study indicated that Mailuoning injection may serve as a viable therapeutic option for thrombophlebitis, potentially aiding in the improvement of wound healing by virtue of its anti-inflammatory and blood flow-enhancing characteristics.

The COVID-19 mRNA vaccine Comirnaty induces anaphylactic shock in an anti-PEG hyperimmune large animal model

Abstract Background The polyethylene-glycol (PEG)-containing COVID-19 mRNA vaccines can cause hypersensitivity reactions (HSRs), or rarely, life-threatening anaphylaxis in a small fraction of patients. The role of anti-PEG antibodies (Abs) has been proposed, but causality has not yet been proven in an animal model. Purpose The aim of this study was to provide such evidence using an anti-PEG hyperimmune porcine model wherein the Ab levels are increased by PEGylated liposomes called Doxebo. We also aimed to find evidence for a role of complement activation and thromboxane A2 release in blood. Methods Experimental pigs (n=6) were immunized with 0.1 mg/kg BW Doxebo i.v., and the rise of anti-PEG Abs were measured with ELISA. 2-3 weeks later the animals were injected with 1/3 human dose of Pfizer’s COVID-19 mRNA vaccine Comirnaty i.v., and the hemodynamic, hematological and blood immune mediator changes were measured to assess the severity of hypersensitivity reaction (HSR). The endpoints were PAP, SAP, HR, EtCO2, WBC, granulocyte, lymphocyte and platelet counts, C3a and plasma TXB2. Results The level of anti-PEG IgM rose 5-10-thousand-fold in all pigs immunized with Doxebo by day 10. Each of the 6 animals developed an anaphylactic shock to injection of 1/3 human dose of Comirnaty that required cardiopulmonary resuscitation in 4 cases. The reaction, starting within 1 min involved maximal pulmonary hypertension (PAP 13±2 before vs. 37±6mmHg after Comirnaty, median±MAD, P&amp;lt;0.05) and severe systemic hypotension (SAP 63±8 before vs. 42±11mmHg after Comirnaty, median±MAD, P&amp;lt;0.05), tachycardia, granulo- and thrombocytopenia, and skin flushing or rash. These hemodynamic changes were paralleled by C3a and TXB2 levels in blood. Conclusion We have established the first relevant large animal model of anaphylactic reaction induced by an mRNA vaccine. Consistent with previous studies, our current data show a causal role of anti-PEG Abs in the anaphylaxis to Comirnaty, which involves complement activation, and, hence, it represents CARPA (complement activation-related pseudo-allergy).

Transdermal Flunixin Meglumine as a Pain Relief in Donkeys: A Pharmacokinetics Pilot Study.

Recent approval of transdermal flunixin meglumine (FM) (Banamine®) in cattle has opened the door for the drug's potential application in other species. Transdermal FM could provide a safe and effective form of pain relief in donkeys. In order to evaluate the pharmacokinetics and effects of FM on anti-inflammatory biomarkers in donkeys, a three-way crossover study design was employed. In total, 6 healthy donkeys were administered transdermal (TD) FM at a dosage of 3.3 mg/kg, and oral (PO) and intravenous (IV) doses of 1.1 mg/kg body weight. Blood samples were collected over 96 h to determine the concentration of flunixin, 5OH flunixin, and eicosanoids (TXB2 and PGF2 alpha) using LC-MS/MS. The results indicated that both flunixin and 5OH flunixin were detectable in blood samples collected during TD. The elimination of the drug was slower following the TD route compared to PO and IV. TD administration significantly decreased TXB2 levels in non-stimulated serum from 1 to 96 h post-administration, while IV and PO resulted in TXB2 reduction for 1 to 8 h. A significant reduction in PGF2 alpha was observed in PO and IV 1 h after administration, while TD resulted in a gradual decline from 4 to 72 h. The study concluded that the off-label use of transdermal FM at 3.3 mg/kg could be effective in controlling inflammation in donkeys.

New Insights into Immunopathology Associated to Bothrops lanceolatus Snake Envenomation: Focus on PLA2 Toxin.

The systemic increase in inflammatory mediator levels can induce diverse pathological disorders, including potentially thrombus formation, which may be lethal. Among the clinical conditions in which the formation of thrombi dictates the patient's prognosis, envenomation by Bothrops lanceolatus should be emphasized, as it can evolve to stroke, myocardial infarction and pulmonary embolism. Despite their life-threatening potential, the immunopathological events and toxins involved in these reactions remain poorly explored. Therefore, in the present study, we examined the immunopathological events triggered by a PLA2 purified from B. lanceolatus venom, using an ex vivo human blood model of inflammation. Our results showed that the purified PLA2 from the venom of B. lanceolatus damages human erythrocytes in a dose dependent way. The cell injury was associated with a decrease in the levels of CD55 and CD59 complement regulators on the cell surface. Moreover, the generation of anaphylatoxins (C3a and C5a) and the soluble terminal complement complex (sTCC) indicates that human blood exposure to the toxin activates the complement system. Increased production of TNF-α, CXCL8, CCL2 and CCL5 followed complement activation. The venom PLA2 also triggered the generation of lipid mediators, as evidenced by the detected high levels of LTB4, PGE2 and TXB2. The scenario here observed of red blood cell damage, dysfunctions of the complement regulatory proteins, accompanied by an inflammatory mediator storm, suggests that B. lanceolatus venom PLA2 contributes to the thrombotic disorders present in the envenomed individuals.

Biochemical characteristics and functional performance of cold-stored platelets: an in-vitro comparative study

Background: Platelet refrigeration could eliminate bacterial contamination and improve the hemostatic function even better than already-used room-temperature storage. This study aimed to assess the effect of cold storage, with and without agitation, on the apheresis platelets' hemostatic, metabolic, and functional activity. Materials and methods: The study included 10 healthy volunteer donors to collect Apheresis PLT. They were submitted to careful clinical examination and standard laboratory workup. Collected samples were processed in accordance with American Association of Blood Banks (AABB) guidelines. Every aliquot collected from each volunteer was stored for up to 5 days at one of the following storage conditions: 1. In an FDA-approved-PLT incubator with agitation at room temperature (RT + AG as a group; GI), 2. In an FDA-approved-refrigerator at 4 oC with agitation (4 oC + AG as a group; GII), 3. In an FDA-approved- refrigerator at 4 oC without agitation (4 oC – AG as a group; GIII). The following PLT workup was done; PLT count and mean platelet volume (MPV), metabolic variables, PLT aggregation studies, PLT receptors expression, and PLT pro-inflammatory mediator’s release.Results:All samples had a significant PLT count decline compared to baseline data. No changes in MPV were observed in all groups on day 3 and day 5, meaning that single PLT size remained unchanged. In addition, GI showed a mark of significant increase in metabolic activity when compared to baseline PLTs in contrast to GII, and GIII, which were more metabolically stable and less active.Comparison between the studied groups regarding PLT aggregation revealed significantly higher PLT aggregation response to ADP and collagen in GII and GIII compared to GI on the 3rd and 5th days. Moreover, it was shown that GII and GIII samples had significantly higher CD62p expression when compared with GI on the 3rd and 5th days despite being less active and more stable. While it was found that TXB2 levels were significantly higher, nearly 3-fold, in GI as compared to GII and GIII.Conclusions: Apheresis platelets (AP) cold storage provides a clear advantage over standard conditions regarding biochemical balance and hemostatic performance, which could markedly improve AP's clinical and economic value in different scenarios. Keywords: Platelet aggregation, P-selectin, Thromboxane B2.

Flavokawain mitigates ovalbumin (OVA)-induced asthma by regulating NLRP3 inflammasome and inflammatory markers: In-vitro and in-vivo models

BackgroundAsthma is the most prevalent chronic inflammatory ailment, which has major communal health implications for both adults and children worldwide. Flavokawain is a chalcones present in kawa plant extracts. They have anti-inflammatory, antioxidant, anti-cancer, antimicrobial, and other activities. MethodsIn the present investigation, the potential of flavokawain on the IL-4-induced 16HBE cell line and the ovalbumin (OVA)-induced mouse model was analyzed. ResultsThe findings revealed that the IL-4-induced 16HBE cell line treated with flavokawain reduced cell apoptosis and increased cell proliferation. The NLRP3 inflammasome activity was suppressed by flavokawain. The level of immune cells was increased in the asthmatic mice, which was effectively reduced by the treatment with flavokawain. IgE, eotaxin, TNF-α and TXB levels were decreased by the flavokawain treatment in the asthmatic mice. Imbalances in Th1 and Th2 cytokines were effectively modulated by the flavokawain treatment in the asthmatic mice. The balance between the Th1 cytokines (IL-12 and IFN-γ) and the Th2 cytokines (IL-4, IL-5, IL-6, and IL-13) was maintained by flavokawain. The histopathology study of lungs revealed that the thickness of the airway membrane and inflammatory cell infiltration was reduced by the flavokawain, which indicates the decrease in inflammation. ConclusionThese findings recommend that flavokawain can be used as a potent candidate to treat asthma in the future.

Cyclooxygenase inhibition attenuates peripheral venous distension reflex in healthy humans

Background: Venous distension in limbs evokes a pressor reflex (venous distension reflex, VDR). Group III and IV nerves, innervating peripheral veins, are suggested as the afferent arm of the VDR. Prostaglandins stimulate/sensitize group III/IV nerves. The purpose of this study was to evaluate the effect of cyclooxygenase (COX) blockade on muscle sympathetic nerve activity (MSNA) and blood pressure (BP) responses to venous distension. Hypothesis: We hypothesized that inhibition of prostaglandin synthesis by local COX blockade would attenuate the MSNA and BP responses to peripheral venous distension. Methods: Nineteen healthy volunteers (age, 27 ± 5 years) participated in the study with a randomized, double-blind, placebo-controlled, and crossover study design with two separate visits. To induce venous distension, a volume of solution (saline alone or 9 mg ketorolac tromethamine in saline) was infused into the vein in the antecubital fossa of an arterially occluded forearm. During the procedure, beat-by-beat heart rate (HR), BP, and MSNA were recorded simultaneously. The vein size at the site with the catheter for the infusion was measured with ultrasound. This study is associated with a registered clinical trial (NCT03513770). Data and a summary of the results: In both visits, the venous distension procedure significantly increased the vein size, and the increase in vein size was not different between the visits (P &gt; 0.05). A significant decrease (P &lt; 0.001) in TXB2 level (an index for prostaglandin synthesis) by the procedure was observed only in the ketorolac visit. In both visits, the venous distension procedure significantly increased BP, HR, and MSNA (all, P &lt; 0.05). The increase in mean arterial pressure (MAP) and MSNA in the ketorolac visit was significantly lower than in the control visit (ΔMAP, 7.0 ± 6.2 vs. 13.8 ± 7.7 mmHg; ΔMSNA, 6.0 ± 7.1 vs. 14.8 ± 7.7 bursts/minute; both, P &lt; 0.05). Discussion and conclusion: The presented data show that COX blockade attenuates the responses in MSNA and BP to peripheral venous distension. The results suggest that COX products play a key role in evoking afferent activation responsible for the VDR. Vein size was measured with ultrasonography only at the catheter site. Some members of COX products have vasoactive effects. Future studies evaluating the effect of COX blockade on vein size in more detail using other approaches (e.g., magnetic resonance imaging) are warranted to verify the detailed mechanism of the attenuated VDR by the COX blockade. This study was supported by National Institutes of Health Grants R01 HL144781 (to J.C. and L.I.S). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Co-exposure to particulate matter and humidity increases blood pressure in hypertensive mice via the TRPV4-cPLA2-COX2 pathway

Epidemiological studies have demonstrated that particulate matter (PM) can induce or exacerbate hypertension. High relative humidity has been associated with elevated blood pressure in certain regions. However, the coupling effect of humidity and PM on elevated blood pressure and the underlying mechanisms remain unknown. Herein, we aimed to explore the effects of exposure to PM and/or high relative humidity on hypertension, as well as elucidate underlying mechanisms. Male C57/BL6 mice were intraperitoneally administered NG-nitro-L-arginine methyl ester (L-NAME) to establish a hypertensive mouse model. The hypertensive mice were exposed to PM (0.15 mg/kg/day) and/or different relative humidities (45/90%) for eight weeks. Histopathological changes, systolic blood pressure (SBP), endothelial-derived contracting factors (thromboxane B2 [TXB2], Prostaglandin F2α [PGF2α], endothelin-1 [ET-1], and angiotensin II [Ang II]), and relaxing factors (prostaglandin I2 [PGI2] and nitric oxide [NO]) were measured to assess the effects of PM exposure and humidity on hypertension in mice. Levels of transient receptor potential vanilloid 4 (TRPV4), cytosolic phospholipase A2 (cPLA2), and cyclooxygenase 2 (COX2) were measured to explore their potential mechanisms. Herein, exposure to 90% relative humidity or PM alone had a slight but insignificant effect on hypertension. However, pathological changes and elevated blood pressure were markedly exacerbated following exposure to PM and 90% relative humidity. Levels of PGF2α, TXB2, and ET-1 were significantly increased, whereas the PGI2 level was substantially decreased. HC-067047-mediated blockade of TRPV4 suppressed TRPV4, cPLA2, and COX2 expression and effectively alleviated the increased blood pressure induced by exposure to PM and 90% relative humidity. These results indicate that 90% relative humidity and PM can activate the TRPV4-cPLA2-COX2 ion channel in the aorta, altering the endothelial-derived contracting and relaxing factors and enhancing blood pressure in hypertensive mice.

Quince extract resists atherosclerosis in rats by down-regulating the EGFR/PI3K/Akt/GSK-3β pathway

We identified the effective components and the underlying mechanisms of Quince (Cydonia oblonga Mill, COM) extract against atherosclerosis. The effective components of COM extract were identified with UHPLC-Q-TOF-MS/MS. Network pharmacology was performed. A rat model of atherosclerosis induced by high-fat emulsion combined with vitamin D3 was established. The anti-atherosclerosis effect of COM extract was evaluated from various aspects such as blood lipid regulation, anti-oxidative stress, anti-inflammatory response, and vascular protection function. We identified 14 serum components of COM extract using UHPLC-Q-TOF-MS/MS. Through prediction, 573 targets were obtained, among which 224 targets were atherosclerosis specific targets. The key targets included GSK3β, ESR1, EGFR, and HSP90AA1. The key signaling pathway was PI3K-Akt signaling pathway. Pharmacodynamics analysis showed that COM extract reduced the levels of TC, TG, and LDL-C as well as ALT and AST, while increased the level of HDL-C. Mechanistically, COM extract significantly increased serum SOD and GSH-Px activities, but decreased MDA content in atherosclerosis rats, showing antioxidant effects. Meanwhile, COM extract significantly down-regulated the levels of pro-inflammatory factors IL-1β, IL-6, TNF-α and CRP, but up-regulated anti-inflammatory factor IL-10. Additionally, COM extract increased the levels of NO, eNOS, and 6-keto-PGF1α; whereas, decreased the levels of ET-1 and TXB2. Furthermore, COM extract significantly inhibited the mRNA and protein levels of EGFR, p-PI3K, p-AKT, GSK-3β, Bax, and Caspase-3 as well as the Bax/Bcl-2 ratio. Conclusively, COM extract exerts hypolipidemic, anti-oxidative, anti-inflammatory, anti-thrombotic and vascular endothelium protective effects on atherosclerosis rat model, which may be related to the inhibition of EGFR/PI3K/AKT/GSK-3β signaling pathway.

Beneficial effects of Panax notoginseng (Burkill) F. H. Chen flower saponins in rats with metabolic hypertension by inhibiting the activation of the renin–angiotensin–aldosterone system through complement 3

BackgroundMetabolic hypertension (MH) has become the most common type of hypertension in recent years due to unhealthy eating habits and lifestyles of people, such as over-eating alcohol, high fat, and sugar diets (ACHFSDs). Therefore, effective means to combat MH are needed. Previous studies have shown that Panax notoginseng (Burkill) F. H. Chen flower saponins (PNFS) can lower blood pressure in spontaneously hypertensive rats (SHR). However, whether it acts on MH and its mechanism of action remain unclear. MethodsThe pharmacodynamic effects of PNFS were evaluated in rats with ACHFSDs-induced MH. The blood pressure, blood biochemical, grip strength, face temperature, vertigo time, and liver index were estimated. The histological changes in the liver and aorta were observed using hematoxylin and eosin staining. The levels of ET-1, TXB2, NO, PGI2, Renin, ACE, Ang II, and ALD in plasma were detected using ELISA. The levels of C3, KLF5, LXRα, and Renin in kidney tissues were measured using qRT-PCR.The expression levels of C3, KLF5, LXRα, and Renin in kidney tissues were examined using Western blotting.ResultsIn the present study, PNFS was found to reduce blood pressure, face temperature, and vertigo time, increase grip strength and improve dyslipidemia in rats with MH. In addition, PNFS decreased the plasma levels of ET-1 and TXB2, elevated the levels of NO and PGI2, and improved pathological aortic injury. Meanwhile, PNFS decreased the plasma levels of Renin, ACE, Ang II, and ALD. QRT-PCR and Western bolt showed that PNFS downregulated C3, KLF5, LXRα, and Renin protein and mRNA expression in the kidneys of rats with MH.ConclusionThe finding of the present study suggested that PNFS could downregulate C3 and KLF-5 expression in rats with MH, thereby inhibiting the overactivation of the renin–angiotensin–aldosterone system, while improving vascular endothelial function and ultimately reducing blood pressure in rats with MH.

Smoking Affects the Post-Stroke Inflammatory Response of Lipid Mediators in a Gender-Related Manner.

The main goal of our study was to determine the effect of cigarette smoking on selected derivatives of arachidonic acid, linoleic acid, DHA, and EPA, which may be markers of post-stroke inflammation. The eicosanoid profile was compared in both smoking and non-smoking patients, without division and with division into gender. In the group of non-smokers, we observed higher levels of the linolenic acid derivative (LA) 9S HODE (p ≤ 0.05) than in smokers. However, after dividing the results by sex, it turned out that the level of this derivative was higher in non-smoking women compared to smoking women (p ≤ 0.01) and did not differentiate the group of men. Similarly, the level of the arachidonic acid metabolite LTX A4 (p ≤ 0.05) differed only in the group of women. In this group, we also observed a decreased level of 15S HETE in smoking women, but it was statistically insignificant (p ≤ 0.08). On the other hand, the level of this derivative was statistically significantly higher in the group of non-smoking women compared to male non-smokers. The group of men was differentiated by two compounds: TXB2 and NPD1. Male smokers had an almost two-fold elevation of TXB2 (p ≤ 0.01) compared with non-smokers, and in this group, we also observed an increased level of NPD1 compared with male non-smokers. On the other hand, when comparing female non-smokers and male non-smokers, in addition to the difference in 15S HETE levels, we also observed elevated levels of TXB2 in the group of non-smokers. We also analyzed a number of statistically significant correlations between the analyzed groups. Generally, men and women smokers showed a much smaller amount of statistically significant correlations than non-smokers. We believe that this is related to the varying degrees of inflammation associated with acute ischemic stroke and post-stroke response. On the one hand, tobacco smoke inhibits the activity of enzymes responsible for the conversion of fatty acids, but on the other hand, it can cause the failure of the inflammatory system, which is also the body's defense mechanism. Smoking cigarettes is a factor that increases oxidative stress even before the occurrence of a stroke incident, and at the same time accelerates it and inhibits post-stroke repair mechanisms. This study highlights the effect of smoking on inflammation in both genders mediated by lipid mediators, which makes smoking cessation undeniable.

Specialized Pro-Resolving Mediator (SPM) Profile in Blood from Children with Sickle Cell Disease (SCD): Comparison with Age-, and Race- Matched Controls

Introduction: Specialized Pro-resolving Mediators (SPMs) are lipid mediators, biosynthesized from the n-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA), docosapentaenoic acid (DPA) and eicosapentaenoic acid (EPA); and the n-6 PUFA arachidonic acid (AA). SPMs are grouped into four families of lipid mediators including resolvins, protectins, maresins and lipoxins; and these mediators play a role in the resolution of inflammation. Inflammation plays a crucial role in the pathogenesis of clinical complications in sickle cell disease (SCD). In addition, multiple studies in SCD have documented decreased levels of anti-inflammatory fatty acids DHA and EPA in blood cells and plasma. We assessed whether the decreased DHA and EPA content play a role in modulation of peripheral blood SPM levels in SCD. Methods: Using targeted liquid chromatography (LC)-tandem mass spectrometry (MS/MS), we investigated plasma SPM profile in children with SCD in steady state (n=45, subjects with confirmed SCD genotypes SS and Sβ0 thalassemia, ages 2-20 years), and compared to that from an age- and race-matched control group (n=24). Analysis also included the SPM pathway markers, prostaglandins, thromboxane and leukotriene and their metabolites. (n-3) PUFA content in total blood cells was measured in both groups using capillary gas chromatography. Results: Consistent with the published results, a significant decrease in (n-3) PUFA content (measured as percent of total fatty acids) was noted in children with SCD (2.14% ± 0.47% in SCD vs 2.39% ± 0.45% in control, mean ± SD, P=0.04). In plasma from both SCD and controls, we identified and quantitated 6 SPMs which included DHA-derived resolvin (Rv) D1, RvD3 and 17R-RvD3; EPA-derived RvE2; and DPA(n-3)-derived RvD5(n-3) and protectin D (PD)-1 (n-3). Median RvD1 level (pg/ml) in SCD plasma was significantly elevated (65 in SCD vs 36 in controls, P=0.002). Levels of other SPMs were not significantly different between the two groups. Multiple SPM pathway markers including mono-hydroxy-PUFAs (EPA-derived 5-HEPE, 12-HEPE, 15-HEPE and 18-HEPE; DHA-derived 4-HDHA, 7-HDHA and 14-HDHA; and AA-derived 5-HETE, 12-HETE and 15-HETE) were present in both control and SCD plasma. Levels (median, pg/ml) of 5-lipoxygenase-derived EPA-metabolite 5-HEPE (212 vs 122, P=0.027) and DHA-derived metabolite 7-HDHA (62 vs 39, P=0.036), and 12-lipoxygenase-derived AA-metabolite 12-HETE (16783 vs 11796, P=0.025) were increased significantly in SCD compared to those from the controls. 15-Lipoxygenase derived pathway markers (15-HEPE and 15-HETE) were not different between the two groups. The cyclo-oxygenase-derived eicosanoids identified and quantitated in both groups included PGE2, PGD2 and TxB2 with a significant increase observed in TxB2 level in SCD plasma (1213 vs 970 pg/ml, P=0.038). Leukotriene (LT) B4 (an AA-derived pro-inflammatory mediator) and lipoxins (AA-derived SPMs), and their metabolites 20-OH-LTB4 and 20-COOH-LTB4 were absent in plasma from both SCD and control. Conclusions: Our results taken together demonstrate that the decreased bioavailability of (n-3) PUFA in children with SCD does not appear to limit SPM production in steady state as documented with normal/increased plasma levels of SPMs and their pathway markers. Whether the SPM profile in circulation is different in SCD pathologies associated with inflammation including vaso-occlusive pain crisis and acute chest syndrome, compared to steady state disease, require further investigation.

Pro-thrombotic changes associated with exposure to ambient ultrafine particles in patients with chronic obstructive pulmonary disease: roles of lipid peroxidation and systemic inflammation

BackgroundExposure to particulate matter air pollution is associated with an increased risk of cardiovascular mortality in patients with chronic obstructive pulmonary disease (COPD), but the underlying mechanisms are not yet understood. Enhanced platelet and pro-thrombotic activity in COPD patients may explain their increased cardiovascular risk. We aim to explore whether short-term exposure to ambient particulate matter is associated with pro-thrombotic changes in adults with and without COPD, and investigate the underlying biological mechanisms in a longitudinal panel study. Serum concentration of thromboxane (Tx)B2 was measured to reflect platelet and pro-thrombotic activity. Lipoxygenase-mediated lipid peroxidation products (hydroxyeicosatetraenoic acids [HETEs]) and inflammatory biomarkers (interleukins [ILs], monocyte chemoattractant protein-1 [MCP-1], tumour necrosis factor alpha [TNF-α], and macrophage inflammatory proteins [MIPs]) were measured as potential mediating determinants of particle-associated pro-thrombotic changes.Results53 COPD and 82 non-COPD individuals were followed-up on a maximum of four visits conducted from August 2016 to September 2017 in Beijing, China. Compared to non-COPD individuals, the association between exposure to ambient ultrafine particles (UFPs) during the 3–8 days preceding clinical visits and the TxB2 serum concentration was significantly stronger in COPD patients. For example, a 103/cm3 increase in the 6-day average UFP level was associated with a 25.4% increase in the TxB2 level in the COPD group but only an 11.2% increase in the non-COPD group. The association in the COPD group remained robust after adjustment for the levels of fine particulate matter and gaseous pollutants. Compared to the non-COPD group, the COPD group also showed greater increases in the serum concentrations of 12-HETE (16.6% vs. 6.5%) and 15-HETE (9.3% vs. 4.5%) per 103/cm3 increase in the 6-day UFP average. The two lipid peroxidation products mediated 35% and 33% of the UFP-associated increase in the TxB2 level of COPD patients. UFP exposure was also associated with the increased levels of IL-8, MCP-1, MIP-1α, MIP-1β, TNF-α, and IL-1β in COPD patients, but these inflammatory biomarkers did not mediate the TxB2 increase.ConclusionsShort-term exposure to ambient UFPs was associated with a greater pro-thrombotic change among patients with COPD, at least partially driven by lipoxygenase-mediated pathways following exposure.Trial registrationChiCTR1900023692. Date of registration June 7, 2019, i.e. retrospectively registered.

The Sodium-Glucose Co-Transporter-2 (SGLT2) Inhibitors Reduce Platelet Activation and Thrombus Formation by Lowering NOX2-Related Oxidative Stress: A Pilot Study.

Sodium–glucose co-transporter-2 inhibitors or gliflozins, the newest anti-hyperglycemic class, induce cardioprotective benefits in patients with type 2 diabetes (T2D). As platelet activation and oxidative stress play a key role in atherothrombotic-related complications, we hypothesized that gliflozins might modulate oxidative stress, platelet activation and thrombus formation. We performed an interventional open-label single-arm before-after study in 32 T2D patients on top of their ongoing metformin therapy. The population was divided into two groups: treatment with GLP-1 receptor agonists (GLP-1RA, Group A) and gliflozins (Group B). Oxidative stress, platelet activation and thrombus growth were assessed before and after 15 days of treatment. Compared to the baseline, gliflozins treatment significantly decreased sNOX2-dp (−45.2%, p < 0.001), H2O2 production (−53.4%, p < 0.001), TxB2 (−33.1%, p < 0.001), sP-selectin (−49.3%, p < 0.001) and sCD40L levels (−62.3%, p < 0.001) as well as thrombus formation (−32%, p < 0.001), whereas it potentiated anti-oxidant power (HBA, +30.8%, p < 0.001). Moreover, a significant difference in oxidative stress, platelet activation and thrombus formation across groups A and B was found. In addition, an in vitro study on stimulated platelets treated with gliflozins (10–30 μM) showed a reduction in oxidative stress, platelet activation and thrombus growth. Our results showed that gliflozins have antiplatelet and antithrombic activity related to an NOX2 down-regulation, suggesting a new mechanism responsible for cardiovascular protection.

Inhibition of P2X7 receptors by Lu AF27139 diminishes colonic hypersensitivity and CNS prostanoid levels in a rat model of visceral pain.

Visceral pain is a prominent feature of various gastrointestinal diseases. The P2X7 receptor is expressed by multiple cell types including dorsal root ganglion satellite glial cells, macrophages, and spinal microglia, all of which have been implicated in nociceptive sensitization. We have used the selective and CNS penetrant P2X7 receptor antagonist Lu AF27139 to explore this receptor's role in distinct rat models of inflammatory and visceral hypersensitivity. Rats injected with CFA in the hindpaw displayed a marked reduction in hindpaw mechanical threshold, which was dose-dependently reversed by Lu AF27139 (3-30mg/kg, p.o.). In rats injected with TNBS in the proximal colon, the colorectal distension threshold measured distally was significantly lower than sham treated rats at 7days post-injection (P < 0.001), indicative of a marked central sensitization. Colonic hypersensitivity was also reversed by Lu AF27139 (10-100mg/kg) and by the κ-opioid receptor agonist U-50,488H (3mg/kg, s.c.). Moreover, both Lu AF27139 and U-50,488H prevented a TNBS-induced increase in spinal and brain levels of PGE2 and LTB4, as well as an increase in brain levels of PGF2α and TXB2. Lu AF27139 was well tolerated as revealed by a lack of significant effect on rotarod motor function and coordination at all doses tested up to 300mg/kg. Thus, P2X7 receptor antagonism is efficacious in a rat model of visceral pain, via a mechanism which potentially involves attenuation of microglial function within spinal and/or supraspinal pain circuits, albeit a peripheral site of action cannot be excluded.

Effect of low molecular weight heparin and ulinastatin as a combined therapy on soluble myeloid cell expression and intestinal mucosal function in patients with severe pancreatitis

Purpose: To investigate the effect of low molecular weight heparins (LMWHs) and ulinastatin on soluble myeloid cells and intestinal mucosal function (IMF) in patients with severe pancreatitis.&#x0D; Methods: A total of 107 patients with severe pancreatitis were divided into two groups: control group (CG, n = 53) and study group (SG, n = 54). The CG was treated with LMWH while SG was similarly treated but in addition received ulinastatin simultaneously. The following parameters were evaluated in the two groups: treatment effects, IMF, time for various indicators to normalize, vascular endothelial function, complication symptoms, T lymphoid subgroup indicators, inflammatory factors, anti-inflammatory factors, soluble B7-H2, and soluble myeloid cell receptor-1 level changes.&#x0D; Results: After treatment, SG showed lower levels of L/M value, DAO and D-lactic acid than in CG (p &lt; 0.05). Gastrointestinal function, leukocytes, amylase, and body temperature in SG had a shorter time to return to normal than in CG (p &lt; 0.05). The levels of IL-10 in SG were higher than in CG, while sB7-H2, TNF-α, sTREM-1 and IL-1 levels were lower than those in the CG (p &lt; 0.05). After treatment, NO levels in SG were higher, but TXB2, vWF and ET levels were lower than in CG (p &lt; 0.05). In addition, CD4+, CD4+/CD8+ indicators were higher and CD8+ lower in SG than in CG (p &lt; 0.05).&#x0D; Conclusion: Ulinastatin + LMWHs improves IMF in patients suffering from severe pancreatitis, shortens the time for various indicators to normalize, and reduces incidence of complications. However, further clinical trials are required to ascertain this therapeutic strategy for the management of severe pancreatitis.&#x0D; Keywords: Low molecular weight heparin; Ulinastatin; Severe pancreatitis; Soluble myeloid cell expression; Intestinal mucosal function; Treatment effect

Pharmacological Investigation of Asphodelus tenuifolius Cav. for its Potential Against Thrombosis in Experimental Models.

BackgroundThrombosis is a major disorder which is an outcome of an imbalance in the hemostatic system that develop undesirable blood clot and hinder blood circulation.PurposeThe current study was designed to verify the potential of aqueous methanolic crude extract of Asphodelus tenuifolius Cav. (At.Cr), used traditionally as remedy in circulatory problems.Research DesignAntioxidant activity, FTIR, and HPLC analysis were performed. In-vitro clot lysis assay was performed on human blood samples, and in-vivo acute pulmonary thromboembolism model was developed by administering the mixture of collagen and epinephrine in tail vein of mice. Carrageenan-induced thrombosis and FeCl3-induced carotid arterial thrombosis models were developed in rats.ResultsAt.Cr demonstrated significant increase in lysis of human blood clot. Bleeding and clotting times were increased dose-dependently. Lungs histology showed clear alveolar spaces with decreased red blood cells congestion. Reduction in infarcted tail length, augmentation in prothrombin time, and activated partial thromboplastin time with decrease in platelet count were observed. At.Cr also prolonged the arterial occlusion time and reduced the weight of thrombus and TXB2 levels dose-dependently.ConclusionsThe results demonstrated the antithrombotic and thrombolytic potential of At.Cr due to activation of coagulation factors through extrinsic and intrinsic pathways.