Background: Flavonoids have been characterized as a prominent class of compounds to treat thrombotic diseases through the inhibition of thiol isomerases. Syzygium cumini is a flavonoid-rich medicinal plant that contains myricetin and gallic acid. Little is known about the potential antiplatelet properties of S. cumini and its constituent flavonoids.Objective: To evaluate the antiplatelet effects and mechanism of action of a polyphenolrich extract (PESc) from S. cumini leaf and its most prevalent polyphenols, myricetin and gallic acid.Methods: PESc, myricetin, and gallic acid were incubated with platelet-rich plasma and washed platelets to assess platelet aggregation and activation. In vitro platelet adhesion and thrombus formation as well as in vivo bleeding time were performed. Finally, myricetin was incubated with recombinant thiol isomerases to assess its potential to bind and inhibit these, while molecular docking studies predicted possible binding sites.Results: PESc decreased platelet activation and aggregation induced by different agonists. Myricetin exerted potent antiplatelet effects, whereas gallic acid did not. Myricetin reduced the ability of platelets to spread on collagen, form thrombi in vitro without affecting hemostasis in vivo. Fluorescence quenching studies suggested myricetin binds to different thiol isomerases with similar affinity, despite inhibiting only protein disulfide isomerase (PDI) and ERp5 reductase activities. Finally, molecular docking studies suggested myricetin formed non-covalent bonds with PDI and ERp5.Conclusions: PESc and its most abundant flavonoid myricetin strongly inhibit platelet function. Additionally, myricetin is a novel inhibitor of ERp5 and PDI, unveiling a new therapeutic perspective for the treatment of thrombotic disorders.
Obesity and metabolic syndrome are the common causes of reproductive and fertility disorders in women. In particular, polycystic ovary syndrome, which is clinically characterized by hyperandrogenism, oligo/anovulation, and polycystic ovarian morphology, has been increasingly associated with metabolic disorders. However, given the broad interplay between metabolic and reproductive functions, this remains a field of intense research. In this study, we investigated the effect of monosodium l-glutamate (MSG)-induced obesity on reproductive biology of female rats. Newborn female rats were subcutaneously injected with MSG (4 g/kg/day) or equiosmolar saline (CTR) each 2 days up to postnatal day (pnd) 10. On pnd 60, estrous cycle was evaluated using vaginal smears twice a day for 15 days, which showed MSG rats to be oligocyclic. Thereafter, animals were killed on estrous phase for blood and tissue collection. MSG rats had increased body mass, accumulation of retroperitoneal and visceral fat pads, and visceral adipocyte hypertrophy compared with CTR rats. MSG rats were also dyslipidemic and hyperinsulinemic but were normoglycemic and normoandrogenic. Ovarian morphology analysis showed that MSG rats had a two-fold decrease in oocyte count but a six-fold increase on ovarian follicular cysts, along with a higher number of total primordial and atretic follicles. Moreover, MSG rats had a four-fold increase in anti-Müllerian hormone immunohistochemical staining on antral follicles. Taken together, data presented here characterize MSG obesity as a unique model to study the metabolic pathways underlying reproductive disorders in the absence of overactivated hypothalamic-pituitary-gonadal axis.
Background Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.
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