Capparis cartilaginea decne (capparaceae): isolation of flavonoids by high-speed countercurrent chromatography and their anti-inflammatory evaluation.

Introduction: Capparis cartilaginea Decne. (CC) originates from the dry regions of Asia and the Mediterranean basin. In traditional medicine, tea of CC leaves is commonly used to treat inflammatory conditions such as rheumatism, arthritis, and gout. Due to the limited studies on the phytochemistry and biological activity of CC compared to other members of the Capparaceae family, this work aims to: 1) Identify the chemical composition of CC extract and 2) Investigate the potential anti-inflammatory effect of CC extract, tea and the isolated compounds. Methods: To guarantee aim 1, high-speed countercurrent chromatography (HSCC) method; Nuclear Magnetic Resonance (NMR) and High-Performance Liquid Chromatography coupled to Electrospray Ionisation and Quadrupole Time-of-Flight Mass Spectrometry (HPLC-ESIQTOF-MS/MS) were employed for this purpose. To guarantee aim 2, we studied the effect of the isolated flavonoids on matrix metalloproteinases (MMPs) -9 and -2 in murine macrophages. Molecular docking was initially performed to assess the binding affinity of the isolated flavonoids to the active site of MMP-9. Results and discussion: In silico model was a powerful tool to predict the compounds that could strongly bind and inhibit MMPs. CC extract and tea have shown to possess a significant antioxidant and anti-inflammatory effect, which can partially explain their traditional medicinal use.

Fyn-kinase and caveolin-1 in the alveolar epithelial junctional adherence complex contribute to the early stages of pulmonary fibrosis.

Current pathophysiological findings indicate that damage to the alveolar epithelium plays a decisive role in the development of idiopathic pulmonary fibrosis (IPF). The available pharmacological interventions (i.e., oral pirfenidone and nintedanib) only slow down progression of the disease, but do not offer a cure. In order to develop new drug candidates, the pathophysiology of IPF needs to be better understood on a molecular level. It has previously been reported that a loss of caveolin-1 (Cav-1) contributes to profibrotic processes by causing reduced alveolar barrier function and fibrosis-like alterations of the lung-parenchyma. Conversely, overexpression of caveolin-1 appears to counteract the development of fibrosis by inhibiting the inflammasome NLRP3 and the associated expression of interleukin-1ß. In this study, the interaction between Fyn-kinase and caveolin-1 in the alveolar epithelium of various bleomycin (BLM)/TGF-ß damage models using precision-cut lung slices (PCLS), wildtype (WT) and caveolin-1 knockout (KO) mice as well as the human NCI-H441 cell line, were investigated. In WT mouse lung tissues, strong signals for Fyn-kinase were detected in alveolar epithelial type I cells, whereas in caveolin-1 KO animals, expression shifted to alveolar epithelial type II cells. Caveolin-1 and Fyn-kinase were found to be co-localized in isolated lipid rafts of NCI-H441 cell membrane fractions. These findings were corroborated by co-immunoprecipitation studies in which a co-localization of Cav-1 and Fyn-kinase was detected in the cell membrane of the alveolar epithelium. After TGF-ß and BLM-induced damage to the alveolar epithelium both in PCLS and cell culture experiments, a decrease in caveolin-1 and Fyn-kinase was found. Furthermore, TEER (transepithelial electrical resistance) measurements indicated that TGF-ß and BLM have a damaging effect on cell-cell contacts and thus impair the barrier function in NCI-H441 cell monolayers. This effect was attenuated after co-incubation with the Fyn-kinase inhibitor, PP-2. Our data suggest an involvement of Fyn-kinase and caveolin-1 in TGF-ß/bleomycin-induced impairment of alveolar barrier function and thus a possible role in the early stages of pulmonary fibrosis. Fyn-kinase and/or its complex with caveolin-1 might, therefore, be novel therapeutic targets in IPF.

A novel aryl-guanidinium derivative, VP79s, targets the signal transducer and activator of transcription 3 signaling pathway, downregulates myeloid cell leukaemia-1 and exhibits preclinical activity against multiple myeloma.

AIMS: We have recently described a novel guanidinium-based compound, VP79s, which induces cytotoxicity in various cancer cell lines. Here, we aim to investigate the activity of VP79s and associated mechanisms of action in multiple myeloma (MM) cells in vitro and ex vivo. MAIN METHODS: The effects of VP79s on cell viability and induction of apoptosis was examined in a panel of drug-sensitive and drug-resistant MM cell lines, as well as ex vivo patient samples and normal donor lymphocytes and platelets. Cell signaling pathways associated with the biological effects of VP79s were analysed by immunoblotting and flow cytometry. Gene expression changes were assessed by quantitative real-time PCR analysis. KEY FINDINGS: VP79s was found to rapidly inhibit both constitutively active and IL-6-induced STAT3 signaling with concurrent downregulation of the IL-6 receptors, CD130 and CD126. VP79s induced a rapid and dose-dependent downregulation of anti-apoptotic Bcl-2 family member, myeloid cell leukaemia-1 (MCL-1). VP79s enhanced bortezomib induced cell death and was also found to overcome bone marrow stromal cell induced drug resistance. VP79s exhibited activity in ex vivo patient samples at concentrations which had no effect on peripheral blood mononuclear cells, lymphocytes and platelets isolated from healthy donors. SIGNIFICANCE: As VP79s resulted in rapid inhibition of the key IL-6/STAT3 signaling pathway and downregulation of MCL-1 expression with subsequent selective anti-myeloma activity, VP79s may be a potential therapeutic agent with a novel mechanism of action in MM cells.

Effect on Platelet Function of Metal-Based Nanoparticles Developed for Medical Applications.

Nanomaterials have been recently introduced as potential diagnostic and therapeutic tools in the medical field. One of the main concerns in relation to the use of nanomaterials in humans is their potential toxicity profile and blood compatibility. In fact, and due to their small size, NPs can translocate into the systemic circulation even after dermal contact, inhalation, or oral ingestion. Once in the blood stream, nanoparticles become in contact with the different components of the blood and can potentially interfere with normal platelet function leading to bleeding or thrombosis. Metallic NPs have been already used for diagnosis and treatment purposes due to their unique characteristics. However, the potential interactions between metallic NPs and platelets has not been widely studied and reported. This review focuses on the factors that can affect platelet activation and aggregation by metal NPs and the nature of such interactions, providing a summary of the effect of various metal NPs on platelet function available in the literature.

Effects of functionalized silver nanoparticles on aggregation of human blood platelets.

PURPOSE: We studied the effects of silver nanoparticles (AgNPs) on human blood platelet function. We hypothesized that AgNPs, a known antimicrobial agent, can be used as blood-compatible, "ideal material'' in medical devices or as a drug delivery system. Therefore, the aim of the current study was to investigate if functionalized AgNPs affect platelet function and platelets as well as endothelial cell viability in vitro. METHODS: AgNPs, functionalized with reduced glutathione (GSH), polyethylene glycol (PEG) and lipoic acid (LA) were synthesized. Quartz crystal microbalance with dissipation was used to measure the effect of AgNPs on platelet aggregation. Platelet aggregation was measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by phase contrast microscopy. Flow cytometry was used to detect surface abundance of platelet receptors. Lactate dehydrogenase test was used to assess the potential cytotoxicity of AgNPs on human blood platelets, endothelial cells, and fibroblasts. Commercially available ELISA tests were used to measure the levels of thromboxane B2 and metalloproteinases (MMP-1, MMP-2) released by platelets as markers of platelet activation. RESULTS: 2 nm AgNPs-GSH, 3.7 nm AgNPs-PEG both at 50 and 100 µg/mL, and 2.5 nm AgNPs-LA at 100 µg/mL reduced platelet aggregation, inhibited collagen-mediated increase in total P-selectin and GPIIb/IIIa, TXB2 formation, MMP-1, and MMP-2 release. The tested AgNPs concentrations were not cytotoxic as they did not affect, platelet, endothelial cell, or fibroblast viability. CONCLUSION: All tested functionalized AgNPs inhibited platelet aggregation at nontoxic concentrations. Therefore, functionalized AgNPs can be used as an antiplatelet agent or in design and manufacturing of blood-facing medical devices, such as vascular grafts, stents, heart valves, and catheters.

Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia.

BACKGROUND: The development of thrombocytopenia in sepsis is a poor prognostic indicator associated with a significantly increased mortality risk. Mechanisms underlying this phenomenon remain to be clearly elucidated. Matrix metalloproteinases (MMPs) are enzymes that regulate the turnover of the extra-cellular matrix. MMP-2 is recognised as a platelet agonist with MMP-9 proposed as an inhibitor of platelet activation. The existence of MMP-9 in platelets is a subject of debate. There is limited evidence thus far to suggest that toll-like receptor 4 (TLR-4) and platelet-leukocyte aggregate (PLA) formation may be implicated in the development of sepsis-associated thrombocytopenia. OBJECTIVES: To investigate whether MMP -2/-9, toll-like receptor 4 (TLR-4) or platelet-leukocyte aggregate (PLA) formation are implicated in a decline in platelet numbers during septic shock. METHODS: This was an observational study which recruited healthy controls, non-thrombocytopenic septic donors and thrombocytopenic septic donors. MMP-2, MMP-9 and TLR-4 platelet surface expression as well as PLA formation was examined using flow cytometry. In addition MMP-2 and MMP-9 were examined by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) using a 3 compartment model (plasma, intraplatelet and platelet membrane). RESULTS: There was no difference found in MMP-2, MMP-9 or TLR-4 levels between non-thrombocytopenic and thrombocytopenic septic donors. PLA formation was increased in thrombocytopenic patients. MMP-9 was detected in platelets using flow cytometry, gelatin zymography and ELISA techniques. CONCLUSIONS: Platelet consumption into PLAs may account for the development of thrombocytopenia in septic shock. MMP-9 is found in platelets and it is upregulated during septic shock.