Unraveling the Mechanism of Platelet Aggregation Suppression by Thioterpenoids: Molecular Docking and In Vivo Antiaggregant Activity.

Natural monoterpenes and their derivatives are widely considered the effective ingredients for the design and production of novel biologically active compounds. In this study, by using the molecular docking technique, we examined the effects of two series of "sulfide-sulfoxide-sulfone" thioterpenoids containing different (e.g., bornane and pinane) monoterpene skeletons on the platelet's aggregation. Our data revealed that all the synthesized compounds exhibit inhibitory activities on platelet aggregation. For example, compound 1 effectively inhibited platelet activation and demonstrated direct binding with CD61 integrin, a well-known platelet GPIIb-IIIa receptor on platelets. We further examined the antiaggregant activity of the most active compound, 1, in vivo and compared its activity with that of acetylsalicylic acid and an oral GPIIb-IIIa blocker, orbofiban. We found that compound 1 demonstrates antiaggregant activity in rats when administered per os and its activity was comparable with that of acetylsalicylic acid and orbofiban. Moreover, similarly, tirofiban, a well-known GPIIb-IIIa blocker, compound 1, effectively decreased the expression of P-selectin to the values similar to those of the intact platelets. Collectively, here, we show, for the first time, the potent antiaggregant activity of compound 1 both in vitro and in vivo due to its ability to bind with the GPIIb-IIIa receptor on platelets.

Novel O-benzylcinnamic acid derivative L26 treats acute lung injury in mice by MD-2.

Acute lung injury (ALI) is an inflammation-mediated respiratory disease that is associated with a high mortality rate. In this study, a series of novel O-benzylcinnamic acid derivatives were designed and synthesized using cinnamic acid as the lead compound. We tested the preliminary anti-inflammatory activity of the compounds by evaluating their effect on inhibiting the activity of alkaline phosphatase (ALP) in Hek-Blue-TLR4 cells, in which compound L26 showed the best activity and 7-fold more active than CIN. ELISA, immunoprecipitation, and molecular docking indicated that L26 targeted MD-2 protein and competed with LPS to bind to MD-2, which resulted in the inhibition of inflammation. In the LPS-induced mouse model of ALI, L26 was found to decrease ALP activity and inflammatory cytokine TNF-α release to reduce lung injury by inhibiting the NF-κB signaling pathway. Acute toxicity experiments showed that high doses of L26 did not cause adverse reactions in mice, and it was safe in vivo. Also, the preliminary pharmacokinetic parameters of L26 were investigated in SD rats (T1/2 = 4.246 h). In summary, L26 exhibited optimal pharmacodynamic and pharmacokinetic characteristics, which suggested that L26 could serve as a potential agent for the development of ALI treatment.

Discovery of 4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide derivatives as novel anti-inflammatory agents for the treatment of acute lung injury and sepsis.

Acute lung injury (ALI) and sepsis, characterized by systemic inflammatory response syndrome, remain the major causes of death in severe patients. Inhibiting the release of proinflammatory cytokines is considered to be a promising method for the treatment of inflammation-related diseases. In this study, a total of 28 4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide derivatives were designed and synthesized and their anti-inflammatory activities in J774A.1 were evaluated. Among them, derivative 13a was found to significantly inhibit lipopolysaccharide (LPS)-induced expression of the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) on J774A.1, THP-1 and LX-2 cells, and inhibited the activation of the NF-κB pathway. Furthermore, administration of 13ain vivo significantly improved the symptoms in LPS-induced ALI mice, including alleviation of pathological changes in the lung tissue, reduction of pulmonary edema, and inhibition of macrophage infiltration. Moreover, the administration of 13ain vivo significantly promoted survival in LPS-induced sepsis mice. 13a demonstrated favorable pharmacokinetic properties with T1/2 value of 11.8 h and F value of 36.3%. Therefore, this study has identified a novel 4-oxo-N-phenyl-1,4-dihydroquinoline-3-carboxamide derivative, 13a, which is an effective anti-inflammatory agent. The findings have laid a foundation for the further development of agents to treat ALI and sepsis.

Design, synthesis, and bioactivity evaluation of novel 1-(4-(benzylsulfonyl)-2-nitrophenyl) derivatives as potential anti-inflammatory agents against LPS-induced acute lung injury.

Acute lung injury (ALI) is a devastating disease with a high mortality rate of 30%-40%. There is an unmet clinical need owing to limited treatment strategies and little clinical benefit. The pathology of ALI indicates that reducing the inflammatory response could be a highly desirable strategy to treat ALI. In this study, we designed and synthesized 36 novel 1-(4-(benzylsulfonyl)-2-nitrophenyl) derivatives and evaluated their anti-inflammatory activities by measuring the release of cytokines in lipopolysaccharide (LPS)-challenged J774A.1 cells. Compounds 19, 20, and 39 potently reduced the release of IL-6 and TNF-α in J774A.1 cells. Additionally, 39 improved LPS-induced ALI in vivo and inhibited cytokine production in lung tissues. Furthermore, 39 reduced inflammatory infiltration and downregulated p-p65 levels in lung tissues. Thus, compound 39 could serve as a new lead structure for the development of anti-inflammatory drugs to treat ALI.

Schisandrin B protects against LPS-induced inflammatory lung injury by targeting MyD88.

BACKGROUND: Acute lung injury (ALI) is a challenging clinical syndrome that manifests as an acute inflammatory response. Schisandrin B (Sch B), a bioactive lignan from Schisandra genus plants, has been shown to suppress inflammatory responses and oxidative stress. However, the underlying molecular mechanisms have remained elusive. HYPOTHESIS/PURPOSE: This study performed an in-depth investigation of the anti-inflammatory mechanism of Sch B in macrophages and in an animal model of ALI. METHODS: qPCR array was used to probe the differential effects and potential target of Sch B. ALI was induced by intratracheal administration of LPS in experimental mice with or without Sch B treatment. RESULTS: Our studies show that Sch B differentially modulates inflammatory factor induction by LPS in macrophages by directly binding myeloid differentiation response factor-88 (MyD88), an essential adaptor protein in the toll-like receptor-4 (TLR4) pathway. Sch B spares non-MyD88-pathways downstream of TLR4. Such inhibition suppressed key signaling mediators such as TAK1, MAPKs, and NF-κB, and pro-inflammatory factor induction. Pull down assay using biotinylated-Sch B validate the direct interaction between Sch B and MyD88 in macrophages. Treatment of mice with Sch B prior to LPS challenge reduced inflammatory cell infiltration in lungs, induction of MyD88-pathway signaling proteins, and prevented inflammatory cytokine induction. CONCLUSION: In summary, our studies have identified MyD88 as a direct target of Sch B for its anti-inflammatory activity, and suggest that Sch B may have therapeutic value for acute lung injury and other MyD88-dependent inflammatory diseases.

Calix[4]Resorcinarene Carboxybetaines and Carboxybetaine Esters: Synthesis, Investigation of In Vitro Toxicity, Anti-Platelet Effects, Anticoagulant Activity, and BSA Binding Affinities.

As a result of bright complexation properties, easy functionalization and the ability to self-organize in an aqueous solution, amphiphilic supramolecular macrocycles are being actively studied for their application in nanomedicine (drug delivery systems, therapeutic and theranostic agents, and others). In this regard, it is important to study their potential toxic effects. Here, the synthesis of amphiphilic calix[4]resorcinarene carboxybetaines and their esters and the study of a number of their microbiological properties are presented: cytotoxic effect on normal and tumor cells and effect on cellular and non-cellular components of blood (hemotoxicity, anti-platelet effect, and anticoagulant activity). Additionally, the interaction of macrocycles with bovine serum albumin as a model plasma protein is estimated by various methods (fluorescence spectroscopy, synchronous fluorescence spectroscopy, circular dichroic spectroscopy, and dynamic light scattering). The results demonstrate the low toxicity of the macrocycles, their anti-platelet effects at the level of acetylsalicylic acid, and weak anticoagulant activity. The study of BSA-macrocycle interactions demonstrates the dependence on macrocycle hydrophilic/hydrophobic group structure; in the case of carboxybetaines, the formation of complexes prevents self-aggregation of BSA molecules in solution. The present study demonstrates new data on potential drug delivery nanosystems based on amphiphilic calix[4]resorcinarenes for their cytotoxicity and effects on blood components.

Thioterpenoids as Potential Antithrombotic Drugs: Molecular Docking, Antiaggregant, Anticoagulant and Antioxidant Activities.

Natural monoterpenes and their derivatives are widely considered as effective ingredients for the design and production of new biologically active compounds with high antioxidant, antimicrobial and anti-protozoa properties. In this study, we synthesized two series of thiotherpenoids "sulfide-sulfoxide-sulfone", with different bicyclic monoterpene skeleton (bornane and pinane) structures. The effect of the obtained compounds on platelet aggregation was investigated by using the molecular docking technique. The obtained data revealed that all the synthesized compounds may act as potential inhibitors of platelet aggregation. Moreover, the studied sulfides have shown high antioxidant activity as revealed by lipid peroxidation (LPO) process inhibition in a non-cellular substrate containing animal lipids. The sulfides were able to inhibit erythrocyte oxidative hemolysis, to reduce the accumulation of secondary LPO products in cells and to prevent the oxidation of native oxyhemoglobin. Additionally, the corresponding sulfones and sulfoxides exhibited insignificant antioxidant activity. However, the sulfides were found to exhibit significant antiaggregant and anticoagulant effects. These findings suggest as well that the sulfides could serve as a leader compound for future research and possible practical applications.

Heme induces inflammatory injury by directly binding to the complex of myeloid differentiation protein 2 and toll-like receptor 4.

Heme, as an essential component of hemoproteins, is a prosthetic co-factor found in many cells, which is essential for physiologically vital oxygen transport. However, extracellular or circulatory heme is cytotoxic and triggers inflammation. Although the proinflammatory role of heme has been reported to be associated with Toll-like receptor 4 (TLR4) signaling, the exact mechanism remains unknown. Here, we show that heme promotes TLR4 signaling and inflammation via directly physically interacting with TLR4 and its adaptor protein myeloid differentiation protein 2 (MD2). Genetic loss of MD2 ameliorates heme-induced inflammation and inflammatory cytokine production in the spleen of MD2 knockout (MD2-/-) mice. Using mouse macrophage RAW 264.7 cell line, we show that heme induces TLR4 dimerization and MD2/TLR4/MyD88 activation by physically interacting with TLR4 and MD2 in vitro. Genetic loss of MD2 inhibits heme-induced inflammation and MAPK/NF-κB pathway in mouse primary macrophages extracted from MD2-/- mice. Furthermore, pharmacological inhibition of MD2 by L6H9 ameliorates heme-induced inflammation in macrophages. These findings demonstrate that heme causes inflammation by directly binding to MD2/TLR4 complex, leading to activation of TLR4/MAPK/NF-κB signaling pathway and production of downstream effectors of inflammation.

Myeloid differential protein-2 inhibition improves diabetic cardiomyopathy via p38MAPK inhibition and AMPK pathway activation.

Myeloid differential protein-2 (MD2) has been shown to play a critical role in the progression of diabetic cardiomyopathy (DCM). This study aims to explore the non-inflammatory mechanisms mediated by MD2 in DCM and to test the therapeutic effects of MD2 inhibitor C30 on DCM. Streptozotocin (STZ) was used to construct DCM model in wild-type and MD2 knockout mice. The collected heart samples were subjected to RNA-sequencing assay. Gene set enrichment analysis of the RNA-seq data indicated that MD2 knockout was associated with energy metabolism pathways in diabetic mouse heart. Further data showed that AMPK pathway was impaired under high glucose condition, which was mediated by p38MAPK activation. MD2 knockout or pharmacological inhibitor C30 completely rescued AMPK signaling through p38MAPK inhibition. Importantly, C30 treatment significantly prevented myocardial damage and dysfunction in T1DM mice evidenced by improved cardiac function and reduced cardiomyocyte apoptosis and cardiac fibrosis. Furthermore, the therapeutic effect of C30 on DCM was correlated to p38MAPK inhibition and AMPK pathway activation in vivo and in vitro. In conclusion, MD2 inhibition exhibits therapeutic effects on DCM through p38MAPK inhibition and AMPK activation, which enables MD2 a promising target for DCM treatment by suppressing metaflammation and improving cardiac metabolism.

Cardamonin inhibits LPS-induced inflammatory responses and prevents acute lung injury by targeting myeloid differentiation factor 2.

BACKGROUND: Acute lung injury (ALI) is a systemic inflammatory process, which has no pharmacological therapy in clinic. Accumulating evidence has demonstrated that natural compounds from herbs have potent anti-inflammatory efficacy in several disease models, which could be the potential candidates for the treatment of ALI. HYPOTHESIS/PURPOSE: Anti-inflammatory screening from natural product bank may provide new anti-inflammatory compounds for therapeutic target discovery and ALI treatment. METHODS: 165 natural compounds were screened for their anti-inflammatory activity in LPS-stimulated macrophages. PCR array, SPR and ELISA were used to determine the potential target of the most active compound, Cardamonin (CAR). The pharmacological effect of CAR was further evaluated in both LPS-stimulated macrophages and ALI mice model. RESULTS: Out of the screened 165 compounds, CAR significantly inhibited LPS-induced inflammatory cytokine secretion in macrophages. We further showed that CAR significantly inhibited NF-κB and JNK signaling activation, and thereby inflammatory cytokine production via directly interacting with MD2 in vitro. In vivo, our data show that CAR treatment inhibited LPS-induced lung damage, systemic inflammatory cytokine production, and reduced macrophage infiltration in the lungs, accompanied with reduced TLR4/MD2 complex in lung tissues, Treatment with CAR also dose-dependently increased survival in the septic mice induced by DH5α bacterial infection. CONCLUSION: We demonstrate that a natural product, CAR, attenuates LPS-induced lung injury and sepsis by inhibiting inflammation via interacting with MD2, leading to the inactivation of the TLR4/MD2-MyD88-MAPK/NF-κB pathway.

Curcumin analogue C66 attenuates obesity-induced myocardial injury by inhibiting JNK-mediated inflammation.

Obesity has been recognized as a major risk factor for the development of chronic cardiomyopathy, which is associated with increased cardiac inflammation, fibrosis, and apoptosis. We previously developed an anti-inflammatory compound C66, which prevented inflammatory diabetic complications via targeting JNK. In the present study, we have tested the hypothesis that C66 could prevent obesity-induced cardiomyopathy by suppressing JNK-mediated inflammation. High-fat diet (HFD)-induced obesity mouse model and palmitic acid (PA)-challenged H9c2 cells were used to develop inflammatory cardiomyopathy and evaluate the protective effects of C66. Our data demonstrate a protective effect of C66 against obesity-induced cardiac inflammation, cardiac hypertrophy, fibrosis, and dysfunction, overall providing cardio-protection. C66 administration attenuates HFD-induced myocardial inflammation by inhibiting NF-κB and JNK activation in mouse hearts. In vitro, C66 prevents PA-induced myocardial injury and apoptosis in H9c2 cells, accompanied with inhibition against PA-induced JNK/NF-κB activation and inflammation. The protective effect of C66 is attributed to its potential to inhibit JNK activation, which led to reduced pro-inflammatory cytokine production and reduced apoptosis in cardiomyocytes both in vitro and in vivo. In summary, C66 provides significant protection against obesity-induced cardiac dysfunction, mainly by inhibiting JNK activation and JNK-mediated inflammation. Our data indicate that inhibition of JNK is able to provide significant protection against obesity-induced cardiac dysfunction.

Kaempferol attenuates streptozotocin-induced diabetic nephropathy by downregulating TRAF6 expression: The role of TRAF6 in diabetic nephropathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Kaempferia rhizome is a famous traditional herbal medical in tropical and subtropical areas. Kaempferol (KPF) is one of the main bioactive compounds in Kaempferia rhizome, with anti-oxidant/anti-inflammatory effects demonstrated in various disease models, including cancers, obesity and diabetes. AIM OF THE STUDY: Inflammation plays an important role in the pathogenesis of diabetic nephropathy (DN). TRAF6 functions as a signal transducer in toll-like receptor 4 and NF-κB pro-inflammatory signaling pathway. We aimed at investigate whether KPF is able to mitigate inflammatory responses by regulating TRAF6 in DN. MATERIAL AND METHODS: C57BL/6 mice were injected with streptozotocin to induce type 1 DN. NRK-52E, a tubular epithelial cell line, was used for in vitro analysis. TRAF6 was knockdown using siRNA in vitro and AAV2/2-shRNA in vivo. The anti-DN and inflammatory effects of KPF or knockdown of TRAF6 were evaluated by investigating renal filtration index, pathological changes of kidney tissue. Proinflammatory cytokine levels were detected using ELISA. NF-κB pathway and protein levels of related pathways were detected through Western blot. RESULTS: KPF significantly reduced renal inflammation, fibrosis, and kidney dysfunction in diabetic mice. These effects were associated with a downregulation of TRAF6 in diabetic mouse kidneys, indicating the potential role of TRAF6. Knockdown of TRAF6 in mice through AAV2-shTRAF6 confirmed the importance of TRAF6 in DN. In vitro, treatment of KPF in NRK-52E cells attenuated high glucose (HG)-induced inflammatory and fibrogenic responses, associated with downregulated TRAF6 expression. The conclusion was further confirmed in NRK-52E cells by knocking down the expression and by overexpression of TRAF6. CONCLUSION: Our findings provide direct evidence that TRAF6 mediates diabetes-induced inflammation leading to renal dysfunction. We also show that KPF is a potential therapeutic agent to reduce inflammatory responses in DN. Also, TRAF6 may represent an interesting target to combat DN.

The off-label use of drugs for parenteral nutrition as a solvent of substances slightly soluble in water in pharmacological research.

Because of the problem to evaluate biological activity in water-soluble substances in all phases of preclinical and clinical studies, the research work enabled to develop the original solvent for poorly soluble compounds based on substances for parenteral nutrition. The main aim is to examine the impact of the original solvent based on substances for parenteral nutrition on biological systems exemplified by the hemostatic system, characterized by sensitivity and variability of the effects in response to any impact, and its comparison with the solvents that are conventional in pharmacological research. Experimental work is performed according to the "guidance on preclinical research of new pharmacological substances" in vitro. The findings show that traditional solvents at low dosages affect all the researched indicators of the hemostasis system. The smallest effect in respect of the hemostatic system was characterized by ethanol, and the most apparent antiaggregational effect was registered with dioxane. 10% concentration of original blend of lipids made no effect on hemostasis system. Thus, according to their own findings and experience in application of lipid emulsions as substances of parenteral nutrition, they can be considered to be an adequate solvent in all phases of preclinical and clinical studies of new drugs.