Morita-Baylis-Hillman adduct 2-(3-hydroxy-2-oxoindolin-3-yl)acrylonitrile (ISACN) modulates the inflammatory process during LPS-induced acute lung injury.

BACKGROUND: Despite its homeostatic role, inflammation is involved in several pathologies, such as acute lung injury. Morita-Ballys-Hilman adducts (MBHA) are a group of synthetic molecules and present a wide range of biological activities, including anti-inflammatory action. Thus, this study aimed to assess whether ISACN, an MBHA, modulates inflammation during acute lung injury induced by lipopolysaccharide (LPS). METHODS: BALB/c mice were intraperitoneally treated with 24 mg/kg ISACN and challenged with LPS (2.5 mg/kg). On bronchoalveolar lavage fluid (BALF), we assessed the total and differential leukocyte count and measurement of protein leakage, cytokines (IL-1ß, IL-6, and TNF-α), and chemokine (CXCL-1). Additionally, lung histopathology was also performed (H&E staining). In vitro studies were conducted with peritoneal macrophages to assess the possible mechanism of action. They were cultured in the presence of ISACN (5 and 10 µM) and stimulated by LPS (1 µg/mL). RESULTS: ISACN reduced neutrophil migration, protein leakage, and inflammatory cytokines (IL-1ß, IL-6, and TNF-α) without interfering with the production of CXCL1. In addition, ISACN caused a decrease in LPS-induced lung injury as evident from histopathological changes. In peritoneal macrophages, ISACN diminishes the nitric oxide and cytokine levels (IL-1ß, IL-6, and TNF-α). The treatment with ISACN (10 µM) also reduced LPS-induced TLR4, CD69, iNOS overexpression, and the LPS-induced ERK, JNK, and p38 phosphorylation. CONCLUSION: Thus, this work showed for the first time the immunomodulatory action of MBHA in LPS-induced acute lung injury and provided new evidence for the mechanisms related to the anti-inflammatory effect of ISACN.

Antiviral activity of ouabain against a Brazilian Zika virus strain.

Zika virus (ZIKV) is an emerging arbovirus associated with neurological disorders. Currently, no specific vaccines or antivirals are available to treat the ZIKV infection. Ouabain, a cardiotonic steroid known as Na+/K+-ATPase inhibitor, has been previously described as an immunomodulatory substance by our group. Here, we evaluated for the first time the antiviral activity of this promising substance against a Brazilian ZIKV strain. Vero cells were treated with different concentrations of ouabain before and after the infection with ZIKV. The antiviral effect was evaluated by the TCID50 method and RT-qPCR. Ouabain presented a dose-dependent inhibitory effect against ZIKV, mainly when added post infection. The reduction of infectious virus was accompanied by a decrease in ZIKV RNA levels, suggesting that the mechanism of ZIKV inhibition by ouabain occurred at the replication step. In addition, our in silico data demonstrated a conformational stability and favorable binding free energy of ouabain in the biding sites of the NS5-RdRp and NS3-helicase proteins, which could be related to its mechanism of action. Taken together, these data demonstrate the antiviral activity of ouabain against a Brazilian ZIKV strain and evidence the potential of cardiotonic steroids as promising antiviral agents.

Patulin inhibits LPS-induced nitric oxide production by suppressing MAPKs signaling pathway.

Patulin (PAT) is a natural product isolated from several species of fungi. Here, we evaluated the effect of PAT (62.5-4,000 ng/ml) in lipopolysaccharide (LPS)-activated murine peritoneal macrophages. Cell viability assay showed that PAT at concentrations up to 250 ng/ml did not affect macrophage viability. PAT (250 ng/ml) significantly reduced LPS-induced nitric oxide production (by 98.4%), inducible nitric oxide synthase (iNOS) expression (by 83.5%), and iNOS messenger ribonucleic acid expression (by 100.0%). Moreover, PAT significantly reduced LPS-induced interleukin-1ß (by 80.6%), cluster of differentiation (CD) 69 (by 63.1%), and Toll-like receptor (TLR) 4 (by 91.9%) protein expression. Finally, PAT significantly reduced LPS-triggered phosphorylation of all mitogen-activated protein kinases (MAPK) assessed: extracellular signal-regulated kinase (ERK; by 89.5%), c-Jun N-terminal kinase (JNK; by 77.5%), and p38 (by 72.3%). Taken together, these data suggest that PAT downregulates acute inflammatory response, inhibiting nitric oxide production by suppressing CD69-TLR4/ERK-JNK-p38 MAPKs/Nos2/iNOS signaling pathway.

Ouabain inhibits p38 activation in mice neutrophils.

Ouabain is a cardiac steroid hormone with immunomodulatory effects. It inhibits neutrophils migration induced by different stimuli, but little is known about the mechanisms involved in this effect. Thus, the aim of this study was to evaluate the ouabain effect on chemotactic signaling pathways in neutrophils. For that, mice neutrophils were isolated from bone marrow, treated with ouabain (1, 10, and 100 nM) for 2 h, submitted to transwell chemotaxis assay and flow cytometry analysis of Akt, ERK, JNK, and p38 phosphorylation induced by zymosan. Ouabain treatment (1, 10 and, 100 nM) reduces neutrophil chemotaxis induced by chemotactic peptide fMLP, but this substance did not inhibit Akt, ERK, and JNK activation induced by zymosan. However, ouabain (1 and 10 nM) reduced p38 phosphorylation in zymosan-stimulated neutrophils. These results suggest that ouabain may interfere in neutrophil migration through p38 MAPK inhibition.

Relation between ABCB1 overexpression and COX2 and ALOX5 genes in human erythroleukemia cell lines.

This study aimed to characterize the relationship between the COX2 and ALOX5 genes, as well as their link with the multidrug resistance (MDR) phenotype in sensitive (K562) and MDR (K562-Lucena and FEPS) erythroleukemia cells. For this, the inhibitors of 5-LOX (zileuton) and COX-2 (acetylsalicylic acid-ASA) and cells with the silenced ABCB1 gene were used. The treatment with ASA caused an increase in the gene expression of COX2 and ABCB1 in both MDR cell lines, and a decrease in the expression of ALOX5 in the FEPS cells. Silencing the ABCB1 gene induced a decrease in COX2 expression and an increase in the ALOX5 gene. Treatment with zileuton did not alter the expression of COX2 and ABCB1. Cytometry data showed that there was an increase in ABCB1 protein expression after exposure to ASA. In addition, the increased activity of ABCB1 in the K562-Lucena cell line indicates that ASA may be a substrate for this efflux pump, corroborating the molecular docking that showed that ASA can bind to ABCB1. Regardless of the genetic alteration in COX2 and ABCB1, the direct relationship between these genes and the inverse relationship with ALOX5 remained in the MDR cell lines. We assume that ABCB1 can play a regulatory role in COX2 and ALOX5 during the transformation of the parental cell line K562, explaining the increased gene expression of COX2 and decreased ALOX5 in the MDR cell lines.

Neutrophils and COVID-19: The road so far.

The SARS-Cov2 infection triggers a multisystem inflammatory disorder, knowing as COVID-19, a pandemic disease. This disease is characterized by acute respiratory distress syndrome, cytokine-driven hyperinflammation, and leukocytes count changes. The innate immune response has been linked to COVID-19 immunopathogenesis (e.g., dysfunctional IFN response and myeloid inflammation). In this regard, neutrophils have been highlighted as essential effector cells in the development of COVID-19. This review summarized the significant finds about neutrophils and its effector mechanisms (e.g., neutrophils enzymes and cytokines, neutrophil extracellular traps) in COVID-19 so far.

Morita-Baylis-Hillman Adduct 2-(3-Hydroxy-2-oxoindolin-3-yl)acrylonitrile (ISACN) Modulates Inflammatory Process In vitro and In vivo.

Morita-Baylis-Hillman adducts (MBHA) are synthetic molecules with several biological actions already described in the literature. It has been previously described that adduct 2-(3-hydroxy-2-oxoindolin-3-yl)acrylonitrile (ISACN) has anticancer potential in leukemic cells. Inflammation is often associated with the development and progression of cancer. Therefore, to better understand the effect of ISACN, this study aimed to evaluate the anti-inflammatory potential of ISACN both in vitro and in vivo. Results demonstrated that ISACN negatively modulated the production of inflammatory cytokines IL-1ß, TNF-α, and IL-6 by cultured macrophages. In vivo, ISACN 6 and 24 mg/kg treatment promoted reduced leukocyte migration, especially neutrophils, to the peritoneal cavity of zymosan-challenged animals. ISACN displays no anti-edematogenic activity, but it was able to promote a significant reduction in the production of inflammatory cytokines in the peritoneal cavity. These data show, for the first time, that MBHA ISACN negatively modulates several aspects of the inflammatory response, such as cell migration and cytokine production in vivo and in vitro, thus having an anti-inflammatory potential.

The pterocarpanquinone LQB 118 inhibits inflammation triggered by zymosan in vivo and in vitro.

LQB 118, a hydride molecule, has been described as an antineoplastic and antiparasitic drug. Recently, LQB118 was also shown to display anti-inflammatory properties using an LPS-induced lung inflammation model. However, LQB 118 effects on the inflammatory response induced by zymosan has not been demonstrated. In this study, swiss mice were LQB 118 intraperitoneally (i.p.) treated and zymosan was used to induce peritoneal inflammation. Peritoneal fluid was collected and used for cell counting and proinflammatory cytokines quantification (IL-1ß, IL-6, and TNF-α) by immunoenzymatic assay (ELISA). For in vitro studies, peritoneal macrophages zymosan-stimulated were used. Results demonstrated that LQB 118 treatment reduced polymorphonuclear cell migration and TNF-α, IL-1ß, and IL-6 levels in the peritoneal cavity. In macrophages, LQB 118 treatment display no cytotoxic effect and is also able to reduce cytokines levels. To investigate LQB 118 putative mechanism of action, TLR2, CD69, and P-p38 MAPK expression were evaluated. LQB 118 treatment reduced CD69 expression and p38 phosphorylation induced by zymosan. Furthermore, LQB 118 was able to negatively modulate TLR2 expression in the presence of inflammatory stimulus. Thus, our study provide new evidences for the mechanisms related to the anti-inflammatory effect of LQB 118 in vivo and in vitro.