Jacareubin inhibits TLR4-induced lung inflammatory response caused by the RBD domain of SARS-CoV-2 Spike protein.

BACKGROUND: COVID-19, the disease caused by SARS-CoV-2 virus infection, has been a major public health problem worldwide in the last 2 years. SARS-CoV-2-dependent activation of innate immune receptors contributes to the strong local and systemic inflammatory reaction associated with rapid disease evolution. The receptor-binding domain (RBD) of Spike (S) viral protein (S-RBD) is essential for virus infection and its interacting molecules in target cells are still under identification. On the other hand, the search for accessible natural molecules with potential therapeutic use has been intense and remains an active field of investigation. METHODS: C57BL6/J (control) and Toll-like receptor (TLR) 4-deficient (Lps del) mice were nebulized with recombinant S-RBD. Tumor Necrosis Factor-alpha (TNF-α) and Interleukin (IL)-6 production in bronchoalveolar lavages (BALs) was determined by enzyme-linked immunosorbent assay (ELISA). Lung-infiltrating cells recovered in BALs were quantified by hematoxylin-eosin (H&E) stain. In selected groups of animals, the natural compound Jacareubin or dexamethasone were intraperitoneally (ip) administered 2 hours before nebulization. RESULTS: A rapid lung production of TNF-α and IL-6 and cell infiltration was induced by S-RBD nebulization in control but not in Lps del mice. Pre-treatment with Jacareubin or dexamethasone prevented S-RBD-induced TNF-α and IL-6 secretion in BALs from control animals. CONCLUSIONS: S-RBD domain promotes lung TNF-α and IL-6 production in a TLR4-dependent fashion in C57BL6/J mice. Xanthone Jacareubin possesses potential anti-COVID-19 properties that, together with the previously tested anti-inflammatory activity, safety, and tolerance, make it a valuable drug to be further investigated for the treatment of cytokine production caused by SARS-CoV-2 infection.

Mast Cells Localize in Hypoxic Zones of Tumors and Secrete CCL-2 under Hypoxia through Activation of L-Type Calcium Channels.

Hypoxia is a condition that together with low pH, high amounts of reactive oxygen species (ROS), and increased adenosine levels characterize tumor microenvironment. Mast cells (MCs) are part of tumor microenvironment, but the effect of hypoxia on the production of MC-derived cytokines has not been fully described. Using the hypoxia marker pimonidazole in vivo, we found that MCs were largely located in the low-oxygen areas within B16-F1 mice melanoma tumors. In vitro, hypoxia promoted ROS production, a ROS-dependent increase of intracellular calcium, and the production of MCP 1 (CCL-2) in murine bone marrow-derived MCs. Hypoxia-induced CCL-2 production was sensitive to the antioxidant trolox and to nifedipine, a blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). Simultaneously with CCL-2 production, hypoxia caused the ROS-dependent glutathionylation and membrane translocation of the α1c subunit of Cav1.2 LVDCCs. Relationship between ROS production, calcium rise, and CCL-2 synthesis was also observed when cells were treated with H2O2 In vivo, high CCL-2 production was detected on hypoxic zones of melanoma tumors (where tryptase-positive MCs were also found). Pimonidazole and CCL-2 positive staining diminished when B16-F1 cell-inoculated animals were treated with trolox, nifedipine, or the adenosine receptor 2A antagonist KW6002. Our results show that MCs are located preferentially in hypoxic zones of melanoma tumors, hypoxia-induced CCL-2 production in MCs requires calcium rise mediated by glutathionylation and membrane translocation of LVDCCs, and this mechanism of CCL-2 synthesis seems to operate in other cells inside melanoma tumors, with the participation of the adenosine receptor 2A.

Chemoinformatic Analysis of Selected Cacalolides from Psacalium decompositum (A. Gray) H. Rob. & Brettell and Psacalium peltatum (Kunth) Cass. and Their Effects on FcεRI-Dependent Degranulation in Mast Cells.

Cacalolides are a kind of sesquiterpenoids natural compounds synthesized by Psacalium decompositum (A. Gray) H. Rob. & Brettell or Psacalium peltatum (Kunth) Cass. Antioxidant and hypoglycemic effects have been found for cacalolides such as cacalol, cacalone or maturine, however, their effects on inflammatory processes are still largely unclear. The main aim of this study was to investigate the biological activities of secondary metabolites from P. decompositum and P. peltatum through two approaches: (1) chemoinformatic and toxicoinformatic analysis based on ethnopharmacologic background; and (2) the evaluation of their potential anti-inflammatory/anti-allergic effects in bone marrow-derived mast cells by IgE/antigen complexes. The bioinformatics properties of the compounds: cacalol; cacalone; cacalol acetate and maturin acetate were evaluated through Osiris DataWarrior software and Molinspiration and PROTOX server. In vitro studies were performed to test the ability of these four compounds to inhibit antigen-dependent degranulation and intracellular calcium mobilization, as well as the production of reactive oxygen species in bone marrow-derived mast cells. Our findings showed that cacalol displayed better bioinformatics properties, also exhibited a potent inhibitory activity on IgE/antigen-dependent degranulation and significantly reduced the intracellular calcium mobilization on mast cells. These data suggested that cacalol could reduce the negative effects of the mast cell-dependent inflammatory process.

Anandamide inhibits FcεRI-dependent degranulation and cytokine synthesis in mast cells through CB2 and GPR55 receptor activation. Possible involvement of CB2-GPR55 heteromers.

Activation of high affinity receptor for IgE (FcεRI) by IgE/antigen complexes in mast cells (MCs) leads to the release of preformed pro-inflammatory mediators stored in granules by a Ca2+-dependent process known as anaphylactic degranulation. Degranulation inhibition has been proposed as a strategy to control allergies and chronic inflammation conditions. Cannabinoids are important inhibitors of inflammatory reactions but their effects on IgE/Ag-mediated MCs responses are not well described. In this study, we analyzed the effect of the endocannabinoid anandamide (AEA), the selective CB2 receptor agonist HU308, and the GPR55 receptor agonist lysophosphatidylinositol (LPI) on FcεRI-induced activation in murine bone marrow-derived mast cells (BMMCs). Our results show that AEA, HU380 and LPI inhibited FcεRI-induced degranulation in a concentration-dependent manner. This effect was mediated by CB2 and GPR55 receptor activation through a mechanism insensitive to pertussis toxin. Degranulation inhibition was prevented by CB2 and GPR55 antagonism, but not by CB1 receptor blockage. AEA also inhibited calcium-dependent cytokine mRNA synthesis induced by FcεRI crosslinking, without affecting early phosphorylation events. In addition, AEA, HU308 and LPI inhibited intracellular Ca2+ rise in response to IgE/Ag. CB2 and GPR55 receptor antagonism could not prevent the inhibition produced by AEA and HU308, but partially blocked the one caused by LPI. These results indicate that AEA inhibits IgE/Ag-induced degranulation through a mechanism that includes the participation of CB2 and GPR55 receptors acting in close crosstalk, and show that CB2-GPR55 heteromers are important negative regulators of FcεRI-induced responses in MCs.

Mexican Propolis: A Source of Antioxidants and Anti-Inflammatory Compounds, and Isolation of a Novel Chalcone and ε-Caprolactone Derivative.

The propolis produced by bees are used in alternative medicine for treating inflammation, and infections, presumably due to its antioxidant properties. In this context, five propolis from México were investigated to determine their inhibitory lipid peroxidation properties. The ethyl acetate extract from a red propolis from Chiapas State (4-EAEP) was the most potent (IC50 = 1.42 ± 0.07 µg/mL) in the TBARS assay, and selected for further studies. This extract afforded two new compounds, epoxypinocembrin chalcone (6), and an ε-caprolactone derivative (10), as well as pinostrobin (1), izalpinin (2), cinnamic acid (3), pinocembrin (4), kaempherol (5), 3,3-dimethylallyl caffeate in mixture with isopent-3-enyl caffeate (7a + 7b), 3,4-dimethoxycinnamic acid (8), rhamnetin (9) and caffeic acid (11). The HPLC profile, anti-mycobacterial, and antioxidant properties of this extract was also determined. Most of the isolated compounds were also tested by inhibition of reactive oxygen species (ROS) in challenged mouse bone marrow-derived mast cells (BMMCs), and DPPH. Their anti-inflammatory activity was evaluated by TPA, and MPO (myeloperoxidase) activity by ear edema test in mice. The most potent compounds were 7a + 7b in the TBARS assay (IC50 = 0.49 ± 0.06 µM), and 2 which restored the ROS baseline (3.5 µM). Our results indicate that 4-EAEP has anti-oxidant, and anti-inflammatory properties due to its active compounds, suggesting it has anti-allergy and anti-asthma potential.

Protein Tyrosine Kinase Fyn Regulates TLR4-Elicited Responses on Mast Cells Controlling the Function of a PP2A-PKCα/ß Signaling Node Leading to TNF Secretion.

Mast cells produce proinflammatory cytokines in response to TLR4 ligands, but the signaling pathways involved are not fully described. In this study, the participation of the Src family kinase Fyn in the production of TNF after stimulation with LPS was evaluated using bone marrow-derived mast cells from wild-type and Fyn-deficient mice. Fyn(-/-) cells showed higher LPS-induced secretion of preformed and de novo-synthesized TNF. In both cell types, TNF colocalized with vesicle-associated membrane protein (VAMP)3-positive compartments. Addition of LPS provoked coalescence of VAMP3 and its interaction with synaptosomal-associated protein 23; those events were increased in the absence of Fyn. Higher TNF mRNA levels were also observed in Fyn-deficient cells as a result of increased transcription and greater mRNA stability after LPS treatment. Fyn(-/-) cells also showed higher LPS-induced activation of TAK-1 and ERK1/2, whereas IκB kinase and IκB were phosphorylated, even in basal conditions. Increased responsiveness in Fyn(-/-) cells was associated with a lower activity of protein phosphatase 2A (PP2A) and augmented activity of protein kinase C (PKC)α/ß, which was dissociated from PP2A and increased its association with the adapter protein neuroblast differentiation-associated protein (AHNAK, desmoyokin). LPS-induced PKCα/ß activity was associated with VAMP3 coalescence in WT and Fyn-deficient cells. Reconstitution of MC-deficient Wsh mice with Fyn(-/-) MCs produced greater LPS-dependent production of TNF in the peritoneal cavity. Our data show that Fyn kinase is activated after TLR4 triggering and exerts an important negative control on LPS-dependent TNF production in MCs controlling the inactivation of PP2Ac and activation of PKCα/ß necessary for the secretion of TNF by VAMP3(+) carriers.