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1.
Microb Pathog ; 196: 107007, 2024 Nov.
Article in English | MEDLINE | ID: mdl-39395747

ABSTRACT

The present study aimed to evaluate the anti-staphylococcal, antibiofilm, cytotoxicity and trypanocidal activity, mechanisms of parasite death and immunomodulatory effect of CrataBL encapsulated into liposomes (CrataBL-Lipo). CrataBL-Lipo were prepared by the freeze-thaw technique and characterized. Anti-staphylococcal and antibiofilm activities of CrataBL and CrataBL-Lipo were evaluated against standard and clinical strains of Staphylococcus aureus susceptible and resistant. Thus, broth microdilution method was performed to determine the Minimum Inhibitory Concentration (MIC). Antibiofilm activity at subinhibitory concentrations was evaluated using the crystal violet staining method. Cytotoxicity of CrataBL-Lipo was verified in L929 fibroblasts and J774A.1 macrophages by determining the inhibitory concentration necessary to kill 50 % of cells (IC50). Trypanocidal activities of CrataBL-Lipo was evaluated in Trypanosoma cruzi and the efficacy was expressed as the concentration necessary to kill 50 % of parasites (EC50). The mechanisms of parasite death and immunomodulatory effect of CrataBL-Lipo were evaluated using flow cytometry analysis. CrataBL-Lipo presented Ø of 101.9 ± 1.3 nm (PDI = 0.245), ζ of +33.8 ± 1.3 mV and %EE = 80 ± 0.84 %. CrataBL-Lipo presented anti-staphylococcal activity (MIC = 0.56 mg/mL to 0.72 mg/mL). CrataBL-Lipo inhibited 45.4 %-75.6 % of biofilm formation. No cytotoxicity of CrataBL-Lipo was found (IC50 > 100 mg/L). CrataBL-Lipo presented EC50 of 1.1 mg/L, presenting autophagy, apoptosis and necrosis as death profile. In addition, CrataBL-Lipo reduced the production of IL-10 and TNF-α levels, causing an immunomodulatory effect. CrataBL-Lipo has a therapeutic potential for the treatment of staphylococcal infections and Chagas disease exhibiting a high degree of selectivity for the microorganism, and immunomodulatory properties.


Subject(s)
Anti-Bacterial Agents , Biofilms , Liposomes , Microbial Sensitivity Tests , Staphylococcus aureus , Trypanocidal Agents , Trypanosoma cruzi , Biofilms/drug effects , Trypanosoma cruzi/drug effects , Animals , Mice , Staphylococcus aureus/drug effects , Cell Line , Anti-Bacterial Agents/pharmacology , Trypanocidal Agents/pharmacology , Macrophages/drug effects , Lectins/pharmacology , Fibroblasts/drug effects , Inhibitory Concentration 50 , Cell Survival/drug effects
2.
Int J Mol Sci ; 25(19)2024 Oct 08.
Article in English | MEDLINE | ID: mdl-39409130

ABSTRACT

Thyroid hormone binds to specific nuclear receptors, regulating the expression of target genes, with major effects on cardiac function. Triiodothyronine (T3) increases the expression of key proteins related to calcium homeostasis, such as the sarcoplasmic reticulum calcium ATPase pump, but the detailed mechanism of gene regulation by T3 in cardiac voltage-gated calcium (Cav1.2) channels remains incompletely explored. Furthermore, the effects of T3 on Cav1.2 auxiliary subunits have not been investigated. We conducted quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence experiments in H9c2 cells derived from rat ventricular tissue, examining the effects of T3 on the expression of α1c, the principal subunit of Cav1.2 channels, and Cavß4, an auxiliary Cav1.2 subunit that regulates gene expression. The translocation of phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB) by T3 was also examined. We found that T3 has opposite effects on these channel proteins, upregulating α1c and downregulating Cavß4, and that it increases the nuclear translocation of pCREB while decreasing the translocation of Cavß4. Finally, we found that overexpression of Cavß4 represses the mRNA expression of α1c, suggesting that T3 upregulates the expression of the α1c subunit in response to a decrease in Cavß4 subunit expression.


Subject(s)
Calcium Channels, L-Type , Myocytes, Cardiac , Animals , Calcium Channels, L-Type/metabolism , Calcium Channels, L-Type/genetics , Rats , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/drug effects , Triiodothyronine/pharmacology , Triiodothyronine/metabolism , Down-Regulation/drug effects , Thyroid Hormones/metabolism , Cell Line , Up-Regulation/drug effects , Cyclic AMP Response Element-Binding Protein/metabolism , Gene Expression Regulation/drug effects , Protein Subunits/metabolism , Protein Subunits/genetics
3.
PLoS One ; 19(10): e0310699, 2024.
Article in English | MEDLINE | ID: mdl-39356686

ABSTRACT

Hippocampal neurons exhibit activation of both the conventional transmembrane adenylyl cyclases (tmACs) and the non-canonical soluble adenylyl cyclase (sAC) as sources of cyclic AMP (cAMP). These two cAMP sources play crucial roles in mediating signaling pathways downstream of CRHR1 in neuronal and neuroendocrine contexts. In this study, we investigate the involvement of both cAMP sources in the molecular mechanisms triggered by CRHR2α. Here we provide evidence demonstrating that UCN1 and UCN3 exert a neuritogenic effect on HT22-CRHR2α cells, which is solely dependent on the cAMP pool generated by sAC and PKA activity but independent of ERK1/2 activation. Through the characterization of the effectors implicated in neurite elongation, we found that CREB phosphorylation and c-Fos induction rely on PKA activity and ERK1/2 phosphorylation, underscoring the critical role of signaling pathway regulation. These findings strengthen the concept that localized cAMP microdomains actively participate in the regulation of these signaling processes.


Subject(s)
Adenylyl Cyclases , Cyclic AMP-Dependent Protein Kinases , Cyclic AMP , Receptors, Corticotropin-Releasing Hormone , Signal Transduction , Cyclic AMP/metabolism , Receptors, Corticotropin-Releasing Hormone/metabolism , Animals , Adenylyl Cyclases/metabolism , Mice , Phosphorylation , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Urocortins/metabolism , Cell Line , Neurites/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Neurons/metabolism
4.
J Gen Virol ; 105(10)2024 Oct.
Article in English | MEDLINE | ID: mdl-39373166

ABSTRACT

Feline calicivirus (FCV) icosahedral viral capsids are composed of dozens of structural subunits that rely on cellular chaperones to self-assemble in an orderly fashion. Here, we report that the heat shock protein 90 (Hsp90) inhibition significantly reduced FCV particle production, suggesting a role in the replicative cycle. We found that Hsp90 inhibition was not related to the synthesis or stability of the early proteins that translate from the gRNA nor to the minor capsid protein VP2 but with a reduction in the major capsid protein VP1 levels, both translated late in infection from the subgenomic RNAs. Reduction in VP1 levels was observed despite an augment of the leader of the capsid (LC)-VP1 precursor levels, from which the LC and VP1 proteins are produced after proteolytic processing by NS6/7. The direct interaction of VP1 with Hsp90 was observed in infected cells. These results suggest that upon release from the polyprotein precursor, VP1 becomes a client of Hsp90 and that this interaction is required for an efficient FCV replicative cycle.


Subject(s)
Calicivirus, Feline , Capsid Proteins , HSP90 Heat-Shock Proteins , Virus Replication , HSP90 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/genetics , Capsid Proteins/metabolism , Capsid Proteins/genetics , Calicivirus, Feline/metabolism , Calicivirus, Feline/physiology , Calicivirus, Feline/genetics , Cats , Animals , Cell Line , Caliciviridae Infections/virology , Caliciviridae Infections/metabolism
5.
Molecules ; 29(19)2024 Sep 30.
Article in English | MEDLINE | ID: mdl-39407583

ABSTRACT

Visceral leishmaniasis (VL), caused by protozoa of the genus Leishmania, remains a significant public health concern due to its potentially lethal nature if untreated. Current chemotherapy options are limited by severe toxicity and drug resistance. Derivatives of 1,2,4-oxadiazole have emerged as promising drug candidates due to their broad biological activity. This study investigated the effects of novel 1,2,4-oxadiazole derivatives (Ox1-Ox7) on Leishmania infantum, the etiological agent of VL. In silico predictions using SwissADME suggest that these compounds have high oral absorption and good bioavailability. Among them, Ox1 showed the most promise, with higher selectivity against promastigotes and lower cytotoxicity towards L929 fibroblasts and J774.G8 macrophages. Ox1 exhibited selectivity indices of 18.7 and 61.7 against L. infantum promastigotes and amastigotes, respectively, compared to peritoneal macrophages. Ultrastructural analyses revealed severe morphological damage in both parasite forms, leading to cell death. Additionally, Ox1 decreased the mitochondrial membrane potential in promastigotes, as shown by flow cytometry. Molecular docking and dynamic simulations indicated a strong affinity of Ox1 for the L. infantum CYP51 enzyme. Overall, Ox1 is a promising and effective compound against L. infantum.


Subject(s)
Antiprotozoal Agents , Leishmania infantum , Molecular Docking Simulation , Molecular Dynamics Simulation , Oxadiazoles , Protozoan Proteins , Leishmania infantum/drug effects , Oxadiazoles/chemistry , Oxadiazoles/pharmacology , Antiprotozoal Agents/pharmacology , Antiprotozoal Agents/chemistry , Animals , Protozoan Proteins/metabolism , Protozoan Proteins/chemistry , Mice , Leishmaniasis, Visceral/drug therapy , Leishmaniasis, Visceral/parasitology , Cell Line , Membrane Potential, Mitochondrial/drug effects
6.
Int J Mol Sci ; 25(19)2024 Sep 26.
Article in English | MEDLINE | ID: mdl-39408715

ABSTRACT

Linoleic acid (LA), the primary ω-6 polyunsaturated fatty acid (PUFA) found in the epidermis, plays a crucial role in preserving the integrity of the skin's water permeability barrier. Additionally, vegetable oils rich in LA have been shown to notably mitigate ultraviolet (UV) radiation-induced effects, including the production of reactive oxygen species (ROS), cellular damage, and skin photoaging. These beneficial effects are primarily ascribed to the LA in these oils. Nonetheless, the precise mechanisms through which LA confers protection against damage induced by exposure to UVB radiation remain unclear. This study aimed to examine whether LA can restore redox and metabolic equilibria and to assess its influence on the inflammatory response triggered by UVB radiation in keratinocytes. Flow cytometry analysis unveiled the capacity of LA to diminish UVB-induced ROS levels in HaCaT cells. GC/MS-based metabolomics highlighted significant metabolic changes, especially in carbohydrate, amino acid, and glutathione (GSH) metabolism, with LA restoring depleted GSH levels post-UVB exposure. LA also upregulated PI3K/Akt-dependent GCLC and GSS expression while downregulating COX-2 expression. These results suggest that LA induces metabolic reprogramming, protecting against UVB-induced oxidative damage by enhancing GSH biosynthesis via PI3K/Akt signaling. Moreover, it suppresses UVB-induced COX-2 expression in HaCaT cells, making LA treatment a promising strategy against UVB-induced oxidative and inflammatory damage.


Subject(s)
Inflammation , Keratinocytes , Linoleic Acid , Oxidative Stress , Reactive Oxygen Species , Ultraviolet Rays , Keratinocytes/metabolism , Keratinocytes/drug effects , Keratinocytes/radiation effects , Ultraviolet Rays/adverse effects , Humans , Linoleic Acid/pharmacology , Oxidative Stress/drug effects , Oxidative Stress/radiation effects , Reactive Oxygen Species/metabolism , Inflammation/metabolism , Glutathione/metabolism , HaCaT Cells , Signal Transduction/drug effects , Cell Line , Phosphatidylinositol 3-Kinases/metabolism , Oxidation-Reduction/drug effects , Cyclooxygenase 2/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Metabolic Reprogramming
7.
Article in English | MEDLINE | ID: mdl-39326941

ABSTRACT

We have evaluated the induction of complete (i.e., without open ends) and incomplete (i.e., with non-rejoined or open ends) chromosomal aberrations by the radiomimetic antibiotic bleomycin (BLM) in human lymphoblastoid cells immortalized with the Epstein-Barr virus (EBV). An EBV-induced lymphoblastoid cell line (T-37) was exposed to BLM (10-200 µg/mL) for 2 h at 37ºC, and chromosomal aberrations were analyzed 24 h after treatment, using PNA-FISH with pan-telomeric and pan-centromeric probes. Both complete (multicentrics, rings, compound acentric fragments, and interstitial deletions) and incomplete (incomplete chromosomes or IC, and terminal acentric fragments or TAF) chromosomal aberrations increased significantly in BLM-exposed cells, although the concentration-response relationship was non-linear. Of the acentric fragments (ace) induced by BLM, 40 % were compound fragments (CF, ace +/+). TAF (ace, +/-) and interstitial fragments (IAF, ace -/-) were induced at similar frequencies (30 %). 230 ICE were induced by BLM, of which 52 % were IC and 48 % TAF. The average ratio between total incomplete chromosome elements (ICE) and multicentrics was 1.52. These findings suggest that human lymphoblastoid cells exhibit less repair capacity than human lymphocytes, with respect to BLM-induced ICE, and that chromosomal incompleteness is a common event following exposure of these cells to BLM.


Subject(s)
Bleomycin , Chromosome Aberrations , Herpesvirus 4, Human , Lymphocytes , Humans , Chromosome Aberrations/drug effects , Chromosome Aberrations/chemically induced , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/drug effects , Bleomycin/toxicity , Bleomycin/pharmacology , Lymphocytes/drug effects , Lymphocytes/virology , Cell Line, Transformed , Antibiotics, Antineoplastic/toxicity , Antibiotics, Antineoplastic/pharmacology , Cell Transformation, Viral/drug effects , Cell Line
8.
PLoS One ; 19(9): e0310735, 2024.
Article in English | MEDLINE | ID: mdl-39325758

ABSTRACT

Extracellular vesicles (EVs) play crucial roles in cell signaling and communication, transporting molecules that convey a message to target cells. During infectious diseases, EVs can also carry viral molecules that may contribute to viral spread, as previously reported for dengue virus (DENV). EVs from infected endothelial cells (EC) may harbor viral segments and various sets of molecules that could contribute to endothelial dysfunction during severe dengue. However, the effect of these EVs on non-infected EC (NIC) remain unknown. We characterized the EVs produced by the human EC line EA.hy 926 infected with DENV-2 and assessed their functional impact on polarized NIC. Results showed that infection induced an increased in the quantity of produced EVs, which differentially carried proteins mainly involved in proteosome activity, along with a peptide of the NS5 viral protein. Additionally, all types of Y-RNAs were found, accompanied by a set of differentially loaded microRNAs (miRs) that could regulate DENV genome. Pre-treatment of polarized NIC with small EVs (sEVs) from infected EC before DENV-2 infection caused EC activation, a decrease in viral genome replication, and a protective effect against barrier disruption during the first 24h post-infection, suggesting that sEVs could be important in the pathology or resolution of DENV and a promising therapeutic tool for infectious diseases.


Subject(s)
Dengue Virus , Endothelial Cells , Extracellular Vesicles , Virus Replication , Humans , Extracellular Vesicles/virology , Extracellular Vesicles/metabolism , Dengue Virus/physiology , Endothelial Cells/virology , Endothelial Cells/metabolism , Cell Line , Genome, Viral , Dengue/virology , MicroRNAs/genetics , MicroRNAs/metabolism
9.
Int J Mol Sci ; 25(17)2024 Aug 30.
Article in English | MEDLINE | ID: mdl-39273404

ABSTRACT

Astrogliosis is a process by which astrocytes, when exposed to inflammation, exhibit hypertrophy, motility, and elevated expression of reactivity markers such as Glial Fibrillar Acidic Protein, Vimentin, and Connexin43. Since 1999, our laboratory in Chile has been studying molecular signaling pathways associated with "gliosis" and has reported that reactive astrocytes upregulate Syndecan 4 and αVß3 Integrin, which are receptors for the neuronal glycoprotein Thy-1. Thy-1 engagement stimulates adhesion and migration of reactive astrocytes and induces neurons to retract neurites, thus hindering neuronal network repair. Reportedly, we have used DITNC1 astrocytes and neuron-like CAD cells to study signaling mechanisms activated by the Syndecan 4-αVß3 Integrin/Thy-1 interaction. Importantly, the sole overexpression of ß3 Integrin in non-reactive astrocytes turns them into reactive cells. In vitro, extensive passaging is a simile for "aging", and aged fibroblasts have shown ß3 Integrin upregulation. However, it is not known if astrocytes upregulate ß3 Integrin after successive cell passages. Here, we hypothesized that astrocytes undergoing long-term passaging increase ß3 Integrin expression levels and behave as reactive astrocytes without needing pro-inflammatory stimuli. We used DITNC1 cells with different passage numbers to study reactivity markers using immunoblots, immunofluorescence, and astrocyte adhesion/migration assays. We also evaluated ß3 Integrin levels by immunoblot and flow cytometry, as well as the neurotoxic effects of reactive astrocytes. Serial cell passaging mimicked the effects of inflammatory stimuli, inducing astrocyte reactivity. Indeed, in response to Thy-1, ß3 Integrin levels, as well as cell adhesion and migration, gradually increased with multiple passages. Importantly, these long-lived astrocytes expressed and secreted factors that inhibited neurite outgrowth and caused neuronal death, just like reactive astrocytes in culture. Therefore, we describe two DITNC1 cell types: a non-reactive type that can be activated with Tumor Necrosis Factor (TNF) and another one that exhibits reactive astrocyte features even in the absence of TNF treatment. Our results emphasize the importance of passage numbers in cell behavior. Likewise, we compare the pro-inflammatory stimulus versus long-term in-plate passaging of cell cultures and introduce them as astrocyte models to study the reactivity process.


Subject(s)
Astrocytes , Cell Adhesion , Cell Movement , Gliosis , Astrocytes/metabolism , Gliosis/metabolism , Gliosis/pathology , Animals , Thy-1 Antigens/metabolism , Integrin alphaVbeta3/metabolism , Inflammation/metabolism , Inflammation/pathology , Syndecan-4/metabolism , Syndecan-4/genetics , Mice , Cell Line , Humans , Cells, Cultured , Signal Transduction
10.
Int J Mol Sci ; 25(18)2024 Sep 11.
Article in English | MEDLINE | ID: mdl-39337305

ABSTRACT

Graphene nanoplatelets (UGZ-1004) are emerging as a promising biomaterial in regenerative medicine. This study comprehensively evaluates UGZ-1004, focusing on its physical properties, cytotoxicity, intracellular interactions, and, notably, its effects on mesenchymal stem cells (MSCs). UGZ-1004 was characterized by lateral dimensions and layer counts consistent with ISO standards and demonstrated a high carbon purity of 0.08%. Cytotoxicity assessments revealed that UGZ-1004 is non-toxic to various cell lines, including 3T3 fibroblasts, VERO kidney epithelial cells, BV-2 microglia, and MSCs, in accordance with ISO 10993-5:2020/2023 guidelines. The study focused on MSCs and revealed that UGZ-1004 supports their gene expression alterations related to self-renewal and proliferation. MSCs exposed to UGZ-1004 maintained their characteristic surface markers. Importantly, UGZ-1004 promoted significant upregulation of genes crucial for cell cycle regulation and DNA repair, such as CDK1, CDK2, and MDM2. This gene expression profile suggests that UGZ-1004 can enhance MSC self-renewal capabilities, ensuring robust cellular function and longevity. Moreover, UGZ-1004 exposure led to the downregulation of genes associated with tumor development, including CCND1 and TFDP1, mitigating potential tumorigenic risks. These findings underscore the potential of UGZ-1004 to not only bolster MSC proliferation but also enhance their self-renewal processes, which are critical for effective regenerative therapies. The study highlights the need for continued research into the long-term impacts of graphene nanoplatelets and their application in MSC-based regenerative medicine.


Subject(s)
Cell Proliferation , Graphite , Mesenchymal Stem Cells , Cell Proliferation/drug effects , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Animals , Graphite/chemistry , Graphite/pharmacology , Mice , Chlorocebus aethiops , Cell Self Renewal/drug effects , Cell Self Renewal/genetics , Vero Cells , Gene Expression Regulation/drug effects , Nanoparticles/chemistry , Cell Line , Nanostructures/chemistry
11.
Molecules ; 29(18)2024 Sep 23.
Article in English | MEDLINE | ID: mdl-39339496

ABSTRACT

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease in humans. The current antichagasic drugs nifurtimox and benznidazole have inconveniences of toxicity; therefore, the search for alternative therapeutic strategies is necessary. The present study reports the synthesis, drug-likeness predictions, and in vitro anti-trypanosome activity of a series of 14 quinazoline 2,4,6-triamine derivatives. All compounds were tested against T. cruzi (epimastigotes and trypomastigotes) and in HFF1 human foreskin fibroblasts. The bioassays showed that compounds 2-4 containing nitrobenzoyl substituents at 6-position of the quinazoline 2,4,6-triamine nucleus were the most potent on its antiprotozoal activity. The effect was observed at 24 h and it was preserved for at least 5 days. Also, compounds 2-4 were not toxic to the human control cells, showing high selectivity index. The quinazoline nitro derivatives have potential use as antichagasic agents.


Subject(s)
Quinazolines , Trypanocidal Agents , Trypanosoma cruzi , Quinazolines/pharmacology , Quinazolines/chemistry , Quinazolines/chemical synthesis , Humans , Trypanosoma cruzi/drug effects , Trypanocidal Agents/pharmacology , Trypanocidal Agents/chemical synthesis , Trypanocidal Agents/chemistry , Structure-Activity Relationship , Fibroblasts/drug effects , Chagas Disease/drug therapy , Molecular Structure , Cell Line
12.
Cells ; 13(17)2024 Sep 05.
Article in English | MEDLINE | ID: mdl-39273061

ABSTRACT

Zika virus (ZIKV) is an arbovirus with maternal, sexual, and TORCH-related transmission capabilities. After 2015, Brazil had the highest number of ZIVK-infected pregnant women who lost their babies or delivered them with Congenital ZIKV Syndrome (CZS). ZIKV triggers an immune defense in the placenta. This immune response counts with the participation of interleukins and transcription factors. Additionally, it has the potential involvement of human endogenous retroviruses (HERVS). Interleukins are immune response regulators that aid immune tolerance and support syncytial structure development in the placenta, where syncytin receptors facilitate vital cell-to-cell fusion events. HERVs are remnants of ancient viral infections that integrate into the genome and produce syncytin proteins crucial for placental development. Since ZIKV can infect trophoblast cells, we analyzed the relationship between ZIKV infection, HERV, interleukin, and transcription factor modulations in the placenta. To investigate the impact of ZIKV on trophoblast cells, we examined two cell types (BeWo and HTR8) infected with ZIKV-MR766 (African) and ZIKV-IEC-Paraíba (Asian-Brazilian) using Taqman and RT2 Profiler PCR Array assays. Our results indicate that early ZIKV infection (24-72 h) does not induce differential interleukins, transcription factors, and HERV expression. However, we show that the expression of a few of these host defense genes appears to be linked independently of ZIKV infection. Future studies involving additional trophoblastic cell lineages and extended infection timelines will illuminate the dynamic interplay between ZIKV, HERVs, interleukins, and transcription factors in the placenta.


Subject(s)
Endogenous Retroviruses , Interleukins , Transcription Factors , Trophoblasts , Zika Virus Infection , Zika Virus , Humans , Trophoblasts/virology , Trophoblasts/metabolism , Female , Zika Virus Infection/virology , Zika Virus Infection/genetics , Endogenous Retroviruses/genetics , Pregnancy , Interleukins/genetics , Interleukins/metabolism , Transcription Factors/metabolism , Transcription Factors/genetics , Placenta/virology , Placenta/metabolism , Cell Line
13.
Chem Biol Interact ; 403: 111216, 2024 Nov 01.
Article in English | MEDLINE | ID: mdl-39218371

ABSTRACT

Leishmaniasis is a neglected tropical disease caused by parasites of the genus Leishmania and is responsible for more than 1 million new cases and 70,000 deaths annually worldwide. Treatment has high costs, toxicity, complex and long administration time, several adverse effects, and drug-resistant strains, therefore new therapies are urgently needed. Synthetic compounds have been highlighted in the medicinal chemistry field as a strong option for drug development against different diseases. Organic salts (OS) have multiple biological activities, including activity against protozoa such as Leishmania spp. This study aimed to investigate the in vitro leishmanicidal activity and death mechanisms of a thiohydantoin salt derived from l-arginine (ThS) against Leishmania amazonensis. We observed that ThS treatment inhibited promastigote proliferation, increased ROS production, phosphatidylserine exposure and plasma membrane permeabilization, loss of mitochondrial membrane potential, lipid body accumulation, autophagic vacuole formation, cell cycle alteration, and morphological and ultrastructural changes, showing parasites death. Additionally, ThS presents low cytotoxicity in murine macrophages (J774A.1), human monocytes (THP-1), and sheep erythrocytes. ThS in vitro cell treatment reduced the percentage of infected macrophages and the number of amastigotes per macrophage by increasing ROS production and reducing TNF-α levels. These results highlight the potential of ThS among thiohydantoins, mainly related to the arginine portion, as a leishmanicidal drug for future drug strategies for leishmaniasis treatment. Notably, in silico investigation of key targets from L. amazonensis, revealed that a ThS compound from the l-arginine amino acid strongly interacts with arginase (ARG) and TNF-α converting enzyme (TACE), suggesting its potential as a Leishmania inhibitor.


Subject(s)
Arginine , Leishmania , Macrophages , Molecular Docking Simulation , Reactive Oxygen Species , Animals , Arginine/pharmacology , Arginine/chemistry , Arginine/metabolism , Mice , Humans , Leishmania/drug effects , Reactive Oxygen Species/metabolism , Macrophages/drug effects , Macrophages/metabolism , Macrophages/parasitology , Membrane Potential, Mitochondrial/drug effects , Sheep , Antiprotozoal Agents/pharmacology , Antiprotozoal Agents/chemistry , Erythrocytes/drug effects , Erythrocytes/parasitology , Erythrocytes/metabolism , Cell Line , Leishmania mexicana/drug effects , Leishmania mexicana/metabolism , THP-1 Cells , Tumor Necrosis Factor-alpha/metabolism
14.
Methods Mol Biol ; 2851: 213-226, 2024.
Article in English | MEDLINE | ID: mdl-39210185

ABSTRACT

Microorganisms with the ability to modulate the immune system (immunobiotics) have shown to interact with different pattern recognition receptors (PRRs) expressed in nonimmune and immune cells and exert beneficial effects on host's health maintenance and promotion. Suitable assay systems are necessary for an efficient and rapid screening of potential immunobiotic strains. More than a decade of research has allowed us to develop efficient in vitro models based on porcine receptors and cells (porcine immunoassay systems) to study the immunomodulatory effects of lactic acid bacteria (LAB). In addition, detailed studies of model immunobiotic LAB strains with proved abilities to improve immune health in humans (Lactobacillus rhamnosus CRL1505) or pigs (Lactobacillus jensenii TL2937) allowed us to select the most suitable biomarkers that have to be evaluated in those porcine immunoassay systems. Our in vitro models, utilizing transfectant cells expressing PRRs along with an established porcine intestinal epitheliocyte (PIE) cell line, have proven to be valuable tools for immunobiotic selection and for gaining insights into the molecular mechanisms responsible for their beneficial effects.


Subject(s)
Lactobacillales , Animals , Swine , Immunoassay/methods , Lactobacillales/immunology , Probiotics , Cell Line , Humans , Receptors, Pattern Recognition/metabolism , Receptors, Pattern Recognition/immunology , Lactobacillus/immunology
15.
Fitoterapia ; 178: 106183, 2024 Oct.
Article in English | MEDLINE | ID: mdl-39142528

ABSTRACT

Two new heptapeptides, [1-7-NαC]-crocaorbs A1 (1) and A2 (2), were isolated from the latex of Croton campanulatus. Their structures were determined using NMR spectroscopic techniques, ESI-HRMS data, Marfey's method, and further refined using molecular dynamics with simulated annealing (MD/SA). Molecular dynamics calculations of peptides 1 and 2 demonstrated greater stability in simulations using a biological solvent compared to those using DMSO. Compound 1, the most abundant peptide in latex, was assessed for NO production, antiplasmodial and cytotoxicity activities. The peptide significantly increased nitric oxide (NO) production at concentrations of 40, 20 or 10 µM (17.932 ± 1.1, 18.270 ± 0.9, 18.499 ± 0.7, respectively). Its antiplasmodial activity exhibited limited efficacy, with only 5% inhibition of Plasmodium falciparum 3D7 growth at a concentration of 50 µM. Also, it exhibited no cytotoxic effects in the J774A.1 murine macrophages cell line. This study represents the first report of a phytochemical investigation of the species C. campanulatus, which showed orbitides with distinctive sequences in contrast to other peptides described for the genus Croton and contributes to the study of structural diversity within this particular class of compounds.


Subject(s)
Antimalarials , Croton , Latex , Nitric Oxide , Plasmodium falciparum , Croton/chemistry , Animals , Mice , Latex/chemistry , Plasmodium falciparum/drug effects , Molecular Structure , Antimalarials/pharmacology , Antimalarials/isolation & purification , Antimalarials/chemistry , Nitric Oxide/metabolism , Cell Line , Phytochemicals/pharmacology , Phytochemicals/isolation & purification , Macrophages/drug effects
16.
Inflammopharmacology ; 32(5): 3327-3345, 2024 Oct.
Article in English | MEDLINE | ID: mdl-39133352

ABSTRACT

Wounds or chronic injuries are associated with high medical costs so, develop healing-oriented drugs is a challenge for modern medicine. The identification of new therapeutic alternatives focuses on the use of natural products. Therefore, the main goal of this study was to evaluate the healing potential and anti-inflammatory mechanism of action of extracts and the main compounds derived from Myrciaria plinioides D. Legrand leaves. The antimicrobial activity of leaf extracts was analyzed. Cell viability, cytotoxicity and genotoxicity of plant extracts and compounds were also assessed. Release of pro- and anti-inflammatory cytokines and TGF-ß by ELISA, and protein expression was determined by Western Blot. The cell migration and cell proliferation of ethanol and aqueous leaf extracts and p-coumaric acid, quercetin and caffeic acid compounds were also evaluated. The aqueous extract exhibited antibacterial activity and, after determining the safety concentrations in three assays, we showed that this extract induced p38-α MAPK phosphorylation and the same extract and the p-coumaric acid decreased COX-2 and caspase-3, -8 expression, as well as reduced the TNF-α release and stimulated the IL-10 in RAW 264.7 cells. In L929 cells, the extract and p-coumaric acid induced TGF-ß release, besides increasing the process of cell migration and proliferation. These results suggested that the healing properties of Myrciaria plinioides aqueous extract can be associated to the presence of phenolic compounds, especially p-coumaric acid, and/or glycosylated metabolites.


Subject(s)
Anti-Inflammatory Agents , Cell Movement , Plant Extracts , Plant Leaves , Wound Healing , Plant Extracts/pharmacology , Plant Extracts/isolation & purification , Plant Leaves/chemistry , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/isolation & purification , Wound Healing/drug effects , Mice , RAW 264.7 Cells , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Line , Cytokines/metabolism , Myrtaceae/chemistry , Coumaric Acids/pharmacology , Coumaric Acids/isolation & purification , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/isolation & purification
17.
Pharmacol Rep ; 76(5): 1147-1159, 2024 Oct.
Article in English | MEDLINE | ID: mdl-39150661

ABSTRACT

BACKGROUND: Currently, there is no antiviral licensed to treat chikungunya fever, a disease caused by the infection with Alphavirus chikungunya (CHIKV). Treatment is based on analgesic and anti-inflammatory drugs to relieve symptoms. Our study aimed to evaluate the antiviral activity of sulfadoxine (SFX), an FDA-approved drug, and its derivatives complexed with silver(I) (AgSFX), salicylaldehyde Schiff base (SFX-SL), and with both Ag and SL (AgSFX-SL) against CHIKV. METHODS: The anti-CHIKV activity of SFX and its derivatives was investigated using BHK-21 cells infected with CHIKV-nanoluc, a marker virus-carrying nanoluciferase reporter. Dose-response and time of drug-addition assays were performed in order to assess the antiviral effects of the compounds, as well as in silico data and ATR-FTIR analysis for insights on their mechanisms of action. RESULTS: The SFX inhibited 34% of CHIKV replication, while AgSFX, SFX-SL, and AgSFX-SL enhanced anti-CHIKV activity to 84%, 89%, and 95%, respectively. AgSFX, SFX-SL, and AgSFX-SL significantly decreased viral entry and post-entry to host cells, and the latter also protected cells against infection. Additionally, molecular docking calculations and ATR-FTIR analysis demonstrated interactions of SFX-SL, AgSFX, and AgSFX-SL with CHIKV. CONCLUSIONS: Collectively, our findings suggest that the addition of metal ions and/or Schiff base to SFX improved its antiviral activity against CHIKV.


Subject(s)
Antiviral Agents , Chikungunya Fever , Chikungunya virus , Sulfadoxine , Chikungunya virus/drug effects , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Animals , Cell Line , Sulfadoxine/pharmacology , Chikungunya Fever/drug therapy , Chikungunya Fever/virology , Cricetinae , Schiff Bases/pharmacology , Silver/pharmacology , Silver/chemistry , Virus Replication/drug effects , Molecular Docking Simulation , Dose-Response Relationship, Drug , Humans , Aldehydes
18.
Front Immunol ; 15: 1440662, 2024.
Article in English | MEDLINE | ID: mdl-39136016

ABSTRACT

Background: Cardiac arrhythmias are the main cause of sudden death due to Chronic Chagasic Cardiomyopathy (CCC). Here we investigated alterations in connexin 43 (Cx43) expression and phosphorylation in cardiomyocytes as well as associations with cardiac arrhythmias in CCC. Methods: C57Bl/6 mice infected with Trypanosoma cruzi underwent cardiac evaluations at 6 and 12 months after infection via treadmill testing and EKG. Histopathology, cytokine gene expression, and distribution of total Cx43 and its phosphorylated forms Cx43S368 and Cx43S325/328/330 were investigated. Human heart samples obtained from subjects with CCC were submitted to immunofluorescence analysis. In vitro simulation of a pro-inflammatory microenvironment (IL-1ß, TNF, and IFN-γ) was performed in H9c2 cells and iPSC-derived cardiomyocytes to evaluate Cx43 distribution, action potential duration, and Lucifer Yellow dye transfer. Results: Mice chronically infected with T. cruzi exhibited impaired cardiac function associated with increased inflammation, fibrosis and upregulated IL-1ß, TNF, and IFN-γ gene expression. Confocal microscopy revealed altered total Cx43, Cx43S368 and Cx43S325/328/330 localization and phosphorylation patterns in CCC, with dispersed staining outside the intercalated disc areas, i.e., in lateral membranes and the cytoplasm. Reduced co-localization of total Cx43 and N-cadherin was observed in the intercalated discs of CCC mouse hearts compared to controls. Similar results were obtained in human CCC heart samples, which showed Cx43 distribution outside the intercalated discs. Stimulation of human iPSC-derived cardiomyocytes or H9c2 cells with IL-1ß, TNF, and IFN-γ induced alterations in Cx43 localization, reduced action potential duration and dye transfer between adjacent cells. Conclusion: Heart inflammation in CCC affects the distribution and phosphorylation pattern of Cx43, which may contribute to the generation of conduction disturbances in Chagas disease.


Subject(s)
Chagas Cardiomyopathy , Connexin 43 , Mice, Inbred C57BL , Myocytes, Cardiac , Connexin 43/metabolism , Connexin 43/genetics , Animals , Chagas Cardiomyopathy/metabolism , Chagas Cardiomyopathy/pathology , Chagas Cardiomyopathy/immunology , Chagas Cardiomyopathy/parasitology , Humans , Mice , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/parasitology , Myocytes, Cardiac/pathology , Inflammation/metabolism , Phosphorylation , Male , Chronic Disease , Trypanosoma cruzi , Disease Models, Animal , Cell Line , Cytokines/metabolism , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/parasitology , Arrhythmias, Cardiac/immunology , Female
19.
J Biophotonics ; 17(10): e202400124, 2024 Oct.
Article in English | MEDLINE | ID: mdl-39134306

ABSTRACT

The objective of the study was to evaluate the effect of photobiomodulation (PBM) with laser on the inflammatory process in an experimental in vitro model of ACO. The groups were: (1) human bronchial epithelial cells (BEAS-2B); (2) BEAS-2B cells treated with dexamethasone; (3) BEAS-2B cells irradiated with laser; (4) BEAS-2B cells stimulated with cigarette smoke extract (CSE) + House Dust Mite (HDM); (5) BEAS-2B cells stimulated with CSE + HDM and treated with dexamethasone; (6) BEAS-2B cells incubated with CSE + HDM and irradiated with laser. After 24 h, cytokines were quantified. There was a reduction in TNF-α, IL-1ß, IL-6, IL-4, IL-5, IL-13, IL-17, IL-21, IL-23, and an increase in IL-10 and IFN-γ in cells from the laser-irradiated ACO group compared to only ACO group. With these results, we can suggest that photobiomodulation acts in the modulation of inflammation observed in ACO, and may be a treatment option.


Subject(s)
Asthma , Cytokines , Low-Level Light Therapy , Pulmonary Disease, Chronic Obstructive , Humans , Asthma/radiotherapy , Pulmonary Disease, Chronic Obstructive/radiotherapy , Cytokines/metabolism , Animals , Cell Line , Models, Biological , Pyroglyphidae/immunology , Epithelial Cells/radiation effects , Dexamethasone/pharmacology , Smoke/adverse effects
20.
Int J Mol Sci ; 25(16)2024 Aug 07.
Article in English | MEDLINE | ID: mdl-39201292

ABSTRACT

MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression post-transcriptionally and are crucial in lipid metabolism. ATP-binding cassette transporter A1 (ABCA1) is essential for cholesterol efflux from cells to high-density lipoprotein (HDL). Dysregulation of miRs targeting ABCA1 can affect cholesterol homeostasis and contribute to coronary artery disease (CAD). This study aimed to investigate the expression of miRs targeting ABCA1 in human monocytes, their role in cholesterol efflux, and their relationship with CAD. We included 50 control and 50 CAD patients. RT-qPCR examined the expression of miR-33a-5p, miR-26a-5p, and miR-144-3p in monocytes. Logistic regression analysis explored the association between these miRs and CAD. HDL's cholesterol acceptance was analyzed using the J774A.1 cell line. Results showed that miR-26a-5p (p = 0.027) and ABCA1 (p = 0.003) expression levels were higher in CAD patients, while miR-33a-5p (p < 0.001) levels were lower. Downregulation of miR-33a-5p and upregulation of ABCA1 were linked to a lower CAD risk. Atorvastatin upregulated ABCA1 mRNA, and metformin downregulated miR-26a-5p in CAD patients. Decreased cholesterol efflux correlated with higher CAD risk and inversely with miRs in controls. Reduced miR-33a-5p expression and increased ABCA1 expression are associated with decreased CAD risk. miR deregulation in monocytes may influence atherosclerotic plaque formation by regulating cholesterol efflux. Atorvastatin and metformin could offer protective effects by modulating miR-33a-5p, miR-26a-5p, and ABCA1, suggesting potential therapeutic strategies for CAD prognosis and treatment.


Subject(s)
ATP Binding Cassette Transporter 1 , Coronary Artery Disease , MicroRNAs , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter 1/metabolism , Coronary Artery Disease/genetics , Coronary Artery Disease/metabolism , Coronary Artery Disease/blood , Male , Female , Middle Aged , Leukocytes, Mononuclear/metabolism , Gene Expression Regulation , Aged , Cell Line , Cholesterol/metabolism , Cholesterol/blood , Monocytes/metabolism
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