Ethyl pyruvate ameliorates acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome (ARDS) became a focus of intensive research due to its death toll during the Covid-19 pandemic. An uncontrolled and excessive inflammatory response mediated by proinflammatory molecules such as high mobility group box protein 1 (HMGB1), IL-6, and TNF mounts as a response to infection. In this study, ethyl pyruvate (EP), a known inhibitor of HMGB1, was tested in the model of murine ARDS induced in C57BL/6 mice by intranasal administration of polyinosinic:polycytidylic acid (poly(I:C)). Intraperitoneal administration of EP ameliorated the ARDS-related histopathological changes in the lungs of poly(I:C)-induced ARDS and decreased numbers of immune cells in the lungs, broncho-alveolar lavage fluid and draining lymph nodes (DLN). Specifically, fewer CD8+ T cells and less activated CD4+ T cells were observed in DLN. Consequently, the lungs of EP-treated animals had fewer damage-inflicting CD8+ cells and macrophages. Additionally, the expression and production of proinflammatory cytokines, IL-17, IFN-γ and IL-6 were downregulated in the lungs. The expression of chemokine CCL5 which recruits immune cells into the lungs was also reduced. Finally, EP downregulated the expression of HMGB1 in the lungs. Our results imply that EP should be further evaluated as a potential candidate for ARDS therapy.

Acetylsalicylic-acid (ASA) regulation of osteo/odontogenic differentiation and proliferation of human dental pulp stem cells (DPSCs) in vitro.

OBJECTIVE: The study aimed to investigate acetylsalicylic acid (ASA) effects on osteo/odontogenic differentiation and proliferation of dental pulp stem cells (DPSCs) in vitro and the potential involvement of adenosine monophosphate-activated protein kinase (AMPK) pathway in these processes. DESIGN: DPSCs were isolated from third molars pulp tissues of five patients and grown in osteogenic medium alone or supplemented with ASA. Expression of DPSCs markers was tested by flow-cytometry. Cytotoxicity of ASA at concentrations of 10, 50 and 100 µg/ml was tested by MTT and NR assays. Osteo/odontogenic differentiation was analyzed via alizarin red staining and ALP activity. Quantitative PCR (qPCR) was used for osteo/odontogenic markers' (DSPP, BMP2, BMP4, BSP, OCN and RUNX2) and c-Myc expression analysis. AMPK inhibition of ASA-induced osteo/odontogenesis was tested by qPCR of selected markers (DSPP, OCN and RUNX2). RESULTS: Cytotoxicity assays showed that only the highest ASA dose decreased cell viability (89.1 %). The smallest concentration of ASA applied on DPSCs resulted in a remarkable enhancement of osteo/odontogenic differentiation, as judged by increased mineralized nodules' formation, ALP activity and gene expression of analyzed markers (increase between 2 and 30 folds), compared to untreated cells. ASA also increased DPSCs proliferation. Interestingly, AMPK inhibition per se upregulated DSPP, OCN and RUNX2; the gene upregulation was higher when ASA treatment was also included. c-Myc expression level decreased in cultures treated with ASA, indicating undergoing differentiation processes. CONCLUSIONS: Low concentrations of ASA (corresponding to the standard use in cardiovascular patients), were shown to stimulate osteo/odontogenic differentiation of dental pulp stem cells.

Phenethyl ester of rosmarinic acid ameliorates experimental autoimmune encephalomyelitis.

Rosmarinic acid is a polyphenolic compound, abundantly present in herbs of the Lamiaceae family. The aim of the study was to evaluate the immunomodulatory properties of a recently developed phenethyl ester derivative of rosmarinic acid (PERA), with enhanced ability of diffusion through biological membranes, in an animal model of the central nervous system (CNS) autoimmunity. To this end, experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis was used. Daily subcutaneous administration of PERA (30 mg/kg) from day 7 to day 22 after immunization successfully ameliorated EAE induced in Dark Agouti rats, shortening the disease duration and reducing maximal, cumulative and mean clinical score. PERA efficiently reduced production of major encephalitogenic cytokines, interferon (IFN)-γ and interleukin (IL)-17, in immune cells from the CNS or the lymph nodes draining the site of immunization of EAE rats, as well as in CD4+ T cells purified from the lymph nodes. Also, PERA inhibited NO production in the CNS and the lymph nodes, as well as in macrophages and microglial cells. Finally, microglial ability to produce pro-inflammatory cytokines IL-6, and tumor necrosis factor (TNF) were also reduced by PERA. Our results clearly imply that PERA possesses anti-encephalitogenic properties. Thus, further studies on the relevance of the observed effects for the therapy of multiple sclerosis are warranted.

Short term exposure to ethyl pyruvate has long term anti-inflammatory effects on microglial cells.

Ethyl pyruvate (EP) has been increasingly appreciated as an anti-inflammatory and neuroprotective agent with potent pharmacological properties relevant for treatment of various CNS disorders. Microglial cells seem to be particularly sensitive to its effects. In this study, microglial cells were exposed to EP for relatively short periods (10-120min) and inflammatory properties of the cells were determined after 24h of cultivation. Application of EP in the short-term periods inhibited production of interleukin-6, tumor necrosis factor and nitric oxide in microglial cells. At the same time, the effects on cell viability, reactive oxygen species generation and expression of F4/80 and CD40 of microglial cells were minor. NFκB activation was not affected by EP in the cells during the short exposures, thus implying that the observed effect of EP on cytokine and nitric oxide generation was performed in NFκB independent way. Importantly, effects of the short term EP treatment on microglial cells were detected by a real time cell analysis, as well. The observed ability of EP to affect microglial cell function after relatively short time of exposure is relevant for its therapeutic potential against inflammatory disorders of the CNS.

Tumor necrosis factor stimulates expression of CXCL12 in astrocytes.

It has been increasingly appreciated that tumor necrosis factor (TNF) performs various protective and anti-inflammatory functions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Recently, CXCL12 has been identified as a key inhibitor of leukocyte entry into the central nervous system (CNS) and as a regulator of inflammation resulting from the invasion. Here, a positive correlation between expression of TNF and CXCL12 in the CNS samples of EAE rats is presented. Also, it is shown that TNF potentiates CXCL12 expression in astrocytes. These results contribute to a view that TNF produced within the CNS plays a protective role in neuroinflammation.

A comparative analysis of multiple sclerosis-relevant anti-inflammatory properties of ethyl pyruvate and dimethyl fumarate.

Dimethyl fumarate (DMF), a new drug for multiple sclerosis (MS) treatment, acts against neuroinflammation via mechanisms that are triggered by adduct formation with thiol redox switches. Ethyl pyruvate (EP), an off-the-shelf agent, appears to be a redox analog of DMF, but its immunomodulatory properties have not been put into the context of MS therapy. In this article, we examined and compared the effects of EP and DMF on MS-relevant activity/functions of T cells, macrophages, microglia, and astrocytes. EP efficiently suppressed the release of MS signature cytokines, IFN-γ and IL-17, from human PBMCs. Furthermore, the production of these cytokines was notably decreased in encephalitogenic T cells after in vivo application of EP to rats. Production of two other proinflammatory cytokines, IL-6 and TNF, and NO was suppressed by EP in macrophages and microglia. Reactive oxygen species production in macrophages, microglia activation, and the development of Ag-presenting phenotype in microglia and macrophages were constrained by EP. The release of IL-6 was reduced in astrocytes. Finally, EP inhibited the activation of transcription factor NF-κB in microglia and astrocytes. Most of these effects were also found for DMF, implying that EP and DMF share common targets and mechanisms of action. Importantly, EP had in vivo impact on experimental autoimmune encephalomyelitis, an animal model of MS. Treatment with EP resulted in delay and shortening of the first relapse, and lower clinical scores, whereas the second attack was annihilated. Further studies on the possibility to use EP as an MS therapeutic are warranted.

Saquinavir-NO inhibits IL-6 production in macrophages.

Covalent attachment of the nitric oxide (NO) moiety to the HIV protease inhibitor Saquinavir (Saq) produced a new chemical entity, named Saquinavir-NO, (Saq-NO) with reduced toxicity and potent immunoregulatory influence on T lymphocytes. In this study, we have compared head-to-head the effects of Saq-NO and Saq on mouse and rat peritoneal macrophage cytokine secretion and NO production upon in vitro, ex vivo and in vivo conditions. The results demonstrate that Saq-NO, but not Saq, potently decreased interleukin (IL)-10, IL-6 and nitrite accumulation and increased the levels of IL-1ß and tumour necrosis factor (TNF) in supernatants of mouse and rat macrophage cultures in vitro. Treatment of mice with Saq-NO, but not Saq, inhibited ex vivo secretion of IL-6 from macrophages. Consistent with these findings, Saq-NO also reduced blood levels of IL-6 in lipopolysaccharide-treated mice. The observed inhibitory influence of Saq-NO on IL-6 generation in macrophages may be involved in the observed antitumour and immunomodulatory effects of the drug.