Deoxynivalenol (DON), a trichothecene mycotoxin, exerts pro-inflammatory and immunomodulatory activity. Interleukin (IL)-1β serves a crucial part as a gate keeper of inflammation in DON-induced macrophages, but an overview of how DON exposure elicits IL-1β secretion from RAW264.7 cells has not been fully illustrated. Here we found that the cellular phenomenon, involved with a type of programmed cell death known as pyroptosis, contains: 1) increase of pro-IL-1β expression, 2) motivation of caspase-1, 3) caspase-1-dependent maturement of IL-1β, 4) caspase-1 fragmentation of gasdermin D (GSDMD), and 5) IL-1β secretion through GSDMD pore. Mechanistically, the present study certified that DON both as first and second signals engaged in IL-1β release is mediated by purinergic P2X7 receptor (P2X7R)-Src signaling. During this process, P2X7R signal is required for GSDMD pore forming course in ASC-independent manner. Moreover, blocking of K+ efflux, ROS formation, as well as cathepsin B activity decreases IL-1β export. Our data show that exposure to DON does cause pyroptosis and IL-1β secretion via P2X7R signal in RAW264.7 macrophages. Overall, these results provide new mechanistic clue for DON as a pro-inflammatory factor in innate immune signaling events.
- Klíčová slova
- Deoxynivalenol, Gasdermin D, IL-1β secretion, P2X7R, Pyroptosis,
- MeSH
- gasderminy MeSH
- interleukin-1beta * metabolismus MeSH
- kaspasa 1 metabolismus MeSH
- makrofágy * účinky léků metabolismus MeSH
- myši MeSH
- proteiny vázající fosfáty metabolismus MeSH
- purinergní receptory P2X7 * metabolismus MeSH
- pyroptóza * účinky léků MeSH
- RAW 264.7 buňky MeSH
- reaktivní formy kyslíku metabolismus MeSH
- signální transdukce účinky léků MeSH
- trichotheceny * toxicita MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- deoxynivalenol MeSH Prohlížeč
- gasderminy MeSH
- Gsdmd protein, mouse MeSH Prohlížeč
- IL1B protein, mouse MeSH Prohlížeč
- interleukin-1beta * MeSH
- kaspasa 1 MeSH
- proteiny vázající fosfáty MeSH
- purinergní receptory P2X7 * MeSH
- reaktivní formy kyslíku MeSH
- trichotheceny * MeSH
Accumulating evidence suggests that manganese oxide nanoparticles (NPs) show multiple enzyme-mimicking antioxidant activities, which supports their potential in redox-targeting therapeutic strategies for diseases with impaired redox signaling. However, the systemic administration of any NP requires thorough hemocompatibility testing. In this study, we assessed the hemocompatibility of synthesized Mn3O4 NPs, identifying their ability to induce spontaneous hemolysis and eryptosis or impair osmotic fragility. Concentrations of up to 20 mg/L were found to be safe for erythrocytes. Eryptosis assays were shown to be more sensitive than hemolysis and osmotic fragility as markers of hemocompatibility for Mn3O4 NP testing. Flow cytometry- and confocal microscopy-based studies revealed that eryptosis induced by Mn3O4 NPs was accompanied by Ca2+ overload, altered redox homeostasis verified by enhanced intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), and a decrease in the lipid order of cell membranes. Furthermore, Mn3O4 NP-induced eryptosis was calpain- and caspase-dependent.
- Klíčová slova
- calcium signaling, cytotoxicity, eryptosis, nanoparticles, oxidative stress, regulated cell death,
- MeSH
- buněčná membrána * metabolismus účinky léků MeSH
- eryptóza * účinky léků MeSH
- erytrocyty účinky léků metabolismus MeSH
- hemolýza účinky léků MeSH
- kalpain * metabolismus MeSH
- kaspasy * metabolismus MeSH
- lidé MeSH
- nanočástice * chemie MeSH
- oxidy * farmakologie chemie MeSH
- reaktivní formy dusíku * metabolismus MeSH
- reaktivní formy kyslíku * metabolismus MeSH
- sloučeniny manganu * farmakologie chemie MeSH
- vápník * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kalpain * MeSH
- kaspasy * MeSH
- manganese oxide MeSH Prohlížeč
- oxidy * MeSH
- reaktivní formy dusíku * MeSH
- reaktivní formy kyslíku * MeSH
- sloučeniny manganu * MeSH
- vápník * MeSH
Cannabidiol (CBD) is one of the principal constituents of Cannabis Sativa with no psychoactive properties. CBD is a promising neuroprotective compound bearing anti-inflammatory and antioxidant properties. However, considering its low solubility, CBD delivery to the retina represents an unresolved issue. The first aim was to investigate the potential neuroprotective effects of CBD in an in vivo model of retinal excitotoxicity induced by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Rats underwent intravitreal co-injection of AMPA (42 nmol) and CBD (10-4 M). The neuroprotective effect of CBD was investigated with histology and immunohistochemical evaluation of inflammatory and oxidative stress biomarkers. CBD reversed the AMPA-induced total retinal, inner nuclear layer and inner plexiform layer shrinkage and loss of amacrine cells. Moreover, CBD decreased the AMPA induced number of cleaved caspase-3, Iba-1 and nitrotyrosine (NT) positive cells. Based on this evidence, we developed a nanotechnological formulation of CBD to overcome critical issues related to its eye delivery. Particularly, nanostructured lipid carriers (NLC) loaded with CBD were prepared, optimized and characterized. Due to the optimal physicochemical characteristics, CBD-NLC3 has been selected and the in vitro release profile has been investigated. Additionally, CBD-NLC3 was topically administered to rats, and retinal CBD levels were determined. CBD-NLC3 formulation, after a single topical administration, efficiently delivered CBD in the retina (Cmax = 98 ± 25.9 ng/mg; Tmax = 60 min), showing a high translational value. In conclusion, these findings showed a good PD/PK profile of CBD warranting further pre-clinical and clinical evaluation of the new formulation for the treatment of retinal degenerative diseases.
- Klíčová slova
- Amacrine cells, Cannabidiol, Excitotoxicity, Neuroprotection, Retina, Topical ocular formulation,
- MeSH
- kanabidiol * farmakokinetika farmakologie chemie aplikace a dávkování MeSH
- kaspasa 3 metabolismus MeSH
- krysa rodu Rattus MeSH
- kyselina alfa-amino-3-hydroxy-5-methyl-4-isoxazolpropionová toxicita MeSH
- modely nemocí na zvířatech MeSH
- neuroprotektivní látky * farmakokinetika farmakologie aplikace a dávkování MeSH
- nosiče léků chemie MeSH
- oxidační stres účinky léků MeSH
- potkani Wistar MeSH
- retina * účinky léků metabolismus patologie MeSH
- tyrosin analogy a deriváty metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 3-nitrotyrosine MeSH Prohlížeč
- kanabidiol * MeSH
- kaspasa 3 MeSH
- kyselina alfa-amino-3-hydroxy-5-methyl-4-isoxazolpropionová MeSH
- neuroprotektivní látky * MeSH
- nosiče léků MeSH
- tyrosin MeSH
AIM: This work explores the synthesis of new bi-heterocyclic hybrid compounds based on quinoline ring and investigates their potential as anticancer agents. MATERIALS & METHODS: The novel fused quinoline-thiazolo[3,2-a] benzimidazole-3(2 h)one hybrids were prepared by regioselective nucleophilic ring opening of the corresponding quinolinyl-oxiranes. In vitro cytotoxic activity was evaluated against human lung (A549) and gastric (AGS) cancer cell lines. RESULTS: Global results showed that all tested compounds have promising inhibitory properties. Compounds 17 and 18 bearing two methoxy groups on the quinoline ring have exhibited remarkable and interesting activities. The investigation of the cell death process showed that these compounds activated a caspase-dependent apoptosis pathway. Results were further supported by molecular docking studies. CONCLUSION: Both compounds exhibited good drug-like characteristics, which make them promising drug candidates.
- Klíčová slova
- Caspase-3, Quinoline, Thiazolobenzimidazole, cytotoxic activity, epoxide ring opening, hybrid compounds, molecular docking,
- MeSH
- apoptóza * účinky léků MeSH
- benzimidazoly * chemie farmakologie chemická syntéza MeSH
- chinoliny * chemie farmakologie MeSH
- kaspasy * metabolismus MeSH
- lidé MeSH
- molekulární struktura MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- protinádorové látky * farmakologie chemická syntéza chemie MeSH
- screeningové testy protinádorových léčiv MeSH
- simulace molekulového dockingu MeSH
- thiazoly * chemie farmakologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- benzimidazolone MeSH Prohlížeč
- benzimidazoly * MeSH
- chinoliny * MeSH
- kaspasy * MeSH
- protinádorové látky * MeSH
- quinoline MeSH Prohlížeč
- thiazoly * MeSH
Ovulation, fertilization, and embryo development are orchestrated and synchronized processes essential for the optimal health of offspring. Postovulatory aging disrupts this synchronization and impairs oocyte quality. In addition, oocyte aging causes fertilization loss and poor embryo development. This investigation aimed to unravel the endpoint of in vitro oocyte aging in common carp (Cyprinus carpio) to understand the involvement of apoptosis in postovulatory oocyte death. It was observed that the fertilization ability significantly declined (P < 0.001) at 8-h poststripping (HPS), subsequently triggering apoptosis in the advanced stage of oocyte aging, i.e., 48 HPS. This process included an increase in proapoptotic transcripts (fas, bax, cathepsin D, caspase 8, caspase 9, and caspase 3a) (P < 0.05), elevated levels of caspase 3 protein (P < 0.05), and activation of caspase 3 enzyme (P < 0.001), a key player in apoptosis, in aging oocytes. Furthermore, the effects of oocyte aging on the embryonic apoptosis machinery were examined in embryos at 5-h postfertilization (HPF) and 24 HPF derived from fresh and aged oocytes. Expression of apoptotic genes and caspase enzyme activity remained at the basal level in 5 HPF (early blastula embryos) from both fresh and aged oocytes. In contrast, the zymogenic and active forms of caspase 3 increased in 24 HPF embryos from 8-h-aged oocytes (P < 0.01) compared with those from fresh oocytes. Thus, apoptosis intensified in 24 HPF embryos from aged oocytes without affecting the apoptotic machinery of early blastula embryos. Our findings demonstrate that apoptosis initiated by the Fas/FasL system is an important physiological process accompanying oocyte aging in common carp.
The delay in fertilization after ovulation or retention of ovulated oocytes in the fish body causes postovulatory aging. Postovulatory aging leads to time-dependent deterioration of oocyte quality and loss of fertilization capacity. The mechanisms behind losing oocyte quality and developmental capacity due to postovulatory oocyte aging remain elusive. The emerging climate change issues in nature and unfavorable spawning conditions have caused the retention of ovulated oocytes in the female body. Analyzing the apoptotic parameters to understand the fate of these aged oocytes and the consequences of this aging on embryo development was the main objective of this study. The results obtained from this study indicate that aged oocytes die by apoptosis. The embryos from aged oocytes show more apoptosis, stating that oocyte aging affects embryo development by affecting the intensity of apoptosis.
- Klíčová slova
- apoptosis, caspases, early blastula embryos, fish, in-vitro-aged oocytes, spontaneous activation,
- MeSH
- apoptóza * fyziologie MeSH
- embryo nesavčí fyziologie MeSH
- embryonální vývoj * fyziologie MeSH
- kapři * embryologie fyziologie MeSH
- kaspasy metabolismus genetika MeSH
- oocyty * fyziologie MeSH
- stárnutí * fyziologie MeSH
- vývojová regulace genové exprese fyziologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kaspasy MeSH
Caspase-12 is a molecule whose functions are still not well understood. Although its expression has been found in various tissues, specific roles have been described in only a few cases. These include the effect of caspase-12 on murine bone cell differentiation during craniofacial development. This work focused on the development of the limbs taking place through endochondral ossification, which precedes the formation of the cartilaginous growth plate. Caspase-12 was described here for the first time in growth plate chondrocytes during physiological development. Using pharmacological inhibition, caspase-12 was found to affect chondrogenesis. Limb-derived micromass cultures showed a significantly increased area of chondrogenic nodules after caspase-12 inhibition and there were changes in gene expression, the most significant of which was the reduction of Mmp9. These data point to potential new functions of caspase-12 in chondrogenesis.
- Klíčová slova
- Caspase-12, Chondrocyte, Chondrogenesis, Differentiation, Growth plate,
- MeSH
- buněčná diferenciace MeSH
- chondrocyty * MeSH
- chondrogeneze * fyziologie MeSH
- inhibitory kaspas farmakologie MeSH
- kaspasa 12 * metabolismus genetika MeSH
- kultivované buňky MeSH
- matrixová metaloproteinasa 9 metabolismus genetika MeSH
- myši MeSH
- růstová ploténka růst a vývoj MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- inhibitory kaspas MeSH
- kaspasa 12 * MeSH
- matrixová metaloproteinasa 9 MeSH
Nanostructured materials have been suggested to be used as a source of dietary zinc for livestock animals. In this study, we assessed the cytotoxicity of newly synthesized nanostructured zinc carbonate hydroxide (ZnCH) Zn5(CO3)(OH)6microflakes. Cytotoxicity of the microflakes was assessed against murine L929 cell line and rat mature erythrocytes. Viability, motility, cell death pathways, implication of Ca2+, reactive oxygen species and reactive nitrogen species (RNS) signaling, caspases, and alterations of cell membranes following exposure of L929 cells to the microflakes were assessed. To assess hemocompatibility of the Zn-containing microflakes, osmotic fragility and hemolysis assays were performed, as well as multiple eryptosis parameters were evaluated. Our findings indicate a dose-response cytotoxicity of ZnCH microflakes against L929 cells with no toxicity observed for low concentrations (10 mg l-1and below). At high concentrations (25 mg l-1and above), ZnCH microflakes promoted nitrosyl stress, Ca2+- and caspase-dependent apoptosis, and altered lipid order of cell membranes in a dose-dependent manner, evidenced by up to 7-fold elevation of RNS-dependent fluorescence, 2.9-fold enhancement of Fura 2-dependent fluorescence, over 20-fold elevation of caspases-dependent fluorescence (caspase-3, caspase-8, and caspase-9), and up to 4.4-fold increase in the ratiometric index of the NR12S probe. Surprisingly, toxicity to enucleated mature erythrocytes was found to be lower compared to L929 cells. ZnCH microflakes induced eryptosis associated with oxidative stress, nitrosyl stress, Ca2+signaling and recruitment of caspases at 25-50-100 mg l-1. Eryptosis assays were found to be more sensitive than evaluation of hemolysis. Zn5(CO3)(OH)6microflakes show no cytotoxicity at low concentrations indicating their potential as a source of zinc for livestock animals.
- Klíčová slova
- cytotoxicity, eryptosis, erythrocytes, reactive oxygen species, regulated cell death,
- MeSH
- apoptóza účinky léků MeSH
- buněčné linie MeSH
- eryptóza účinky léků MeSH
- erytrocyty * účinky léků MeSH
- hemolýza účinky léků MeSH
- hydroxidy chemie toxicita MeSH
- kaspasy metabolismus MeSH
- krysa rodu Rattus MeSH
- myši MeSH
- nanostruktury * toxicita chemie MeSH
- reaktivní formy dusíku metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- sloučeniny zinku toxicita chemie MeSH
- uhličitany chemie MeSH
- viabilita buněk účinky léků MeSH
- zinek chemie toxicita MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- hydroxidy MeSH
- kaspasy MeSH
- reaktivní formy dusíku MeSH
- reaktivní formy kyslíku MeSH
- sloučeniny zinku MeSH
- uhličitany MeSH
- zinek MeSH
The CYCS gene is highly evolutionarily conserved, with only a few pathogenic variants that cause thrombocytopenia-4 (THC4). Here, we report a novel CYCS variant NM_018947.6: c.59C>T [NP_061820.1:p.(Thr20Ile)] segregating with thrombocytopenia in three generations of a Czech family. The phenotype of the patients corresponds to THC4 with platelets of normal size and morphology and dominant inheritance. Intriguingly, a gradual decline in platelet counts was observed across generations. CRISPR/Cas9-mediated gene editing was used to introduce the new CYCS gene variant into a megakaryoblast cell line (MEG-01). Subsequently, the adhesion, shape, size, ploidy, viability, mitochondrial respiration, cytochrome c protein (CYCS) expression, cell surface antigen expression and caspase activity were analysed in cells carrying the studied variant. Interestingly, the variant decreases the expression of CYCS while increasing mitochondrial respiration and the expression of CD9 cell surface antigen. Surprisingly, the variant abates caspase activation, contrasting with previously known effects of other CYCS variants. Some reports indicate that caspases may be involved in thrombopoiesis; thus, the observed dysregulation of caspase activity might contribute to thrombocytopenia. The findings significantly enhance our understanding of the molecular mechanisms underlying inherited thrombocytopenia and may have implications for diagnosis, prognosis and future targeted therapies.
- Klíčová slova
- CRISPR/Cas9, CYCS, caspase, cytochrome c, mitochondria, thrombocytopenia,
- MeSH
- kaspasy * metabolismus genetika MeSH
- lidé MeSH
- rodokmen MeSH
- trombocytopenie * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kaspasy * MeSH
Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.
- Klíčová slova
- ABHD2, Caspase 9, diaPASEF, migration, osteoblasts, proteomics,
- MeSH
- buněčné linie MeSH
- genový knockout MeSH
- kaspasa 9 * metabolismus genetika MeSH
- myši MeSH
- osteoblasty * metabolismus cytologie MeSH
- pohyb buněk * MeSH
- proteomika * metody MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- Casp9 protein, mouse MeSH Prohlížeč
- kaspasa 9 * MeSH
Caspase-11 is the murine homologue of human caspases-4 and -5 and is involved in mediating the inflammatory response. However, its functions are often confused and misinterpreted with the more important and better described caspase-1. Therefore, this study focused exclusively on the specific roles of caspase-11, both in cartilage formation and in the inflammatory environment. The presence of caspase-11 during mouse limb development and in chondrogenic cell cultures was investigated by immunofluorescence detection. Subsequently, the function of caspase-11 was downregulated and the affected molecules investigated. The expression analysis applied for osteo/chondrogenesis associated factors and inflammatory cytokines. Simultaneously, morphological appearance of the micromass cultures was evaluated. The results revealed that caspase-11 is physiologically present during cartilage development, but its inhibition under physiological conditions has no significant effect on chondrogenic differentiation. However, in an inflammatory environment, inhibition and downregulation of caspase-11 leads to reduced differentiation of cartilage nodules. Additionally, reduced expression of several genes including Col2a1 and Sp7 and conversely increased expression of Mmp9 were observed. In the cytokine expression panel, a significant decrease was found in molecules that, along with the inflammatory function, may also be involved in cartilage differentiation. The findings bring new information about caspase-11 in chondrogenesis and show that its downregulation under inflammatory conditions reduces cartilage formation.
- Klíčová slova
- Cartilage, Caspase-11, Chondrogenesis, Inflammation, Osteoarthritis,
- MeSH
- buněčná diferenciace * MeSH
- chondrocyty metabolismus cytologie MeSH
- chondrogeneze * MeSH
- chrupavka metabolismus MeSH
- cytokiny metabolismus MeSH
- iniciační kaspasy * metabolismus MeSH
- kaspasy metabolismus MeSH
- myši MeSH
- zánět * patologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- Casp4 protein, mouse MeSH Prohlížeč
- cytokiny MeSH
- iniciační kaspasy * MeSH
- kaspasy MeSH
