Differential Mitochondrial, Oxidative Stress and Inflammatory Responses to SARS-CoV-2 Spike Protein Receptor Binding Domain in Human Lung Microvascular, Coronary Artery Endothelial and Bronchial Epithelial Cells.

Recent evidence indicates that the SARS-CoV-2 spike protein affects mitochondria with a cell type-dependent outcome. We elucidate the effect of the SARS-CoV-2 receptor binding domain (RBD) on the mitochondrial network and cristae morphology, oxygen consumption, mitoROS production, and inflammatory cytokine expression in cultured human lung microvascular (HLMVECs), coronary artery endothelial (HCAECs), and bronchial epithelial cells (HBECs). Live Mito Orange staining, STED microscopy, and Fiji MiNa analysis were used for mitochondrial cristae and network morphometry; an Agilent XFp analyser for mitochondrial/glycolytic activity; MitoSOX fluorescence for mitochondrial ROS; and qRT-PCR plus Luminex for cytokines. HLMVEC exposure to SARS-CoV-2 RBD resulted in the fragmentation of the mitochondrial network, mitochondrial swelling, increased cristae area, reduced cristae density, and suppressed mitochondrial oxygen consumption and glycolysis. No significant mitochondrial morphology or oxygen consumption changes were observed in HCAECs and HBECs. SARS-CoV-2 RBD induced mitoROS-mediated expression of cytokines GM-CSF and IL-1ß in all three investigated cell types, along with IL-8 expression in both endothelial cell types. The findings suggest mitochondrial ROS control SARS-CoV-2 RBD-induced inflammation in HLMVECs, HCAECs, and HBECs, with the mitochondria of HLMVECs being more sensitive to SARS-CoV-2 RBD.

The Effect of SARS-CoV-2 Spike Protein RBD-Epitope on Immunometabolic State and Functional Performance of Cultured Primary Cardiomyocytes Subjected to Hypoxia and Reoxygenation.

Cardio complications such as arrhythmias and myocardial damage are common in COVID-19 patients. SARS-CoV-2 interacts with the cardiovascular system primarily via the ACE2 receptor. Cardiomyocyte damage in SARS-CoV-2 infection may stem from inflammation, hypoxia-reoxygenation injury, and direct toxicity; however, the precise mechanisms are unclear. In this study, we simulated hypoxia-reoxygenation conditions commonly seen in SARS-CoV-2-infected patients and studied the impact of the SARS-CoV-2 spike protein RBD-epitope on primary rat cardiomyocytes to gain insight into the potential mechanisms underlying COVID-19-related cardiac complications. Cell metabolic activity was evaluated with PrestoBlueTM. Gene expression of proinflammatory markers was measured by qRT-PCR and their secretion was quantified by Luminex assay. Cardiomyocyte contractility was analysed using the Myocyter plugin of ImageJ. Mitochondrial respiration was determined through Seahorse Mito Stress Test. In hypoxia-reoxygenation conditions, treatment of the SARS-CoV-2 spike RBD-epitope reduced the metabolic activity of primary cardiomyocytes, upregulated Il1ß and Cxcl1 expression, and elevated GM-CSF and CCL2 cytokines secretion. Contraction time increased, while amplitude and beating frequency decreased. Acute treatment with a virus RBD-epitope inhibited mitochondrial respiration and lowered ATP production. Under ischaemia-reperfusion, the SARS-CoV-2 RBD-epitope induces cardiomyocyte injury linked to impaired mitochondrial activity.

The Role of Intracellular Ca2+ and Mitochondrial ROS in Small Aß1-42 Oligomer-Induced Microglial Death.

Alzheimer's disease (AD) is the most common form of dementia worldwide, and it contributes up to 70% of cases. AD pathology involves abnormal amyloid beta (Aß) accumulation, and the link between the Aß1-42 structure and toxicity is of major interest. NMDA receptors (NMDAR) are thought to be essential in Aß-affected neurons, but the role of this receptor in glial impairment is still unclear. In addition, there is insufficient knowledge about the role of Aß species regarding mitochondrial redox states in neurons and glial cells, which may be critical in developing Aß-caused neurotoxicity. In this study, we investigated whether different Aß1-42 species-small oligomers, large oligomers, insoluble fibrils, and monomers-were capable of producing neurotoxic effects via microglial NMDAR activation and changes in mitochondrial redox states in primary rat brain cell cultures. Small Aß1-42 oligomers induced a concentration- and time-dependent increase in intracellular Ca2+ and necrotic microglial death. These changes were partially prevented by the NMDAR inhibitors MK801, memantine, and D-2-amino-5-phosphopentanoic acid (DAP5). Neither microglial intracellular Ca2+ nor viability was significantly affected by larger Aß1-42 species or monomers. In addition, the small Aß1-42 oligomers caused mitochondrial reactive oxygen species (mtROS)-mediated mitochondrial depolarization, glutamate release, and neuronal cell death. In microglia, the Aß1-42-induced mtROS overproduction was mediated by intracellular calcium ions and Aß-binding alcohol dehydrogenase (ABAD). The data suggest that the pharmacological targeting of microglial NMDAR and mtROS may be a promising strategy for AD therapy.

Adipose Tissue-Derived Stem Cell Extracellular Vesicles Suppress Glioblastoma Proliferation, Invasiveness and Angiogenesis.

Extracellular vesicles (EVs) are attractive anticancer drug delivery candidates as they confer several fundamental properties, such as low immunogenicity and the ability to cross biological barriers. Mesenchymal stem cells (MSCs) are convenient producers for high EV yields, and patient-derived adipose tissue MSC-EVs could serve as personalised carriers. However, MSC-EV applications raise critical concerns as their natural cargo can affect tumour progression in both inducing and suppressing ways. In this study, we investigated the effect of adipose tissue-derived mesenchymal stem cell EVs (ASC-EVs) on several glioblastoma (GBM) cell lines to define their applicability for anticancer therapies. ASC-EVs were isolated from a cell-conditioned medium and characterised by size and specific markers. The internalisation of fluorescently labelled ASC-EVs by human GBM cells HROG36, U87 MG, and T98G was evaluated by fluorescent microscopy. Changes in GBM cell proliferation after ASC-EV application were determined by the metabolic PrestoBlue assay. Expression alterations in genes responsible for cell adhesion, proliferation, migration, and angiogenesis were evaluated by quantitative real-time PCR. ASC-EV effects on tumour invasiveness and neoangiogenesis in ovo were analysed on the chicken embryo chorioallantoic membrane model (CAM). ASC-EV treatment reduced GBM proliferation in vitro and significantly downregulated invasiveness-related genes ITGα5 (in T98G and HROG63) and ITGß3 (in HROG36) and the vascularisation-inducing gene KDR (in all GBM lines). Additionally, an approximate 65% reduction in the GBM invasion rate was observed in CAM after ASC-EV treatment. Our study indicates that ASC-EVs possess antitumour properties, reducing GBM cell proliferation and invasiveness, and can be applied as anticancer therapeutics and medicine carriers.

Species-Specific Plant-Derived Nanoparticle Characteristics.

Medicinal and agricultural plants contain numerous phytochemical compounds with pronounced biological effects on human health. They are known to encapsulate most of their characteristic bioactive compounds within membranous elements of intercellular communication known as exosomes. These nanovesicles serve as capsules protecting their biological activity and improving their penetration into the tissue. Therefore, the application of plant exosome preparations holds considerable potential for cosmetics and pharmacy, but the quality and consistency of plant material for exosome isolation is of critical importance. Therefore, in this study, we aimed to evaluate yield, size distribution patterns, and antioxidant properties between nanovesicle preparations of the following portfolio of medicinal plants: Kalanchoe daigremontiana, Artemisia absinthium, Hypericum perforatum, Silybum marianum, Chelidonium majus, and Scutellaria baicalensis. Results showed that nanoparticle yield, size distribution, and antioxidant activities were specific to plant species. Compared to other plants, nanoparticle preparations from Artemisia absinthium were distinguished by remarkably higher yield and concentration, while the highest antioxidant activity of plant-derived nanoparticle preparations per weight and per particle was determined to occur in Chelidonium majus and Hypericum perforatum samples. Results showed no significant correlation in DPPH (2-diphenyl-1-picrylhydrazyl) free radical scavenging activity and FRAP (ferric reducing antioxidant power) between plant material and nanoparticle preparations. More detailed biochemical analysis of exosome preparations is necessary to validate their biological activity and its relation to source plant cells.

Internalisation of RGD-Engineered Extracellular Vesicles by Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most aggressive CNS tumour with no efficient treatment, partly due to the retention of anticancer drugs by the blood-brain barrier (BBB) and their insufficient concentration in tumour cells. Extracellular vesicles (EVs) are attractive drug carriers because of their biocompatibility and ability to cross the BBB. Additional efficiency can be achieved by adding GBM-cell-specific ligands. GBM cells overexpress integrins; thus, one of the most straightforward targeting strategies is to modify EVs with integrin-recognising molecules. This study investigated the therapeutic potential of genetically engineered EVs with elevated membrane levels of the integrin-binding peptide RGD (RGD-EVs) against GBM cells in vitro. For RGD-EV production, stable RGD-HEK 293FT cells were generated by using a pcDNA4/TO-Lamp2b-iRGD-HA expression vector and performing antibiotic-based selection. RGD-EVs were isolated from RGD-HEK 293FT-cell-conditioned medium and characterised by size (Zetasizer), specific markers (ELISA) and RGD expression (Western Blot). Internalisation by human GBM cells HROG36 and U87 MG and BJ-5ta human fibroblasts was assessed by fluorescent EV RNA labelling. The effect of doxorubicin-loaded RGD-EVs on GBM cells was evaluated by the metabolic PrestoBlue viability assay; functional GAPDH gene knockdown by RGD-EV-encapsulated siRNA was determined by RT-qPCR. RGD-EVs had 40% higher accumulation in GBM cells (but not in fibroblasts) and induced significantly stronger toxicity by loaded doxorubicin and GAPDH silencing by loaded siRNA compared to unmodified EVs. Thus, RGD modification substantially increases the specific delivery capacity of HEK 293FT-derived EVs to GBM cells.

Comparison of Microglial Morphology and Function in Primary Cerebellar Cell Cultures on Collagen and Collagen-Mimetic Hydrogels.

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca2+ signalling (intracellular Ca2+ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.

Virus Mimetic Poly (I:C)-Primed Airway Exosome-like Particles Enter Brain and Induce Inflammatory Cytokines and Mitochondrial Reactive Oxygen Species in Microglia.

Viral infections induce extracellular vesicles (EVs) containing viral material and inflammatory factors. Exosomes can easily cross the blood-brain barrier during respiratory tract infection and transmit the inflammatory signal to the brain; however, such a hypothesis has no experimental evidence. The study investigated whether exosome-like vesicles (ELVs) from virus mimetic poly (I:C)-primed airway cells enter the brain and interact with brain immune cells microglia. Airway cells were isolated from Wistar rats and BALB/c mice; microglial cell cultures-from Wistar rats. ELVs from poly (I:C)-stimulated airway cell culture medium were isolated by precipitation, visualised by transmission electron microscopy, and evaluated by nanoparticle analyser; exosomal markers CD81 and CD9 were determined by ELISA. For in vitro and in vivo tracking, particles were loaded with Alexa Fluor 555-labelled RNA. Intracellular reactive oxygen species (ROS) were evaluated by DCFDA fluorescence and mitochondrial superoxide-by MitoSOX. ELVs from poly (I:C)-primed airway cells entered the brain within an hour after intranasal introduction, were internalised by microglia and induced intracellular and intramitochondrial ROS production. There was no ROS increase in microglial cells was after treatment with ELVs from airway cells untreated with poly (I:C). In addition, poly (I:C)-primed airway cells induced inflammatory cytokine expression in the brain. The data indicate that ELVs secreted by virus-primed airway cells might enter the brain, cause the activation of microglial cells and neuroinflammation.

Extracellular Vesicles in Skin Wound Healing.

Each year, millions of individuals suffer from a non-healing wound, abnormal scarring, or injuries accompanied by an infection. For these cases, scientists are searching for new therapeutic interventions, from which one of the most promising is the use of extracellular vesicles (EVs). Naturally, EV-based signaling takes part in all four wound healing phases: hemostasis, inflammation, proliferation, and remodeling. Such an extensive involvement of EVs suggests exploiting their action to modulate the impaired healing phase. Furthermore, next to their natural wound healing capacity, EVs can be engineered for better defined pharmaceutical purposes, such as carrying specific cargo or targeting specific destinations by labelling them with certain surface proteins. This review aims to promote scientific awareness in basic and translational research of EVs by summarizing the current knowledge about their natural role in each stage of skin repair and the most recent findings in application areas, such as wound healing, skin regeneration, and treatment of dermal diseases, including the stem cell-derived, plant-derived, and engineered EVs.

The Role of Mitochondria in Brain Cell Protection from Ischaemia by Differently Prepared Propolis Extracts.

Mitochondria are both the primary targets and mediators of ischaemic damage in brain cells. Insufficient oxygen causes reactive oxygen species that damage the mitochondria, leading to the loss of functionality and viability of highly energy-demanding neurons. We have recently found that aqueous (AqEP), polyethylene glycol-aqueous (Pg-AqEP) and ethanolic propolis extracts (EEP) can modulate mitochondria and ROS production in C6 cells of astrocytic origin. The aim of this study was to investigate the effect of the extracts on viability, mitochondrial efficiency and superoxide generation, and inflammatory cytokine release in primary rat cerebellar neuronal-glial cell cultures affected by ischaemia (mimicked by hypoxia +/- deoxyglucose). AqEP and Pg-AqEP (15-60 µg/mL of phenolic compounds, or PC) significantly increased neuronal viability in ischaemia-treated cultures, and this was accompanied by a reduction in mitochondrial superoxide levels. Less extended protection against ischaemia-induced superoxide production and death was exhibited by 2 to 4 µg/mL of PC EEP. Both Pg-AqEP and Ag-EP (but not EEP) significantly protected the cultures from hypoxia-induced elevation of TNF-α, IL-1ß and IL-6. Only Pg-AqEP (but not AqEP or EEP) prevented hypoxia-induced loss of the mitochondrial basal and ATP-coupled respiration rate, and significantly increased the mitochondrial respiratory capacity. Summarising, the study revealed that hydrophilic propolis extracts might protect brain cells against ischaemic injury by decreasing the level of mitochondrial superoxide and preventing inflammatory cytokines, and, in the case of Pg-AqEP, by protecting mitochondrial function.

Investigation of Phenolic Composition and Anticancer Properties of Ethanolic Extracts of Japanese Quince Leaves.

Glioblastoma multiforme is an aggressive and invasive disease with no efficient therapy available, and there is a great need for finding alternative treatment strategies. This study aimed to investigate anticancer activity of the extracts of the Japanese quince (JQ) cultivars 'Darius', 'Rondo', and 'Rasa' leaf extracts on glioblastoma C6 and HROG36 cells. As identified by ultra high performance liquid chromatography electrospray ionization tandem mass spectrometry, the extracts contained three prevailing groups of phenols: hydroxycinnamic acid derivatives; flavan-3-ols; and flavonols. Sixteen phenols were detected; the predominant compound was chlorogenic acid. The sum of detected phenols varied significantly between the cultivars ranging from 9322 µg/g ('Rondo') to 17,048 µg/g DW ('Darius'). Incubation with the extracts decreased the viability of glioblastoma HROG36 cells with an efficiency similar to temozolomide, a drug used for glioblastoma treatment. In the case of C6 glioblastoma cells, the extracts were even more efficient than temozolomide. Interestingly, primary cerebellar neuronal-glial cells were significantly less sensitive to the extracts compared to the cancer cell lines. The results showed that JQ leaf ethanol extracts are rich in phenolic compounds, can efficiently reduce glioblastoma cell viability while preserving non-cancerous cells, and are worth further investigations as potential anticancer drugs.

Investigation of Cancer Cell Migration and Proliferation on Synthetic Extracellular Matrix Peptide Hydrogels.

Chemical and mechanical properties of a tumor microenvironment are essential players in cancer progression, and it is important to precisely control the extracellular conditions while designing cancer in vitro models. The study investigates synthetic hydrogel matrices from multi-arm polyethylene glycol (PEG) functionalized with collagen-like peptide (CLP) CG(PKG)4(POG)4(DOG)4 alone and conjugated with either cell adhesion peptide RGD (mimicking fibronectin) or IKVAV (mimicking laminin). Human glioblastoma HROG36, rat C6 glioma cells, and A375 human melanoma cells were grown on the hydrogels and monitored for migration, proliferation, projected cell area, cell shape index, size and number, distribution of focal contacts in individual cells, and focal adhesion number. PEG-CLP-RGD induced migration of both glioma cell lines and also stimulated proliferation (assessed as metabolic activity) of HROG36 cells. Migration of C6 cells were also stimulated by PEG-CLP-IKVAV. These responses strongly correlated with the changes in adhesion and morphology parameters of individual cells - projected cell area, cell shape index, and focal contact number. Melanoma A375 cell proliferation was increased by PEG-CLP-RGD, and this was accompanied by a decrease in cell shape index. However, neither RGD nor IKVAV conjugated to PEG-CLP stimulated migratory capacity of A375 cells. Taken together, the study presents synthetic scaffolds with extracellular matrix (ECM)-mimicking peptides that allow for the exploration of the effect of ECM signaling to cancer cells.

Comparison of Effects of Metformin, Phenformin, and Inhibitors of Mitochondrial Complex I on Mitochondrial Permeability Transition and Ischemic Brain Injury.

Damage to cerebral mitochondria, particularly opening of mitochondrial permeability transition pore (MPTP), is a key mechanism of ischemic brain injury, therefore, modulation of MPTP may be a potential target for a neuroprotective strategy in ischemic brain pathologies. The aim of this study was to investigate whether biguanides-metformin and phenformin as well as other inhibitors of Complex I of the mitochondrial electron transfer system may protect against ischemia-induced cell death in brain slice cultures by suppressing MPTP, and whether the effects of these inhibitors depend on the age of animals. Experiments were performed on brain slice cultures prepared from 5-7-day (premature) and 2-3-month old (adult) rat brains. In premature brain slice cultures, simulated ischemia (hypoxia plus deoxyglucose) induced necrosis whereas in adult rat brain slice cultures necrosis was induced by hypoxia alone and was suppressed by deoxyglucose. Phenformin prevented necrosis induced by simulated ischemia in premature and hypoxia-induced-in adult brain slices, whereas metformin was protective in adult brain slices cultures. In premature brain slices, necrosis was also prevented by Complex I inhibitors rotenone and amobarbital and by MPTP inhibitor cyclosporine A. The latter two inhibitors were protective in adult brain slices as well. Short-term exposure of cultured neurons to phenformin, metformin and rotenone prevented ionomycin-induced MPTP opening in intact cells. The data suggest that, depending on the age, phenformin and metformin may protect the brain against ischemic damage possibly by suppressing MPTP via inhibition of mitochondrial Complex I.

Proteomic analysis links alterations of bioenergetics, mitochondria-ER interactions and proteostasis in hippocampal astrocytes from 3xTg-AD mice.

The pathogenesis of Alzheimer's disease (AD), a slowly-developing age-related neurodegenerative disorder, is a result of the action of multiple factors including deregulation of Ca2+ homeostasis, mitochondrial dysfunction, and dysproteostasis. Interaction of these factors in astrocytes, principal homeostatic cells in the central nervous system, is still poorly understood. Here we report that in immortalized hippocampal astrocytes from 3xTg-AD mice (3Tg-iAstro cells) bioenergetics is impaired, including reduced glycolysis and mitochondrial oxygen consumption, and increased production of reactive oxygen species. Shotgun proteomics analysis of mitochondria-ER-enriched fraction showed no alterations in the expression of mitochondrial and OxPhos proteins, while those related to the ER functions and protein synthesis were deregulated. Using ER- and mitochondria-targeted aequorin-based Ca2+ probe we show that, in 3Tg-iAstro cells, ER was overloaded with Ca2+ while Ca2+ uptake by mitochondria upon ATP stimulation was reduced. This was accompanied by the increase in short distance (≈8-10 nm) contact area between mitochondria and ER, upregulation of ER-stress/unfolded protein response genes Atf4, Atf6 and Herp, and reduction of global protein synthesis rate. We suggest that familial AD mutations in 3Tg-iAstro cells induce mitochondria-ER interaction changes that deregulate astrocytic bioenergetics, Ca2+ homeostasis and proteostasis. These factors may interact, creating a pathogenic loop compromising homeostatic and defensive functions of astroglial cells predisposing neurons to dysfunction.

Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels.

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell-cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

Elsholtzia ciliata (Thunb.) Hyl. Extracts from Different Plant Parts: Phenolic Composition, Antioxidant, and Anti-Inflammatory Activities.

Polyphenols play an important role on the health-promoting properties of humans. Plants belonging to Lamiaceae family are known as rich source of phenolic compounds. The current work aimed to evaluate the phenolic compounds, antioxidant, and anti-inflammatory activity of Elsholtzia ciliata (Thunb.) Hyl. ethanolic extracts from leaf, stem, flower, and whole herb. Twelve compounds were identified in ethanolic extracts using high-performance liquid chromatography (HPLC). The HPLC analysis revealed that chlorogenic acid, rosmarinic acid, and rutin were predominant compounds in ethanolicic extracts. Using HPLC-ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) post-column assay, avicularin, chlorogenic, and rosmarinic acids were identified as the predominant radical scavengers in all ethanolic extracts. All tested preparations significantly reduced the level of secretion of proinflammatory cytokines TNF-α, IL-6, and prostaglandin E2 induced by lipopolysaccharide treatment in mouse peritoneal macrophage cell culture. Stem and flower extracts were most efficient in reducing cytokine release, but leaf extract demonstrated stronger effect on prostaglandin E2 secretion. This is the first study exploring antioxidant efficiency by HPLC-ABTS post-column method and investigating anti-inflammatory activity of ethanolic extracts from E. ciliata different plant parts.

The Essential Oil and Hydrolats from Myristica fragrans Seeds with Magnesium Aluminometasilicate as Excipient: Antioxidant, Antibacterial, and Anti-inflammatory Activity.

Nutmeg (Myristica fragrans) essential oil has antimicrobial, antiseptic, antiparasitic, anti-inflammatory, and antioxidant properties. We have recently demonstrated that hydrodistillation of nutmeg essential oil by applying magnesium aluminometasilicate as an excipient significantly increases both the content and amount of bioactive substances in the oil and hydrolats. In this study, we aimed to compare the antioxidant, antimicrobial, and anti-inflammatory activity of hydrolats and essential oil obtained by hydrodistillation in the presence and absence of magnesium aluminometasilicate as an excipient. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method revealed that magnesium aluminometasilicate did not significantly improved antioxidant activity of both essential oil and hydrolat. Antibacterial efficiency was evaluated by monitoring growth of 15 bacterial strains treated by a range of dilutions of the essential oil and the hydrolats. Essential oil with an excipient completely inhibited the growth of E. faecalis, S. mutans (referent), and P. multocida, whereas the pure oil was only efficient against the latter strain. Finally, the anti-inflammatory properties of the substances were assessed in a fibroblast cell culture treated with viral dsRNR mimetic Poly I:C. The essential oil with an excipient protected cells against Poly I:C-induced necrosis more efficiently compared to pure essential oil. Also, both the oil and the hydrolats with aluminometasilicate were more efficient in preventing IL-6 release in the presence of Poly I:C. Our results show that the use of magnesium aluminometasilicate as an excipient might change and in some cases improve the biological activities of nutmeg essential oil and hydrolats.

Investigation of Antibacterial and Antiinflammatory Activities of Proanthocyanidins from Pelargonium sidoides DC Root Extract.

The study explores antibacterial, antiinflammatory and cytoprotective capacity of Pelargonium sidoides DC root extract (PSRE) and proanthocyanidin fraction from PSRE (PACN) under conditions characteristic for periodontal disease. Following previous finding that PACN exerts stronger suppression of Porphyromonas gingivalis compared to the effect on commensal Streptococcus salivarius, the current work continues antibacterial investigation on Staphylococcus aureus, Staphylococcus epidermidis, Aggregatibacter actinomycetemcomitans and Escherichia coli. PSRE and PACN are also studied for their ability to prevent gingival fibroblast cell death in the presence of bacteria or bacterial lipopolysaccharide (LPS), to block LPS- or LPS + IFNγ-induced release of inflammatory mediators, gene expression and surface antigen presentation. Both PSRE and PACN were more efficient in suppressing Staphylococcus and Aggregatibacter compared to Escherichia, prevented A. actinomycetemcomitans- and LPS-induced death of fibroblasts, decreased LPS-induced release of interleukin-8 and prostaglandin E2 from fibroblasts and IL-6 from leukocytes, blocked expression of IL-1ß, iNOS, and surface presentation of CD80 and CD86 in LPS + IFNγ-treated macrophages, and IL-1ß and COX-2 expression in LPS-treated leukocytes. None of the investigated substances affected either the level of secretion or expression of TNFα. In conclusion, PSRE, and especially PACN, possess strong antibacterial, antiinflammatory and gingival tissue protecting properties under periodontitis-mimicking conditions and are suggestable candidates for treatment of the disease.

Comparison of the Effect of Native 1,4-Naphthoquinones Plumbagin, Menadione, and Lawsone on Viability, Redox Status, and Mitochondrial Functions of C6 Glioblastoma Cells.

BACKGROUND: 1,4-naphthoquinones, especially juglone, are known for their anticancer activity. However, plumbagin, lawsone, and menadione have been less investigated for these properties. Therefore, we aimed to determine the effects of plumbagin, lawsone, and menadione on C6 glioblastoma cell viability, ROS production, and mitochondrial function. METHODS: Cell viability was assessed spectrophotometrically using metabolic activity method, and by fluorescent Hoechst/propidium iodide nuclear staining. ROS generation was measured fluorometrically using DCFH-DA. Oxygen uptake rates were recorded by the high-resolution respirometer Oxygraph-2k. RESULTS: Plumbagin and menadione displayed highly cytotoxic activity on C6 cells (IC50 is 7.7 ± 0.28 µM and 9.6 ± 0.75 µM, respectively) and caused cell death by necrosis. Additionally, they increased the amount of intracellular ROS in a concentration-dependent manner. Moreover, even at very small concentrations (1-3 µM), these compounds significantly uncoupled mitochondrial oxidation from phosphorylation impairing energy production in cells. Lawsone had significantly lower viability decreasing and mitochondria-uncoupling effect, and exerted strong antioxidant activity. CONCLUSIONS: Plumbagin and menadione exhibit strong prooxidant, mitochondrial oxidative phosphorylation uncoupling and cytotoxic activity. In contrast, lawsone demonstrates a moderate effect on C6 cell viability and mitochondrial functions, and possesses strong antioxidant properties.

Efficacy of Proanthocyanidins from Pelargonium sidoides Root Extract in Reducing P. gingivalis Viability While Preserving Oral Commensal S. salivarius.

Bacterial resistance to antibiotics and the disruption of beneficial microbiota are key problems in contemporary medicine and make the search for new, more efficient infection treatment strategies among the most important tasks in medicine. Multicomponent plant-derived preparations with mild antibacterial activity created by many simultaneous mechanisms together with anti-inflammatory, innate immune and regenerative capacity-stimulating properties are good candidates for this therapy, and proanthocyanidins are among the most promising compounds of this sort. In this study, we have isolated proanthocyanidins from Pelargonium sidoides DC root extract and characterized and compared the composition, antioxidant properties and antibacterial activity of the proanthocyanidin fraction with those of the whole extract. The results revealed that proanthocyanidins had significantly stronger antioxidant capacity compared to the root extract and exhibited a unique antibacterial action profile that selectively targets Gram-negative keystone periodontal and peri-implant pathogenic strains, such as Porphyromonas gingivalis, while preserving the viability of beneficial oral commensal Streptococcus salivarius. The finding suggests that proanthocyanidins from Pelargonium sidoides root extract are good candidates for the prolonged and harmless treatment of infectious diseases.