This study describes 14 novel D2AAK1 derivatives exhibiting enhanced neuroprotective properties. These derivatives were tested for their inhibitory effects on AChE and MAO-B, as well as their effect on cell viability under normal conditions and under oxidative stress. D2AAK1 derivatives show strong cytoprotective effects, increasing cell viability by up to 80% under normal conditions and up to 60% under oxidative stress. These effects may involve modulation of MAPK p38 and Nrf2 pathway interactions, known to promote antioxidant and anti-apoptotic responses. In vivo studies indicated a beneficial effect of the tested derivative on memory processes in the novel object recognition test. These findings identify D2AAK1 derivatives as promising lead compounds for further development as potential treatments of memory deficits.

Flavonols are dietary polyphenols whose biological activity is influenced by structural modifications such as O-methylation. This study compared two quercetin derivatives, isorhamnetin (3 '-O-methylquercetin) and rhamnetin (7-O-methylquercetin). Antioxidant activity was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 '-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), cupric reducing antioxidant capacity (CUPRAC), and ferric reducing antioxidant power (FRAP) assays. Cyclooxygenase-2 (COX-2) inhibitory activity was assessed in vitro and supported by molecular docking simulations. In vivo effects included developmental toxicity, behavioral assessment, and locomotor responses in a 6-hydroxydopamine (6-OHDA) model. The results demonstrated that rhamnetin exhibited significantly stronger radical-scavenging and reducing activity in DPPH, ABTS, and FRAP assays, whereas no significant differences were observed in the CUPRAC assay. Isorhamnetin showed stronger COX-2 inhibition, with docking results suggesting a different mode of binding when analyzing possible interactions with enzyme active site. In zebrafish larvae, rhamnetin showed lower observable developmental toxicity within the tested concentration range, whereas isorhamnetin induced developmental abnormalities at higher concentrations. Both flavonols attenuated 6-OHDA-associated locomotor deficits and modulated antioxidant enzyme activity under oxidative stress conditions. In conclusion, our findings indicate that the position of O-methylation influences flavonol antioxidant properties, COX-2 interactions, and organism-level responses.

Abstract The clinical translation of RNA interference (RNAi) therapeutics remains limited by inefficient delivery and cancer-target accumulation. Here, we report the development of a new cationic liposome (CLP) nanocarrier engineered for delivery and controlled-release of small interfering RNA (siRNA) targeting the epidermal growth factor receptor (EGFR) in human colorectal cancer. CLPs were synthesized from ethylphosphocholine-based lipids and PEGylated components, with folic acid (FA) tissue-specific ligand and fluorophore labelling. These nanocarriers exhibited robust physicochemical stability across a broad pH and temperature range, efficient siRNA complexation, and nuclease-protection of siRNA. Functional studies revealed that CLP-siEGFR achieved effective cytosolic siRNA cargo release and EGFR silencing in vitro, proving to be more effective than conventional lipid-based transfection systems. In human xenograft models, intravenously administered CLP-siEGFR showed enhanced tumor localization, prolonged siRNA retention, and significant tumor growth suppression, accompanied by marked downregulation of EGFR. Importantly, systemic dosing was well-tolerated, with no evidence of hepatotoxicity, nephrotoxicity, or hematological abnormalities. These results position CLP nanocarriers as an effective platform for targeted RNAi therapeutics, offering translational potential for precision oncology applications.

BACKGROUND AND PURPOSE:Recognition and procedural memory are modulated by the noradrenergic system, whose terminals release noradrenaline along with neuropeptide Y, acting via Y1 and Y2 receptors. This study aims to investigate the role of Y1 and Y2 receptor antagonists (BMS-193885 and SF-11) on recognition and procedural memory, including their co-administration with clenbuterol, a β2-adrenoceptor agonist, with additional focus on the molecular mechanisms underlying memory-related behavioural changes. EXPERIMENTAL APPROACH:Recognition memory was assessed in mice using the novel object recognition task. Motor learning and procedural memory were evaluated in the accelerating rotarod test. Memory-related behavioural outcomes were complemented by analyses of NPY levels, the phosphorylation status of CaMKII and ERK1/2, along with the concentrations of 5-HT, dopamine, noradrenaline and its metabolite in selected brain regions, including the prefrontal cortex, hippocampus and cerebellum. KEY RESULTS:SF-11 at 5 mg·kg-1 impaired recognition memory, prolonged procedural memory retention and inhibited CaMKII, but at 20 mg·kg-1, SF-11 disrupted both recognition and procedural memory increasing NPY. BMS-193885, at 5 mg·kg-1, prolonged procedural memory retention, but at 20 mg·kg-1, it impaired long-term recognition memory, prolonged procedural memory retention, increased NPY and decreased phospho-CaMKII. Clenbuterol enhanced procedural memory retention, but this pro-cognitive effect was diminished by co-administration with either Y receptor antagonist, which correlated with decreased noradrenaline levels. CONCLUSIONS AND IMPLICATIONS:NPY receptor antagonists modulate memory processes in a dose- and memory-type-dependent manner, and these effects appear to be linked to hippocampal function. NPY receptors may serve as therapeutic targets to treat cognitive impairments.

The global rise in life expectancy has been accompanied by a growing prevalence of neurodegenerative diseases, such as Alzheimer's disease (AD). These complex disorders arise from multiple pathogenic factors and biological pathways, necessitating the development of multi-target therapeutic strategies. D2AAK1, discovered by our group, has emerged as a promising candidate due to its cytoprotective, antioxidant, and procognitive properties. This study aimed to further elucidate the mechanisms underlying the action of D2AAK1 and its derivatives, with a focus on their potential for neuroprotection and cognitive enhancement. The effect of D2AAK1 on cell viability was evaluated under normal conditions and during H2O2-induced oxidative stress using the resazurin assay. p38 MAPK activity was measured through cell-based ELISA. mRNA expression was analyzed using a two-step quantitative PCR method, and enzymatic effects were assessed via photometric, fluorescence, and luminescence techniques. Behavioral studies in murine models were performed to investigate the influence of the compounds on memory processes. It was found that D2AAK1 and its derivatives significantly enhanced cell viability, with some derivatives exhibiting greater potency than D2AAK1. In vivo, one derivative notably improved memory performance and reversed scopolamine-induced memory impairment in the novel object recognition test in male Swiss mice. Mechanistic studies revealed that D2AAK1 increased the expression of cytoprotective proteins such as Bcl-2 and HO-1, while concurrently reducing the expression and activity of pro-apoptotic factors, including caspase-3, p38 MAPK, and MAO-B. These dual actions culminated in enhanced cellular resilience and viability, translating into improved cognitive outcomes. The findings suggest that D2AAK1 and its derivatives, through their multi-factor mechanism of action, hold promise as therapeutic agents for the treatment of neurodegenerative diseases.

The aim of this work is to provide a comprehensive set of biological tests to assess the biomedical potential of novel osteochondral scaffolds with methods proposed to comply with the 3Rs principle, focusing here on a biphasic Curdlan-based osteochondral scaffold as a promising model biomaterial. In vitro experiments include the evaluation of cytotoxicity, mutagenicity, and genotoxicity referring to ISO standards, the assessment of the viability and proliferation of human chondrocytes and osteoblasts, and the estimation of inflammation after direct contact of biomaterials with human macrophages. Ex vivo experiments include assessments of the response of the surrounding osteochondral tissue after incubation with the implanted biomaterial. In vivo experiments involve an evaluation of the toxicity and regenerative potential of the biomaterial in zebrafish (larvae and adults) and in osteochondral defects in dogs (veterinary patients). The applied set of tests allows us to show that the Curdlan-based scaffold does not induce cytotoxicity (cell viability close to 100%), mutagenicity (the level of reversion is not 2× higher compared to the control), and genotoxicity (it does not exhibit any change in chromosomal aberration; the frequency of micronuclei, micronucleated binucleated cells, and cytokinesis-block proliferation index is comparable to the control; moreover, it does not cause the formation of comets in cells). This biomaterial also promotes the viability and proliferation of chondrocytes and osteoblasts (the OD values between the fourth and seventh day of incubation increase by approximately 1.6×). The Curdlan-based scaffold stimulates only a transient inflammatory response in vitro and ex vivo. This biomaterial does not cause Danio rerio larvae malformation and also enables proper regeneration of the caudal fin in adults. Finally, it supports the regeneration of an osteochondral defect in veterinary patients. Thus, this is a proposal to use alternative methods for biological assessment of osteochondral scaffolds as opposed to commonly used tests using large numbers of laboratory animals.

The aim of this work was to produce bone scaffolds containing whey protein isolate and pearl powder and to conduct a preliminary assessment of the biomedical potential in vitro and in vivo. This included analysis of structural, physicochemical, mechanical, and biological properties, which revealed that biomaterials containing pearl powder exhibited an enhanced porous structure, increasing absorptive properties, and decreasing proteolytic capacity with increasing inorganic component content. Pearl powder content in the biomaterials did not clearly influence their mechanical properties or their ability to release calcium ions, as well as proteins. Extracts obtained from all tested biomaterials showed no cytotoxicity in vitro. The surfaces of all biomaterials promoted normal human osteoblast growth, proliferation, and osteogenic differentiation. Furthermore, all biomaterials did not display toxicity in vivo, but no changes in Danio rerio were observed after evaluation of the biomaterial containing the highest amount of pearl powder-10% v/w (marked as WPI/P10). Taking all the obtained results into account, it appears that this biomaterial can be promising for bone scaffolds and similar applications, thanks to its porous structure, high cytocompatibility in vitro, and lack of toxicity in vivo. However, advanced studies will be conducted in the future.

This work aimed to compare the biomedical potential of thermally obtained hydrogels consisting of curdlan and a curdlan-whey protein isolate mixture. The purpose of this paper was also to investigate and explain the mechanisms responsible for their different cyto- and biocompatibility. To do this, mechanical and structural properties, wettability, and protein adsorption of biomaterials were analysed and compared. Biological experiments in vitro were carried out on normal human chondrocytes, osteoblasts, and macrophages. Moreover, in vivo studies were performed using the Danio rerio (zebrafish). Our studies showed that in comparison to the curdlan/whey protein isolate hydrogel, the curdlan hydrogel had lower mechanical properties. Its surface was less rough and more hydrophobic and adsorbed a greater amount of blood proteins, but most probably induced their conformational changes. In addition, the curdlan hydrogel exhibited lower cytocompatibility towards normal human osteoblasts and chondrocytes. It also induced greater inflammation in human macrophages in vitro and did not enhance the re-growth of the amputated caudal fin in the zebrafish larvae model when compared to the curdlan/whey protein isolate hydrogel. Hence, our studies demonstrated that a commonly used curdlan hydrogel has a significantly lower biomedical potential compared to the curdlan/whey protein isolate hydrogel proposed by us.

Platelet concentrates such as platelet-rich plasma, platelet-rich fibrin or concentrated growth factors are cost-effective autologous preparations containing various growth factors, including platelet-derived growth factor, transforming growth factor β, insulin-like growth factor 1 and vascular endothelial growth factor. For this reason, they are often used in regenerative medicine to treat wounds, nerve damage as well as cartilage and bone defects. Unfortunately, after administration, these preparations release growth factors very quickly, which lose their activity rapidly. As a consequence, this results in the need to repeat the therapy, which is associated with additional pain and discomfort for the patient. Recent research shows that combining platelet concentrates with biomaterials overcomes this problem because growth factors are released in a more sustainable manner. Moreover, this concept fits into the latest trends in tissue engineering, which include biomaterials, bioactive factors and cells. Therefore, this review presents the latest literature reports on the properties of biomaterials enriched with platelet concentrates for applications in skin, nerve, cartilage and bone tissue engineering.

Citral, a common monoterpene found in numerous plants, is an interesting compound that has been shown to have various biological activities. Although it is widely distributed in nature and there are many studies presenting its biological activities, its anti-neurodegenerative activity, especially under in vivo conditions, is very poorly understood. Thus, this paper aimed to deepen knowledge about citral activity towards factors and symptoms of neurodegeneration. To accomplish this, several comprehensive tests were conducted, including the estimation of butyrylcholinesterase inhibition, the evaluation of hepatotoxicity and the detection of oxidative stress and lipid peroxidation in vitro, as well as an in vivo behavioral assessment using mice models. Additionally, ex vivo determination of level of the compound in the brain and blood of a tested animal was undertaken. The results obtained revealed that citral is able to inhibit butyrylcholinesterase activity and protect hepatic cells against oxidative stress and lipid peroxidation in vitro. Moreover, behavioral tests in vivo indicated that citral (50 mg/kg) improves memory processes associated with acquisition (passive avoidance test), both in acute and subchronic administration. Additionally, we found that the administration of citral at 25 mg/kg and 50 mg/kg did not significantly affect the locomotor activity. Beyond the aforementioned, gas chromatography-mass spectrometry analysis revealed the presence of the compound in the blood and brain after subchronic administration of citral. Taken together, the results obtained in vitro, in vivo and ex vivo clearly indicate that citral is a promising monoterpene that can potentially be used towards cognition improvement.

Mephedrone is a psychoactive drug that increases dopamine, serotonin and noradrenaline levels in the central nervous system via interaction with transporters or monoamines. The aim of the presented study was to assess the role of the GABA-ergic system in the expression of mephedrone-induced reward. For this purpose, we conducted (a) a behavioral evaluation of the impact of baclofen (a GABA(B) receptors agonist) and GS39783 (a positive allosteric modulator of GABA(B) receptors) on the expression of mephedrone-induced conditioned place preference (CPP) in rats, (b) an ex vivo chromatographic determination of the GABA level in the hippocampi of rats subchronically treated with mephedrone and (c) an in vivo evaluation of GABA hippocampal concentration in rats subchronically administered with mephedrone using magnetic resonance spectroscopy (MRS). The results show that GS39783 (but not baclofen) blocked the expression of CPP induced by (20 mg/kg of) mephedrone. The behavioral effect was consistent with chromatographic analysis, which showed that mephedrone (5 and 20 mg/kg) led to a decrease in GABA hippocampal concentration. Altogether, the presented study provides a new insight into the involvement of the GABA-ergic system in the rewarding effects of mephedrone, implying that those effects are at least partially mediated through GABA(B) receptors, which suggests their potential role as new targets for the pharmacological management of mephedrone use disorder.

Chronic wounds, among others, are mainly characterized by prolonged inflammation associated with the overproduction of reactive oxygen species and pro-inflammatory cytokines by immune cells. As a consequence, this phenomenon hinders or even precludes the regeneration process. It is known that biomaterials composed of biopolymers can significantly promote the process of wound healing and regeneration. The aim of this study was to establish whether curdlan-based biomaterials modified with hop compounds can be considered as promising candidates for the promotion of skin wound healing. The resultant biomaterials were subjected to an evaluation of their structural, physicochemical, and biological in vitro and in vivo properties. The conducted physicochemical analyses confirmed the incorporation of bioactive compounds (crude extract or xanthohumol) into the curdlan matrix. It was found that the curdlan-based biomaterials improved with low concentrations of hop compounds possessing satisfactory hydrophilicity, wettability, porosity, and absorption capacities. In vitro, tests showed that these biomaterials were non-cytotoxic, did not inhibit the proliferation of skin fibroblasts, and had the ability to inhibit the production of pro-inflammatory interleukin-6 by human macrophages stimulated with lipopolysaccharide. Moreover, in vivo studies showed that these biomaterials were biocompatible and could promote the regeneration process after injury (study on Danio rerio larvae model). Thus, it is worth emphasizing that this is the first paper demonstrating that a biomaterial based on a natural biopolymer (curdlan) improved with hop compounds may have biomedical potential, especially in the context of skin wound healing and regeneration.

Repeated maternal separation (MS) is a useful experimental model in rodents for studying the long-term influence of early-life stress on brain neurophysiology. In our work, we assessed the effect of repeated MS (postnatal day (PND)1–21, 180 min/day) on the postnatal development of rat brain regions involved in memory using proton magnetic resonance spectroscopy (1HMRS) for tissue volume and the level of amino acids such as glutamate, aspartate, glutamine, glycine and gamma-aminobutyric acid (GABA) in the hippocampus. We assessed whether these effects are sex dependent. We also use novel object recognition (NOR) task to examine the effect of MS on memory and the effect of ethanol on it. Finally, we attempted to ameliorate postnatal stress-induced memory deficits by using VU-29, a positive allosteric modulator (PAM) of the metabotropic glutamate type 5 (mGlu5) receptor. In males, we noted deficits in the levels of glutamate, glycine and glutamine and increases in GABA in the hippocampus. In addition, the values of perirhinal cortex, prefrontal cortex and insular cortex and CA3 were decreased in these animals. MS females, in contrast, demonstrated significant increase in glutamate levels and decrease in GABA levels in the hippocampus. Here, the CA1 values alone were increased. VU-29 administration ameliorated these cognitive deficits. Thus, MS stress disturbs amino acids levels mainly in the hippocampus of adult male rats, and enhancement of glutamate neurotransmission reversed recognition memory deficits in these animals.

This review aims to summarize the literature data regarding the effects of different toothpaste compounds in the zebrafish model. Danio rerio provides an insight into the mechanisms of the ecotoxicity of chemicals as well as an assessment of their fate in the environment to determine long-term environmental impact. The regular use of adequate toothpaste with safe active ingredients possessing anti-bacterial, anti-inflammatory, anti-oxidant, and regenerative properties is one of the most effective strategies for oral healthcare. In addition to water, a typical toothpaste consists of a variety of components, among which three are of predominant importance, i.e., abrasive substances, fluoride, and detergents. These ingredients provide healthy teeth, but their environmental impact on living organisms are often not well-known. Each of them can influence a higher level of organization: subcellular, cellular, tissue, organ, individual, and population. Therefore, it is very important that the properties of a chemical are detected before it is released into the environment to minimize damage. An important part of a chemical risk assessment is the estimation of the ecotoxicity of a compound. The zebrafish model has unique advantages in environmental ecotoxicity research and has been used to study vertebrate developmental biology. Among others, the advantages of this model include its external, visually accessible development, which allows for providing many experimental manipulations. The zebrafish has a significant genetic similarity with other vertebrates. Nevertheless, translating findings from zebrafish studies to human risk assessment requires careful consideration of these differences.

The aim of this study is to determine whether type I and type II positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (nAChRs) induce antidepressant-like activity in mice after acute, subchronic, and chronic treatments, and to assess whether α7-PAMs inhibit neurotransmitter transporters and activate mTOR (mammalian target of rapamycin) and/or ERK (extracellular signal-regulated protein kinases) signaling. The forced swim (FST) and tail suspension (TST) test results indicated that NS-1738 (type I PAM), PNU-120596 and PAM-2 (type II PAMs) induce antidepressant-like activity after subchronic treatment, whereas PAM-2 was also active after chronic treatment. Methyllycaconitine (α7-antagonist) inhibited the observed effects, highlighting the involvement of α7 nAChRs in this process. Drug interaction studies showed synergism between PAM-2 and bupropion (antidepressant), but not between PAM-2 and DMXBA (α7-agonist). The studied PAMs showed no high affinity (< 1 µM) for the human dopamine, serotonin, and noradrenaline transporters, suggesting that transporter inhibition is not the underlying mechanism for the observed activity. To assess whether mTOR and ERK signaling pathways are involved in the activity of α7-PAMs, the phosphorylation status of key signaling nodes was determined in prefrontal cortex and hippocampus from mice chronically treated with PAM-2. In conclusion, the antidepressant-like activity of type I and type II PAMs is mediated by a mechanism involving α7 potentiation but not α7 desensitization or neurotransmitter transporter blockade, and is correlated with activation of both mTOR and ERK signaling pathways. These results support the view that α7-PAMs might be clinically used to ameliorate depression disorders .

Rationale Mephedrone is a frequently overused drug of abuse that belongs to the group of novel psychoactive substances. Although its mechanism of action, as well as toxic and psychoactive effects, has been widely studied, the role of different factors that could contribute to the increased vulnerability to mephedrone abuse is still poorly understood. Objectives The aim of the presented study was to assess the impact of several factors (sex differences, social-conditioning, and chronic mild unpredictable stress - CMUS) on the liability to mephedrone-induced reward in Wistar rats. Methods The rewarding effects of mephedrone in male and female rats were assessed using the conditioned place preference (CPP) procedure. Furthermore, the impact of social factor and stress was evaluated in male rats using social-CPP and CMUS-dependent CPP, respectively. Results Mephedrone induced classic-CPP in female (10 mg/kg), as well as in male (10 and 20 mg/kg) rats. However, the impact of mephedrone treatment during social-CPP was highly dose-dependent as the rewarding effects of low dose of mephedrone (5 mg/kg; non-active in classic-CPP) were potentiated when administered during social-conditioning. Interestingly, social-conditioning with a higher dose of 20 mg/kg (that induced classic-CPP) was able to reverse these effects. Finally, CMUS potentiated rewarding effects of a low dose of mephedrone (5 mg/kg) and increased the level of corticosterone in rats' prefrontal cortex and hippocampus. Conclusions Altogether, the presented results give new insight into possible factors underlying the vulnerability to mephedrone abuse and can serve as a basis for further studies assessing mechanisms underlying observed effects.

Mephedrone is a widely used drug of abuse, exerting its effects by interacting with monoamine transporters. Although this mechanism has been widely studied heretofore, little is known about the involvement of glutamatergic transmission in mephedrone effects. In this study, we comprehensively evaluated glutamatergic involvement in rewarding effects of mephedrone using an interdisciplinary approach including (1) behavioural study on effects of memantine (non-selective NMDA antagonist) on expression of mephedrone-induced conditioned place preference (CPP) in rats; (2) evaluation of glutamate concentrations in the hippocampus of rats following 6 days of mephedrone administration, using in vivo magnetic resonance spectroscopy (MRS); and (3) determination of glutamate levels in the hippocampus of rats treated with mephedrone and subjected to MRS, using ion-exchange chromatography. In the presented research, we confirmed priorly reported mephedrone-induced rewarding effects in the CPP paradigm and showed that memantine (5 mg/kg) was able to reverse the expression of this effect. MRS study showed that subchronic mephedrone administration increased glutamate level in the hippocampus when measured in vivo 24 h (5 mg/kg, 10 mg/kg and 20 mg/kg) and 2 weeks (5 mg/kg and 20 mg/kg) after last injection. Ex vivo chromatographic analysis did not show significant changes in hippocampal glutamate concentrations; however, it showed similar results as obtained in the MRS study proving its validity. Taken together, the presented study provides new insight into glutamatergic involvement in rewarding properties of mephedrone.

Neuroinflammation is a key process in the pathogenesis of many neurological disorders, i.e. Alzheimer's disease and Parkinson's disease. However, there are no anti-inflammatory medical interventions recommended so far in the treatment of neuroinflammation-related brain disorders. Therefore, the burden of searching for effective and safe antineuroinflammatory agents is well founded, especially in the aging society. Compounds of plant origin, mainly (poly)phenols, have attracted considerable attention in recent years. Notably, the role of flavonoids in ameliorating neuroinflammation is in the limelight. Thus, we used comprehensive literature retrieval to summarize the effects and active components of edible fruits and their phenolic compounds. As a result, this review presents a valuable summary of results of in vitro, ex vivo, and in vivo studies on the antineuroinflammatory effects of edible fruits and their (poly)phenolic extracts as well as dietary flavonoids and other selected (poly)phenols based on the detailed description of foregoing studies. Additionally, problems resulting from the limited bioavailability of (poly)phenols were discussed.

Intracellular signalling pathways have been extensively studied as therapeutic targets for the treatment of mental diseases. Our attention has been caught by two kinases potentially involved in anxiety, ERK1/2 and CaMKII. The study aimed to examine changes in the activation of ERK1/2 and CaMKII concerning anxiolytic-like behaviours in mice. To evaluate anxiety-related response in mice, we used the open field test and the elevated plus maze test. Behavioural studies were complemented with the immunoblotting analysis to identify proteins of interest in the cortex, hippocampus, and striatum. We analysed the phosphorylation status of ERK1/2 and CaMKII in mice treated with a well-known anxiolytic drug - diazepam. Next, the blockade of ERK1/2 pathway by SL-327, a selective MEK1/2 inhibitor, was checked for anxiolytic action. Finally, the co-administration of subeffective doses of diazepam and SL-327 was investigated for a potential synergistic anxiolytic effect. Anxiolytic effects of acute diazepam are accompanied by decreased p-ERK1/2 and upregulation of p-CaMKII. Subchronic treatment with SL-327 leads to the manifestation of anxiolytic-like behaviours and changes in the phosphorylation status of both kinases in a diazepam-like manner. Co-administration of subeffective doses of SL-327 and diazepam induces anxiolysis, which is CaMKII-independent and correlates to selectively decreased phosphoactive ERK1/2 in the hippocampus. The MEK-ERK pathway is significantly involved in anxiolytic action of diazepam and its prolonged inhibition produces anxiolytic-like phenotype in mice. ERK inhibition could be used to manage anxiety symptoms in a benzodiazepine-sparing regimen for treatment of anxiety.

There are several reports indicating that the roots of the Carlina acaulis L. used to be commonly applied as a treatment measure in skin diseases and as an antiparasitic agent, starting from antiquity to the 19th century; however, nowadays, it has lost its importance. Currently, numerous studies are being conducted assessing the possibility of reintroducing C. acaulis-derived extracts to phytotherapy. Determining the safety profile of the main constituents of the plant material is crucial for achieving this goal. Here, we aimed to determine the toxicity profile of carlina oxide, one of the most abundant components of the C. acaulis root extract. We obtained the carlina oxide by distillation of C. acaulis roots in the Deryng apparatus. The purity of the standard was evaluated using GC-MS, and the identity was confirmed by IR, Raman, and NMR spectroscopy. In vitro cytotoxicity was assessed using a panel of human cell lines of skin origin, including BJ normal fibroblasts and UACC-903, UACC-647, and C32 melanoma cells. This was accompanied by an in vivo zebrafish acute toxicity test (ZFET). In vitro studies showed a toxic effect of carlina oxide, as demonstrated by an induction of apoptosis and necrosis in both normal and melanoma cells. Decreased expression of AKT kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) was noted in the UACC-647 melanoma cell line. It was also observed that carlina oxide modified the expression of programmed cell death-ligand 1 (PD-L1) in tested cell lines. Carlina oxide exhibited high in vivo toxicity, with LC50 = 10.13 µg/mL upon the 96 h of exposure in the ZFET test. Here, we demonstrate that carlina oxide displays toxic effects to cells in culture and to living organisms. The data indicate that C. acaulis-based extracts considered for therapeutic use should be completely deprived of carlina oxide.