Ahmet Davut Aksu1, Fatemeh Bahadori1, Leyla Elmas2,3, Ahmet Ceyhan Gören4, Erhan Ayşan5
Ethnic use of
Depending on the experimental outcome, (a) a decrease in t3 levels and (c) an increase in TSH levels can be seen for the LAG group compared to the L group. No significant difference was observed in (b) t4 levels between these two groups. *
Emil Babiak, Gabriel Dóka, Peter Křenek, Ján Klimas
Patients with heart diseases might have a worse prognosis if they also suffer from local intracellular hypothyroidism, which is observed by reduced activity of the thyroid signalling pathway in the myocardium. We studied mRNA expression of thyroid receptors, hormone transporters, and genes transcriptionally regulated by these hormones with other components of this pathway by RT-qPCR method. We used experimental rat models of streptozotocin-induced diabetic heart and isoprenaline-induced cardiac hypertrophy. In addition, we also implemented a meta-analysis of transcriptomic data from 535 explanted hearts of heart failure patients reposited in the Gene Expression Omnibus database. mRNA expression of several thyroid pathway components was altered in heart damage, most notably thyroid hormone receptors THRA and THRB (−24% to −34%), transporters MCT10 and P-GP (−12% to −15%), and intracellular thyroid-hormone sequestrant CRYM (+58%). The impact of impaired thyroid pathway was confirmed by transcriptional dysregulation of thyroid-directed cardiac genes, most notably MYH6 and SERCA2A (−22% to −85%), important components of cardiac contractile apparatus. Conclusively, local intracellular hypothyroidism in cardiac tissue could contribute to pathological changes in heart damage.
Linda Bartošová1, Csaba Horváth1, Peter Galis1, Kristína Ferenczyová2, Barbora Kaločayová2, Adrián Szobi1, Adriana Duriš-Adameová1,2, Monika Barteková2,3, Tomáš Rajtík1,2
Quercetin (QUE) is a widely known polyphenolic flavonoid that possesses a notable antioxidative, anti-inflammatory, and antiapoptotic activities that were also recorded by multiple studies on cardiovascular pathologies. Therefore, it is rational to employ QUE in a study that comprises both diabetes mellitus 2 (DM2) and obesity as a base for cardiovascular disease development, given that such an experimental model has not been explored yet. The exact role of QUE in either DM2 or cardiac remodelling remains elusive, even though published reports suggest a protective role of QUE against heart remodelling or diabetes.
In this study, 1-year-old male Zucker diabetic fatty rats were randomised into two groups: nontreated sham (Dia) and QUE-treated (DiaQ). Wistar rats were used as control groups: nontreated (C) and QUE-treated (CQ) groups. QUE treatment was administered orally at a dose of 20 mg/kg/day for 6 weeks. Echocardiography (GE Healthcare Vivid E9) was performed to assess heart parameters before the onset of treatment and at the end of the experiment. Hydroxyproline assay was used to measure total levels of collagen in left ventricles (LVs) and protein content was analysed by western blot.
QUE normalised E/A ratio in the DiaQ group, which indicates an improvement of diastolic dysfunction. Moreover, QUE reduced interventricular septum diameter and left ventricular posterior wall thickness, resulting in reduced relative wall thickness. Similarly, total LV collagen content was decreased in the DiaQ group compared to the Dia group. Furthermore, protein level of myocyte enhancer factor-2 (MEF2) was significantly decreased in the DiaQ group when compared to the Dia group.
QUE treatment alleviated diastolic dysfunction of the heart accompanied by an overall reduction of ventricular mass and collagen content in LV. This observation was further supported by decreased expression of remodelling-associated MEF2. Our results indicate that QUE might exert potential cardioprotective and antiremodelling effects in experimental DM2.
Michaela Benesová1, Marek Krivošík2, Stanislava Kosírová1, Tatiana Foltánová1
Every year stroke affects 15 million people worldwide. It is fatal for 5 million of them and disabling for another 5 million. Anticoagulant treatment is a well-known stroke prevention measure. The aim of this work was to compare the results of the success in recanalization treatment (NIHSS) in patients with and without antithrombotic treatment in the anterior condition and to evaluate any change in pharmacotherapy after recanalization treatment in patients with antithrombotic treatment in predisease.
We analysed data from 100 patients (59% men, 41% women) with an average age of 66.4 (
Note: also published as short communication
Eur. Pharm. J. 2022, 69(s1), 78–81
Daniel Bobek1, Martin Šíma1, Petra Cihlářová2, Pavel Ryšánek1, Jaroslava Roušarová1, Jan Beroušek3, Martin Kuchař2, Tomáš Vymazal3, Ondřej Slanař1
Ciprofloxacin belongs to a group of fluoroquinolone antibiotics efficacious on a broad spectrum of bacteria. It is an important agent for the treatment of serious and life-threatening infections. The objectives of this work were to describe metabolic ratios of ciprofloxacin and its main active metabolites in critically ill patients and to examine demographic, laboratory, and genetic factors that could potentially affect them. A further aim was to develop a population pharmacokinetic (PK) model for a metabolite mostly associated with the potential covariates. A total of 29 patients were treated with an intravenous infusion of ciprofloxacin and enrolled to this open-label (laboratory blinded) prospective PK trial. Blood samples for PK analysis were taken at 1, 4, and 11.5 hr following completion of the infusion. Sex, age, body weight, height, serum creatinine and bilirubin levels, and creatinine clearance (CLCR) and 24-hr urine output were recorded. Polymorphisms rs2032582 and rs1045642 have been analysed in the MDR1 gene, rs4148977 in the SLCO1A2 gene, and rs762551 in the CYP1A2 gene. Median (IQR) metabolite/parent ratios of desethylene ciprofloxacin, formyl ciprofloxacin, and oxociprofloxacin were 5.86% (4.09–9.87%), 4.08% (3.38–6.92%), and 5.91% (3.42–13.65%), respectively. Desethylene ciprofloxacin/ciprofloxacin metabolic ratio was positively associated with height (
Julius Brtko1, Dana Strouhalova2, Lucia Toporova1, Dana Macejova1, Janette Bobalova2
Nuclear retinoic acid receptors (RARs) and nuclear retinoid X receptors (RXRs) are retinoid/rexinoid inducible transcription factors. Trialkyltins and triaryltins, a class of organometallic compounds, function as nuclear RXR agonists due to their capability to bind to the ligand-binding domain of RXR subtypes and function as transcriptional activa tors. In this study, we present the proteomic data confirming that selected triorganotin compounds affect expression of proteins associated with tumour progression in human breast cancer MDA-MB-231 cells. Proteomic strategies based on a bottom-up method were applied in this study. The total MDA-MB-231 human cell proteins were extracted, separated on 2D SDS-PAGE and their characterization was achieved by MALDI-TOF/TOF MS/MS. Employing PDQuest™ software, we identified more than 30 pro teins differently affected by the above compounds. Specific proteins associated either with metabolic pathway (glyceraldehyde-3-phosphate dehydrogenase) or cellular processes such as apoptosis, regulation of gene transcription, or epithelial–mesenchymal transition (annexin 5, nucleoside diphosphate kinase B, and vimentin) have been selected for further analyses. We found that treatment of MDA-MB-231 cells with selected triorganotin compounds reduced the expression of studied proteins. Moreover, the treatment of MDA-MB-231 cells with selected triorganotin compounds together with ATRA resulted in an additional reduction of annexin 5, vimentin, and nucleoside diphosphate kinase B. These results demonstrate that an RXR/RAR heterodimer might act under this experimental design as a permissive heterodimer allowing direct activation of RXRs by triorganotins.
This work was supported by institutional support RVO:68081715 of the Institute of Analytical Chemistry of the CAS, APVV-15-0372, APVV-19-0093, APVV-20-0314, and VEGA 2/0116/21 grants.
Alejandro Carazo1, Marcel Hrubša1, Pavel Skořepa2,3, Lenka Javorská4, Lenka Kujovská-Krčmová4,5, Vladimír Blaha2, Přemysl Mladěnka1
The process of platelet aggregation is often conditioned by personal circumstances such as age, gender, lifestyle, and the presence of concomitant diseases. An additional factor is the resistance to current antiplatelet drugs developed by some patients, although the reason is not always obvious. In this work, we enrolled a total of 53 overall healthy women and men donors with ages ranging from 20 to 66 years for a cross-sectional study. Blood samples were taken, and aggregation was induced with a total of seven different compounds (ristocetin, thrombin receptor activating protein-6 [TRAP], arachidonic acid [AA], platelet activating factor-16 [PAF], ADP, collagen, or thromboxane A2 analogue U46619) ex vivo. In addition, some samples were pretreated with clinically used antiplatelet drugs (vorapaxar, ticagrelor, or acetylsalicylic acid [ASA]). Our results showed a pattern in which increasing age decreased the aggregatory responses to AA and TRAP in both genders, whereas responses to ADP, U-46619, and PAF were affected by age only in women. In addition, women had stronger responses to some aggregation inducers (ADP, TRAP), as well as lower benefit from antiplatelet drugs (ASA, vorapaxar). Physiological levels of blood lipids and glucose, as well as body mass index, had mostly no effect on platelet aggregation in this study. On the contrary, several strong positive correlations between different aggregation triggers were observed. The different responses in women, in relation to age, are of note for future studies.
This study was supported by the Czech Health Research Council (NU21J-02-00021).
Anna Ďurinová1, Lucie Smutná1, Pavel Bárta2, František Trejtnar1, Petr Pávek1
Low-density lipoprotein receptor-related protein 2 (LRP2, megalin) plays an important role in many different tissues. In the kidneys, LRP2 is responsible for trafficking structurally varied high-molecular-weight substances (including albumin, apolipoproteins, antibiotics, etc.) from primary urine to cytoplasm of proximal tubular cells. Human cell lines HK-2 (proximal tubular cell line) and JEG-3 (human placental choriocarcinoma cell line) originally expressing a high level of megalin were used within this project for gene modification.
The knockout of the LRP2 gene was performed using the CRISPR/Cas9 technique. sgRNA sequences were specifically designed to target crucial sites for regulation of function and trafficking ligands (NPMY motif), phosphorylation (PPPSP motif), or transmembrane domain of LRP2. The function of LRP2 after editing was verified experimentally using viability and accumulation studies. Higher viability after treatment with a cytotoxic concentration of aminoglycoside antibiotic gentamicin and lower fluorescence level of standard substrate FITC-albumin were observed in LRP2 knockout cell lines, indicating harmed function of LRP2 in edited cells.
To separate modified cells exerting low activity of megalin from unmodified cells, the fluorescence-activated cell sorting (FACS) method was involved after transfection of HK-2 and JEG-3 cell lines. Using the accumulation of the known substrate LRP2 FITC-albumin and the fluorescent dye of viable cells with rhodamine, we obtained the target group of viable cells without FITC-albumin accumulation, cells with the LRP2 gene mutation.
Developed cells therefore can be used as a suitable model for further testing of potential ligands of LRP2 (e.g., radiolabeled polypeptides), which can lead to improvement of LRP2 substrate characterisation.
This study was supported by EFSA-CDN CZ.02.1.01/0.0/0.0/16_019/0000841, GAUK150120, and SVV260549.
Zdeněk Dvořák1, Aneta Grycová1, Vítězslav Maier2
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays multiple roles in many physiological and pathophysiological processes. Therapeutic targeting of the AhR is an emerging strategy in the treatment of various diseases, and novel AhR ligands are needed. A large number of the AhR ligands, including xenobiotics (natural, dietary, synthetic) and endogenous compounds including those produced by commensal microbiota, were described.
2-(1′ Underlying its discovery, ITE was detected and identified in porcine lungs. To date, no attempts have been made to detect or determine if ITE is found in human tissues. Indothiazinone, a structural backbone of ITE, was found in cultures of myxobacteria belonging to the Sorangiineae family. Related indothiazinones, including ITE, were identified in Thermosporothrix hazakensis, which are bacteria found in ripe compost. In this study, we performed targeted metabolomic analyses of human sera obtained from 20 healthy human subjects, covering men and women equally, of evenly distributed ages from 20 to 70 years. We did not detect ITE or its metabolic product ITE-COOH in any examined sample, applying the limit of detection of 70 pM.
Underlying its discovery, ITE was detected and identified in porcine lungs. To date, no attempts have been made to detect or determine if ITE is found in human tissues.
Indothiazinone, a structural backbone of ITE, was found in cultures of myxobacteria belonging to the Sorangiineae family. Related indothiazinones, including ITE, were identified in Thermosporothrix hazakensis, which are bacteria found in ripe compost.
In this study, we performed targeted metabolomic analyses of human sera obtained from 20 healthy human subjects, covering men and women equally, of evenly distributed ages from 20 to 70 years. We did not detect ITE or its metabolic product ITE-COOH in any examined sample, applying the limit of detection of 70 pM.
In conclusion, our data and existing literature evidence reveal that ITE is a bacterial endosymbiont or a product of its precursor found in porcine lungs rather than a human endogenous metabolite.
Financial support from the Czech Health Research Council (NV19-05-00220) is acknowledged.
Katarina Hadova, Eva Kralova, Gabriel Doka, Lenka Bies Pivackova, Zuzana Kmecova, Peter Krenek,
Myocardial hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are known to play a role in arrhythmogenesis in some cardiac pathologies and thus they could participate in electrophysiological remodelling of the diabetic heart as well. We aimed to investigate HCN2 channels expression in several anatomical areas of diabetic rat heart and in a differentiated H9c2 cell line. To induce diabetes mellitus, rats were injected by single dose of streptozotocin (STZ, 55 mg/kg body weight, i.p.) and after 6 weeks of diabetes mellitus ECG was measured in anaesthetised rats in basal conditions. Protein expression was analysed by western blotting in the left and right atrium, left and right ventricle free walls, and intraventricular septum walls. Differentiated H9c2 cells were cultivated in high-glucose medium (33 mM), insulin (100 nM), or HNMPA(AM)3 insulin receptor inhibitor (100 nM). mRNA expression in H9c2 cells, mRNA and microRNA 1 and 133a in the left ventricle of diabetic rats were measured using the RT-qPCR method. Six weeks of diabetes resulted in reduced heart rate and prolongation of QRS complex, QT interval, and T-wave compared to controls. HCN2 expression declined exclusively in ventricle tissue of diabetic rats. No changes in HCN2 expression in atria and H9c2 cells were observed. MicroRNA levels were stable in diabetic ventricles. Our results suggest that ventricular but not atrial HCN2 channels might be an integral part of electrophysiological remodelling of diabetic heart, which could project onto changed cardiac electrical stability.
Tibor Hodbod, Kristína Szmicseková, Dávid Murčo, Zuzana Kiliánová, Dominika Dingová, Anna Hrabovská
The latest research showed that individuals with lower butyrylcholinesterase (BChE) activity are more prone to obesity. In connection with this finding, we decided to look closer at a possible link between BChE activity and obesity in rats. In our experiment, adult male Wistar rats were fed with a standard diet (SD) or high-fat diet (HFD), with or without parallel treatment with a BChE inhibitor, isoOMPA, dissolved in drinking water for 13 weeks. Food and water intake and change in body weight were monitored during the experiment. Selected tissues were collected post mortem for further anatomical, morphological, and biochemical analyses. Cholinesterase activity was measured using Ellman’s method, relative expression of cholinesterases was assessed by RT-qPCR, and epididymal adipose tissue was cut in a cryostat, stained with hematoxyline and eosine staining, and the size of adipocytes was observed by light microscopy. In rats fed with HFD, we observed higher body weight gain when compared to rats on SD. Despite higher body weight, the weight of adipose tissue did not increase, but size and area of the adipocytes were significantly higher in both HFD groups. Moreover, higher BChE activities were present in serum. Although BChE relative expression was increased in the liver, its activity level did not change in this tissue, suggesting secretion of the de novo synthesised BChE into the bloodstream. Surprisingly, systemic inhibition of BChE activity in rats on SD caused weight gain comparable to that observed in rats on HFD. No morphological changes in epididymal adipocytes were, however, observed. Our results suggest a reciprocal relationship between serum BChE activity and obesity, as obesity caused by HFD led to an increase in serum BChE activity and inhibition of BChE activity-induced weight gain in Wistar rats.
This project was supported by VEGA 1/0283/22, 1/0815/21, and SK-FR-19-0005.
Maria Hrickova, Petra Amchova, Yevheniia Babenko, Jana Ruda-Kucerova
Addiction is a major global problem and the cure for this chronic disease is still limited. Therefore, finding an effective and safe treatment is an urgent need. In recent years, addiction research has focused not only on influencing the dopaminergic pathway of reward, but also on reducing craving by reducing glutamate levels in the brain. The aims of our studies were to evaluate the effect of two different AMPA/kainate receptor antagonists, NBQX and CNQX, on nicotine and methamphetamine intravenous (IV) self-administration in rats. When animals achieved a stable intake of the drug, we administered a single IV injection of NBQX or CNQX 10 min before the session to evaluate its effect on drug intake. Subsequently, the rats were kept in their home cages for 2 weeks of forced abstinence. This period was followed by a reinstatement session, where the drug was no longer available and NBQX or CNQX was administered to evaluate the effect on drug seeking. In the case of NBQX we also assessed its effects on nicotine intake when administered for 10 consecutive days in the same manner.
There were contrasting effects of NBQX and CNQX in the nicotine dependence studies. In the acute intake phase, CNQX but not NBQX significantly reduced drug intake. Conversely, in the reinstatement, NBQX exerted a suppressing effect on nicotine seeking, unlike CNQX. Therefore, we decided to test the (sub)chronic effect of NBQX on nicotine intake and we confirmed no effect. Neither NBQX nor CNQX affected methamphetamine intake or reinstatement.
NBQX and CNQX are both antagonists of glutamatergic AMPA/kainate receptors but have considerable pharmacodynamic differences. CNQX also modulates the glycine site of the NMDA receptor. Our results may be interpreted as different involvement of individual glutamatergic receptors in different phases of the dependence model.
This report was written with the support of the Specific University Research (MUNI/A/1292/2019) provided by MŠMT.
Orsolya Hrubá1, Lucia Žigová1, Katarína Vranecová1, Petra Massarová1, Nikola Chomaničová2, Adriana Adamčíková2, Simona Valášková2, Martin Kužma3, Peter Jackuliak3, Juraj Payer3, Jan Kyselovič2,4, Andrea Gažová1
The significant increase in life expectancy seen in Slovakia in recent decades led to a change in the profile of patients being most frequently admitted in internal medicine wards. We intend to focus on the number of hospitalisations, mean age of deceased patients, and mortality rate by causes of death of patients admitted to an internal medicine ward. A retrospective study was done of patients managed in a Department of Internal Medicine between January 2010 and December 2020. Data were obtained from the database of the department. A total of 4,560 patients were included with 51.3% of them females. The outcomes of deceased patients were compared with nationwide data retrieved from the Health Statistics Yearbook of the Slovak Republic (2010–2020). The aim of this study was to analyse the demographic data, compare the hospitalised patients’ characteristics, and predictors of mortality in patients. Results of this study could contribute to reducing complications and mortality rate and easier decision making about the best care for patients.
Marta Chalupová1, Jarmila Klusáková1, Gabriela Kuzmínová1, Alžběta Kružicová1, Lucy Vojtová2, Pavel Suchý1
Surgical techniques in urology require effective hemostasis to achieve rapid control of bleeding. Therefore, different topical hemostatic agents based on various forms and mechanisms of action were introduced, but they are not universally applicable due to their specific properties. The development of new materials, which are expected to have high hemostatic efficacy, biocompatibility, and easy handling, comes to the fore. The aim of the study was evaluation of newly synthesised combined materials with collagen and carboxymethyl cellulose. Porcine collagen, equine collagen, carboxymethylcellulose, and combined materials consisting of collagen and carboxymethylcellulose were evaluated and compared with conventionally used material (Tachosil®) in a rat partial nephrectomy model. The assessed parameters included time to hemostasis and the dynamics of the tissue reaction 3 and 30 days after the procedure based on histopathological and immunohistochemical examination.
The evaluated hemostatic agents were shown to provide rapid and effective hemostasis, comparable to the reference agent. However, the results of the histopathological examination indicated a lower absorbability of carboxymethylcellulose agents and their fibrous component was probably the source of a fibroproductive, even granulomatous inflammatory reaction. Because of good hemostatic properties but poorer biodegradability, the indication of a nonimplantable hemostatic agent in clinical use can be assumed.
This work was supported by the TAČR Project TH04020540.
Nikola Chomaničová1, Adriana Adamičková1, Simona Valášková1, Miroslava Molitorisová1, Andrea Gažová2, Ján Kyselovic1,3
Currently published data suggest that the specific transcription factors GATA4 and MEF2C play a crucial role in the development of pathological cardiac hypertrophy. The synergistic effect of these two transcription factors was observed mainly with a calcineurin/NFAT signalling pathway that induces the expression of genes associated with hypertrophy. It is assumed that statins can affect the process of cardiac fibrosis and subsequent heart remodelling through cardioprotective pleiotropic effects, which are still not sufficiently examined.
The aim of the study was to investigate the intervention of atorvastatin into molecular mechanisms of cardiac fibrosis and pathological cardiac hypertrophy by monitoring changes of the expression of selected genes in human cardiac fibroblasts (HCF) in vitro. Experimental cell cultures of HCF were treated with atorvastatin (10 µM) for 24 hr, 48 hr, and 72 hr. Changes in the expression of selected genes were assessed by RT-qPCR.
Our findings reflect the occurrence of several changes in HCF after their treatment with atorvastatin. A significant reduction (
Grant support was received from APPV-18-0103, VEGA 1/0378/21.
Martin Chrastina1,2, Katarína Pružinská1,2, Ľudmila Pašková3, Veronika Vyletelová3, Karol Švík1, Katarína Bauerová1, Silvester Poništ1
In this study we investigated the potential antioxidant effect of carnosic acid and its combination with methotrexate (MTX) on mRNA expression of catalase (CAT) and heme oxygenase 1 (HO-1) in the liver as key parameters of oxidative stress in rat adjuvant arthritis (AA). We also examined arthritic score (AS) as representative clinical parameter monitored in this animal model. MTX was administrated in a subtherapeutic dose twice a week of 0.3 mg/kg b.w. The carnosic acid (CA) was administered daily in a dose of 100 mg/kg b.w., alone and in combination with MTX (CA+MTX). CA and MTX were administrated orally by gastric gavage. Plasma samples were collected on the 28th experimental day and used for PCR determination. The 28th experimental day was also used for AS analysis. We observed a significant decrease of CAT expression in the liver in the nontreated AA animal group, which indicated successful induction of AA. In MTX and CA groups, we did not observe any significant increment of CAT, but in the group with combination therapy, there was a significant increase of CAT expression compared to the AA group and to the MTX group itself. Expression of HO-1 was significantly increased in the AA group compared to the control group. MTX and CA groups did not significantly improve the levels of HO-1, but in CA+MTX we observed a significant decrease compared to the AA group. In the clinical parameter AS, we observed a very similar result as with markers of oxidative stress, meaning that CA+MTX was the only group that significantly lowered AS compared to the AA group. Monotherapy with CA as well as with MTX had no significant effect in our experiment on all evaluated parameters. Combination of CA and MTX has been proven to be more efficient than MTX in monotherapy, which might have promising effects in reducing side effects of MTX treatment by lowering the dose of MTX.
This work was supported by VEGA 2/136/20, VEGA 2/0115/19, APVV-15-0308.
Izabela Jarabicova, Csaba Horvath, Eva Velasova, Lenka Bies Pivackova, Jana Veteskova, Jan Klimas, Peter Krenek, Adriana Adameova
Pulmonary arterial hypertension (PAH) is a progressive disease with high mortality, particularly due to right ventricular (RV) failure development. Its underlying mechanisms, especially in terms of necrosis-like cell death involvement, remain poorly defined. Therefore, we investigated the role of both necroptosis and pyroptosis in the lung and RV tissue damage under the conditions of PAH and the circulating levels of receptor-interacting protein kinase 3 (RIP3), associated with both cell death modalities, were also examined.
To induce PAH, male Wistar rats were injected with monocrotaline (MCT, 60 mg/kg, s.c.) and the control group received vehicle. Based on health status, the MCT-treated animals were divided into two subgroups: the MCT group sacrificed 28 days after MCT administration and the ptMCT group terminated prematurely (on average on Day 24, 11 ± 0.7) due to showing signs of terminal PAH stage. Worsened vital functions, RV enlargement, and increased cardiac hemodynamic stress confirmed the development of PAH in the respective groups.
Immunoblotting analysis revealed increased expression of pThr231/Ser232-RIP3 in both tissue types of both PAH stages, irrespective of the disease severity. In the RVs, upregulated pThr231/Ser232-RIP3 proceeded to the activation of mixed linkage kinase domain-like protein (MLKL), as evidenced by elevated pSer345-MLKL levels, thereby indicating necroptosis execution. Contrary, in the lungs, pThr231/Ser232-RIP3 was instead associated with the pyroptotic caspase-1–interleukin-1β–gasdermin D signalling activation. The circulating RIP3 levels were elevated in both PAH stages although more evident in the terminal stage. A positive correlation between the circulating RIP3 and RV hypertrophy was found, but no association between cardiac hemodynamic stress and the plasma RIP3 was observed. Taken together, this is the first study showing a prominent role of pThr231/Ser232-RIP3 in mediating two different necrosis-like cell death forms that might underlie organ damage in PAH and suggesting the plasma RIP3 as an additional diagnostic and prognostic marker of RV injury due to PAH.
This work was supported by Grants APVV-20-0242, APVV-19-0458, APVV-15-0685, VEGA 1/0203/19, VEGA 1/0016/20.
Lenka Kalmanova, Lukas Smiesko, Jarmila Palencarova, Miroslava Molitorisova, Sona Franova
The experimental pharmacotherapy of asthma is interested in molecules with concomitant bronchodilatory, anti-inflammatory, and antiremodelling activity. More recently a new trend has emerged, focusing on using compound derivatives of natural origin. In this study, we evaluated the antiasthmatic properties of dihydroxyflavone chrysin on an experimental model of allergic asthma. Our experiments were aimed at evaluating the bronchodilatory, antitussive, and anti-inflammatory efficiency of chrysin after chronic 21 days of administration to ovalbumin (OVA) sensitised guinea pigs. The long-acting beta2 receptors agonist (LABA) salmeterol and glucocorticoid budesonide were used as antiasthmatic standards. By these measures, we evaluated the following parameters: the specific airway resistance to histamine via an in vivo method; the sensitivity of a chemically induced cough reflex via an in vivo method; and the activity of chrysin on the ciliary beat frequency (CBF) of respiratory epithelium via an in vitro method. The degree of inflammation of the airways was confirmed by assessing the leucocyte count, inflammatory cytokines concentrations Th2 (IL-4, IL-5, IL-13), Th1 (GM-CSF, INF-γ, IL-12) in the BALF, and the level of transforming growth factor beta 1 (TGF-β1) in lung homogenate.
Chronic chrysin (30 mg/kg/day for 21 days) administration to OVA-sensitised guinea pigs showed comparable bronchodilatory activity with LABA salmeterol. Chrysin revealed antitussive efficiency, but was not able to abolish the negative effect of OVA on CBF. Chrysin managed to ameliorate the progression of chronic airway inflammation. The acquired results support the complex antiasthmatic profile of chrysin that could represent an attractive compound for further studies concerning prevention or a future asthma treatment.
This work was financially supported by Grant APVV-19-033, VEGA 1/0314/21, VEGA 1/0253/19. This publication was created thanks to support under the Operational Programme Integrated Infrastructure for the project Research and Development in Preclinical Testing of Chemical Substances for Use in Healthcare, ITMS2014+ project code: 313011T433, cofinanced by the European Regional Development Fund.
Petra Karabinová1, Zdenko Pirník2,3,4, Monika Fedorová5
Activation of the renin angiotensin aldosterone system during chronic heart failure (CHHF) leads to increased ventricular preload, afterload, and myocardial wall stress, which induces production of pre-pro B-type natriuretic peptide (BNP). Pre-pro BNP is cleaved by neprilysin to active BNP (that promotes natriuresis and vasodilatation) and inactive N-terminal proBNP (NT-proBNP). The main goal of this study was to compare the efficacy of 1 year of treatment of CHHF by angiotensin receptor neprilysin inhibitors (ARNI; sacubitrile/valsartan), angiotensin-converting enzyme inhibitors (ACEI; perindopril and ramipril, respectively), and angiotensin II type 1 receptor blockers (AT1RB; losartan and candesartan, respectively) in patients with reduced left ventricular ejection fraction (LVEF). Thirty patients were randomised: Half of them received ARNI (age 63 years ± 10 years, LVEF 30.0% ± 5.2%, NT-proBNP 977 ng/l ± 577 ng/l) and the others received ACEI or AT1RB (age 60 years ± 13 years, LVEF 31.7% ± 10.0%, NT-proBNP 918.1 ng/l ± 861.9 ng/l). There was a statistically significant increase in LVEF of CHHF patients after ARNI treatment (34.7% ± 6.5%, 95% CI [−9.07, −0.33],
Lucia Karailievova1, Agnesa Puhova1, Harald Murck2,3, Daniela Jezova1
Chronic stress contributes to the development and course of several pathological states, such as major depression.
This work was supported by APVV-18-0283.
Tereza Kauerová1, Magdaléna Onuščáková2, Pavel Bobáľ2, Peter Kollár1
Histone deacetylase inhibitors (iHDACs), a class of epigenetic regulators, are substances with anticancer activity that is associated with inhibition of cell proliferation, induction of programmed cell death, inhibition of angiogenesis, and induction of the cytodifferentiation process in cancer cells. Hydroxamic acid derivatives are currently the most important group of iHDACs. Of these, three are registered as anticancer drugs: vorinostat for the treatment of advanced cutaneous T-cell lymphoma (CTCL), panobinostat for the treatment of multiple myeloma in combination with bortezomib and dexamethasone, and belinostat for the treatment of peripheral T-cell lymphoma (PTCL). However, these drugs are still under intensive research in terms of their possible use in other indications, mainly in the field of hematologic malignancies.
Our study was focused on the evaluation of biological activity of new hydroxamic acids derivates as potential iHDACs designed and synthesised by the Department of Chemical Drugs in Faculty of Pharmacy at Masaryk University. These new compounds are structurally derived from hydroxamates that effectively inhibit histone deacetylases (HDACs) and thus they possess the basic structural requirements for the ability to inhibit HDACs. Our in vitro research studied their potential to affect histone acetylation, to inhibit proliferation, and to induce apoptosis in cancer cells. Our results so far suggest that the novel group of hydroxamic acids might represent an interesting model structure for development of novel iHDACs that merits further investigation on the molecular basis of the possible anticancer effect. The results obtained in this study are beneficial for research of new substances with anticancer potential derived from vorinostat, as the biological activity of these novel hydroxamic acids was evaluated in vitro for the first time.
This study was supported by the project MUNI/A/1598/2020.
Sasan Khademnematolahi1,2, Mohsen Taghdisiesfejir1, Katarína Pružinská1, Silvester Poništ1, Karol Švík1, Jana muchová3, Katarína Bauerová1
Recent studies have demonstrated anti-inflammatory and chondroprotective properties of hyaluronic acid (HA) in rheumatoid arthritis. This study aimed to examine the combination of HA and methotrexate (MTX) in rats suffering from adjuvant arthritis (AA). In our studies, we used male Lewis rats and the arthritis was induced with
This work was supported by VEGA 2/0136/20, APVV-15-0308.
Stanislava Kosirova, Jan Klimas, Tatiana Foltanova
Potentially inappropriate medicines (PIMs) are a prevalent problem in older persons, making it challenging to improve a patient’s safety. The main objective of this study was to assess PIM use in different settings: nursing homes (NH) versus ambulatory care and home care settings (OUT). PIMs were identified according to the EU(7) PIM list. In 904 elderly patients, 50.30% NH and 49.70% OUT, women were overrepresented (70% vs. 30%). NH patients were significantly older compared to OUT patients (76.12 ± 0.33 years vs. 79.96 ± 0.38 years,
Maria Karakitsiou, Miriam Petrova, Milan Kriska, Robert Vojtko, Vasil Hricak, Kristina Hudecova, Viera Kristova
Analgesics including nonsteroidal anti-inflammatory drugs (NSAIDs) belong to one of the most frequently used medicaments worldwide. Many of them are available over the counter and could lead to gastrointestinal, cardiovascular, and renal toxicity during especially prolonged use. The aim of our study was to examine the frequency and length of analgesic use in a specific group of patients with a focus on risk perception.
A structured questionnaire was delivered to 278 patients hospitalised at the Department of Surgery of a rural general hospital in Greece. Data were evaluated using descriptive statistics. A lower percentage of patients (12.58%) used analgesics because of chronic diseases such as inflammatory musculoskeletal disorders, whereas most patients used NSAIDs for temporary sources of pain (87.42%): headache, menstrual problems, backache, toothache, or occasional joint pain. The study also revealed that most patients did not take analgesics regularly, but were rather occasional users. A visual analogue scale evaluating the risk of these drugs was marked predominantly with a score of 5. Despite the fact that many patients suffered from comorbidities that increased the incidence of adverse effects, the overall knowledge of potential risk was relatively low.
This work was supported by Grant VEGA 1/0024/21.
Michaela Krivosova1, Eva Baranovicova1, Anna Bobcakova2, Maria Skerenova1, Lubica Jesenska3, Dusan Dobrota3, Matus Dohal4, Juraj Mokry4, Peter Liptak5, Robert Rosolanka6, Zuzana Dankova1, Erika Halasova1
A positive association between anemia and all-cause mortality and cardiovascular mortality independent of age, gender, and history of cardiovascular diseases has been confirmed. Disturbed iron metabolism might also play a role in the prognosis of patients with COVID-19. Moreover, alterations of iron homeostasis can persist long after COVID-19 onset and could be associated with impaired physical performance. We aimed to evaluate the levels of parameters associated with iron metabolism in patients hospitalised with COVID-19 during a 1-week period. In our study, 53 patients were included and they were further divided into two groups according to the outcome: positive (recovery and discharge from hospital) or negative (aggravation, exitus, or both). Their blood samples were collected on Days 1, 3, and 7 during hospitalization and basic laboratory analyses were performed, including measurement of iron metabolism parameters. All patients had pathologically increased plasmatic levels of ferritin and decreased levels of transferrin during the whole observation period. We have not found any correlation between levels of these markers and patients’ prognosis. However, levels of ferritin significantly decreased and levels of transferrin significantly increased on the seventh day only in patients with a positive outcome. Further studies with a longer observation period are warranted to evaluate the period needed for reestablishment of iron homeostasis.
This project was supported by Grants APVV-18-0084, VEGA-1/0093/22, and Integrated Infrastructure Operational Program for the project New Possibilities for Laboratory Diagnostics and Massive Screening of SARS-CoV-2 and Identification of Mechanisms of Virus Behaviour in Human Body, ITMS: 313011AUA4, cofinanced by the European Regional Development Fund.
Alžběta Kružicová1, Marta Chalupová1, Gabriela Kuzmínová1, Jarmila Klusáková1, Tomáš Sopuch2, Pavel Suchý1
Wound healing is a complex and time-consuming process that is influenced by many factors. Due to internal and external factors, complications that prolong the healing time can occur, representing not only a health but also an economic problem. By choosing a suitable dressing, we try to make treatment more effective, shorten the total healing time, and minimise possible complications. It is necessary to constantly develop new materials that have suitable properties, actively promote wound healing, bring comfort to the patient, and are more affordable than currently used dressings.
Functional collagen-based wound dressings for chronic (stagnant) wounds and wounds in the phase of granulation and epithelialisation, including bleeding wounds, were prepared. To monitor biological efficacy, lyophilized pure equine and porcine collagen foams with the required physical and application properties, high absorbency, and haemostatic properties were tested in an acute excision wound in a laboratory rat. The wound size was recorded at 2, 7, and 14 days after surgery. Presence of inflammatory infiltrate and granulomatous reaction were evaluated histologically. Levels of IL-6, TGF beta, and VEGF, which play a key role in the phases of wound healing and affect the healing dynamics, were determined by western blotting.
This study was funded by the project TAČR (TH04020540).
Hana Lastuvkova1, Fatemeh Alaei Faradonbeh1, Jolana Schreiberova J.1, Milos Hroch2, Hana Faistova3, Jaroslav Mokry4, Radomir Hyspler5, Alzbeta Stefela6, Petr Pavek6, Stanislav Micuda1
Bile acids (BAs) play a significant role as signalling molecules in the pathophysiology of nonalcoholic steatohepatitis (NASH). The expression of some transporters responsible for BAs homeostasis in hepatocytes is stimulated by beta-adrenoreceptors, but their effect on BAs metabolomics is unknown. Carvedilol, nonselective beta-adrenoreceptor blocker, is routinely used for therapy of cardiovascular complications accompanying NASH. Therefore, the present study aims to investigate the effect of carvedilol on the mechanisms of BAs homeostasis using a relevant mice model of NASH. In mice, liver damage was induced by a continuous 24-week high-fat diet (HFD) with glucose/fructose in drinking water. At week 21, individual groups of animals received carvedilol (10 mg/kg/day, p.o.) for three weeks. The bile acid spectrum was thereafter analysed in bile, stool, and plasma by LC-MS method together with molecular diagnostics of BAs-related enzymes and transporters. Biochemical and histological analysis has shown the effectiveness of a high-fat diet in inducing NASH. Untreated NASH led to increased BA concentrations in plasma in association with their reduced biliary secretion. This was consistent with reduced blood-to-bile BA transport via repressed major transporters for BAs, Ntcp, and Bsep. Compensatory reaction developed in the ileum where downregulation of Asbt reabsorption transporter for BAs led to their increased fecal excretion. Administration of carvedilol significantly attenuated the severity of NASH based on the histopathological and biochemical evaluation. However, the drug did not aggravate BA retention in plasma of NASH mice consistent with unchanged BA biliary secretion or fecal output. In conclusion, carvedilol demonstrates significant hepatoprotective activity in NASH without harmful effects on BAs metabolomics. These results support the convenient use of this agent as a part of cardiovascular regimens in patients with metabolic syndrome and a high risk of NASH development.
This project was supported by Grant SVV 260543/2020.
Lucia Polyáková1, Andrea Šoltýsová2, Kristína Szmicseková1, Dominika Dingová1, Anna Hrabovská1
Butyrylcholinesterase (BChE), also known as pseudoesterase or plasma cholinesterase, is mainly a detoxification enzyme, responsible for hydrolysis of numerous xenobiotics, including drugs. More than 70 BChE mutations have been described so far in humans. Despite changed BChE activity, there is no obvious phenotype under physiological conditions, unless the individual is exposed to the BChE substrate, such as succinylcholine. The aim of this work was to assess plasma BChE in a patient who showed abnormal (prolonged) response to succinylcholine application during surgery as well as in other family members. BChE activity was assessed by Ellman’s method using butyrylthiocholine as a substrate and by ELISA using B2 18-5 primary antibodies. All samples were tested for BChE mutations by targeted sequencing. Our results revealed 20% BChE activity in the patient with succinylcholine sensitivity. The patient carried the two most common BChE mutations (A- and K-variants) and one unreported mutation (G115C). Her son who carried A- and K-variants showed a 20% decrease in BChE activity. One of his daughters with normal BChE activity harbored the K-variant, whereas the other daughter with a 20% decrease carried an F- variant that she inherited from her mother. In conclusion, we detected the most common BChE mutations in the studied family and we also detected a novel BChE mutation that leads to an 80% decrease in BChE activity.
Lucia Markusková1, Zuzana Javorová-Rihová2,3, Tomáš Fazekaš4, Anna Martinkovičová1, Daria Rábarová5, Mária Soľavová6, Dominika Dingová1, Anna Paul Hrabovská1,3
The COVID-19 disease caused by SARS-CoV-2 viral infection represents an excessive burden on health care systems in many countries of the world. Despite the implementation of public health measures and the accessibility of vaccination, their effectiveness in Slovakia is low and with the spread of new variants of the SARS-CoV-2 virus, there is still an unmet need for early and effective identification of people who are likely to develop severe to critical disease. Although several biochemical parameters associated with severe COVID-19 disease progression have been identified to date, the presence of a suitable prognostic marker of disease progression remains absent. Butyrylcholinesterase (BChE), an enzyme regulating the activity of the cholinergic system, has been investigated mainly in the past from a toxicological point of view in organophosphate poisoning and from a pharmacological point of view in the context of the metabolism of drugs such as succinylcholine or mivacurium. Its physiological function is still not well understood despite its high activity in serum. In recent years, BChE has been investigated in metabolic disease development such as obesity, hyperlipidemia, Type 2 diabetes mellitus, and inflammatory processes, factors identified as risks for the development of a more severe course of COVID-19. In our monocentric clinical study, we have focused on the relationships between changes in serum BChE activity, demographic data, and biochemical parameters in relation to disease severity, present comorbidities, pharmacotherapy, and clinical outcomes. Further understanding of these relationships provides promise for the use of new prognostic markers useful for practice.
This project was supported by VEGA 1/0283/22, 1/0815/21, and APVV SK-FR-19-0005.
Anna Martinkovičová1, Lucia Markusková1, Zuzana Javorová-Rihová2,3, Tomáš Fazekaš4, Daria Rábarová5, Mária Soľavová6, Dominika Dingová1, Anna Paul Hrabovská1,3
In early 2019, the coronavirus pandemic became a global concern, especially due to an increased burden of severe cases on the health care system. Several research teams have therefore focused on recognising the susceptible groups for a severe course of COVID-19. The aim of our project was to analyse suggested risk factors of severe COVID-19. Namely, we assessed whether certain sociodemographic data, nonvaccination, and certain comorbidities were associated with a higher risk of a severe course of COVID-19. The health status, pharmacotherapy, and demographic data were obtained from electronic medical records of patients hospitalised in the University Hospital Trnava in the Department of Anaesthesiology and Intensive Care Medicine and Department of Infectology. Patients were divided into three groups according to their outcome during hospitalization: transfer to another ward, discharge from the hospital, and death. We categorised comorbidities into groups according to the involved organ systems. Chronic pharmacotherapy was divided into subgroups using the ATC system. Consistent with previous results, nonsurvivors, compared to survivors, showed higher age, higher nonvaccination rates, and a greater number of comorbidities. The relationship between higher mortality and gender was, however, not confirmed in our cohort. Cardiovascular diseases, obesity, and Type 2 diabetes mellitus were the most frequent diagnoses in patients with severe COVID-19. Our results highlight the necessity for early monitoring of high-risk patients and implementation of appropriate treatment that could ultimately save lives and contribute to more cost-effective pandemic management.
This project was supported by VEGA 1/0283/22, 1/0815/21, and SK-FR-19-0005.
Petra Massarová1, Lucia Žigová1, Katarína Vranecová1, Orsolya Hrubá1, Nikola Chomaničová2, Adriana Adamičková2, Simona Valášková2, Jan Kyselovic2,3, Andrea Gažová1
Recently, the pleiotropic extraskeletal effects of vitamin D have received more attention in addition to its well-known importance for bone health. Research suggests that vitamin D deficiency is as associated with the risk of developing various malignancies. Increased expression of vitamin D receptors and vitamin D metabolism enzymes is observed in cancer cells. There is growing evidence that vitamin D exhibits anticancer activity via different pathways, for example, inhibition of proliferation or induction of cell apoptosis. The aim of this work was to evaluate the effect of vitamin D (in the form of cholecalciferol) on viability and relative expression of apoptosis regulators BAX and Bcl-2 in human cervical cancer cell line HeLa. Experimental cultures in eighth passage were treated with 1300 nM and 130 nM cholecalciferol for 24 hr. Viability of the cells was analysed by flow cytometry and relative expression of BAX and Bcl-2 proteins by western blotting.
Our preliminary results showed that viability of HeLa cells after cholecalciferol treatment did not change significantly compared to the control condition. The amount of BAX protein decreased more in cholecalciferol-treated cells compared to the control condition, but no significant difference was observed. HeLa cells treated with cholecalciferol also indicated slight alteration in Bcl-2 expression and BAX/Bcl-2 ratio without statistical significance. In conclusion, the treatment of HeLa cells with cholecalciferol at the used concentrations did not have a significant effect on the apoptotic signalling pathway.
This work was supported by Grants APPV-18-0103, VEGA 1/0378/21, and UK/96/2022.
Jana Mlíchová1, Ondřej Šimandl1, Eva Kmoníčková1, Zoltán Paluch2
Analgesics are among the most widely used groups of drugs. Improperly chosen therapy leads to the occurrence of side effects, worsens the quality of life of patients, and increases the economic burden on patients. We evaluated the consumption and side effects of the over-the-counter analgesics in the Czech Republic: dexketoprofen, diclofenac, ibuprofen, acetylsalicylic acid, naproxen, and paracetamol. The aim of our evaluation was to analyse spontaneous reports of suspected adverse reactions (ADRs) of monitored substances registered in the pharmacovigilance database of the State Institute for Drug Control (SÚKL) in the period from 2015 to 2020. Data on the occurrence of ADRs were extracted and categorised. In our analysis, we included the ADRs directly associated with the administration of a specific medication. We extracted and analysed a total of 186 reports: dexketoprofen, 2 (1.0%) reports; diclofenac, 38 (20.4%) reports; ibuprofen, 77 (41.4%) reports; acetylsalicylic acid, 19 (10.2%) reports; naproxen 4, (2.0%) reports; and 46 (25.0%) reports were for paracetamol, of which 13 (7.0%) were due to overdose. In terms of categorization, the most common ADRs were skin related, gastrointestinal, and central. The skin ADRs were primarily drug rashes and photosensitivity reactions. Among analgesics, consumption was clearly dominated by ibuprofen. The analysis provided us with information on the severity and frequency of ADRs. Contraindications, age and medical history of the patient, individual efficacy and tolerability, and potential drug interactions must be taken into account when selecting a specific substance. Our findings can help to select a suitable analgesic for a particular patient and contribute to safety.
Henrieta Oravcová1,2, Jana Trebatická3, Barbora Katrenčíková4, Zdeňka Ďuračková4, Daniela Ježová1
Mood disorders in children and adolescents are a serious global problem for the health care field and society. Stress hormones cortisol and aldosterone are thought to be involved in the pathophysiology of depressive disorders. There is evidence that the supplementation of omega-3 fatty acids can be helpful in the prevention and treatment of these disorders. This study examines the effects of supplementation with omega-3 fatty acids on cortisol and aldosterone concentrations in saliva obtained in the sample of children described previously. The study group involves boys and girls between the age of 7 and 18 years suffering from either mixed anxiety and depressive disorder or depressive disorder. Patients were randomly divided into two groups to receive either omega-3 fatty acids (referred to as the Om3 group) or omega-6 fatty acids as an active comparator (referred to as the Om6 control group) in addition to their antidepressant therapy. As a source of omega-3 fatty acids, a fish oil emulsion was used. A sunflower oil was used as a source for omega-6. Patients in both groups received 20 ml of oil daily for 12 weeks. Six saliva samples were collected from each patient. Sampling took place in the morning and at midday at three specific time intervals, namely at the beginning of the study (Week 0), at the middle of the intervention with fatty acids (Week 6), and at the end of the intervention with fatty acids (Week 12). Cortisol and aldosterone concentrations will be measured using ELISA and our own methodology.
The study was supported by APVV-18-0283.
Jarmila Palenčárová, Miroslava Molitorisová, Lenka Kalmanová, Lukáš Smieško, Martina Šutovská, Soňa Fraňová
This work was financially supported by Grant APVV-19-033, VEGA 1/0314/21, and VEGA 1/0253/19. This publication was created thanks to support under the Operational Programme Integrated Infrastructure for the project Research and Development in Preclinical Testing of Chemical Substances for Use in Healthcare, ITMS2014+ project code: 313011T433, cofinanced by the European Regional Development Fund.
Iveta Pavlova1,3, Eva Drazanova1,2, Lucie Kratka1, Petra Amchova2, Ondrej Macicek1, Jana Starcukova1, Zenon Starcuk1, Jana Ruda-Kucerova2
A general brain metabolic state, its detection, and quantification might give us a useful insight into major depressive disorder with the potential for developing innovative antidepressants. Two nuclear magnetic resonance techniques were used to examine and assess brain metabolic state in the olfactory bulbectomised (OBX) rat model of depression and sham-operated control male rats. Moreover, the left and right sides of bilateral brain regions were compared within the two examined groups.
The blood–brain perfusion was detected by the arterial spin labelling (ASL) experiment, which found no differences in the group comparison. The lateral differences of cerebral blood flow were shown in the thalamus of the control group and in the sensorimotor cortex of the OBX group, indicating neuronal asymmetry in these brain regions. Information about relative brain metabolite concentration was obtained by proton magnetic resonance spectroscopy (1H MRS). Two volumes of interest were examined: left and right hippocampus. The spectra with creatine and phosphocreatine (total creatine: tCr), N-acetyl-aspartate, choline, glutamate, glutamine, myo-inositol, and taurine as key brain metabolites were processed, and the relative concentrations were calculated. We observed the following differences in the metabolite ratio levels: lower choline to tCr and lower choline to N-acetyl-aspartate in lesioned rats, which might reflect the role of choline in acetylcholine production and release, thus a lower ability of memory storage in OBX rats. The lateral differences of metabolite ratios in the hippocampus were found within both examined groups: myo-inositol to tCr in the control group, indicating its lateral distribution in the hippocampus of the healthy brain; and choline to N-acetyl-aspartate, N-acetyl-aspartate to tCr, and taurine to tCr in the lesioned group, suggesting different neuronal activity and density between the left and the right side of the hippocampus of the model group.
The study was suported by MEYS Grants MUNI/A/1440/2021, LM2015062, and CZ.02.1.01/0.0/0.0/18_046/00
Katarína Pružinská1,2, Martin Chrastina1,2, Silvester Poništ1, František Dráfi1, Mohsen Taghdisiesfejir1, Sasan Khademnematolahi1, Karol Švík1, Katarína Bauerová1
During rheumatoid arthritis (RA), there is a shift in redox balance. Natural compounds play a major role as an adjuvant in reducing the symptoms of RA. In this study we evaluated the effect of carotenoids (astaxanthin, beta-carotene, and beta-cryptoxanthin) in monotherapy or in combination therapy with methotrexate (MTX) on clinical parameters during adjuvant arthritis (AA). Male Lewis rats were divided into 8 groups. The AA was induced by an injection of
This work was supported by Grants VEGA 2/0136/20, APVV-15-0308, and APVV SK-CN-21-0039.
Zuzana Romanova1,2, Eva Kornanova3, Alexandra Garafova1,4, Miroslav Borovsky3, Daniela Jezova1
Induced abortion from genetic indication or for other medical reasons in women in the second trimester is a strong stressor and can lead to mental disorders. This study will examine the stressfulness of the life events related to a medical induction abortion by measurement of cumulative concentrations of cortisol in hair. The women undergoing medical induction abortion will be recruited at the First Department of Obstetrics and Gynecology, University Hospital, School of Medicine, Bratislava, Slovakia. The follow-up visit will be held at the Research Clinic in the Pavilion of Medical Sciences, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia, 2 months after the intervention. The saliva and hair samples (2 cm proximally from the scalp) will be collected during each visit. Hair samples 2 cm long reflect 2-month retrograde cumulative cortisol secretion. Cortisol extraction from hair will be performed according to our own modification of the methodology and analysed using ELISA (IBL, International, Hamburg, Germany). Examinations of mental state will be done 1 day before and 2 months after the intervention. To avoid the influence of daily rhythm, the saliva samples will be collected in the afternoon between 13:00 and 16:00. Salivary and hair concentrations of cortisol will be correlated with scores obtained on questionnaires evaluating stress coping.
The study will be supported by Grants VEGA 2/0022/19 and APVV-18-0283.
Note: also published as short communication
Eur. Pharm. J. 2022, 69(s1), 82–83
Žofia Sládečková, Anna Paul Hrabovská, Dominika Dingová
Butyrylcholinesterase (BChE) is a serine hydrolase with broad distribution in the body. Although its physiological function remains unknown, its activity change in biological samples was observed in various pathologies. Usually, biological samples need to be sent to laboratories for determination of BChE activity. However, papers published online refer to varying storage conditions, without any relevant data about enzyme stability. The aim of this project was to determine the time-dependent stability of BChE activity in biological samples under different storage conditions. We have stored human blood serum and tissue extracts from rat heart, mouse brain, and mouse lungs at 4°C, −20°C, and −80°C for 6 months. BChE activity in samples stored at 4°C was measured daily for 10 days, weekly for 1 month, and monthly for 6 months by modified Ellman’s assay at 412 nm using butyrylthiocholine as substrate. BChE activity in frozen samples was measured weekly for 1 month and monthly for 6 months by the method previously described. Effect of the freezing and defrosting of the biological samples on enzyme stability was also tested. The results showed no change in BChE activity of all tested samples stored at 4°C for the first 10 days. Prolonged storage at 4°C, −20°C, and −80°C for 6 months also did not affect BChE activity. Surprisingly, we did not observe any change in the activity of BChE in the samples that were frozen and defrosted in the five-round cycle. The results showed that BChE activity in human blood serum, rat heart, mouse brain, and mouse lungs is highly stable and samples can be used for long-term measurements without loss of its activity.
This project was supported by Grants VEGA 1/0283/22, 1/0815/21, and SK-FR-19-0005.
Lukas Smiesko, Michaela Molitorisova, Jarmila Palencarova, Lenka Kalmanova, Martina Sutovska, Sona Franova
The Rho-kinase signalling pathway plays a significant role in regulating allergic inflammation. Rho-associated kinases belong to the Ser/Thr-protein kinases family, and two isoforms of ROCK—ROCK1 and ROCK2—have been identified. Cytokines involved in allergic inflammation produced by Th2 cells such as IL-4, IL-5, IL-9, and IL-13 increase the Rho-kinase pathway activity. Rho-kinase signalling presumably contributes to profibrotic processes of both epithelial and endothelial cells in response to tissue damage. This assumption has been confirmed in idiopathic pulmonary fibrosis. However, Rho-kinases are supposed to be involved even in other processes; for example, airway remodelling in allergic asthma. Based on the existing knowledge, this study aimed to evaluate the pharmacological profile of Rho-kinase inhibitor hydroxyfasudil in conditions of experimentally induced allergic inflammation in guinea pigs after 14 days of its systemic administration.
Chronic allergic airway inflammation was induced by 28-day sensitisation of guinea pigs with allergen ovalbumin (OVA). The effect of the Rho-kinase inhibitor on airway reactivity and cough reflex sensitivity was evaluated in vivo, and the cilia beating frequency (CBF) was tested in vitro. The multiplex method (BioPlex) and the ELISA method were used to determine the levels of inflammatory cytokines, factors involved in tissue remodelling, and transcription factors. The results of our experiments demonstrated the bronchodilatory, anti-inflammatory, and antiremodelling effects of hydroxyfasudil after its systemic administration. These findings indicate that RhoA/Rho-kinase is involved in the pathophysiology of allergic airway inflammation and suggest that Rho-kinase inhibitors have therapeutic potential for the treatment of asthma.
This work was financially supported by Grants APVV-19-033, VEGA 1/0314/21, and VEGA 1/0253/19. This publication was created thanks to support under the Operational Programme Integrated Infrastructure for the project Research and Development in Preclinical Testing of Chemical Substances for Use in Healthcare, ITMS2014+ project code: 313011T433, cofinanced by the European Regional Development Fund.
Tomáš Smutný,1 Lucie Smutná,1 Lukáš Lochman,2 Zdeňka Krajníková,1 Petr Pávek1
Pregnane X receptor (PXR) is the pivotal liver-enriched transcription factor. It is a well-recognised xenobiotic sensor that is activated by many compounds. Of them, rifampicin (RIF) is a clear-cut ligand of human PXR. RIF is metabolised by arylacetamide deacetylase (AADAC) yielding 25-deacetyl metabolite. It is also a subject of nonenzymatic autoxidation to produce RIF quinone. Other reported degradation products are 3-formylrifamycin and RIF N-oxide. In our work, we observed RIF spontaneous instability in cultivation medium, in which the concentration of RIF quinone increased in a time-dependent manner. Because it is scarcely covered by the literature, we decided to compare the potency of RIF derivatives to activate PXR employing two different gene reporter assays. Among the tested RIF derivatives, 25-desacetyl RIF showed the lowest affinity toward PXR. On the other hand, RIF N-oxide, 3-formylrifamycin, and RIF quinone revealed a similar activation of PXR to that of RIF. As RIF quinone is produced in a significant extent in medium under standard cultivation conditions, it can participate in PXR activation along with RIF in in vitro experiments.
Next, RIF increases its own metabolism, which is nearly completed within a week of repeated RIF dosing, leading to a decrease in its half-life. This is believed to be caused by enhanced deacetylation. Therefore, we used hepatocyte spheroids, which maintain hepatic relevant phenotype for several weeks, to study changes in AADAC expression within a long exposure period with RIF. Although AADAC expression was slightly downregulated in the early treatment period, expression remained at basal levels on Day 7. We can hypothesise that other factors such as enhancement in biliary transport could underlie autoinduction of RIF elimination.
Overall, our data demonstrate that RIF is spontaneously transformed to RIF quinone in medium to contribute to PXR activation. A reduction in RIF half-life after repeated administration might be explained by upregulated biliary transport rather than enhancement of AADAC expression.
Ondřej Šimandl1, Jana Mlíchová1, Eva Kmoníčková1, Zoltán Paluch2
Inhaled corticosteroids (ICS) are one of the basic pillars of pharmacotherapy for many lung diseases. However, their use in clinical practice is also associated with the risk of adverse drug reactions (ADRs). This includes gingivitis, which is “rare” in patients treated with ICS according to the Summary of Product Characteristics (SmPC). Using questionnaires attached to medicines dispensed in the pharmacy, we tried to evaluate the incidence of selected ARDs—especially sores, tooth decay, and gingivitis—and the degree of their occurrence in the way of oral care. After 6 months of data collection (143 respondents, 63.6% used ICS), we concluded that the majority (92.3%) of patients performed some form of oral care, most often mouthwash (52.7%), after inhalation of an ICS-containing medicinal product. Teeth cleaning (7.7%) was the least used practice. Gingivitis was present in 11 (12.1%) of them. Gingivitis was the only ADR that bothered patients in 63.6% of cases. Sores (7.7%) and tooth decay (4.4%) were recorded to a lesser extent in the monitored population, which corresponded to the incidence according to SmPC. As our study showed, it can be assumed that the incidence of gingivitis as a side effect associated with ICS treatment is likely to be higher than indicated in the SmPC, perhaps very common instead of rare. However, further research is needed to confirm our partial conclusions based on real practice. It is already clear, though, that proper oral care can help minimise the risk of gingivitis in patients treated with ICS.
Iveta Štempeľová1,2, Monika Fedorová1, Tímea Oršuláková1
Drugs contain a complex of substances with different irreplaceable functions. Excipients have been considered inert, but currently the influence of excipients with a known action or effect on health is known. Excipients’ variability, potential interactions, medication overuse, and patients’ predisposition can lead to the manifestation of adverse reactions. Therefore requirements for excipients’ safety increase. Informing pharmacists and patients about the effects and risks of excipients with a known action or effect on health can improve pharmacotherapy safety and patients’ compliance.
This work deals with sweeteners and colorants in authorised medicines in Slovakia available without medical prescription, which contain active substances from the ATC group of Stomatological Preparations (A01) and Throat Preparations (R02). Examples include aspartame, maltitol, sunset yellow, and azorubine, which according to the annex to the European Commission guideline, “Excipients in the labelling and package leaflet of medicinal products for human use” (EMA/CHMP/302620/2017), can be unsuitable for specific groups of patients. Work also contains pharmacists’ opinions (103 pharmacists worked in pharmacies in Slovakia) on this topic. Seventy-four percent of respondents considered informing patients about excipients with a known action or effect and their effects on health important. Twenty-four percent of pharmacists had experience with patients’ interest in the effects of excipients in medicinal products.
Martina Šutovská, Jozef Mažerik, Soňa Fraňová
The role of TRP ion channels in the regulation and activation of inflammatory processes associated with allergic asthma is well known. Morphological studies have also confirmed presence of TRPV4 channels on both excitable and nonexcitable airway cells. The aim of this study is to determine the effects of the TRPV4 blocker on airway defence mechanism and chronic inflammation in an experimental model of ovalbumin-induced allergic asthma. The TRPV4 blocker was administered by inhalation and its effect was compared with the control antiasthmatics budesonide and salbutamol. The airway defence reflexes analysis consisted of airway reactivity analysed by specific airway resistance (sRaw), contraction strength of isolated airway smooth muscles, and mucociliary clearance expressed by ciliary beating frequency (CBF). The immuno-biochemical markers of chronic inflammation were evaluated by BioPlex and ELISA assays. The processed data show a significant antiasthmatic effect of the TRPV4 blocker associated with a decrease in sRaw values and inhibition on acetylcholine-induced contraction of isolated airway smooth muscle. The anti-inflammatory effect is demonstrated by the suppression of Th2 cytokine synthesis. Effect of the inhaled TRPV4 blocker on CBF was negligible. Inhaled TRPV4 blockers have the potential to be used in the treatment of allergic asthma, as their administration has been shown to suppress chronic inflammation and airway hyperresponsiveness without a concomitant negative effect on mucociliary clearance.
This work was financially supported by Grants APVV-19-033, VEGA 1/0314/21, and VEGA 1/0253/19. This publication was created thanks to support under the Operational Programme Integrated Infrastructure for the project Research and Development in Preclinical Testing of Chemical Substances for Use in Healthcare, ITMS2014+ project code: 313011T433, cofinanced by the European Regional Development Fund.
Magdalena Sustkova, Chrysostomos Charalambous, Anna Khryakova, Alina Certilina
Cannabinoid-associated use disorders and dependence are increasing at an alarming rate; however, an effective treatment is lacking. Recently, the growth hormone secretagogue receptor (GHS-R1A) antagonism was suggested as a promising mechanism for drug addiction therapy. Nevertheless, the role of GHS-R1A and its endogenous ligand ghrelin in cannabinoid abuse remains unclear. Similar to the case with other addictive drugs, cannabinoid-induced behavioural stimulation is considered to be a sign of dopaminergic nigrostriatal pathway activation that contributes to drug dependence. The aim of our research was to investigate whether the GHS-R1A antagonist JMV2959 could reduce the behavioural stimulation induced by tetrahydrocannabinol (THC) the synthetic cannabinoid WIN55,212-2, or both in rats.
We used the LABORAS cage, which is a fully automated system for continuous behaviour recognition and tracking of rodents. The following behavioural categories were evaluated: time spent in locomotion, immobile, rearing, distance, and average speed. Rats were placed into the LABORAS cage immediately after i.p. injection of saline or JMV2959 (1 or 3 mg/kg) for habituation. Twenty minutes later, 0.1 mg/kg THC or WIN55,212-2 or vehicle was administered i.p., and 20 min later, the monitoring period started. Behaviour changes were thus measured 20–40 min after cannabinoid, when significant cannabinoid-induced behavioural stimulation could be observed. The vehicle + saline group served as a control group. One-way analysis of variance followed by the Holm–Sidak post hoc test was used for statistical analysis.
The locomotion, rear, distance traveled, and average speed after both cannabinoids administration were significantly increased, whereas immobility was decreased in comparison to the control group. The 1 or 3 mg/kg JMV2959 administered 20 min before THC significantly and dose-dependently reduced the THC-induced changes in all monitored parameters. In the case of WIN55,212-2, only pretreatments with 3 mg/kg JMV2959 reached significance in effects. Both doses of JMV2959 administered alone or with the vehicle did not cause significant changes in rat behaviour. Our results demonstrated that the GHS-R1A receptor antagonism reduced both cannabinoids-induced behavioural stimulation, which encourages further research of the GHS-R1A antagonism as a potential new approach to cannabinoid addiction treatment.
The investigation was supported by Grants GACR 21-30795S, SVVV 260533/SVV/2022, COO38, and COO41.
František Trejtnar, Maixnerová Jana, Eva Teichmanová
An important part of low-molecular drugs are organic cationic compounds. Organic cation transporter (OCT2) localised mainly in the kidney can be responsible for renal excretion of this type of drugs via the kidney. The significant role of OCT2 in renal elimination has also been proven in an important agent against HIV and hepatitis B virus, lamivudine. The drug is used in patients with various concomitant diseases treated with other medicines such as cardiovascular drugs or anti-infectives. Many of these drugs are also substrates of the renal OCT2. Consequently, drug–drug interactions during renal excretion in the kidney could potentially disturb lamivudine pharmacokinetics. The aim of this study was to analyse possible interaction of lamivudine with two drug OCT2 substrates, a beta blocker nadolol and an anti-infective agent pentamidine, using a perfused rat kidney model. Rat kidney was perfused in a recirculation regimen at a constant pressure. A therapeutic concentration of lamivudine and therapeutic concentrations of nadolol and pentamidine were tested. The analysis of excretion parameters of lamivudine in the perfused rat kidney confirmed the expected significant contribution of secretion in the renal tubules to lamivudine renal excretion. The excretion rate of lamivudine was not significantly affected at therapeutic concentrations of nadolol or pentamidine. However, both tested OCT2 substrates slightly reduced the renal clearance of lamivudine. In conclusion, the results confirm the mode of lamivudine renal excretion and suggest the relative safety of combining lamivudine with nadolol or pentamidine regarding the renal OCT2-mediated drug–drug interactions.
The study was supported by the Charles University Grant Progres Q42.
Eva Velasova1, Katarina Lelkova1, Jana Veteskova1, Zuzana Kmecova1, Margareta Marusakova1, Gabriel Doka1, Lenka Bies Pivackova1, Jan Klimas1, Peter Krenek1
PAH – pulmonary arterial hypertension leads to a progressive increase in pulmonary vascular resistance, right heart failure and adverse cardiorenal interactions. Preclinical studies have shown an association between elevated serotonin (5-HT) levels and disease progression, however, the local pathogenic roles of individual components of 5-HT system in the lung in PAH are not well known. Short treatment of experimental PAH with riociguat may have an impact on the 5-HT system. We also examined whether riociguat could alleviate renal damage associated with cardiorenal interactions in PAH. We used rat monocrotaline model of PAH (60 mg/kg, s.c.). Rats were divided into 4 groups: CON-control, MCT-rats with established PAH, RIO-healthy rats with administered riociguat, MCT+RIO-rats with established PAH treated with riociguat. 2 weeks after MCT, we started treatment with riociguat (10 mg/kg/d, p.o.). In forth week rats were sacrificed. We used RT-qPCR to monitor mRNA changes of components of serotoninergic system in the lungs and right ventricle, and renal damage markers: Kim1, Nphs1, Epo and Ren. MCT caused RV hypertrophy and increased lung weight (p<0.05 vs CON). RIO did not revert these changes. MCT in the lungs increased the expression Tph1, Htr1b and Htr2b genes and decreased that of Sert. Riociguat treatment (MCT+RIO) normalised these changes (p<0.05 vs MCT) with the exception of decreased Sert expression. In the kidney, we observed a 5-fold increase in expression of Kim1 and a 50% increase in Nphs1 mRNA by MCT (p<0.05 vs CON), expression of both genes were normalised in MCT+RIO (p<0.05 vs MCT). 5-HT - related genes alterations in the lungs of PAH rats are indicative of increased 5-HT signalling and they are partly normalised after of treatment with riociguat. At the same time, riociguat had beneficial effects on renal damage markers in PAH.
Katarína Vranecová1, Orsolya Hrubá1, Petra Massarová1, Lucia Žigová1, Adriana Adamičková2, Matúš Adamička3, Nikola Chomaničová2, Ján Kyselovič2,4, Andrea Gažová1
Cell therapy represents an innovative approach in treatment for many injuries. Stem cells can also be applied in dental treatment to regenerate bone tissue in the craniofacial region. It has been shown that vascular endothelial growth factor A (VEGFA) and hepatocyte growth factor (HGF) play an important role in this type of regeneration. However, due to complications such as pain, it is necessary to study the effect of pharmacotherapy used because of its potential influence on applied stem cells and subsequently molecular regenerative processes.
The main goal of this work was to evaluate the effect of ibuprofen on human dental pulp stem cells (DPSC) in vitro and to identify relative mRNA expression levels of growth factors, VEGFA and HGF. DPSC in ninth passage was incubated with 300 µM ibuprofen for 24, 48, and 72 hr. Total RNA was extracted using phenol-chloroform extraction. The relative mRNA expression levels of VEGFA and HGF were measured by RT-qPCR method. Our preliminary data showed a significant increase in relative mRNA expression levels of VEGFA and HGF after influence of DPSC by ibuprofen at all time intervals. This suggests that the possible use of nonsteroidal anti-inflammatory drugs in parallel with cell therapy could be beneficial, but to confirm this phenomenon further research is needed.
This work was supported by Grants UK/156/2022, VEGA 1/0378/21, and APVV-18-0103.
Martin Wawruch1, Jan Murin2, Tomas Tesar3, Miriam Petrova1, Robert Vojtko1
Antiplatelet therapy constitutes one of the most important secondary prevention measures in patients after a transient ischemic attack (TIA). This study was aimed at evaluating nonpersistence with antiplatelet medications in older patients after a TIA and identifying patient-related characteristics associated with the probability of nonpersistence during the follow-up period. Data for our study were collected from the database of the largest health insurance provider in Slovakia. Our study cohort included 1,298 patients (62.1% of them women) in whom antiplatelet therapy was initiated after a TIA. Patients were followed for 3 years from the index date. Patients with a treatment gap, defined as a 6-month period without any antiplatelet medication prescription, were classified as nonpersistent. The Cox regression model was applied to identify patient-related characteristics associated with the patient’s likelihood of nonpersistence. At the end of the follow-up period, 647 (49.8%) of the 1,298 patients were found to be nonpersistent with antiplatelet medication. Older age (≥ 65 years; hazard ratio [HR] = 0.54), switch in the antiplatelet medication use (HR = 0.57), and certain comorbid conditions: Arterial hypertension (HR = 0.70), diabetes mellitus (HR = 0.69), hypercholesterolemia (HR = 0.72), and dementia (HR = 0.70) represented factors associated with a decreased probability for a patient becoming nonpersistent. The results of our study suggest that TIA patients younger than age 65 and those without certain comorbid conditions or switching antiplatelet medication require special attention aimed at improving their persistence with this medication.
This study was funded by a grant from the Scientific Grant Agency of the Ministry of Education, Science,
Petra Zatovkaňuková, Jiří Slíva
Pharmacovigilance is an integral part of medicine. Using a medicinal product always comes with a risk of side effects, which can in most cases be quite trivial, but sometimes also life-threatening or even fatal. The burden of adverse reactions falls not only on the health of the patient, but also on the health and social system and its costs. Therefore, the imperative of current clinical practice is not only to strive for maximum effectiveness of the administered drug, but also its safety, which means reducing the likelihood of occurrence and intensity of adverse effects or drug interactions. Requirements for pharmacovigilance practice vary in different regions of the world, however. In our work, we are currently focusing on a comparison of the current system in the Czech Republic and other European (EU and non-EU) and non-European (Canada, Japan, and China) countries. This approach could lead to improved general awareness of pharmacovigilance among health care professionals as well as the general public.
Lucia Žigová1, Katarína Vranecová1, Petra Massarová1, Orsolya Hrubá1, Nikola Chomaničová2, Adriana Adamičková2, Simona Valášková2, Martin Kužma3, Peter Jackuliak3, Juraj Payer3, Jan Kyselovic2,4, Andrea Gažová1
The global pandemic caused by the SARS-COV2 virus persists. Coronaviruses causing COVID-19 disease interact with ACE-2 receptors and penetrate host cells by endocytosis. This process can lead to a rapid release of proinflammatory mediators, which is one of the factors responsible for the development of one serious complication of the disease, acute respiratory distress syndrome. The altered regulation of specific cytokines and growth factors that affect various physiological processes, such as immune responses, can exacerbate the progression of viral diseases and contribute to the pathogenesis of COVID-19 disease. The aim of this study was to compare relative expression of selected growth factors (IGF-1, FGF-2, VEGF-A) in plasma samples from patients with and without COVID-19 disease. To measure relative expression RT-qPCR was used.
Our data showed that relative expressions of selected growth factors in 3 groups of patients (6 patients cured from the disease, 6 patients who succumbed to the disease, 6 COVID-19 negative patients) are altered. The relative expression of VEGF-A was increased in patients who died of COVID-19 disease. A decrease in the relative expression of FGF-2 was observed in patients who overcame the disease compared to patients who succumbed to the disease. Differences in relative expression of IGF-1 were very slight in all three patient groups. These changes all suggest the importance of examining specific growth factors and their levels in relation to the development of COVID-19 disease.
This work was supported by the Grants APVV-20-0101 and UK/165/2022.