Pelargonidin, a natural anthocyanidin, is known for its anti-inflammatory, antioxidant, and cytoprotective properties, however its role in myocardial fibrosis remains unclear. This study explored the therapeutic potential of pelargonidin in a mouse model of isoproterenol (ISO)-induced myocardial fibrosis. Male C57BL/6 mice were treated with ISO and subsequently administered either a low (20 mg/kg/day) or high (40 mg/kg/day) dose of pelargonidin, with captopril (15 mg/kg/day) serving as a reference control. Histological analysis revealed that pelargonidin significantly reduced collagen deposition in the myocardium in a dose-dependent manner. Molecular assessments showed decreased protein expression of alpha-SMA, COL3A1, and FN1, along with downregulation of TGF-beta/Smad2/3 signaling, as evidenced by reduced levels of TGF-beta and phosphorylated Smad2/3. Additionally, pelargonidin suppressed the expression of extracellular matrix-related genes and decreased circulating levels of Th2 cytokines IL-4 and IL-13. These findings indicate that pelargonidin mitigates myocardial fibrosis by targeting the TGF-beta/Smad2/3 pathway and modulating Th2-mediated immune responses. Overall, the results suggest that pelargonidin may serve as a promising therapeutic agent for myocardial fibrosis and related cardiovascular disorders. Key words Pelargonidin " Myocardial fibrosis " Transforming Growth Factor-beta " Extracellular matrix " Cytokines.
The present study was aimed to investigate whether trimethylamine-N-oxide (TMAO) contributed to kidney aging by activating necroptosis. Male C57BL/6J mice were randomly divided into Control group (3 months old) and Old group (18 months old), compared to 3-month-old controls, 18-month-old male C57BL/6J mice showed significant increases in plasma creatinine (Cre) and blood urea nitrogen (BUN) (P<0.05), enhanced renal fibrosis (P<0.001), elevated plasma TMAO (P<0.01), and upregulation of senescence markers p53, p21, and p16 (P<0.05, P<0.01, and P<0.001, respectively). In order to investigate the effects of TMAO on kidney aging, the mice were intraperitoneally injected with TMAO for one to three months, mice showed time-dependent increases in Cre and BUN (P<0.05, respectively), progressive fibrosis, and gradual upregulation of senescence markers, ZBP1, and phosphorylation of RIPK3 and MLKL (P<0.05, respectively). In addition, three months of DMB treatment (inhibitor for TMAO formation) significantly reduced the plasma Cre and BUN levels (P<0.001 and P<0.05), downregulated the senescence markers expression, and improved kidney fibrosis (P<0.001 or P<0.05, respectively). In conclusion, our studies revealed that TMAO induced kidney aging by activating ZBP1-mediated necroptosis. Moreover, the inhibition of TMAO generation might be a potential treatment for kidney aging. Key words Kidney aging " Trimethylamine-N-oxide " ZBP1 " Necroptosis " DMB.
The aim of the study was to analyze changes in platelet count and function in hospitalized COVID-19 patients and compare them with non-COVID-19 patients, focusing on the association between platelet indices and mortality beyond the known link between neutrophil-to-lymphocyte ratio (NLR) and COVID-19 severity. The study sample consisted of 572 patients, out of which 472 were hospitalized with COVID-19 infection from 15th October 2021 to 30th April 2022 in Louis Pasteur University Hospital Kosice, Slovak Republic and 100 represented the control group without COVID-19 infection. COVID-19 positive patients (n=472) had significantly larger size of platelets (MPV 9.2+/-1.4 vs. 8.8+/-1.2, p=0.002) and therefore a higher percentage of platelets larger than 12 fl (P-LCR 33.7 % vs. 24.8 %, p=0.002) than patients in the control group (non-COVID19). The statistically significant relationship was between mortality in patients with COVID-19 infection (n=122) and the larger size of the platelets (MPV), higher platelet large cell ratio, (P-LCR), higher PLT/MPV ratio, higher platelet distribution width to plateletcrit ratio (PDW/PCT), higher neutrophil-to-lymphocyte ratio (NLR) (p<0.001, respectively) and lower platelet count (PLT) and lower plateletcrit (PCT) (p=0.006; p=0.028; respectively). In multivariable logistic regression analysis, a significant positive correlation between mortality, MPV (OR 2.29; 95 % CI 1.70-3.08, p<0.001) and age (OR 1.06; 1.03-1.08, p<0.001) was observed. When NLR was included into this model, MPV was stronger predictor of mortality (OR 2.48; 95 % CI 1.79-3.43, p<0.001) compared to NLR (OR 1.06; 95 % CI 1.03-1.08, p<0.001) and age (OR 1.04; 95 % CI 1.02-1.07, p<0.001). MPV is a strong and independent predictor of mortality in hospitalized COVID-19 patients, demonstrating superior prognostic value compared to established association between neutrophil-to-lymphocyte ratio (NLR) and COVID-19 severity. As a simple and routinely available parameter from standard blood count, MPV may serve as a practical and accessible tool for early risk stratification in the clinical management of COVID-19. Key words Platelets " COVID-19 " Mean platelet volume " Neutrophil to lymphocyte ratio " Mortality.
Heart failure (HF) is a prevalent cardiovascular condition among the elderly population, with an incidence rate that continues to rise annually, highlighting the urgent need for effective therapeutic interventions. Sustained activation of Toll-like receptor 4 (TLR4) may contribute to left ventricular dysfunction and adverse cardiac remodeling through the induction of myocardial inflammation and oxidative stress - pathological processes that closely align with the hallmark features of HF. Preclinical studies in animal models have demonstrated that TLR4 deficiency improves cardiac function in aged mice; however, the precise role and underlying mechanisms of TLR4 in human HF remain poorly understood. This study aims to test the central hypothesis that TLR4 serves as a critical molecular link between chronic inflammation and the pathophysiology of HF. HF was induced in 18-month-old male C57BL/6J mice via continuous subcutaneous infusion of isoproterenol (ISO, 30 mg/kg/day) over a period of 3 weeks. Thereafter, mice received daily intraperitoneal injections of the TLR4 inhibitor TAK-242 (2 mg/kg), deoxyribonuclease I (DNase I, 5 mg/kg), or the peptidylarginine deiminase 4 (PAD4) inhibitor GSK484 (4 mg/kg) for 7 consecutive days. Cardiac function was assessed using a ultrasound imaging system. HE staining and Masson staining were employed to evaluate myocardial pathological changes and collagen deposition. ELISA was performed to measure serum levels of myeloperoxidase-DNA (MPO-DNA), neutrophil elastase-DNA (NE-DNA), cTnI, NT-proBNP, IL-1beta, IL-6 and TNF-alpha. Immunofluorescence staining was performed to detect the co-localization levels of Ly6G with myeloperoxidase (MPO) and citrullinated histone H3 (cit-H3) in myocardial tissue, in order to assess the formation level of neutrophil extracellular traps (NETs). Western blot were utilized to determine the expression level of TLR4 protein. The expression of TLR4 was significantly upregulated in the myocardial tissue of aged HF mice. Inhibition of TLR4 not only markedly improved cardiac function but also alleviated pathological damage to myocardial tissue and reduced collagen fiber deposition. Concurrently, it also decreased the serum levels of MPO-DNA, NE-DNA, NT-proBNP, cTnI, and inflammatory factors. Moreover, the colocalization levels of Ly6G with MPO or cit-H3 in myocardial tissue was also diminished. These findings were consistent with the effects observed following DNase I and GSK484 interventions. Targeting TLR4 can mitigate inflammatory responses and enhance cardiac function in HF mice by inhibiting NETs formation. Key words Heart failure " Cardiac function " Inflammation " Toll-like receptor 4 " Neutrophil extracellular traps.
The aim of this study was to investigate the mechanisms by which glucagon-like peptide-2 (GLP-2) improves metabolic dysfunction-associated steatotic liver disease (MASLD) induced by free fatty acids (FFAs) in HepG2 cells, with a focus on the regulation of the adiponectin (ADPN) signaling axis and the downstream AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor alpha (PPARalpha) pathway. An MASLD model was established in HepG2 cells by FFA exposure. Following GLP-2 treatment, improvements in lipid metabolism were evaluated using the Cell Counting Kit-8, Oil Red O staining, and biochemical assays. Differential gene expression was examined using RNA sequencing, and potential mechanisms were evaluated through Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were performed to assess the expression of key molecular components within the signaling pathway. FFA treatment led to significant lipid accumulation in HepG2 cells, whereas GLP-2 reduced intracellular lipid droplet formation (p<0.01) and decreased triglyceride and total cholesterol levels in a dose-dependent manner (p<0.05). KEGG enrichment analysis indicated that GLP-2 acted on the adipokine, AMPK, and PPARalpha pathways. Western blotting and RT-PCR confirmed that GLP-2 restored protein expression (p<0.01) and mRNA expression (p<0.05) of adiponectin receptor 1, adiponectin receptor 2, and downstream signaling molecules AMPK and PPARalpha in FFA-treated HepG2 cells. GLP-2 alleviated FFA-induced hepatocyte steatosis by modulating the AMPK/PPARalpha pathway through the regulation of ADPN and its receptors. These findings provide a theoretical foundation for the potential use of gut hormones in the treatment of MASLD. Keywords Adiponectin " AMPK " Glucagon-like peptide-2 " Metabolic dysfunction-associated steatotic liver disease " PPARalpha.
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic progressive disorder characterized by an excess accumulation of lipids in the liver. The aim of this study was to examine the role of bilirubin (BR), the catabolic heme product and a putative peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, in an in vitro model of MASLD. In our study, we used human hepatoblastoma HepG2 cells exposed to oleic (OA)/palmitic acid (PA) (2:1 ratio, 24 h) with subsequent treatment with BR or fenofibrate (FF, a clinically used PPARalpha agonist) at clinically relevant concentrations. A significant increase in total cellular lipid content after OA/PA treatment (p<0.05) was observed. When treated with BR and FF, intracellular concentrations of OA and PA decreased significantly (p<0.05). Changes in lipid content were attenuated by GW6471 (a PPARalpha antagonist) indicating the importance of PPARalpha pathway in a mechanism of action of BR and FF. Furthermore, we observed a significant increase in the gene expression of a pyruvate dehydrogenase kinase 4 after treatment with FF; FF also increased mitochondrial respiration. Collectively, our data indicate that both BR and FF reduce the accumulation of OA/PA in HepG2 cells exposed to these fatty acids, presumably by up-regulating fatty acid oxidation via PPARalpha pathway. Key words Bilirubin " Fatty acids " Fenofibrate " MASLD " PPARalpha.
This study investigates the protective effect of opuntiol, a naturally occurring flavonoid, against lipopolysaccharide (LPS)-induced acute kidney injury in mice. Acute kidney injury (AKI) is a serious clinical complication characterized by inflammation, oxidative stress, and apoptosis, often resulting in high morbidity and mortality. Male mice were divided into six groups and administered opuntiol (25, 50 and 100 mg/kg b. wt.) intraperitoneally prior to LPS (10 mg/kg b. wt.) administration. The most effective dose was 50 mg/kg b. wt., as indicated in the dose-finding study. Kidney function markers (urea, creatinine, blood urea nitrogen (BUN), uric acid), antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), cyclooxygenase-2 (COX-2), and gene expression levels (pro-inflammatory, apoptotic, and antioxidant genes) were analyzed using biochemical assays and qRT-PCR. Opuntiol significantly reduced elevated levels of serum urea, creatinine, BUN, and uric acid compared to the LPS group. Further, opuntiol restored antioxidant enzyme activities and MDA levels were significantly decreased. Opuntiol also downregulated inflammatory markers and gene expressions (TNF-alpha, NF-kappaB, TLR4, Bax, Caspase-3, etc.) while upregulating anti-apoptotic (Bcl-2) and antioxidant (Nrf-2) genes. Opuntiol offers significant protection against LPS-induced AKI by mitigating oxidative damage, inflammation, and apoptotic signaling. It enhances renal function and promotes antioxidant defense. These findings support the therapeutic potential of opuntiol as a novel nephroprotective agent in managing sepsis-associated kidney injury and encourage further preclinical and clinical investigations. Key words Opuntiol " Acute kidney injury " Lipopolysaccharide " Oxidative stress " Inflammation " Apoptosis.
Dysfunction of pulmonary artery endothelial cells (PAECs) contributes to the pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH). However, the role of mitochondrial metabolism in this process remains unclear. The present study evaluated whether the tetrameric form of pyruvate kinase muscle isoform 2 (PKM2) regulates PAEC mitochondrial metabolism through peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1alpha) and mitochondrial transcription factor A (mtTFA), thereby influencing arterial intimal remodeling in CTEPH. A CTEPH rat model was established by repeated injections of autologous thrombi. Activation of PKM2 tetramer expression was achieved through synthetic pyruvate kinase M2 activator (TEPP-46) administration. Pulmonary artery pressure (PAP), thrombus pathology, and protein expression levels of PKM2, mtTFA, and PGC-1alpha were assessed. Plasma lactate concentrations and tumor necrosis factor alpha (TNF-alpha) levels were measured. Rats with CTEPH demonstrated thrombotic obstruction, elevated PAP, and reduced expression of the PKM2 tetramer, mtTFA, and PGC-1alpha. Treatment with TEPP-46 was associated with a reduction in thrombus burden, lower PAP, and restoration of mitochondrial protein expression, accompanied by decreased lactate concentrations and TNF-alpha levels. In the CTEPH rat model, increased inflammation and elevated lactate concentrations were observed, along with decreased expression of mtTFA and PGC-1alpha in the pulmonary artery intima, which is indicative of mitochondrial dysfunction. The PKM2 tetramer may play a role in modulating PAEC mitochondrial function, reducing pulmonary artery pressure, and improving pulmonary arterial intimal remodeling in CTEPH. Key words Chronic thromboembolic pulmonary hypertension " Lactic acid " Mitochondrial transcription factor A " Peroxisome proliferator-activated receptor gamma coactivator 1alpha " Pyruvate kinase muscle.
Excessive inflammatory responses represent one of the primary causes of sepsis- associated acute kidney injury (S-AKI). The activation of the nuclear factor-kappa B (NF-kappaB) signaling pathway plays a critical role in the pathogenesis and progression of S-AKI. Previous studies have demonstrated that ribosomal protein S3 (RPS3) serves as a pivotal regulator of the NF-kappaB pathway; however, its specific biological functions in the context of S-AKI remain to be fully elucidated. This study aims to elucidate the regulatory mechanisms of RPS3 in S-AKI-associated inflammation and to explore the underlying molecular pathways. First, we conducted an analysis of RPS3 level in urine and TNF-alpha level in serum from S-AKI patients recruited at our hospital. Second, we established a murine model of S-AKI by intraperitoneal injection of LPS, followed by the evaluation of renal function, inflammatory response, RPS3 expression, and NF-kappaB activation in renal tissues. Finally, we explored the regulatory role and underlying mechanism of RPS3 in the LPS-induced inflammatory response in HK-2 cells through RPS3 knockdown and the introduction of an NF-kappaB agonist. The results demonstrated that urinary RPS3 and serum TNF-alpha levels were significantly elevated in patients with S-AKI, with a positive correlation observed between these two parameters. In LPS-induced S-AKI mice, renal function was impaired, accompanied by a robust inflammatory response, increased RPS3 protein expression, and enhanced NF-kappaB activation in kidney tissue. Knockdown of RPS3 in HK-2 cells mitigated LPS-induced the inflammatory response and suppressed NF-kappaB activation. However, the effects of RPS3 silencing were partially reversed upon intervention with an NF-kappaB agonist. Collectively, these findings indicate that RPS3 plays a critical role in the inflammatory response of S-AKI via activation of the NF-kappaB signaling pathway, suggesting its potential as a novel therapeutic target for S-AKI. Key words Sepsis-associated acute kidney injury " Inflammatory response " Ribosomal protein S3 " Nuclear factor-kappa B.
Mitochondria participate in regulating cytosolic Ca2+ signaling by their Ca2+ handling via mitochondrial Ca2+ uniporter (MCU) and mitochondrial Na+/Ca2+ exchanger (mitoNCX). In this study, we examined how agonist-triggered cytosolic Ca2+ oscillations in human alveolar type 2 A549 cells were affected by an MCU inhibitor (MCU-i4), MCU activator (kaempferol) and mitoNCX inhibitor (CGP-37157). Whilst inhibition of MCU did not significantly repress Ca2+ oscillations, MCU activation by kaempferol considerably dampened oscillatory activities. Inhibition of mitochondrial Ca2+ efflux by CGP-37157 also suppressed Ca2+ oscillations; the suppressive effects of kaempferol and CGP 37157 were not additive. Both kaempferol and CGP-37157 caused a rise in mitochondrial matrix Ca2+ level, but their effects were not additive. Taken together, our results suggest Ca2+ oscillations in alveolar type 2 A549 cells were regulated by stimulating Ca2+ uptake into, and preventing Ca2+ efflux from, the mitochondria, with both cases resulting in disturbed Ca2+ traffic and Ca2+ accumulation in the mitochondrial matrix. Key words Mitochondria " Ca2+ oscillations " Mitochondrial Ca2+ uniporter " Mitochondrial Na+/Ca2+ exchanger " A549 cells.
Gluten-free diet is currently recommended for people with gluten-related diseases; however some studies document their positive effects also in other diseases. Oppositely gluten is often vilified in nutrition, but serious results about their negative effects in healthy are missing, or controversial. The objective of this study is to compare the effects of different types of diets on ovarian, testicular, and thyroid morphology in an experimental mouse model. Forty-eight (n=48) laboratory mice of the BALB/c line were included in the experiment, divided into 4 groups, and maintained on special diets for 5 weeks. The control group, (6 females, 6 males) was fed a gluten-free diet. The first (E1), second (E2) and third (E3) experimental groups, (6 females, 6 males) were fed a mixture of casein hydrolysate combined with E1: pure extracted gluten in a 30 %:70 % ratio. E2: gliadins at a ratio of 30 %:70 % and E3: avenin at a ratio of 30 %:70 %. At the end of the experiment, the mice were euthanized and ovaries, testes, and thyroid glands were sampled. The samples were fixed in a 10 % formalin solution and processed into hematoxylin-eosin-stained slides. The oocyte and follicle widths of the ovaries were measured; as well as the germinal epithelium and the width of the seminiferous tubules of the testes; as well as the follicle epithelium width and the follicle width of the thyroid gland. The results showed significant differences in the width of oocytes, follicles, testicular seminiferous tubule epithelium, testicular tubules, thyroid follicle epithelium as well as differences in the width of thyroid follicles. Concentrated gluten and gliadin-based diets showed positive results compared to concentrated avenin and gluten-free diets. On the basis of animal experiment using histological methods, it seems that gluten may not be for healthy population harmful and is not recommended to be avoided outside groups of people with gluten-related disorders. Key words Celiac disease " Gluten " Non-celiac gluten sensitivity " Cereals o Nutrition.
NMDA receptor hypofunction can lead to behavioral and cognitive disturbances, including hyperlocomotion, and is considered a core pathophysiological mechanism underlying cognitive and negative symptoms in schizophrenia. This study examined whether treatment with the mGlu2/3 receptor agonist LY379268 (1 and 2 mg/kg) could counteract such disruptions induced by the NMDA antagonist MK-801 (0.1 mg/kg). Rats were tested under two conditions: an aversive learning task (active place avoidance on a rotating arena) and a non-aversive open field test. Additionally, local field potentials were recorded from the medial prefrontal cortex during the open field test and later under urethane anesthesia. Contrary to expectations, LY379268 did not consistently alleviate MK-801-induced impairments. In the aversive learning context, the combination of MK-801 with LY379268 (2 mg/kg) paradoxically led to exacerbated hyperlocomotion and impaired navigational performance. In contrast, the 1 mg/kg dose of LY379268 had a modest beneficial effect in the non-aversive setting, slightly reducing MK-801-induced hyperactivity. Electrophysiological recordings revealed that MK-801, alone or in combination with LY379268 (1 mg/kg), disrupted theta-high gamma phase-amplitude coupling in the open field test, indicating impaired neural processing. Under anesthesia, MK-801 increased low gamma power. LY379268 did not reverse this alteration. These findings highlight the task- and dose-dependent nature of LY379268's effects. While it offered limited improvement in a non-aversive environment, it failed to mitigate and sometimes exacerbated deficits in more challenging, aversive tasks. This complexity underscores the need for further research to refine the therapeutic potential of mGlu2/3 modulation in conditions associated with glutamatergic dysfunction. Key words MK-801 " LY379268 " Electrophysiology " Medial prefrontal cortex " Hyperlocomotion.
Osteoarthritis (OA) is a severe chronic inflammatory disorder with limited treatment options. Naringin (nar) has been shown to protect against OA; however, its mechanisms of action on OA remain poorly understood. This study aims to investigate the molecular mechanism of nar in treating OA via network pharmacology and experiments. Differentially expressed genes (DEGs) were identified using GSE283079 dataset. Protein-protein interaction (PPI) network was constructed using STRING database, and protein interactions were analyzed. Network pharmacology was employed to investigate the molecular interaction network influenced by nar in OA, and molecular docking was applied to predict the binding interactions between nar and core genes. The OA mouse models were constructed using anterior cruciate ligament transection (ACLT) to explore the action of nar in vivo. The OA damage was examined using Hematoxylin and Eosin (HE) and Safranin-O/Fast Green staining, along with Osteoarthritis Research Society International (OARSI) scoring for quantitative histopathological evaluation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive rate and inflammation factor (tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta), and reactive oxygen species (ROS) levels were detected using corresponding assay kits. The protein expression was analyzed using western blot. Cell viability and cell apoptosis were examined using cell counting kit 8 (CCK8) assay kit and flow cytometry assays. In GSE283079 dataset, the up-regulation of DEGs was enriched in immune response activation, cartilage development, collagen metabolic process, and leukocyte proliferation. Additionally, matrix metalloproteinase 13 (MMP13), MMP1, and phospholipase A2 group IIA (PLA2G2A) may be the core genes for nar-protected OA. The binding energy of nar and MMP13 was strongest. In vivo OA models, nar mitigated OA progression and reduced OARSI scores. Mechanistically, nar suppressed cell apoptosis, inflammation factor productions, extracellular matrix (ECM) degradation, and ROS production via decreasing MMP13. Nar alleviates OA malignant progression via reducing MMP13. Key words Osteoarthritis " Naringin " Network pharmacology " MMP13 " Molecular mechanism.
We performed a mechanical analysis of the commonly used needles for ultrasound-guided interventions in the musculoskeletal system. Specifically, focusing on the ability to absorb potential physical loads and the degree of deformation during the procedure, the needle gauge best suited for those procedures is evaluated. A customized tensile-compression device was used for an experimental buckling strength test on three commonly used needle types with specific gauge sizes. The loss of structural integrity, loss of needle stability, and buckling load were modeled also theoretically using finite element analysis software. Theoretical software needle buckling analysis detected the load for the first buckling mode of the needle, when the highest value was reached for the G20 needle with the load 18.8 N. The load for G21 needle was 9.7 N and for G23 8 N. Experimental data with customized tensile-compression device aligned theoretical data when the highest value was reached for the G20 needle with the load 19.7±1.9 N. The load for G21 needle was 10.6+/-2.7 N and for G23 7.9+/-0.7 N. Theoretical and practical experiments have shown that the standard G20 needle model exhibits the highest mechanical tolerance for potential interventions in the musculoskeletal system. Key words Interventional ultrasound " Needle " Buckling strength.
The objective of this in vitro study was to examine the impact of metformin (MET) at different concentrations (0.1, 1, 10, 50, and 100 mM) on rat primary osteoblasts, as the results obtained so far are inconsistent. Osteoblast apoptosis, viability, alkaline phosphatase (ALPL) activity, production of osteoblast-specific biomarkers, including ALPL, osteocalcin (BGLAP), type I collagen alpha 1 (COL1A1), integrin-binding sialoprotein (IBSP), bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2), vascular endothelial growth factor (VEGF), tumor necrosis factor ligand superfamily member 11 (TNFSF11 or RANKL), as well as calcium/collagen deposition were assessed. Our results revealed that a dose of 100 mM was cytotoxic to osteoblasts and resulted in a complete loss of their viability. Therefore, this concentration was excluded from further analyses. In general, MET exhibited a dose-dependent impact on multiple osteoblast-specific functional biomarkers, with beneficial effects noted on ALPL activity (at 0.1 and 1 mM) as well as on the levels of ALPL (0.1 and 1 mM), BGLAP (at 0.1-50 mM), IBSP (at 0.1-50 mM), BMP2 (at 0.1, 10 and 50 mM), VEGF (at 0.1 and 1 mM), and RANKL (at 0.1 mM). Calcium/collagen deposition at concentrations of 0.1 and 1 mM reached the same level as control cells, higher doses (10 and 50 mM) dramatically reduced cell viability after 21 days and the aforementioned parameters could not be evaluated. It can be concluded that MET at concentrations up to 1 mM can promote osteoblast viability, osteogenic differentiation, angiogenic signaling, and reduce osteoclastogenesis. Key words Metformin " Osteoblasts " Bone health " In vitro.
Mirogabalin is a newly developed gabapentinoid drug. Several in vivo and clinical studies have demonstrated the potent analgesic effects of mirogabalin in neuropathic pain. This study aims to investigate the impact of mirogabalin on visceral pain and inflammation. Adult male Balb/c mice (20-25 g) were used in the study (n=7). Mirogabalin was administered intraperitoneally at 10, 20, and 40 mg/kg doses. Inflammatory visceral pain was induced by intraperitoneal administration of acetic acid. The number of writhing was observed after acetic acid administration, and the effective dose of mirogabalin was determined. In the second phase of the study, the effects of mirogabalin on locomotor activity and leukocyte infiltration into peritoneal tissue were examined. IL-6, GSH levels, and SOD activity were investigated biochemically. Statistical analyses were performed in the GraphPad Prism (v8.0.1) program. Mirogabalin was significantly antinociceptive at all three doses (p<0.001). Histopathologic examination showed that the effective dose of mirogabalin decreased leukocyte infiltration into the peritoneum. Mirogabalin did not affect total distance moved and mean speed in the open field test. There was no significant difference between the groups in terms of IL-6, GSH levels, and SOD activity. Our results demonstrated a significant antinociceptive effect of mirogabalin against visceral pain. In addition, anti-inflammatory effects were revealed by decreasing leukocyte infiltration. However, the fact that mirogabalin did not alter antioxidant systems and IL-6 levels suggests that other mechanisms are responsible for its anti-inflammatory effects. Key words Mirogabalin " Pain " Inflammation " Visceral " GABA.
Early-life stressful stimuli, such as social isolation, alter brain neurochemistry and lead to negative behavioral outcomes in adulthood. Isolated animals are deprived of social interactions, which results in impaired brain development. Post-weaning isolation rearing deregulates various brain processes and may affect nitric oxide (NO) signaling. The aim of our study was to determine time-dependent impact of social isolation on behavioral and biochemical parameters in Wistar Kyoto rats. At the age of 21 days, male rats were randomly assigned into four groups reared in isolation or socially for 10 or 29 weeks. At the end of the rearing, open-field and prepulse inhibition (PPI) tests were carried out. Furthermore, in several brain areas we assessed NO synthase (NOS) activity, protein expression of nNOS and iNOS isoforms and the concentration of conjugated dienes (CD), a marker of lipid peroxidation. The number of entries into the central zone of the open field test decreased significantly only after 29 weeks of isolation. Isolated rats (IR) rats exhibited impaired habituation of the acoustic startle response after prolonged social isolation. While cerebellar NOS activity and nNOS protein expression decreased significantly in IR rats after 29 weeks of isolation, the expression of nNOS and iNOS was increased in the hippocampus. 10-week and 29-week social isolation led to increased CD concentration in the brain. Our results suggest that the duration of social isolation plays an important role in the development of behavioral and biochemical changes in the brain. The decreased NO bioavailability may result from lipid peroxidation, oxidative stress, and inflammatory responses.
Intermittent fasting (IF) represents one of the dietary regimens being effectively used in non-pharmacological prevention and treatment of cardiometabolic disorders. The aim of the present study was to detect the retained alterations at the level of arterial function caused by a 5-week-lasting period of IF in adult male Wistar-Kyoto rats after their switching back to ordinary feeding (4 weeks of ad libitum regimen). The rats were administered a diet containing normal or high percentage of fat. Control rat groups were fed continuously ad libitum. The decreased weekly calorie intake in rats during IF period was associated with the discontinuation of body weight gain, irrespective of the type of diet; moreover, rats fed with a high-fat diet had significantly increased systolic blood pressure in comparison with the other groups. At the end of the experiment, large and small arteries were isolated from the rats and arterial rings with intact or removed perivascular adipose tissue (PVAT) were prepared for isometric tension recording. In the rat groups exposed to IF period, the aorta rings with intact PVAT showed a significant increase in relaxation responses when compared to groups without IF. The effect of IF was also manifested in the increase in sensitivity of arterial preparations to noradrenaline which was, however, mostly attenuated by the enhanced anticontractile influence of PVAT. These results indicate that the improvement of PVAT properties could represent one of the mechanisms by which IF-induced beneficial effects on vascular function might be preserved even after the return to ad libitum regimen.
Stroke survivors frequently present with impaired trunk control, which is a key determinant of mobility, balance, and independence in activities of daily living (ADL). Reliable clinical tools are therefore needed to evaluate postural stability, particularly in patients unable to stand. This randomized controlled study assessed the applicability of the Trunk Impairment Scale (TIS) and Stroke Impact Scale (SIS) in post-stroke patients after completion of a complementary sensory intervention targeting sitting postural stability. Forty inpatients in the subacute stage after stroke were randomized into an Experimental group receiving daily postural training with visual biofeedback in addition to standard physiotherapy, and a Control group receiving standard physiotherapy only. Assessments included TIS and SIS at baseline and post-intervention. Both groups demonstrated significant improvements in trunk control, mobility, strength and ADL over time, as reflected by higher TIS and SIS scores. However, the Experimental group achieved greater gains, with the most pronounced effects observed in TIS, as well as SIS Mobility, and SIS ADL domains. Mobility improvements were strongly associated with enhanced ADL performance, underscoring the relevance of trunk control rehabilitation. These findings confirm the clinical sensitivity of TIS and SIS in capturing meaningful postural changes associated with functional recovery after stroke. This study demonstrates that targeted trunk-focused interventions with complementary sensory input can significantly enhance both motor and functional outcomes in stroke survivors. Combining TIS and SIS provides a comprehensive evaluation of clinical performance and patient-reported outcomes, offering valuable insight for rehabilitation strategies aimed at improving independence and quality of life.
The human endometrium undergoes dynamic hormonal and structural changes throughout the menstrual cycle. Their aim is to create an environment essential for embryo implantation. Successful implantation depends on the proper composition of the endometrial microenvironment, including cytokine synthesis and local immune responses. During the first trimester, uterine natural killer (uNK) cells play a key role in regulating trophoblast invasion, vascular remodelling, and establishing embryo tolerance, with nitric oxide (NO) also contributing to these processes. The study aimed to evaluate the expression patterns of NOS2 and NOS3 and their relationship to the infiltration of endometrium by uterine natural killer (uNK) cells during different menstrual phases. The endometrial tissue samples representing proliferative, early secretory, late secretory, menstrual, and hypersecretory phases were analysed by immunohistochemistry and fluorescence microscopy. NOS2 and NOS3 showed distinct cyclic patterns. NOS3 expression peaked in the early secretory phase, supporting tissue remodelling, while NOS2 expression increased progressively, reaching its maximum in the late secretory and menstrual phases. The number of uNK cells paralleled NOS2 expression, with a positive correlation suggesting a possible NO-related immunomodulatory mechanism. Elevated NOS2 expression and increased clustering of CD56+ uNK cells were observed in some cases of proliferative endometrium, possibly reflecting phase-inappropriate immune activation. These findings indicate that NOS activity and uNK cell dynamics may jointly contribute to the cyclic regulation of the endometrial microenvironment. Understanding NOS regulation and its hormonal and immune interactions may offer new insights into implantation mechanisms.
