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Abstract Astaxanthin due to its strong antioxidant activity is believed to reduce oxidative stress and therefore is considered as feed additive in pathological conditions and also for the athletes. It is promoted by several equine web portals, however, data supporting that concept in horses is limited. Thus, the aim of this study was to evaluate the effect of astaxanthin supplementation on the parameters of oxidative status in 3 years old, racing Arabian horses during long term observation and the changes related to a single training session of high intensity. Six horses were supplemented with astaxanthin at a dose of 0.52–0.58 mg/kg BW and 7 received no supplementation. Astaxanthin supplementation resulted in the increase in total antioxidant status by 31.5%, accompanied by decreases in the amount of total thiobarbituric acid-reactive substances -TBARS and glutathione reductases - GR values by 34.5% and 45.4%, respectively, after 1 month and this effect persisted until the end of the observation. After individual training session the activities of glutathione peroxidases and GR were lower by 69% and 46%, respectively, and TBARS lower by 38% in supplemented horses. These results directly confirmed the beneficial effects of astaxanthin supplementation on the antioxidant status of race horses. Astaxanthin partially counterbalance the training-related oxidative stress, save the horse natural antioxidant defense, and shift the redox status towards a more reducing environment. At the same time, exercise-induced reactive oxygen species production at certain level was maintained and so that contributed to training progress.

BackgroundMusculoskeletal injuries and chronic degenerative diseases pose significant challenges in equine health, impacting performance and overall well-being. Sex Hormone-Binding Globulin (SHBG) is a glycoprotein determining the bioavailability of sex hormones in the bloodstream, and exerting critical metabolic functions, thus impacting the homeostasis of many tissues including the bone.MethodsIn this study, we investigated the potential role of SHBG in promoting osteogenesis and its underlying mechanisms in a model of equine adipose-derived stromal cells (ASCs). An SHBG-knocked down model has been established using predesigned siRNA, and cells subjected to osteogenic induction medium in the presence of exogenous SHBG protein. Changes in differentiation events where then screened using various analytical methods.ResultsWe demonstrated that SHBG treatment enhances the expression of key osteoconductive regulators in equine ASCs CD34+ cells, suggesting its therapeutic potential for bone regeneration. Specifically, SHBG increased the cellular expression of BMP2/4, osteocalcin (OCL), alkaline phosphatase (ALP), and osteopontin (OPN), crucial factors in early osteogenesis. Furthermore, SHBG treatment maintained adequate apoptosis and enhanced autophagy during osteogenic differentiation, contributing to bone formation and remodeling. SHBG further targeted mitochondrial dynamics, and promoted the reorganization of the mitochondrial network, as well as the expression of dynamics mediators including PINK, PARKIN and MFN1, suggesting its role in adapting cells to the osteogenic milieu, with implications for osteoblast maturation and differentiation.ConclusionOverall, our findings provide novel insights into SHBG’s role in bone formation and suggest its potential therapeutic utility for bone regeneration in equine medicine.

Corneal ulcers, characterized by severe inflammation of the cornea, can lead to serious, debilitating complications and may be vision-threatening for horses. In this study, we aimed to investigate the role of endoplasmic reticulum (ER) stress in corneal stem progenitor cell (CSSC) dysfunction and explore the potential of equine adipose-derived stromal stem cell (ASC)-derived extracellular vesicles (EVs) to improve corneal wound healing. We showed that CSSCs expressed high levels of CD44, CD45, and CD90 surface markers, indicating their stemness. Supplementation of the ER-stress-inducer tunicamycin to CSSCs resulted in reduced proliferative and migratory potential, accumulation of endoplasmic reticulum (ER)-stressed cells in the G0/G1 phase of the cell cycle, increased expression of proinflammatory genes, induced oxidative stress and sustained ER stress, and unfolded protein response (UPR). Importantly, treatment with EVs increased the proliferative activity and number of cells in the G2/Mitosis phase, enhanced migratory ability, suppressed the overexpression of proinflammatory cytokines, and upregulated the anti-inflammatory miRNA-146a-5p, compared to control and/or ER-stressed cells. Additionally, EVs lowered the expression of ER-stress master regulators and effectors (PERK, IRE1, ATF6, and XBP1), increased the number of mitochondria, and reduced the expression of Fis-1 and Parkin, thereby promoting metabolic homeostasis and protecting against apoptosis in equine CSSCs. Our findings demonstrate that MSCs-derived EVs represent an innovative and promising therapeutic strategy for the transfer of bioactive mediators which regulate various cellular and molecular signaling pathways.

Abstract Background Equine metabolic syndrome (EMS), which encompasses insulin resistance, low-grade inflammation and predisposition to laminitis is a critical endocrine disorder among the most prevalent conditions affecting horses from different breeds. According to the most recent research, low human sex hormone-binding globulin (SHBG) serum levels correlate with an increased risk of obesity, insulin resistance and diabetes, and may contribute to overall metabolic dysregulations. This study aimed to test whether exogenous SHBG could protect EMS affected adipose-derived stromal stem cells (EqASCEMS) from apoptosis, oxidative stress, ER stress and thus improve insulin sensitivity. Methods EqASCEMS wells were treated with two different concentrations (50 and 100 nM) of exogenous SHBG, whose biocompatibility was tested after 24, 48 and 72 h of incubation. Several parameters including cell viability, apoptosis, cell cycle, reactive oxygen species levels, ER stress, Pi3K/MAPK activation and insulin transducers expression were analysed. Results Obtained data demonstrated that exogenous SHBG treatment significantly promoted ASCs cells proliferation, cell cycle and survival with reduced expression of p53 and p21 pro-apoptotic mediators. Furthermore, SHBG alleviated the oxidative stress caused by EMS and reduced the overaccumulation of intracellular ROS, by reducing ROS + cell percentage and regulating gene expression of endogenous antioxidant enzymes (Sod 1, Cat, GPx), SHBG treatment exhibited antioxidant activity by modulating total nitric oxide (NO) levels in EMS cells as well. SHBG treatment dampened the activation of ER stress sensors and effectors in EqASCEMS cells via the upregulation of MiR-7a-5p, the decrease in the expression levels of ATF-6, CHOP and eiF2A and the restoration of PDIA3 chaperone protein levels. As a consequence, SHBG application substantially improved insulin sensitivity through the modulation of Pi3K/Akt/Glut4 insulin signalling cascades. Conclusion Our results suggest that the SHBG is endowed with crucial beneficial effects on ASCs metabolic activities and could serve as a valuable therapeutic target for the development of efficient EMS treatment protocols. Video Abstract

Equine metabolic syndrome (EMS) is a critical endocrine condition in horses, characterized by hyperinsulinemia, hyperlipidemia, and insulin resistance, posing a significant threat to their health. This study investigates the efficacy of supplementing EMS-affected horses with Arthrospira platensis enriched with Cr(III), Mg(II), and Mn(II) ions using biosorption process in improving insulin sensitivity and glucose tolerance, reducing inflammation, and mitigating obesity-related fat accumulation. Our results demonstrate that Arthrospira supplementation reduces baseline insulin and glucose levels, contributing to decreased adipose tissue inflammation. Furthermore, Arthrospira supplementation results in a decrease in body weight and improvements in overall body condition scores and cresty neck scores. Additionally, administration of Arthrospira leads to reduced levels of triglycerides and aspartate aminotransferase, indicating a decrease in hepatic adiposity and inflammation. These findings suggest that Arthrospira, enriched with essential micro- and macroelements, can be an advanced feed additive to enhance insulin sensitivity, promote weight reduction, and alleviate inflammatory processes, thereby improving the overall condition of horses affected by EMS. The use of Arthrospira as a feed additive has the potential to complement conventional management strategies for EMS.

This study aimed to evaluate the oral supplementation of astaxanthin (ATX) on inflammatory markers in 3-year-old Arabian racehorses. Despite the recognized antioxidant and anti-inflammatory properties of ATX observed in vitro in rodent models and in human athletes, the effects in equine subjects remain unknown. This study involved a controlled trial with 14 horses receiving either ATX (six horses) or a placebo (eight horses), monitored over four months of race training. Inflammatory cytokines: TNFα, IFNγ, IL-6, IL-10, and prostaglandin E (PGE), were measured monthly to assess the impact of ATX on the inflammatory response. The results indicated no significant differences in measured parameters between the ATX and the control group during the study. However, a significant time-dependent decrease in TNFα and IFNγ levels (p = 0.001) was observed in both groups, suggesting that regular training naturally modulates inflammatory responses. Moreover, positive correlations were noted between TNFα and IFNγ (p < 0.001) in the early phase of the study and between IL-6 and IL-10 (p = 0.008) in the later phase. Hematological parameters remained stable and within reference ranges, indicating no adverse effects of ATX supplementation. Performance metrics, including the number of races completed and wins, showed no significant differences between groups, suggesting that ATX did not enhance athletic performance under the study conditions. Overall, while ATX supplementation affected neither cytokine levels nor performance in Arabian racehorses, the natural anti-inflammatory effects of regular training were evident. Further research is needed to explore potential benefits of ATX supplementation under different conditions, such as in horses with subclinical inflammation or varying training regimens, to fully clarify its role and applications in equine sports medicine.

Equine metabolic syndrome (EMS) is a steadily growing endocrine disorder representing a real challenge in veterinary practice. As a multifactorial condition, EMS is characterized by three main metabolic abnormalities including insulin resistance, increased adiposity or obesity and hoof laminitis. Adipose tissue dysfunction is recognized as a core pathophysiological determinant of EMS, as it strongly participates to lipotoxicity and systemic metaflammation, both of which have been closely linked to the development of generalized insulin resistance. Besides, sex hormone binding globulin (SHBG) is an important sex steroids transporters that has been recently proposed as an important metabolic mediator. Therefore, the aim of this study was to verify whether SHBG treatment may ameliorate subcutaneous adipose tissue metabolic failure under EMS condition in terms of lipidome homeostasis, lipid metabolism programs, insulin signalling and local inflammation. Subcutaneous adipose tissue (SAT) biopsies were collected post-mortem from healthy (n = 3) and EMS (n = 3) slaughtered horses. SHBG protein has been applied to SAT samples from EMS horses for 24 h at a final concentration of 50 nM, while control groups (healthy and untreated EMS) were cultured in the presence of SHBG-vehicle only. Tissues from all groups were afterwards secured for downstream analysis of gene expression using RT-qPCR, protein levels by Western blot and ELISA assay and lipidomics through GC-MS technique. Obtained results showcased that SHBG intervention efficiently normalized the altered fatty acids (FAs) profiles by lowering the accumulation of saturated and trans FAs, as well as the pro-inflammatory arachidonic and linoleic acids. Moreover, SHBG showed promising value for the regulation of adipocyte lipolysis and engorgement by lowering the levels of perilipin-1. SHBG exerted moderated effect toward SCD1 and FASN enzymes expression, but increased the LPL abundance. Interestingly, SHBG exhibited a negative regulatory effect on pro-adipogenic stimulators and induced higher expression of KLF3, IRF3 and β-catenin, known as strong adipogenesis repressors. Finally, SHBG protein showed remarkable ability in restoring the insulin signal transduction, IR/IRS/Pi3K/AKT phosphorylation events and GLUT4 transporter abundance, and further attenuate pro-inflammatory response by lowering IL-6 tissue levels and targeting the PDIA3/ERK axis. Overall, the obtained data clearly demonstrate the benefice of SHBG treatment in the regulation of adipose tissue metabolism in the course of EMS and provide new insights for the development of molecular therapies with potential translational application to human metabolic disorders.

Abstract Background Progression of senile osteoporosis is associated with deteriorated regenerative potential of bone marrow-derived mesenchymal stem/stromal cells (BMSCs). According to the recent results, the senescent phenotype of osteoporotic cells strongly correlates with impaired regulation of mitochondria dynamics. Moreover, due to the ageing of population and growing osteoporosis incidence, more efficient methods concerning BMSCs rejuvenation are intensely investigated. Recently, miR-21-5p was reported to play a vital role in bone turnover, but its therapeutic mechanisms in progenitor cells delivered from senile osteoporotic patients remain unclear. Therefore, the goal of this paper was to investigate for the first time the regenerative potential of miR-21-5p in the process of mitochondrial network regulation and stemness restoration using the unique model of BMSCs isolated from senile osteoporotic SAM/P6 mice model. Methods BMSCs were isolated from healthy BALB/c and osteoporotic SAM/P6 mice. We analysed the impact of miR-21-5p on the expression of crucial markers related to cells’ viability, mitochondria reconstruction and autophagy progression. Further, we established the expression of markers vital for bone homeostasis, as well as defined the composition of extracellular matrix in osteogenic cultures. The regenerative potential of miR-21 in vivo was also investigated using a critical-size cranial defect model by computed microtomography and SEM–EDX imaging. Results MiR-21 upregulation improved cells’ viability and drove mitochondria dynamics in osteoporotic BMSCs evidenced by the intensification of fission processes. Simultaneously, miR-21 enhanced the osteogenic differentiation of BMSCs evidenced by increased expression of Runx-2 but downregulated Trap, as well as improved calcification of extracellular matrix. Importantly, the analyses using the critical-size cranial defect model indicated on a greater ratio of newly formed tissue after miR-21 application, as well as upregulated content of calcium and phosphorus within the defect site. Conclusions Our results demonstrate that miR-21-5p regulates the fission and fusion processes of mitochondria and facilitates the stemness restoration of senile osteoporotic BMSCs. At the same time, it enhances the expression of RUNX-2, while reduces TRAP accumulation in the cells with deteriorated phenotype. Therefore, miR-21-5p may bring a novel molecular strategy for senile osteoporosis diagnostics and treatment.

Propenylbenzenes, including isosafrole, anethole, isoeugenol, and their derivatives, are natural compounds found in essential oils from various plants. Compounds of this group are important and valuable, and are used in the flavour and fragrance industries as well as the pharmaceutical and cosmetic industries. The aim of this study was to develop an efficient process for synthesising oxygenated derivatives of these compounds and evaluate their potential biological activities. In this paper, we propose a two-step chemo-enzymatic method. The first step involves the synthesis of corresponding diols 1b–5b from propenylbenzenes 1a–5avia lipase catalysed epoxidation followed by epoxide hydrolysis. The second step involves the microbial oxidation of a diasteroisomeric mixture of diols 1b–5b to yield the corresponding hydroxy ketones 1c–4c, which in this study was performed on a preparative scale using Dietzia sp. DSM44016, Rhodococcus erythropolis DSM44534, R. erythropolis PCM2150, and Rhodococcus ruber PCM2166. Application of scaled-up processes allowed to obtain hydroxy ketones 1-4c with the following yield range 36–62.5%. The propenylbenzene derivatives thus obtained and the starting compounds were tested for various biological activities, including antimicrobial, antioxidant, haemolytic, and anticancer activities, and their impact on membrane fluidity. Fungistatic activity assay against selected strains of Candida albicans results in MIC50 value varied from 37 to 124 μg/mL for compounds 1a, 3a–c, 4a,b, and 5a,b. The highest antiradical activity was shown by propenylbenzenes 1-5a with a double bond in their structure with EC50 value ranged from 19 to 31 μg/mL. Haemolytic activity assay showed no cytotoxicity of the tested compounds on human RBCs whereas, compounds 2b–4b and 2c–4c affected the fluidity of the RBCs membrane. The tested compounds depending on their concentration showed different antiproliferative activity against HepG2, Caco-2, and MG63. The results indicate the potential utility of these compounds as fungistatics, antioxidants, and proliferation inhibitors of selected cell lines.

Abstract The purpose of our study was to investigate if vascular injury in immature epiphyses affects cartilage repair outcomes of matrix-associated stem cell implants (MASI). Porcine bone marrow mesenchymal stromal stem cells (BMSCs) suspended in a fibrin glue scaffold were implanted into 24 full-thickness cartilage defects (5 mm ø) of the bilateral distal femur of six Göttingen minipigs (n = 12 defects in 6 knee joints of 3 immature pigs; age 3.5–4 months; n = 12 defects in 6 knee joints of 3 mature control pigs; age, 21–28 months). All pigs underwent magnetic resonance imaging (MRI) at 2, 4, 12 (n = 24 defects), and 24 weeks (n = 12 defects). After the last imaging study, pigs were sacrificed, joints explanted and evaluated with VEGF, H&E, van Gieson, Mallory, and Safranin O stains. Results of mature and immature cartilage groups were compared using the Wilcoxon signed-rank test. Quantitative scores for subchondral edema at 2 weeks were correlated with quantitative scores for cartilage repair (MOCART score and ICRS score) at 12 weeks as well as Pineda scores at end of the study, using linear regression analysis. On serial MRIs, mature joints demonstrated progressive healing of cartilage defects while immature joints demonstrated incomplete healing and damage of the subchondral bone. The MOCART score at 12 weeks was significantly higher for mature joints (79.583 ± 7.216) compared to immature joints (30.416 ± 10.543, p = 0.002). Immature cartilage demonstrated abundant microvessels while mature cartilage did not contain microvessels. Accordingly, cartilage defects in immature joints showed a significantly higher number of disrupted microvessels, subchondral edema, and angiogenesis compared to mature cartilage. Quantitative scores for subchondral edema at 2 weeks were negatively correlated with MOCART scores (r = − 0.861) and ICRS scores (r = − 0.901) at 12 weeks and positively correlated with Pineda scores at the end of the study (r = 0.782). Injury of epiphyseal blood vessels in immature joints leads to subchondral bone defects and limits cartilage repair after MASI.

Equine metabolic syndrome (EMS) is a significant global health concern in veterinary medicine. There is increasing interest in utilizing molecular agents to modulate hepatocyte function for potential clinical applications. Recent studies have shown promising results in inhibiting protein tyrosine phosphatase (PTP1B) to maintain cell function in various models. In this study, we investigated the effects of the inhibitor Trodusquemine (MSI-1436) on equine hepatic progenitor cells (HPCs) under lipotoxic conditions. We examined proliferative activity, glucose uptake, and mitochondrial morphogenesis. Our study found that MSI-1436 promotes HPC entry into the cell cycle and protects them from palmitate-induced apoptosis by regulating mitochondrial dynamics and biogenesis. MSI-1436 also increases glucose uptake and protects HPCs from palmitate-induced stress by reorganizing the cells’ morphological architecture. Furthermore, our findings suggest that MSI-1436 enhances 2-NBDG uptake by increasing the expression of SIRT1, which is associated with liver insulin sensitivity. It also promotes mitochondrial dynamics by modulating mitochondria quantity and morphotype as well as increasing the expression of PINK1, MFN1, and MFN2. Our study provides evidence that MSI-1436 has a positive impact on equine hepatic progenitor cells, indicating its potential therapeutic value in treating EMS and insulin dysregulation.

BackgroundEquine metabolic syndrome (EMS) is a multifactorial pathology gathering insulin resistance, low-grade inflammation and past or chronic laminitis. Among the several molecular mechanisms underlying EMS pathogenesis, increased negative insulin signalling regulation mediated by protein tyrosine phosphatase 1 B (PTP1B) has emerged as a critical axis in the development of liver insulin resistance and general metabolic distress associated to increased ER stress, inflammation and disrupted autophagy. Thus, the use of PTP1B selective inhibitors such as MSI-1436 might be considered as a golden therapeutic tool for the proper management of EMS and associated conditions. Therefore, the present investigation aimed at verifying the clinical efficacy of MSI-1436 systemic administration on liver metabolic balance, insulin sensitivity and inflammatory status in EMS affected horses. Moreover, the impact of MSI-1436 treatment on liver autophagy machinery and associated ER stress in liver tissue has been analysed.MethodsLiver explants isolated from healthy and EMS horses have been treated with MSI-1436 prior to gene and protein expression analysis of main markers mediating ER stress, mitophagy and autophagy. Furthermore, EMS horses have been intravenously treated with a single dose of MSI-1436, and evaluated for their metabolic and inflammatory status.ResultsClinical application of MSI-1436 to EMS horses restored proper adiponectin levels and attenuated the typical hyperinsulinemia and hyperglycemia. Moreover, administration of MSI-1436 further reduced the circulating levels of key pro-inflammatory mediators including IL-1β, TNF-α and TGF-β and triggered the Tregs cells activation. At the molecular level, PTP1B inhibition resulted in a noticeable mitigation of liver ER stress, improvement of mitochondrial dynamics and consequently, a regulation of autophagic response. Similarly, short-term ex vivo treatment of EMS liver explants with trodusquemine (MSI-1436) substantially enhanced autophagy by upregulating the levels of HSC70 and Beclin-1 at both mRNA and protein level. Moreover, the PTP1B inhibitor potentiated mitophagy and associated expression of MFN2 and PINK1. Interestingly, inhibition of PTP1B resulted in potent attenuation of ER stress key mediators’ expression namely, CHOP, ATF6, HSPA5 and XBP1. ConclusionPresented findings shed for the first time promising new insights in the development of an MSI-1436-based therapy for proper equine metabolic syndrome intervention and may additionally find potential translational application to human metabolic syndrome treatment.

Abstract Background Protein tyrosine phosphatase 1B (PTP1B) and low molecular weight protein tyrosine phosphatase (LMPTP) are implicated in the development of metabolic disorders. Yet, their role in progenitor stem cell adipogenic differentiation and modulation of mitochondrial dynamics remains elusive. Methods In this study, we decided to investigate whether inhibition of PTP1B and LMPTP enhance adipogenic differentiation of metabolically impaired progenitor stem cells via modulation of mitochondrial bioenergetics and dynamics. Cells were cultured under adipogenic conditions in the presence of PTP1B and LMPTP inhibitors, and were subjected to the analysis of the main adipogenic-related and mitochondrial-related genes using RT-qPCR. Protein levels were established with western blot while mitochondrial morphology with MicroP software. Results Selective inhibitors of both PTP1B and MPTP enhanced adipogenic differentiation of metabolically impaired progenitor stem cells. We have observed enhanced expression of PPARy and adiponectin in treated cells. What is more, increased antioxidative defence and alternations in mitochondrial bioenergetics were observed. We have found that inhibition of PTP1B as well as C23 activates oxidative phosphorylation and enhances mitochondrial fusion contributing to enhanced adipogenesis. Conclusions The presented data provides evidence that the application of PTP1B and LMPTP inhibitors enhances adipogenesis through the modulation of mitochondrial dynamics. Video abstract

Protein tyrosine phosphatase PTP1B is considered as a key metabolic enzyme that has been reported to be associated with insulin resistance onset, and underlying cellular metabolic malfunctions, including ER stress and mitochondrial failure. In this study, effects of selective PTP1B inhibition using MSI-1436 on cellular apoptosis, oxidative stress, mitochondrial dysfunction and ER stress have been assessed using an in vitro model of Tunicamycin induced ER stress in HepG2 cell line. Inhibition of PTP1B using MSI-1436 significantly increased cell viability and reduced the number of apoptotic cells as well as the expression of key apoptosis initiators and effectors. MSI-1436 further mitigated ER stress, by downregulating the expression of IRE1, ATF6 and PERK transcripts, all being key ER stress sensors. Interestingly, MSI-1436 inhibited the XBP1 splicing, and thus its UPR-associated transcriptional activity. PTP1B inhibition further enabled to restore proper mitochondrial biogenesis, by improving transmembrane potential, and diminishing intracellular ROS while restoring of endogenous antioxidant enzymes genes expression. PTP1B inhibition using MSI-1436 could improve cellular apoptosis and metabolic integrity through the mitigation of ER and mitochondrial stress signalling pathways, and excessive ROS accumulation. This strategy may be useful for the treatment of metabolic disorders including IR, NAFLD and diabetes.

Background. Hyperactivation of protein tyrosine phosphatase (PTP1B) has been associated with several metabolic malfunctions ranging from insulin resistance, metaflammation, lipotoxicity, and hyperglycaemia. Liver metabolism failure has been proposed as a core element in underlying endocrine disorders through persistent inflammation and highly fibrotic phenotype. Methods. In this study, the outcomes of PTP1B inhibition using trodusquemine (MSI-1436) on key equine metabolic syndrome (EMS)-related alterations including inflammation, fibrosis, and glucose uptake have been analyzed in liver explants collected from EMS-affected horses using various analytical techniques, namely, flow cytometry, RT-qPCR, and Western blot. Results. PTP1B inhibition using trodusquemine resulted in decreased proinflammatory cytokines (IL-1β, TNF-α, and IL-6) release from liver and PBMC affected by EMS and regulated expression of major proinflammatory microRNAs such as miR-802 and miR-211. Moreover, MSI-1436 enhanced the anti-inflammatory profile of livers by elevating the expression of IL-10 and IL-4 and activating CD4+CD25+Foxp3+ regulatory T cells in treated PBMC. Similarly, the inhibitor attenuated fibrogenic pathways in the liver by downregulating TGF-β/NOX1/4 axis and associated MMP-2/9 overactivation. Interestingly, PTP1B inhibition ameliorated the expression of TIMP-1 and Smad7, both important antifibrotic mediators. Furthermore, application of MSI-1436 was found to augment the abundance of glycosylated Glut-2, which subsequently expanded the glucose absorption in the EMS liver, probably due to an enhanced Glut-2 stability and half-life onto the plasma cell membranes. Conclusion. Taken together, the presented data suggest that the PTP1B inhibition strategy and the use of its specific inhibitor MSI-1436 represents a promising option for the improvement of liver tissue integrity and homeostasis in the course of EMS and adds more insights for ongoing clinical trials for human MetS management.

Abstract Mesenchymal stem cells (MSCs), known from their key role in the regeneration process of tissues, and their abilities to release bioactive factors like extracellular vesicles (EVs) could be considered as a potential, modern tool in the treatment of AKI (acute kidney injury) in both human and veterinary patients. The complex pathophysiology of a renal function disorder (AKI) makes difficult to find a universal therapy, but the treatment strategy is based on MSCs and derived from them, EVs seem to solve this problem. Due to their small size, the ability of the cargo transport, the ease of crossing the barriers and the lack of the ability to proliferate and differentiate, EVs seem to have a significant impact on the development such therapy. Their additional impact associated with their ability to modulate immune response and inflammation process, their strong anti-fibrotic and anti-apoptotic effects and the relation with the releasing of the reactive oxygen species (ROS), that pivotal role in the AKI development is undoubtedly, limits the progress of AKI. Moreover, the availability of EVs from different sources encourages to extend research with using EVs from MSCs in AKI treatment in felines; in that, the possibilities of kidney injuries treatment are still limited to the classical therapies burdened with dangerous side effects. In this review, we underline the significance of the processes, in whose EVs are included during the AKI in order to show the potential benefits of EVs-MSCs-based therapies against AKI in felines.

Abstract Background Chronic superphysiological glucose and insulin concentrations are known to trigger several tissue and organ failures, including insulin resistance, oxidative stress and chronic low-grade inflammation. Hence, the screening for molecules that may counteract such conditions is essential in current existing therapeutic strategies, thereby the use of medicinal plant derivatives represents a promising axis in this regard. Methods In this study, the effect of a selected traditional medicinal plant, Hyoscyamus albus from which, calystegines have been isolated, was investigated in an experimental model of hyperinsulinemia and hyperglycemia induced on HepG2 cells. The mRNA and protein expression levels of different insulin signaling, gluconeogenic and inflammatory pathway- related molecules were examined. Additionally, cell viability and apoptosis, oxidative stress extent and mitochondrial dysfunctions were assayed using flow cytometric and qRT-PCR techniques. Results Treatment of IR HepG2 cells with calystegines strongly protected the injured cells from apoptosis, oxidative stress and mitochondrial integrity loss. Interestingly, nortropane alkaloids efficiently regulated the impaired glucose metabolism in IR HepG2 cells, through the stimulation of glucose uptake and the modulation of SIRT1/Foxo1/G6PC/mTOR pathway, which is governing the hepatic gluconeogenesis. Furthermore, the alkaloidal extract restored the defective insulin signaling pathway, mainly by promoting the expression of Insr at the mRNA and protein levels. What is more, treated cells exhibited significant mitigated inflammatory response, as evidenced by the modulation and the regulation of the NF- κB/JNK/TLR4 axis and the downstream proinflammatory cytokines recruitment. Conclusion Overall, the present investigation demonstrates that calystegines from Hyoscyamus albus provide cytoprotection to the HepG2 cells against insulin/glucose induced insulin resistance and apoptosis due to the regulation of SIRT1/Foxo1/G6PC/mTOR and NF-κB/JNK/TLR4 signaling pathways. Video Abstract

Abstract Background The senescence of dermal fibroblasts (DFLs) leads to an imbalance in the synthesis and degradation of extracellular matrix (ECM) proteins, presenting so-called senescence-associated secretory phenotype (SASP), which ultimately leads to skin aging. Recently, mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been recognized as a promising cell-free therapy for degenerative diseases, which opens a new avenue for skin aging treatment. Methods In this study, we utilized chitosan (CS) hydrogel for effective loading and sustained release of EVs. In vitro, we explored the rejuvenation effects of CS hydrogel-incorporated EVs (CS-EVs) on replicative senescence DFLs through a series of experiments such as senescence-associated β-galactosidase (SA-β-gal) staining, RT-PCR, and Western blot analysis. Besides, we employed local multi-site subcutaneous injection to treat skin aging of naturally aged mice with CS-EVs and DiI fluorescent dye was used to label EVs to achieve in vivo real-time tracking. Results CS-EVs can significantly improve the biological functions of senescent fibroblasts, including promoting their proliferation, enhancing the synthesis of ECM proteins, and inhibiting the overexpression of matrix metalloproteinases (MMPs). Moreover, CS hydrogel could prolong the release of EVs and significantly increase the retention of EVs in vivo. After CS-EVs subcutaneous injection treatment, the aging skin tissues showed a rejuvenation state, manifested explicitly as the enhanced expression of collagen, the decreased expression of SASP-related factors, and the restoration of tissue structures. Conclusions CS hydrogel-encapsulated EVs could delay the skin aging processes by ameliorating the function of aging DFLs. Our results also highlight the potential of CS hydrogel-encapsulated EVs as a novel therapeutic strategy for improving aging skin to rejuvenation.

Abstract Background Protein tyrosine phosphatase 1B (PTP1B) is one of the major negative regulators of leptin and insulin signaling, and has been strongly implicated in insulin resistance development in the course of obesity and metabolic syndrome conditions; however, its exact role in controlling adipose tissue biogenesis is still poorly understood. Objectives This investigation aimed to elucidate whether selective inhibition of PTP1B using MSI-1436 compound may improve and restore the defective adipogenicity of ASCs isolated from EMS-affected horses. Methods Equine ASC EMS cells were cultured under adipogenic conditions in the presence of PTP1B inhibitor and were subsequently tested for expression of the main adipogenic-related genes using RT-qPCR, changes in free fatty acid profiles by means of GC-MS technique, and for mitochondrial dynamics improvement through the analysis of mitochondrial transmembrane potential and oxidative stress. Results Selective inhibition of PTP1B in equine ASC EMS cells improved substantially adipogenic differentiation by promoting cellular proliferation and normalizing expression of C/EBPalpha, PPARγ, and Adipoq markers that are critical for proper adipogenesis. Levels of secreted adiponectin and PPARγ were also shown to be increased in MSI-1436-conditioned cells, while total leptin levels markedly dropped under the same conditions. Moreover, MSI-1436 treatment enabled the regulation of metabolic-related transcripts that are crosslink to adipogenesis, namely Akt1, Akt2, and SHBG. The obtained results demonstrated also an obvious reduction in intracellular accumulated ROS and NO, as well as mitigated ER stress through the downregulation of Chop, Perk, Atf6, Ire1, and Xbp1 transcripts upon PTP1B inhibition. Furthermore, general fluctuations in FFA composition of all differentiated groups have been highlighted, where palmitic acid, palmitoleic acid, stearic acid, and linolelaidic acid that are known to be associated with the development of metabolic disorders were found to be normalized upon PTP1B inhibition during adipogenic differentiation. Conclusion The presented data provides the evidence that the use of PTP1B inhibitor may be successful in controlling and enhancing adipogenic differentiation of impaired equine ASCs affected by metabolic syndrome, and thus offers new insights for the management of obesity through the regulation of adipose tissue dynamics. Graphical abstract

Emerging knowledge indicates that non-coding RNAs, including microRNAs (miRNAs) and long-noncoding RNAs (lncRNAs), have a pivotal role in bone development and the pathogenesis of bone-related disorders. Most recently, miRNAs have started to be regarded as potential biomarkers or targets for various sets of diseases, while lncRNAs have gained attention as a new layer of gene expression control acting through versatile interactions, also with miRNAs. The rapid development of RNA sequencing techniques based on next-generation sequencing (NGS) gives us better insight into molecular pathways regulated by the miRNA-lncRNA network. In this review, we summarize the current knowledge related to the function of miRNAs and lncRNAs as regulators of genes that are crucial for proper bone metabolism and homeostasis. We have characterized important non-coding RNAs and their expression signatures, in relationship to bone. Analysis of the biological function of miRNAs and lncRNAs, as well as their network, will pave the way for a better understanding of the pathogenesis of various bone disorders. We also think that this knowledge may lead to the development of innovative diagnostic tools and therapeutic approaches for bone-related disorders.

Abstract Background Progressive loss of cell functionality caused by an age-related impairment in cell metabolism concerns not only mature specialized cells but also its progenitors, which significantly reduces their regenerative potential. Adipose-derived stem cells (ASCs) are most commonly used in veterinary medicine as an alternative treatment option in ligaments and cartilage injuries, especially in case of high-value sport horses. Therefore, the main aim of this study was to identify the molecular alternations in ASCs derived from three age-matched horse groups: young ( 15 years old). Methods ASCs were isolated from three age-matched horse groups using an enzymatic method. Molecular changes were assessed using qRT-PCR, ELISA and western blot methods, flow cytometry-based system, and confocal and scanning electron microscopy. Results Our findings showed that ASCs derived from the middle-aged and old groups exhibited a typical senescence phenotype, such as increased percentage of G1/G0-arrested cells, binucleation, enhanced β-galactosidase activity, and accumulation of γH2AX foci, as well as a reduction in cell proliferation. Moreover, aged ASCs were characterized by increased gene expression of pro-inflammatory cytokines and miRNAs (interleukin 8 (IL-8), IL-1β, tumor necrosis factor α (TNF-α), miR-203b-5p, and miR-16-5p), as well as apoptosis markers (p21, p53, caspase-3, caspase-9). In addition, our study revealed that the protein level of mitofusin 1 (MFN1) markedly decreased with increasing age. Aged ASCs also displayed a reduction in mRNA levels of genes involved in stem cell homeostasis and homing, like TET-3, TET-3 (TET family), and C-X-C chemokine receptor type 4 (CXCR4), as well as protein expression of DNA methyltransferase (DNMT1) and octamer transcription factor 3/4 (Oct 3/4). Furthermore, we observed a higher splicing ratio of XBP1 (X-box binding protein 1) mRNA, indicating elevated inositol-requiring enzyme 1 (IRE-1) activity and, consequently, increased endoplasmic reticulum (ER) stress. We also observed reduced levels of glucose transporter 4 (GLUT-4) and insulin receptor (INSR) which indicated impaired insulin sensitivity. Conclusions Obtained data suggest that ASCs derived from horses older than 5 years old exhibited several molecular alternations which markedly limit their regenerative capacity. The results provide valuable information that allows for a better understanding of the molecular events occurring in ASCs in the course of aging and may help to identify new potential drug targets to restore their regenerative potential. Graphical abstract

Abstract Background Chondrogenesis represents a highly dynamic cellular process that leads to the establishment of various types of cartilage. However, when stress-related injuries occur, a rapid and efficient regeneration of the tissues is necessary to maintain cartilage integrity. Mesenchymal stem cells (MSCs) are known to exhibit high capacity for self-renewal and pluripotency effects, and thus play a pivotal role in the repair and regeneration of damaged cartilage. On the other hand, the influence of certain pathological conditions such as metabolic disorders on MSCs can seriously impair their regenerative properties and thus reduce their therapeutic potential. Objectives In this investigation, we attempted to improve and potentiate the in vitro chondrogenic ability of adipose-derived mesenchymal stromal stem cells (ASCs) isolated from horses suffering from metabolic syndrome. Methods Cultured cells in chondrogenic-inductive medium supplemented with Cladophora glomerata methanolic extract were experimented for expression of the main genes and microRNAs involved in the differentiation process using RT-PCR, for their morphological changes through confocal and scanning electron microscopy and for their physiological homeostasis. Results The different added concentrations of C. glomerata extract to the basic chondrogenic inductive culture medium promoted the proliferation of equine metabolic syndrome ASCs (ASCsEMS) and resulted in chondrogenic phenotype differentiation and higher mRNA expression of collagen type II, aggrecan, cartilage oligomeric matrix protein, and Sox9 among others. The results reveal an obvious inhibitory effect of hypertrophy and a strong repression of miR-145-5p, miR-146-3p, and miR-34a and miR-449a largely involved in cartilage degradation. Treated cells additionally exhibited significant reduced apoptosis and oxidative stress, as well as promoted viability and mitochondrial potentiation. Conclusion Chondrogenesis in EqASCsEMS was found to be prominent after chondrogenic induction in conditions containing C. glomerata extract, suggesting that the macroalgae could be considered for the enhancement of ASC cultures and their reparative properties.

Abstract Background In athlete horses, suspensory ligament (SL) injuries are the most common cause of lameness. Healing of SL injury is still problematic, and even proper rehabilitation and pharmacological therapy do not guarantee returning to the initial performance level. In our previous studies, we have shown that a combination of 5-azacytidine (AZA) and resveratrol (RES) exerts beneficial, rejuvenating effects on metabolic syndrome derived adipose-derived stem cells (ASCs). Thus, in the presented research, we investigate whether not only rejuvenated ASC but also microvesicles (MVsAZA/RES) secreted by them possess enhanced regenerative properties in SL injury. Methods In the presented study, a 6-year-old Dutch Warmblood gelding, working in jumping, was diagnosed with SL injury using ultrasonography, Doppler, real-time elastography and thermography. As a therapeutic strategy, the affected animal was treated with extracellular microvesicles derived from ASC treated with the combination of 5-azacytydine (AZA) and resveratrol (RES) (MVsAZA/RES). Results First, anti-apoptotic effects of MVsAZA/RES were tested in co-culture with metabolic syndrome derived ASC. The proliferation of cells and expression of pro-apoptotic genes were investigated. Then, MVsAZA/RES were injected directly into the injured SL of the Dutch Warmblood gelding. In vitro assays revealed that MVsAZA/RES enhance the proliferation of ASC and exert an anti-apoptotic effect. In the affected horse, the application of MVsAZA/RES resulted in increased lesion filling and improvement of angiogenesis and elasticity in injured tissue. Conclusions As MVsAZA/RES mimic several of the biological actions exerted by ASC, they have become an alternative for stem cell-based therapies and can be effectively applied for the treatment of SL injury in horses.

Abstract Background Cervical vertebral stenotic myelopathy (CVSM) remains one of the most important abnormalities of the cervical spine resulting in neurological deficits in horses. The aim of the following study was to compare the results of the clinical and neurological examination, the results of myelography and the post mortem anatomical and histological appearance of the spinal cord and cervical vertebrae in a horse with CVSM. Case presentation The following study describes a clinical case of an eight-month-old stallion with ataxia. Plain cervical radiographs indicated narrowing of the spinal canal. Conservative therapy using NSAIDs did not result in any improvement in the gait of the horse. Due to economic constraints, surgical intervention was excluded. The owner chose to humanely euthanise the horse. Immediately after euthanasia, post mortem myelography was performed, and measurements of the myelographic dye column were taken. They revealed a 67% DMC reduction and a 64% DD reduction at the C3/C4 level. Afterwards, an anatomical dissection was performed. The cervical vertebrae and vertebral canal were macroscopically inspected and measured and indicated a 44% narrowing of the canal at the C3/C4 level. The spinal cord was removed and underwent histological evaluation after staining. Microscopic lesions were visible at the level of the compression and included axonal degeneration with partial or complete loss of myelin in the white matter of the lateral and dorsal funiculi as well as the formation of dysfunctional so-called “spongy structures”. An increase in the number of microglial cells and collagen was also observed. The formation of glial scars was excluded. Immunohistochemical studies revealed a negative transmembrane glycoprotein CD68(−) – monocyte response and a negative tumor necrosis alpha TNFα (−) reaction. Conclusions CVSM may be difficult to diagnose, even for experienced veterinary surgeons. Currently, an ex vivo histopathologic examination of the spinal cord is thought to be the gold standard in the diagnosis of CVSM. Our histological examination revealed no CVSM-specific glial scar formation and a CD68(−) negative and TNF-α negative reaction, which have not been previously reported. Histological lesions in CVSM may vary depending show inter-individual variability and on the treatment, which further hinders ex-vivo diagnostics.

Abstract Objectives Equine metabolic syndrome (EMS) refers to a cluster of associated abnormalities and metabolic disorders, including insulin resistance and adiposity. The numerous biological properties of mesenchymal stem cells (MSCs), including self-renewal and multipotency, have been the subject of many in-depth studies, for the management of EMS; however, it has been shown that this cell type may be affected by the condition, impairing thus seriously their therapeutic potential. Therefore, an attempt to rescue EMS adipose-derived stem cells (ASCs) with calystegines (polyhydroxylated alkaloids) that are endowed with strong antioxidant and antidiabetic abilities was performed. Methods ASCs isolated from EMS horses were subsequently treated with various concentrations of total calystegines. Different parameters were then assessed using flow cytometry, confocal as well as SE microscopy, and RT-qPCR. Results Our results clearly demonstrated that calystegines could improve EqASC viability and proliferation and significantly reduce apoptosis, via improvement of mitochondrial potentiation and functionality, regulation of pro- and anti-apoptotic pathways, and suppression of ER stress. Furthermore, nortropanes positively upregulated GLUT4 and IRS transcripts, indicating a possible sensitizing or mimetic effect to insulin. Most interesting finding in this investigation lies in the modulatory effect of autophagy, a process that allows the maintenance of cellular homeostasis; calystegines acted as pharmacological chaperones to promote cell survival. Conclusion Obtained data open new perspectives in the development of new drugs, which may improve the metabolic dynamics of cells challenged by MS.

Cyclosporine A (CsA) is a selective and reversible immunosuppressant agent that is widely used as a medication for a wide spectrum of diseases in humans such as graft versus host disease, non-infectious uveitis, rheumatoid arthritis, psoriasis, and atopic dermatitis. Furthermore, the CsA is used to treat keratoconjunctivitis sicca, chronic superficial keratitis, immune-mediated keratitis and equine recurrent uveitis in animals. The selective activity of Cyclosporine A (CsA) was demonstrated to be an immunomodulation characteristic of T-lymphocyte proliferation and inhibits cytokine gene expression. Moreover, the lipophilic characteristics with poor bioavailability and low solubility in water, besides the side effects, force the need to develop new formulations and devices that will provide adequate penetration into the anterior and posterior segments of the eye. This review aims to summarize the effectiveness and safety of cyclosporine A delivery platforms in veterinary ophthalmology.

Astaxanthin is gaining recognition as a natural bioactive component. This study aimed to test whether astaxanthin could protect adipose-derived stromal stem cells (ASCs) from apoptosis, mitochondrial dysfunction and oxidative stress. Phaffia rhodozyma was used to extract astaxanthin, whose biocompatibility was tested after 24, 48 and 72 h of incubation with the cells; no harmful impact was found. ASCs were treated with optimal concentrations of astaxanthin. Several parameters were examined: cell viability, apoptosis, reactive oxygen levels, mitochondrial dynamics and metabolism, superoxide dismutase activity, and astaxanthin’s antioxidant capacity. A RT PCR analysis was performed after each test. The astaxanthin treatment significantly reduced apoptosis by modifying the normalized caspase activity of pro-apoptotic pathways (p21, p53, and Bax). Furthermore, by regulating the expression of related master factors SOD1, SOD2, PARKIN, PINK 1, and MFN 1, astaxanthin alleviated the oxidative stress and mitochondrial dynamics failure caused by EMS. Astaxanthin restored mitochondrial oxidative phosphorylation by stimulating markers associated with the OXPHOS machinery: COX4I1, COX4I2, UQCRC2, NDUFA9, and TFAM. Our results suggest that astaxanthin has the potential to open new possibilities for potential bio-drugs to control and suppress oxidative stress, thereby improving the overall metabolic status of equine ASCs suffering from metabolic syndrome.

The study aimed to investigate the influence of obesity on cellular features of equine endometrial progenitor cells (Eca EPCs), including viability, proliferation capacity, mitochondrial metabolism, and oxidative homeostasis. Eca EPCs derived from non-obese (non-OB) and obese (OB) mares were characterized by cellular phenotype and multipotency. Obesity-induced changes in the activity of Eca EPCs include the decline of their proliferative activity, clonogenic potential, mitochondrial metabolism, and enhanced oxidative stress. Eca EPCs isolated from obese mares were characterized by an increased occurrence of early apoptosis, loss of mitochondrial dynamics, and senescence-associated phenotype. Attenuated metabolism of Eca EPCs OB was related to increased expression of pro-apoptotic markers (CASP9, BAX, P53, P21), enhanced expression of OPN, PI3K, and AKT, simultaneously with decreased signaling stabilizing cellular homeostasis (including mitofusin, SIRT1, FOXP3). Obesity alters functional features and the self-renewal potential of endometrial progenitor cells. The impaired cytophysiology of progenitor cells from obese endometrium predicts lower regenerative capacity if used as autologous transplants.

Hyperglycaemia and its resulting glucotoxicity are among the most prominent hallmarks of diabetes mellitus (DM) development. Persistent hyperglycaemia further leads to oxidative stress via mitochondrial dysfunction and subsequent ER stress onset, while associated hyperlipidaemia triggers the adipose tissue to secrete pro-inflammatory cytokines. In this study, the effect of calystegines has been investigated in an experimental model of hyperglycaemia induced on human ASCs cells. Different cellular pathways including apoptosis, oxidative and ER stress, inflammation as well as Pi3K/AKT/mTOR metabolic-associated axis have been evaluated by means on RT-qPCR, western blot, and flow cytometry techniques. Treatment of HuASCs cells with calystegines strongly promoted the hyperglycaemic cells survival and significantly diminished oxidative stress, mitochondrial dynamics failure and ER stress, while improving the endogenous cellular antioxidant defenses. Interestingly, nortropane alkaloids efficiently prevented the hyperglycaemia-mediated inflammatory response, as evidenced by the regulation of the pro- and anti-inflammatory response in HuASCs cells. Finally, we evidenced that calystegines may exert their protective effect on HuASCs cells metabolic functions through the restoration of the defective PI3K/AKT/mTOR pathway. Overall, the present investigation demonstrated that calystegines possess important abilities to protect HuASCs against hyperglycaemia-induced cellular dysfunction, and it evidenced that the observed effects are associated to the promotion of PI3K/AKT/mTOR pathway.

Despite multiple research studies regarding metabolic syndrome and diabetes, the full picture of their molecular background and pathogenies remains elusive. The latest studies revealed that sex hormone-binding globulin (SHBG)—a serum protein released mainly by the liver—may participate in metabolic dysregulation, as its low serum level correlates with a risk for obesity, metabolic syndrome, and diabetes. Yet, the molecular phenomenon linking SHBG with these disorders remains unclear. In the presented study, we investigate how exogenous SHBG affects metabolically impaired hepatocytes with special attention to endoplasmic reticulum stress (ER stress) and lipid metabolism both in vitro and ex vivo. For that reason, palmitate-treated HepG2 cells and liver tissue samples collected post mortem were cultured in the presence of 50 nM and 100 nM SHBG. We found that SHBG protects against ER stress development and its progression. We have found that SHBG decreased the expression levels of inositol-requiring enzyme 1 (IRE1α), activating transcription factor 6 (ATF6), DNA damage-inducible transcript 3 (CHOP), and immunoglobulin heavy chain-binding protein (BIP). Furthermore, we have shown that it regulates lipolytic gene expression ex vivo. Additionally, herein, we deliver a novel large-animal model to study SHBG in translational research. Our data provide new insights into the cellular and molecular mechanisms by which SHBG modulates hepatocyte metabolism and offer a new experimental approach to study SHBG in human diseases.

In the present study, a nanoapatite-mediated delivery system for imatinib has been proposed. Nanohydroxyapatite (nHAp) was obtained by co-precipitation method, and its physicochemical properties in combination with imatinib (IM) were studied by means of XRPD (X-ray Powder Diffraction), SEM-EDS (Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy), FT-IR (Fourier-Transform Infrared Spectroscopy), absorption spectroscopy as well as DLS (Dynamic Light Scattering) techniques. The obtained hydroxyapatite was defined as nanosized rod-shaped particles with high crystallinity. The amorphous imatinib was obtained by conversion of its crystalline form. The beneficial effects of amorphous pharmaceutical agents have been manifested in the higher dissolution rate in body fluids improving their bioavailability. Imatinib-to-hydroxyapatite interactions on the surface were confirmed by SEM images as well as absorption and FT-IR spectroscopy. The cytotoxicity of the system was tested on NI-1, L929, and D17 cell lines. The effectiveness of imatinib was not affected by nHAp modification. The calculated IC50 values for drug-modified nHAp were similar to those for the drug itself. However, higher cytotoxicity was observed at higher concentrations of imatinib, in comparison with the drug alone.

Bioactive compounds of natural origin are gaining increasing popularity. High biological activity and bioavailability, beneficial effects on health and safety of use are some of their most desirable features. Low production and processing costs render them even more attractive. Microorganisms have been used in the food, medicinal, cosmetic and energy industries for years. Among them, microalgae have proved to be an invaluable source of beneficial compounds. Haematococcus pluvialis is known as the richest source of natural carotenoid called astaxanthin. In this paper, we focus on the cultivation methods of this green microalga, its chemical composition, extraction of astaxanthin and analysis of its antioxidant, anti-inflammatory, anti–diabetic and anticancer activities. H. pluvialis, as well as astaxanthin can be used not only for the treatment of human and animal diseases, but also as a valuable component of diet and feed.

MiR-21 is being gradually more and more recognized as a molecule regulating bone tissue homeostasis. However, its function is not fully understood due to the dual role of miR-21 on bone-forming and bone-resorbing cells. In this study, we investigated the impact of miR-21 inhibition on pre-osteoblastic cells differentiation and paracrine signaling towards pre-osteoclasts using indirect co-culture model of mouse pre-osteoblast (MC3T3) and pre-osteoclast (4B12) cell lines. The inhibition of miR-21 in MC3T3 cells (MC3T3inh21) modulated expression of genes encoding osteogenic markers including collagen type I (Coll-1), osteocalcin (Ocl), osteopontin (Opn), and runt-related transcription factor 2 (Runx-2). Inhibition of miR-21 in osteogenic cultures of MC3T3 also inflected the synthesis of OPN protein which is essential for proper mineralization of extracellular matrix (ECM) and anchoring osteoclasts to the bones. Furthermore, it was shown that in osteoblasts miR-21 regulates expression of factors that are vital for survival of pre-osteoclast, such as receptor activator of nuclear factor κB ligand (RANKL). The pre-osteoclast cultured with MC3T3inh21 cells was characterized by lowered expression of several markers associated with osteoclasts’ differentiation, foremost tartrate-resistant acid phosphatase (Trap) but also receptor activator of nuclear factor-κB ligand (Rank), cathepsin K (Ctsk), carbonic anhydrase II (CaII), and matrix metalloproteinase (Mmp-9). Collectively, our data indicate that the inhibition of miR-21 in MC3T3 cells impairs the differentiation and ECM mineralization as well as influences paracrine signaling leading to decreased viability of pre-osteoclasts.

In the present study, a comprehensive approach to the biosorption process was proposed. Biosorption of Cr(III), Mn(II) and Mg(II) ions by a freshwater macroalga Cladophora glomerata was examined using several advanced techniques including FTIR (Fourier Transform Infrared Spectroscopy), ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometry) and SEM-EDX (Scanning Electron Microscopy with Energy Dispersive X-Ray Spectroscopy). The enriched biomass can become a valuable, bioactive feed additive for different breeds of animals. Additionally, the collected algal biomass was soaked in water in order to reduce the content of carbohydrate, what is especially important for animals with metabolic disorders. The content of starch was reduced by 22% but additionally some elements-mainly Si, K and P were removed from the biomass. It was shown that the natural macroalga had better biosorption properties than soaked. Cr(III) ions were sorbed by the biomass in the highest extent, then Mn(II) and finally Mg(II) ions. The content of chromium in the enriched algal biomass increased almost ~200 000 times, manganese ~75 times and magnesium ~4.5 times (both for Mg(II) ions used from magnesium sulphate, as well as from magnesium chloride) when compared to the natural Cladophora glomerata. In the case of the soaked biomass the increase of the content of elements in the enriched biomass was as follows ~17 165 times for Cr, ~25 times for Mn and for Mg ~3.5 times for chloride and 3.8 times for sulphate. The type of magnesium salt (chloride or sulphate) had no significant effect on the algal sorption capacity. The proposed mechanism of the biosorption is ion exchange in which mainly potassium participated. The applied FTIR analysis enabled the identification of the functional groups that participated in the biosorption process-mainly carboxyl and hydroxyl. The main changes in the appearance of the spectra were observed for the following wavenumbers- 3300-3400; 2900; 1700; 1400-1500 and 1200-1300 cm-1. The application of SEM-EDX proved that the metal ions were sorbed on the surface of both tested algae.

Metabolic syndrome (MetS) is highly associated with a modern lifestyle. The prevalence of MetS has reached epidemic proportion and is still rising. The main cause of MetS and finally type 2 diabetes occurrence is excessive nutrient intake, lack of physical activity, and inflammatory cytokines secretion. These factors lead to redistribution of body fat and oxidative and endoplasmic reticulum (ER) stress occurrence, resulting in insulin resistance, increase adipocyte differentiation, and much elevated levels of proinflammatory cytokines. Cellular therapies, especially mesenchymal stem cell (MSC) transplantation, seem to be promising in the MetS and type 2 diabetes treatments, due to their immunomodulatory effect and multipotent capacity; adipose-derived stem cells (ASCs) play a crucial role in MSC-based cellular therapies. In this review, we focused on etiopathology of MetS, especially on the crosstalk between chronic inflammation, oxidative stress, and ER stress and their effect on MetS-related disease occurrence, as well as future perspectives of cellular therapies. We also provide an overview of therapeutic approaches that target endoplasmic reticulum and oxidative stress.

Mesenchymal stem cells (MSCs) are frequently used in both human and veterinary medicine because their unique properties, such as modulating the immune response and differentiating into multiple lineages, make them a valuable tool in cell-based therapies. However, many studies have indicated the age-, lifestyle-, and disease-related deterioration of MSC regenerative characteristics. However, it still needs to be elucidated how the patient’s health status affects the effectiveness of MSC differentiation. In the present study, we isolated mesenchymal stem cells from adipose tissue (adipose-derived mesenchymal stem cells (ASCs)) from horses diagnosed with equine metabolic syndrome (EMS), a common metabolic disorder characterized by pathological obesity and insulin resistance. We investigated the metabolic status of isolated cells during adipogenic differentiation using multiple research methods, such as flow cytometry, PCR, immunofluorescence, or transmission and confocal microscopy. The results indicated the impaired differentiation potential of ASCEMS. Excessive ROS accumulation and ER stress are most likely the major factors limiting the multipotency of these cells. However, we observed autophagic flux during differentiation as a protective mechanism that allows cells to maintain homeostasis and remove dysfunctional mitochondria.

Diphosphate compounds (KYP2O7) co-doped with Yb3+ and Er3+ ions were obtained by one step urea assisted combustion synthesis. The experimental parameters of synthesis were optimized using an experimental design approach related to co-dopants concentration and heattreatment as well as annealing time. The obtained materials were studied with theinitial requirements showing appropriate morphological (X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM)) and spectroscopic properties (emission, luminescence kinetics). Moreover, the effect of Er3+ and Yb3+ ions doped KYP2O7 on morphology, proliferative and metabolic activity and apoptosis in MC3T3-E1 osteoblast cell line and 4B12osteoclasts cell line was investigated. Furthermore, the expression of the common pro-osteogenic markers in MC3T3-E1 osteoblast as well as osteoclastogenesis related markers in 4B12 osteoclasts was evaluated. The extensive in vitro studies showed that KYP2O7 doped with 1 mol% Er3+ and 20 mol% Yb3+ ions positively affected the MC3T3-E1 and 4B12 cells activity without triggering their apoptosis. Moreover, it was shown that an activation of mTOR and Pi3k signaling pathways with 1 mol% Er3+, 20 mol% Yb3+: KYP2O7 can promote the MC3T3-E1 cells expression of late osteogenic markers including RUNX and BMP-2. The obtained data shed a promising light for KYP2O7 doped with Er3+ and Yb3+ ions as a potential factors improving bone fracture healing as well as in bioimaging (so-called in theranostics).