(-)-epicatechin treatment did not modify the thermogenic pathway in the gastrocnemius muscle of male rat offspring obeses by programming.

The aim of this study was to analyse the expression of genes related to the regulation of energy metabolism in skeletal muscle tissue by comparing male offspring in two age groups [at 110 and 245 postnatal days (pnd)] from a mother with obesity induced by a high-fat diet and (-)-epicatechin (Epi) administration. Four groups of six male offspring from different litters were randomly selected for the control groups [C and offspring of mothers with maternal obesity (MO)] or Epi intervention groups. We evaluated the effect of Epi on gastrocnemius tissue by analysing the mRNA and protein expression levels of Fndc5/irisin, Pgc-1α, Ucp3, and Sln. Epi significantly increased the Pgc-1α protein in the MO group of offspring at 110 pnd (p < 0.036, MO vs. MO+Epi), while at 245 pnd, Epi increased Fndc5/irisin mRNA expression in the MO+Epi group versus the MO group (p = 0.006).No differences were detected in Fndc5/irisin, Ucp3 or Sln mRNA or protein levels (including Pgc-1α mRNA) in the offspring at 110 pnd or in Pgc-1α, Ucp3, or Sln mRNA or protein levels (including Fndc5/irisin protein) at 245 pnd among the experimental groups. In conclusion, (-)-epicatechin treatment increased Fndc5/irisin mRNA expression and Pgc-α protein levels in the gastrocnemius muscle of offspring at postnatal days 110 and 245. Furthermore, it is suggested that the flavonoid effect in a model of obesity and its impact on thermogenesis in skeletal muscle are regulated by a different pathway than Fndc5/irisin.

Trolox and recombinant Irisin as a potential strategy to prevent neuronal damage induced by random positioning machine exposure in differentiated HT22 cells.

Neuronal death could be responsible for the cognitive impairments found in astronauts exposed to spaceflight, highlighting the need to identify potential countermeasures to ensure neuronal health in microgravity conditions. Therefore, differentiated HT22 cells were exposed to simulated microgravity by random positioning machine (RPM) for 48 h, treating them with a single administration of Trolox, recombinant irisin (r-Irisin) or both. Particularly, we investigated cell viability by MTS assay, Trypan Blue staining and western blotting analysis for Akt and B-cell lymphoma 2 (Bcl-2), the intracellular increase of reactive oxygen species (ROS) by fluorescent probe and NADPH oxidase 4 (NOX4) expression, as well as the expression of brain-derived neurotrophic factor (BDNF), a major neurotrophin responsible for neurogenesis and synaptic plasticity. Although both Trolox and r-Irisin manifested a protective effect on neuronal health, the combined treatment produced the best results, with significant improvement in all parameters examined. In conclusion, further studies are needed to evaluate the potential of such combination treatment in counteracting weightlessness-induced neuronal death, as well as to identify other potential strategies to safeguard the health of astronauts exposed to spaceflight.

mir-150-5p inhibits the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by targeting irisin to regulate the p38/MAPK signaling pathway.

PURPOSE: To study the effect of miR-150-5p on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and further explore the relationship between its regulatory mechanism and irisin. METHODS: We isolated mouse BMSCs, and induced osteogenic differentiation by osteogenic induction medium. Using qPCR to detect the expression of osteogenic differentiation-related genes, western blot to detect the expression of osteogenic differentiation-related proteins, and luciferase reporter system to verify that FNDC5 is the target of miR-150-5p. Irisin intraperitoneal injection to treat osteoporosis in mice constructed by subcutaneous injection of dexamethasone. RESULTS: Up-regulation of miR-150-5p inhibited the proliferation of BMSCs, and decreased the content of osteocalcin, ALP activity, calcium deposition, the expression of osteogenic differentiation genes (Runx2, OSX, OCN, OPN, ALP and BMP2) and protein (BMP2, OCN, and Runx2). And down-regulation of miR-150-5p plays the opposite role of up-regulation of miR-150-5p on osteogenic differentiation of BMSCs. Results of luciferase reporter gene assay showed that FNDC5 gene was the target gene of miR-150-5p, and miR-150-5p inhibited the expression of FNDC5 in mouse BMSCs. The expression of osteogenic differentiation genes and protein, the content of osteocalcin, ALP activity and calcium deposition in BMSCs co-overexpressed by miR-150-5p and FNDC5 was significantly higher than that of miR-150-5p overexpressed alone. In addition, the overexpression of FNDC5 reversed the blocked of p38/MAPK pathway by the overexpression of miR-150-5p in BMSCs. Irisin, a protein encoded by FNDC5 gene, improved symptoms in osteoporosis mice through intraperitoneal injection, while the inhibitor of p38/MAPK pathway weakened this function of irisin. CONCLUSION: miR-150-5p inhibits the osteogenic differentiation of BMSCs by targeting irisin to regulate the/p38/MAPK signaling pathway, and miR-150-5p/irisin/p38 pathway is a potential target for treating osteoporosis.

Irisin protects against cardiac injury by inhibiting NLRP3 inflammasome-mediated pyroptosis during remodeling after infarction.

This study aimed to explore the cardioprotective mechanism of irisin in the context of cardiac injury. Utilizing a myocardial infarction (MI) mouse model, we investigated the therapeutic potential of recombinant human irisin (rhIrisin) administered for 28 days post-infarction. The efficacy of irisin treatment was evaluated through echocardiographic assessment of cardiac function and serum analysis of myocardial injury markers. Our research provided novel insights into the impacts of irisin on the NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation and pyroptosis, assessed both in vivo in MI mice and in vitro in hypoxia/reoxygenation-treated H9C2 cells. Remarkably, irisin treatment significantly reduced levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), and troponin I, indicating reduced myocardial injury. Echocardiography highlighted substantial improvements in left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and dimensions (LVIDd and LVIDs) in irisin-treated mice, underscoring enhanced cardiac function. Moreover, irisin was shown to significantly suppress the mRNA and protein expressions of key components involved in NLRP3 inflammasome pathway (NLRP3, ASC, caspase-1 (p20), and interleukin-18 (IL-18)) both in MI-induced mice and hypoxia/reoxygenation-treated cells. This study firstly reveals that the cardioprotective effect of irisin is mediated through the attenuation of NLRP3 inflammasome activation and pyroptosis, positioning irisin as a promising therapeutic agent for cardiac injury.

Alantolactone Attenuates Renal Fibrosis via Inhibition of Transforming Growth Factor ß/Smad3 Signaling Pathway.

BACKGRUOUND: Renal fibrosis is characterized by the accumulation of extracellular matrix proteins and interstitial fibrosis. Alantolactone is known to exert anticancer, anti-inflammatory, antimicrobial and antifungal effects; however, its effects on renal fibrosis remains unknown. Here, we investigated whether alantolactone attenuates renal fibrosis in mice unilateral ureteral obstruction (UUO) and evaluated the effect of alantolactone on transforming growth factor (TGF) signaling pathway in renal cells. METHODS: To evaluate the therapeutic effect of alantolactone, cell counting kit-8 (CCK-8) assay, histological staining, Western blot analysis, and real-time quantitative polymerase chain reaction were performed in UUO kidneys in vivo and in TGF-ß-treated renal cells in vitro. RESULTS: Alantolactone (0.25 to 4 µM) did not affect the viability of renal cells. Mice orally administered 5 mg/kg of alantolactone daily for 15 days did not show mortality or liver toxicity. Alantolactone decreased UUO-induced blood urea nitrogen and serum creatinine levels. In addition, it significantly alleviated renal tubulointerstitial damage and fibrosis and decreased collagen type I, fibronectin, and α-smooth muscle actin (α-SMA) expression in UUO kidneys. In NRK-49F cells, alantolactone inhibited TGF-ßstimulated expression of fibronectin, collagen type I, plasminogen activator inhibitor-1 (PAI-1), and α-SMA. In HK-2 cells, alantolactone inhibited TGF-ß-stimulated expression of collagen type I and PAI-1. Alantolactone inhibited UUO-induced phosphorylation of Smad3 in UUO kidneys. In addition, it not only decreased TGF-ß secretion but also Smad3 phosphorylation and translocation to nucleus in both kidney cell lines. CONCLUSION: Alantolactone improves renal fibrosis by inhibiting the TGF-ß/Smad3 signaling pathway in obstructive nephropathy. Thus, alantolactone is a potential therapeutic agent for chronic kidney disease.

Exercise ameliorating myocardial injury in type 2 diabetic rats by inhibiting excessive mitochondrial fission involving increased irisin expression and AMP-activated protein kinase phosphorylation.

PURPOSE: Though exercise generates beneficial effects on diabetes-associated cardiac damage, the underlying mechanism is largely unclear. Therefore, we prescribed a program of 8-week treadmill training for type 2 diabetes mellitus (T2DM) rats and determined the role of irisin signaling, via interacting with AMP-activated protein kinase (AMPK), in mediating the effects of exercise on myocardial injuries and mitochondrial fission. METHODS: Forty 8-week-old male Wistar rats were randomly divided into groups of control (Con), diabetes mellitus (DM), diabetes plus exercise (Ex), and diabetes plus exercise and Cyclo RGDyk (ExRg). Ex and ExRg rats received 8 weeks of treadmill running, and the rats in the ExRg group additionally were treated with a twice weekly injection of Cyclo RGDyk, an irisin receptor-αV/ß5 antagonist. At the end of the experiment, murine blood samples and heart tissues were collected and analyzed with methods of ELISA, Western blot, real-time quantitative polymerase chain reaction, as well as immunofluorescence staining. RESULTS: Exercise effectively mitigated T2DM-related hyperglycemia, hyperinsulinemia, lipid dysmetabolism, and inflammation, which could be diminished by Cyclo RGDyk treatment. Additionally, exercise alleviated T2DM-induced myocardial injury and excessive mitochondrial fission, whereas the beneficial effects were blocked by the administration of Cyclo RGDyk. T2DM significantly decreased serum irisin concentrations and fibronectin type III domain-containing protein 5 (FNDC5)/irisin gene and protein expression levels in the rat heart, whereas exercise could rescue T2DM-reduced FNDC5/irisin expression. Blocking irisin receptor signaling diminished the exercise-alleviated mitochondrial fission protein expression and elevated AMPK phosphorylation. CONCLUSION: Exercise is effective in mitigating diabetes-related insulin resistance, metabolic dysfunction, and inflammation. Irisin signaling engages in exercise-associated beneficial effects on myocardial injury and excessive mitochondrial fission in diabetes rats involving elevated AMPK phosphorylation.

Effects of irisin and exercise on adropin and betatrophin in a new metabolic syndrome model.

Metabolic syndrome (MetS) is a prevalent public health problem. Uric acid (UA) is increased by MetS. We investigated whether administration of UA and 10% fructose (F) would accelerate MetS formation and we also determined the effects of irisin and exercise. We used seven groups of rats. Group 1 (control); group 2 (sham); group 3 (10% F); group 4 (1% UA); group 5 (2% UA); group 6 (10% F + 1% UA); and Group 7, (10% F + 2% UA). After induction of MetS (groups 3 -7), Group 3 was divided into three subgroups: 3A, no further treatment; 3B, irisin treatment; 3C, irisin treatment + exercise. Group 4, 1% UA, which was divided into three subgroups: 4A, no further treatment; 4B, irisin treatment; 4C, Irisin treatment + exercise. Group 5, 2% UA, which was divided into three subgroups: 5A, no further treatment; 5B, irisin treatment; 5C, irisin treatment + exercise. Group 6, 10% F + 1% UA, which was divided into three subgroups: 6A, no further treatment; 6B, irisin treatment; 6C, irisin treatment + exercise. Group 7, 10% F + 2% UA, which was divided into three subgroups: 7A, no further treatment; 7B, irisin treatment; 7C, irisin treatment + exercise., Irisin was administered 10 ng/kg irisin intraperitoneally on Monday, Wednesday, Friday, Sunday each week for 1 month. The exercise animals (in addition to irisin treatment) also were run on a treadmill for 45 min on Monday, Wednesday, Friday, Sunday each week for 1 month. The rats were sacrificed and samples of liver, heart, kidney, pancreas, skeletal muscles and blood were obtained. The amounts of adropin (ADR) and betatrophin in the tissue supernatant and blood were measured using an ELISA method. Immunohistochemistry was used to detect ADR and betatrophin expression in situ in tissue samples. The duration of these experiments varied from 3 and 10 weeks. The order of development of MetS was: group 7, 3 weeks; group 6, 4 weeks; group 5, 6 weeks; group 4, 7 weeks; group 3, 10 weeks. Kidney, liver, heart, pancreas and skeletal muscle tissues are sources of adropin and betatrophin. In these tissues and in the circulation, adropin was decreased significantly, while betatrophin was increased significantly due to MetS; irisin + exercise reversed this situation. We found that the best method for creating a MetS model was F + UA2 supplementation. Our method is rapid and simple. Irisin + exercise was best for preventing MetS.

Impact of Human Recombinant Irisin on Tissue-Engineered Skeletal Muscle Structure and Function.

Tissue engineering of exogenous skeletal muscle units (SMUs) through isolation of muscle satellite cells from muscle biopsies is a potential treatment method for acute volumetric muscle loss (VML). A current issue with this treatment process is the limited capacity for muscle stem cell (satellite cell) expansion in cell culture, resulting in a decreased ability to obtain enough cells to fabricate SMUs of appropriate size and structural quality and that produce native levels of contractile force. This study determined the impact of human recombinant irisin on the growth and development of three-dimensional (3D) engineered skeletal muscle. Muscle satellite cells were cultured without irisin (control) or with 50, 100, or 250 ng/mL of irisin supplementation. Light microscopy was used to analyze myotube formation with particular focus placed on the diameter and density of the monotubes during growth of the 3D SMU. Following the formation of 3D constructs, SMUs underwent measurement of maximum tetanic force to analyze contractile function, as well as immunohistochemical staining, to characterize muscle structure. The results indicate that irisin supplementation with 250 ng/mL significantly increased the average diameter of myotubes and increased the proliferation and differentiation of myoblasts in culture but did not have a consistent significant impact on force production. In conclusion, supplementation with 250 ng/mL of human recombinant irisin promotes the proliferation and differentiation of myotubes and has the potential for impacting contractile force production in scaffold-free tissue-engineered skeletal muscle.

Irisin limits amyloid-ß buildup in Alzheimer's disease.

Mounting evidence suggests that physical exercise protects the brain against neurodegenerative disease. In a recent paper in Neuron, Kim et al. reported that the exercise-induced hormone irisin curbs amyloid-ß buildup by promoting secretion of astrocyte-derived neprilysin. These findings may help explain the neuroprotection by irisin and exercise in Alzheimer's disease.

Irisin protected hemopoietic stem cells and improved outcome of severe bone marrow failure.

Acquired aplastic anemia (AA) is a bone marrow failure (BMF) disease, characterized by fatty bone marrow (BM) and BM hypocellularity resulted from auto-immune dysregulated T cells-mediated destruction of BM haemopoietic stem cells (HPSC). The objective of this study was to investigate potential therapeutic effect of irisin, a molecule involved in adipose tissue transition, on AA mouse model. Our results showed that the concentration of irisin in serum was lower in AA patients than in healthy controls, suggesting a role of irisin in the pathogenesis of AA. In the AA mice, irisin administration prolonged the survival rate, prevented or attenuated peripheral pancytopenia, and preserved HPSC in the BM. Moreover, irisin also markedly reduced BM adipogenesis. In vitro results showed that irisin increased both cell proliferation and colony numbers of HPSC. Furthermore, our results demonstrated that irisin upregulated the expression of mitochondrial ATPase Inhibitory Factor 1 (IF1) in HPSC, inhibited the activation of mitochondrial fission protein (DRP1) and enhanced aerobic glycolysis. Taken together, our findings indicate novel roles of irisin in the pathogenesis of AA, and in the protection of HPSC through stimulation of proliferation and regulation of mitochondria function, which provides a proof-of-concept for the application of irisin in AA therapy.

Influence of Extracellular Matrix Components on the Differentiation of Periodontal Ligament Stem Cells in Collagen I Hydrogel.

Regeneration of periodontal tissues requires an integrated approach to the restoration of the periodontal ligament, cementum, and alveolar bone surrounding the teeth. Current strategies in endogenous regenerative dentistry widely use biomaterials, in particular the decellularized extracellular matrix (dECM), to facilitate the recruitment of populations of resident cells into damaged tissues and stimulate their proliferation and differentiation. The purpose of our study was to evaluate the effect of the exogenous components of the extracellular matrix (hyaluronic acid, laminin, fibronectin) on the differentiation of periodontal ligament stem cells (PDLSCs) cultured with dECM (combinations of decellularized tooth matrices and periodontal ligament) in a 3D collagen I hydrogel. The immunohistochemical expression of various markers in PDLSCs was assessed quantitatively and semi-quantitatively on paraffin sections. The results showed that PDLSCs cultured under these conditions for 14 days exhibited phenotypic characteristics consistent with osteoblast-like and odontoblast-like cells. This potential has been demonstrated by the expression of osteogenic differentiation markers (OC, OPN, ALP) and odontogenic markers (DSPP). This phenomenon corresponds to the in vivo state of the periodontal ligament, in which cells at the interface between bone and cementum tend to differentiate into osteoblasts or cementoblasts. The addition of fibronectin to the dECM most effectively induces the differentiation of PDLSCs into osteoblast-like and odontoblast-like cells under 3D culture conditions. Therefore, this bioengineered construct has a high potential for future use in periodontal tissue regeneration.

Inhibition of integrin alpha v/beta 5 mitigates the protective effect induced by irisin in hemorrhage.

INTRODUCTION: Irisin plays an important role in regulating tissue stress, cardiac function, and inflammation. Integrin αvß5 was recently identified as a receptor for irisin to elicit its physiologic function. It remains unknown whether integrin αvß5 is required for irisin's function in modulating the physiologic response to hemorrhage. The objective of this study is to examine if integrin αvß5 contributes to the effects of irisin during the hemorrhagic response. METHODS: Hemorrhage was induced in mice by achieving a mean arterial blood pressure of 35-45 mmHg for one hour, followed by two hours of resuscitation. Irisin (0.5  µg/kg) was administrated to assess its pharmacologic effects in hemorrhage. Cilengitide, a cyclic Arg-Gly-Asp peptide (cRGDyK) which is an inhibitor of integrin αvß5, or control RGDS (1 mg/kg) was administered with irisin. In another cohort of mice, the irisin-induced protective effect was examined after knocking down integrin ß5 with nanoparticle delivery of integrin ß5 sgRNA using CRSIPR/Cas-9 gene editing. Cardiac function and hemodynamics were measured using echocardiography and femoral artery catheterization, respectively. Systemic cytokine releases were measured using Enzyme-linked immunosorbent assay (ELISA). Histological analyses were used to determine tissue damage in myocardium, skeletal muscles, and lung tissues. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was carried out to assess apoptosis in tissues. RESULTS: Hemorrhage induced reduction of integrin αvß5 in skeletal muscles and repressed recovery of cardiac performance and hemodynamics. Irisin treatment led to significantly improved cardiac function, which was abrogated by treatment with Cilengitide or knockdown of integrin ß5. Furthermore, irisin resulted in a marked suppression of tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1), muscle edema, and inflammatory cells infiltration in myocardium and skeletal muscles, which was attenuated by Cilengitide or knockdown of integrin ß5. Irisin-induced reduction of apoptosis in the myocardium, skeletal muscles, and lung, which were attenuated by either the inhibition of integrin αvß5, or knockdown of integrin ß5. CONCLUSION: Integrin αvß5 plays an important role for irisin in modulating the protective effect during hemorrhage.

Phytohormones Affect Differentiation Status of Human Skin Fibroblasts via UPR Activation.

Normalization of secretory activity and differentiation status of mesenchymal cells, including fibroblasts, is an important biomedical problem. One of the possible solutions is modulation of unfolded protein response (UPR) activated during fibroblast differentiation. Here, we investigated the effect of phytohormones on the secretory activity and differentiation of cultured human skin fibroblasts. Based on the analysis of expression of genes encoding UPR markers, abscisic acid (ABA) upregulated expression of the GRP78 and ATF4 genes, while gibberellic acid (GA) upregulated expression of CHOP. Evaluation of the biosynthetic activity of fibroblasts showed that ABA promoted secretion and synthesis of procollagen I and synthesis of fibronectin, as well as the total production of collagen and non-collagen proteins of the extracellular matrix (ECM). ABA also stimulated the synthesis of smooth muscle actin α (α-SMA), which is the marker of myofibroblasts, and increased the number of myofibroblasts in the cell population. On the contrary, GA increased the level of fibronectin secretion, but reduced procollagen I synthesis and the total production of the ECM collagen proteins. GA downregulated the synthesis of α-SMA and decreased the number of myofibroblasts in the cell population. Our results suggest that phytohormones modulate the biosynthetic activity of fibroblasts and affect their differentiation status.

Irisin inhibits neutrophil extracellular traps formation and protects against acute pancreatitis in mice.

INTRODUCTION: Irisin is a newly discovered myokine which links exercise to inflammation and inflammation-related diseases through macrophage regulation. However, the effect of irisin on the activity of inflammation related immune cells (such as neutrophils) has not been clearly described. OBJECTIVES: The objective of our study was to explore the effect of irisin on the neutrophil extracellular traps (NETs) formation. METHODS: Phorbol-12-myristate-13-acetate (PMA) was used to construct a classic neutrophil inflammation model that was used to observe the formation of NETs in vitro. We studied the effect of irisin on NETs formation and its regulation mechanism. Subsequently, acute pancreatitis (AP) was used to verify the protective effect of irisin in vivo, which was an acute aseptic inflammatory response disease model closely related to NETs. RESULTS: Our study found that addition of irisin significantly reduced the formation of NETs via regulation of the P38/MAPK pathway through integrin αVß5, which might be the one of key pathways in NETs formation, and which could theoretically offset the immunoregulatory effect of irisin. Systemic treatment with irisin reduced the severity of tissue damage common in the disease and inhibited the formation of NETs in pancreatic necrotic tissue of two classical AP mouse models. CONCLUSION: The findings confirmed for the first time that irisin could inhibit NETs formation and protect mice from pancreatic injury, which further elucidated the protective effect of exercise on acute inflammatory injury.

CRISPRa-based activation of Fgf21 and Fndc5 ameliorates obesity by promoting adipocytes browning.

BACKGROUND: Skeletal muscle-secreted myokines widely participate in lipids metabolism through autocrine, paracrine and endocrine actions. The myokines represented by FGF21 and Irisin can promote the browning of adipocytes and serve as promising targets for treating obesity. Although recombinant myokines replacement therapy and AAV (adeno-associated virus)-based myokines overexpression have shown a definite effect in ameliorating obesity, novel myokine activation strategies with higher efficacy and safety are still in pressing need. This study aimed to evaluate the therapeutic potential of a novel CRISPR-based myokines activation strategy in obesity treatments. METHODS: In this study, we used lentivirus and a single AAV vector containing dCas9-VP64 with a single-guide RNA to selectively activate Fgf21 and Fndc5 expression in skeletal muscles both in vitro and in vivo. The activation efficacy of the CRISPRa system was determined by qRT-PCR, Western blotting and ELISA. The treatment effect of CRISPR-based myokines activation was tested in 3T3-L1-derived adipocytes and diet-induced obese (DIO) mice (male C57BL/6 mice, induced at 6-week-old for 10 weeks). RESULTS: The virus upregulates myokines expression in both mRNA and protein levels of muscle cells in vitro and in vivo. Myokines secreted by muscle cells promoted browning of 3T3-L1-derived adipocytes. In vivo activation of myokines by AAVs can reduce body weight and fat mass, increase the adipocytes browning and improve glucose tolerance and insulin sensitivity in DIO mice. CONCLUSIONS: Our study provides a novel CRISPR-based myokines activation strategy that can ameliorate obesity by promoting adipocytes browning.

FN1 mediated activation of aspartate metabolism promotes the progression of triple-negative and luminal a breast cancer.

BACKGROUND: Breast cancer (BC) is regarded as one of the most common cancers diagnosed among the female population and has an extremely high mortality rate. It is known that Fibronectin 1 (FN1) drives the occurrence and development of a variety of cancers through metabolic reprogramming. Aspartic acid is considered to be an important substrate for nucleotide synthesis. However, the regulatory mechanism between FN1 and aspartate metabolism is currently unclear. METHODS: We used RNA sequencing (RNA seq) and liquid chromatography-mass spectrometry to analyze the tumor tissues and paracancerous tissues of patients. MCF7 and MDA-MB-231 cells were used to explore the effects of FN1-regulated aspartic acid metabolism on cell survival, invasion, migration and tumor growth. We used PCR, Western blot, immunocytochemistry and immunofluorescence techniques to study it. RESULTS: We found that FN1 was highly expressed in tumor tissues, especially in Lumina A and TNBC subtypes, and was associated with poor prognosis. In vivo and in vitro experiments showed that silencing FN1 inhibits the activation of the YAP1/Hippo pathway by enhancing YAP1 phosphorylation, down-regulates SLC1A3-mediated aspartate uptake and utilization by tumor cells, inhibits BC cell proliferation, invasion and migration, and promotes apoptosis. In addition, inhibition of FN1 combined with the YAP1 inhibitor or SLC1A3 inhibitor can effectively inhibit tumor growth, of which inhibition of FN1 combined with the YAP1 inhibitor is more effective. CONCLUSION: Targeting the "FN1/YAP1/SLC1A3/Aspartate metabolism" regulatory axis provides a new target for BC diagnosis and treatment. This study also revealed that intratumoral metabolic heterogeneity plays an important role in the progression of different subtypes of breast cancer.

Matrix-Binding, N-Cadherin-Targeting Chimeric Peptide Inhibits Intimal Thickening but Not Endothelial Repair in Balloon-Injured Carotid Arteries.

BACKGROUND: Treatment of occluded vessels can involve angioplasty, stenting, and bypass grafting, which can be limited by restenosis and thrombosis. Drug-eluting stents attenuate restenosis, but the current drugs used are cytotoxic, causing smooth muscle cell (SMC) and endothelial cell (EC) death that may lead to late thrombosis. N-cadherin is a junctional protein expressed by SMCs, which promotes directional SMC migration contributing to restenosis. We propose that engaging N-cadherin with mimetic peptides can act as a cell type-selective therapeutic strategy to inhibit polarization and directional migration of SMCs without negatively impacting ECs. METHODS: We designed a novel N-cadherin-targeting chimeric peptide with a histidine-alanine-valine cadherin-binding motif, combined with a fibronectin-binding motif from Staphylococcus aureus. This peptide was tested in SMC and EC culture assays of migration, viability, and apoptosis. Rat carotid arteries were balloon injured and treated with the N-cadherin peptide. RESULTS: Treating scratch-wounded SMCs with the N-cadherin-targeting peptide inhibited migration and reduced polarization of wound-edge cells. The peptide colocalized with fibronectin. Importantly, EC junction, permeability, or migration was not impacted by peptide treatment in vitro. We also demonstrated that the chimeric peptide persisted for 24 hours after transient delivery in the balloon-injured rat carotid artery. Treatment with the N-cadherin-targeting chimeric peptide reduced intimal thickening in balloon-injured rat carotid arteries at 1 and 2 weeks after injury. Reendothelialization of injured vessels after 2 weeks was unimpaired by peptide treatment. CONCLUSIONS: These studies show that an N-cadherin-binding and fibronectin-binding chimeric peptide is effective in inhibiting SMC migration in vitro and in vivo and limiting neointimal hyperplasia after balloon angioplasty without affecting EC repair. These results establish the potential of an advantageous SMC-selective strategy for antirestenosis therapy.

Effects of atorvastatin on the function of Tenon's capsule fibroblasts in human eyes.

PURPOSE: This study aimed to explore the role of atorvastatin (ATO) in the prevention and treatment of the scarring of filtration channels after glaucoma surgery. METHODS: Human Tenon's capsule fibroblasts (HTFs) were co-cultured with various concentrations of ATO. First, Cell Counting Kit-8 assay was performed to evaluate the effects of various concentrations of ATO on the viability of HTFs. Then, after the ATO stimulated the HTFs for 24 h, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to evaluate the apoptosis of HTFs. Transwell assay was also performed to evaluate the migration of HTFs. Moreover, enzyme-linked immunosorbent assay (ELISA) was performed to detect the protein expression levels of transforming growth factor-ß1 (TGF-ß1) and TGF-ß2 in the cell culture supernatant of HTFs. Western blot was carried out to detect the protein expression levels of smooth muscle actin (SMA), p38, Smad3, fibronectin, collagen I and collagen III in different groups. RESULTS: The results revealed that ATO could inhibit the proliferation and migration of HTFs. Based on the TUNEL assay, 100 µM and 150 µM ATO could induce cell apoptosis. The ELISA results indicated that ATO could down-regulate the expression level of TGF-ß2, and western blot analysis revealed that the protein expression levels of SMA, p38, Smad3, fibronectin, collagen I and collagen III in the TGF-ß2 group were all up-regulated compared with the control group, whereas the addition of ATO could reverse this up-regulation. CONCLUSIONS: ATO could inhibit the proliferation and migration of HTFs and induce their apoptosis. It was preliminary proven that ATO could inhibit the signaling pathway induced by TGF-ß. It is suggested that ATO could be a basis for the treatment of the scarring of filtration channels after glaucoma surgery.

Irisin Protects against Loss of Trabecular Bone Mass and Strength in Adult Ovariectomized Mice by Stimulating Osteoblast Activity.

Irisin is a peptide secreted by skeletal muscle that plays a major role in bone metabolism. Experiments in mouse models have shown that administration of recombinant irisin prevents disuse-induced bone loss. In this study, we aimed to evaluate the effects of irisin treatment for the prevention of bone loss in the ovariectomized (Ovx) mouse, the animal model commonly used to investigate osteoporosis caused by estrogen deficiency. Micro-Ct analysis conducted on Sham mice (Sham-veh) and Ovx mice treated with vehicle (Ovx-veh) or recombinant irisin (Ovx-irisn) showed bone volume fraction (BV/TV) decreases in femurs (Ovx-veh 1.39± 0.71 vs. Sham-veh 2.84 ± 1.23; p = 0.02) and tibia at both proximal condyles (Ovx-veh 1.97 ± 0.68 vs. Sham-veh 3.48 ± 1.26; p = 0.03) and the subchondral plate (Ovx-veh 6.33 ± 0.36 vs. Sham-veh 8.18 ± 0.41; p = 0.01), which were prevented by treatment with a weekly dose of irisin for 4 weeks. Moreover, histological analysis of trabecular bone showed that irisin increased the number of active osteoblasts per bone perimeter (Ovx-irisin 32.3 ± 3.9 vs. Ovx-veh 23.5 ± 3.6; p = 0.01), while decreasing osteoclasts (Ovx-irisin 7.6 ± 2.4 vs. Ovx-veh 12.9 ± 3.04; p = 0.05). The possible mechanism by which irisin enhances osteoblast activity in Ovx mice is upregulation of the transcription factor Atf4, one of the key markers of osteoblast differentiation, and osteoprotegerin, thereby inhibiting osteoclast formation.

Biomimetic Electrospun Scaffold-Based In Vitro Model Resembling the Hallmarks of Human Myocardial Fibrotic Tissue.

Adverse remodeling post-myocardial infarction is hallmarked by the phenotypic change of cardiac fibroblasts (CFs) into myofibroblasts (MyoFs) and over-deposition of the fibrotic extracellular matrix (ECM) mainly composed by fibronectin and collagens, with the loss of tissue anisotropy and tissue stiffening. Reversing cardiac fibrosis represents a key challenge in cardiac regenerative medicine. Reliable in vitro models of human cardiac fibrotic tissue could be useful for preclinical testing of new advanced therapies, addressing the limited predictivity of traditional 2D cell cultures and animal in vivo models. In this work, we engineered a biomimetic in vitro model, reproducing the morphological, mechanical, and chemical cues of native cardiac fibrotic tissue. Polycaprolactone (PCL)-based scaffolds with randomly oriented fibers were fabricated by solution electrospinning technique, showing homogeneous nanofibers with an average size of 131 ± 39 nm. PCL scaffolds were then surface-functionalized with human type I collagen (C1) and fibronectin (F) by dihydroxyphenylalanine (DOPA)-mediated mussel-inspired approach (PCL/polyDOPA/C1F), in order to mimic fibrotic cardiac tissue-like ECM composition and support human CF culture. BCA assay confirmed the successful deposition of the biomimetic coating and its stability during 5 days of incubation in phosphate-buffered saline. Immunostaining for C1 and F demonstrated their homogeneous distribution in the coating. AFM mechanical characterization showed that PCL/polyDOPA/C1F scaffolds, in wet conditions, resembled fibrotic tissue stiffness with an average Young's modulus of about 50 kPa. PCL/polyDOPA/C1F membranes supported human CF (HCF) adhesion and proliferation. Immunostaining for α-SMA and quantification of α-SMA-positive cells showed HCF activation into MyoFs in the absence of a transforming growth factor ß (TGF-ß) profibrotic stimulus, suggesting the intrinsic ability of biomimetic PCL/polyDOPA/C1F scaffolds to sustain the development of cardiac fibrotic tissue. A proof-of-concept study making use of a commercially available antifibrotic drug confirmed the potentialities of the developed in vitro model for drug efficacy testing. In conclusion, the proposed model was able to replicate the main hallmarks of early-stage cardiac fibrosis, appearing as a promising tool for future preclinical testing of advanced regenerative therapies.