BACKGROUND: Recent studies have proved the role of autophagy in mesenchymal stem cell (MSCs) function and regenerative properties. How and by which mechanism autophagy modulation can affect the juxtacrine interaction of MSCs should be addressed. Here, the role of autophagy was investigated in the formation of tunneling nanotubes (TNTs) and homotypic mitochondrial donation.
METHODS: MSCs were incubated with 15 µM Metformin (Met) and/or 3 µM 3-methyladenine (3-MA) for 48 h. The formation of TNTs was assessed using bright-field and SEM images. The mitochondria density and ΔΨ values were monitored using flow cytometry analysis. Using RT-PCR and protein array, the close interaction and shared mediators between autophagy, apoptosis, and Wnt signaling pathways were also monitored. The total fatty acid profile was assessed using gas chromatography.
RESULTS: Data indicated the increase of TNT length and number, along with other cell projections after the induction of autophagy while these features were blunted in 3-MA-treated MSCs (p < 0.05). Western blotting revealed the significant reduction of Rab8 and p-FAK in 3-MA-treated MSCs (p < 0.05), indicating the inhibition of TNT assembly and vesicle transport. Likewise, the stimulation of autophagy increased autophagic flux and mitochondrial membrane integrity compared to 3-MA-treated MSCs. Despite these findings, protein levels of mitochondrial membrane Miro1 and 2 were unchanged after autophagy inhibition/stimulation (p > 0.05). We found that the inhibition/stimulation of autophagy can affect the protein, and transcription levels of several mediators related to Wnt and apoptosis signaling pathways involved in different cell bioactivities. Data confirmed the profound increase of mono and polyunsaturated/saturated fatty acid ratio in MSCs exposed to autophagy stimulator.
CONCLUSIONS: In summary, autophagy modulation could affect TNT formation which is required for homotypic mitochondrial donation. Thus, the modulation of autophagy creates a promising perspective to increase the efficiency of cell-based therapies.

Lifestyle influences physical and cognitive development during the period of adolescence greatly. The most important of these lifestyle factors are diet and stress. Therefore, the aim of this study was to investigate the impact of high fat diet (HFD) and chronic mild stress on cognitive function and anxiety-like behaviors in young rats and to study the role of caffeic acid as a potential treatment for anxiety and cognitive dysfunction. Forty rats were assigned into 4 groups: control, HFD, HFD + stress, and caffeic acid-treated group. Rats were sacrificed after neurobehavioral testing. We detected memory impairment and anxiety-like behavior in rats which were more exaggerated in stressed rats. Alongside the behavioral changes, there were biochemical and histological changes. HFD and/or stress decreased hippocampal brain-derived neurotrophic factor (BDNF) levels and induced oxidative and inflammatory changes in the hippocampus. In addition, they suppressed Wnt/β-catenin pathway which was associated with activation of glycogen synthase kinase 3β (GSK3β). HFD and stress increased arginase 1 and inducible nitric oxide synthase (iNOS) levels as well. These disturbances were found to be aggravated in stressed rats than HFD group. However, caffeic acid was able to reverse these deteriorations leading to memory improvement and ameliorating anxiety-like behavior. So, the current study highlights an important neuroprotective role for caffeic acid that may guard against induction of cognitive dysfunction and anxiety disorders in adolescents who are exposed to HFD and/or stress.

Mechanical forces are of major importance in regulating vascular homeostasis by influencing endothelial cell behavior and functions. Adherens junctions are critical sites for mechanotransduction in endothelial cells. β-catenin, a component of adherens junctions and the canonical Wnt signaling pathway, plays a role in mechanoactivation. Evidence suggests that β-catenin is involved in flow sensing and responds to tensional forces, impacting junction dynamics. The mechanoregulation of β-catenin signaling is context-dependent, influenced by the type and duration of mechanical loads. In endothelial cells, β-catenin\'s nuclear translocation and signaling are influenced by shear stress and strain, affecting endothelial permeability. The study investigates how shear stress, strain, and surface topography impact adherens junction dynamics, regulate β-catenin localization, and influence endothelial barrier properties. Insight box Mechanical loads are potent regulators of endothelial functions through not completely elucidated mechanisms. Surface topography, wall shear stress and cyclic wall deformation contribute overlapping mechanical stimuli to which endothelial monolayer respond to adapt and maintain barrier functions. The use of custom developed flow chamber and bioreactor allows quantifying the response of mature human endothelial to well-defined wall shear stress and gradients of strain. Here, the mechanoregulation of β-catenin by substrate topography, wall shear stress, and cyclic stretch is analyzed and linked to the monolayer control of endothelial permeability.

Cartilage tissue engineering aims to develop functional substitutes for treating cartilage defects and osteoarthritis. Traditional two-dimensional (2D) cell culture systems lack the complexity of native cartilage, leading to the development of 3D regenerative cartilage models. In this study, we developed a 3D model using Gelatin Methacryloyl (GelMA)-based hydrogels seeded with Y201 cells, a bone marrow mesenchymal stem cell line. The model investigated chondrogenic differentiation potential in response to Wnt3a stimulation within the GelMA scaffold and validated using known chondrogenic agonists. Y201 cells demonstrated suitability for the model, with increased proteoglycan content and upregulated chondrogenic marker expression under chondrogenic conditions. Wnt3a enhanced cell proliferation, indicating activation of the Wnt/β-catenin pathway, which plays a role in cartilage development. GelMA hydrogels provided an optimal scaffold, supporting cell viability and proliferation. The 3D model exhibited consistent responses to chondrogenic agonists, with TGF-β3 enhancing cartilage-specific extracellular matrix (ECM) production and chondrogenic differentiation. The combination of Wnt3a and TGF-β3 showed synergistic effects, promoting chondrogenic differentiation and ECM production. This study presents a 3D regenerative cartilage model with potential for investigating cartilage biology, disease mechanisms, and drug screening. The model provides insights into complex cartilage regeneration mechanisms and offers a platform for developing therapeutic approaches for cartilage repair and osteoarthritis treatment.

UNASSIGNED: Based on the secreted frizzled-related protein 2 (SFRP2)-Wnt/β-catenin signaling pathway, this study explored the effect and mechanism of Cuiru Keli (CRKL) in the treatment of postpartum hypogalactia.
UNASSIGNED: A rat model of postpartum hypogalactia was established by gavaging 2 mL of 1.6 mg/mL bromocriptine mesylate to female rats on the third day after delivery. Female rats with a delivery time difference of less than 48 hours were selected and randomly assigned to 7 groups, including a normal group (without any modeling or medication), a model group, a CRKL low-dose group of model group model rats receiving CRKL at the dose of 3 g/kg, a CRKL medium-dose group of model rats receiving CRKL at the dose of 6 g/kg, a CRKL high-dose group of model rats receiving CRKL at the dose of 9 g/kg, a positive drug group of model rats receiving domperidone at the dose of 3 mg/kg, and a negative control (NC) group of model rats receiving normal saline. Each group contained 6 rats. Except for the normal and model groups, the remaining 5 groups were continuously administered with the respective intervention drugs at the specified doses by gavage once a day for 10 days. Changes in the total litter mass of the offspring in the 7 groups within 10 days were measured, and HE staining was performed to identify pathological changes in the mammary tissue (MT). Six groups of rats (excluding the positive control group) were used to observe the pathological changes of eosinophils in pituitary tissue. ELISA was performed to determine the content of prolactin (PRL) in serum, immunohistochemical staining was used to determine the expression of prolactin receptor (PRLR) in MT, and RT-qPCR was used to determine the mRNA expression of genes related to lactation in MT. Network pharmacology and molecular docking were used to study the therapeutic effect and mechanism of CRKL on postpartum hypogalactia, particularly whether it acted through the SFRP2-Wnt/β-catenin signaling pathway. The mechanism of CRKL treatment was further validated by detecting mRNA (RT-qPCR) and protein expression (Western blot) of related pathway genes. Cell experiments were conducted using primary culture rat mammary epithelial cells (RMEC) from rat MT. RMEC were divided into four groups, including a normal group (primary culture RMEC, untreated), SFRP2 overexpression group (primary cultured RMEC treated with SFRP2 overexpression vector), SFRP2 overexpression+CRKL group (receiving treatment for SFRP2 overexpression group plus 10% drug-containing serum), and negative control group (primary culture RMEC treated with empty vector). The effect of CRKL on the expression of lactation-related genes FASN, CSN2, and GLUT1 mRNA after SFRP2 overexpression was detected by RT-qPCR.
UNASSIGNED: In this study, CRKL was administered at a dose of 3 g/kg in the CRKL low-dose group, 6 g/kg in the medium-dose group, and 9 g/kg in the high-dose group (P<0.05 or P<0.01). Compared with the model group, CRKL at all doses significantly increased the total litter weight gain of the offsprings within 10 days (P<0.05 or P<0.01), and effectively increased lactation (P<0.01), the area of mammary lobules, and the size and filling of acinar cavities. CRKL at all doses also increased the number of eosinophils that secreted PRL in the pituitary gland of the postpartum hypogalactia rat model, and increased the content of PRL in the serum (P<0.05 or P<0.01). CRKL promoted the secretion and expression of PRL in postpartum hypogalactic model rats. In addition, it significantly promoted the expression of genes related to milk fat, milk protein, and lactose synthesis in MT (P<0.05 or P<0.01). Network pharmacology predicted that the Wnt signaling pathway might be a key pathway for CRKL in treating postpartum hypogalactia. The molecular docking results showed that related chemical components in CRKL had good binding ability with CCND1 and SFRP2. Compared with the model group, CRKL at all doses inhibited the expression of SFRP2 gene in vivo (P<0.01) and activated the mRNA and protein expression of CCND1 and c-Myc in the Wnt/β-catenin signaling pathway in MT (P<0.05 or P<0.01). Cell experiments showed that, compared to the normal group, SFRP2 overexpression reduced the mRNA expression of milk synthesis-related genes FASN, CSN2, and GLUT1 in RMEC (P<0.01). The CCK8 results indicated that 10% of the drug-containing serum was the effective concentration administered to cells (P<0.01). After administering drug-containing serum, the expression of the lactation-related genes FASN, CSN2, and GLUT1 were up-regulated (compared with the SFRP2 overexpression group, P<0.01).
UNASSIGNED: CRKL alleviates postpartum hypogalactia through the SFRP2-Wnt/β-catenin signaling pathway. SFRP2 might be a potential new target for the diagnosis and treatment of postpartum hypogalactia. This reveals a new mechanism of CRKL in treating postpartum hypogalactia and promotes its clinical application.

UNASSIGNED: Osteochondral defects (OCDs) are localized areas of damaged cartilage and underlying subchondral bone that can produce pain and seriously impair joint function. Literature reports indicated that icariin (ICA) has the effect of promoting cartilage repair. However, its mechanism remains unclear. Here, we explored the effects of icariin and extracellular vesicles (EVs) from rabbit synovial-derived mesenchymal stem cells (rSMSCs) on repairing of OCDs.
UNASSIGNED: Rabbit primary genicular chondrocytes (rPGCs), knee skeletal muscle cells (rSMCKs), and rSMSCs, and extracellular vesicles derived from the latter two cells (rSMCK-EVs and rSMSC-EVs) were isolated and identified. The rPGCs were stimulated with ICA, rSMSC-EVs either separately or in combination. The rSMCK-EVs were used as a control. After stimulation, chondrogenic-related markers were analyzed by quantitative RT-PCR and western blotting. Cell proliferation was determined by the CCK-8 assay. The preventative effects of ICA and SMSC-EVs in vivo were determined by H&E and toluidine blue staining. Immunohistochemical analyses were performed to evaluate the levels of COL2A1 and β-catenin in vivo. Results. In vitro, the proliferation of rPGCs was markedly increased by ICA treatment in a dose-dependent manner. When compared with ICA or rSMSC-EVs treatment alone, combined treatment with ICA and SMSC-EVs produced stronger stimulative effects on cell proliferation. Moreover, combined treatment with ICA and rSMSC-EVs promoted the expression of chondrogenic-related gene, including COL2A1, SOX-9, and RUNX2, which may be via the activation of the Wnt/β-catenin pathway. In vivo, combined treatment with rSMSC-EVs and ICA promoted cartilage repair in joint bone defects. Results also showed that ICA or rSMSC-EVs both promoted the COL2A1 and β-catenin protein accumulation in articular cartilage, and that was further enhanced by combined treatment with rSMSC-EVs and ICA.
UNASSIGNED: Our findings highlight the promising potential of using combined treatment with ICA and rSMSC-EVs for promoting osteochondral repair.

UNASSIGNED: Chondrocyte viability, apoptosis, and migration are closely related to cartilage injury in osteoarthritis (OA) joints. Exosomes are identified as potential therapeutic agents for OA.
UNASSIGNED: This study aimed to investigate the role of exosomes derived from osteocytes in OA, particularly focusing on their effects on cartilage repair and molecular mechanisms.
UNASSIGNED: An injury cell model was established by treating chondrocytes with IL-1β. Cartilage repair was evaluated using cell counting kit-8, flow cytometry, scratch test, and Western Blot. Molecular mechanisms were analyzed using quantitative real-time PCR, bioinformatic analysis, and Western Blot. An OA mouse model was established to explore the role of exosomal DLX2 in vivo.
UNASSIGNED: Osteocyte-released exosomes promoted cell viability and migration, and inhibited apoptosis and extracellular matrix (ECM) deposition. Moreover, exosomes upregulated DLX2 expression, and knockdown of DLX2 activated the Wnt pathway. Additionally, exosomes attenuated OA in mice by transmitting DLX2.
UNASSIGNED: Osteocyte-derived exosomal DLX2 alleviated IL-1β-induced cartilage repair and inactivated the Wnt pathway, thereby alleviating OA progression. The findings suggested that osteocyte-derived exosomes may hold promise as a treatment for OA.

Osteoarthritis (OA) is a chronic disease that involves chondrocyte injury. ADAMTS5 has been confirmed to mediate chondrocyte injury and thus regulate OA progression, but its underlying molecular mechanisms remain unclear. In the present study, interleukin‑1β (IL‑1β)‑induced chondrocytes were used to mimic OA in vitro. Cell proliferation and apoptosis were assessed by MTT assay, EdU assay and flow cytometry, and protein levels of ADAMTS5, specificity protein 1 (SP1), matrix‑related markers and Wnt/β‑catenin pathway‑related markers were examined using western blotting. In addition, ELISA was performed to measure the concentrations of inflammation factors, and oxidative stress was evaluated by detecting SOD activity and MDA levels. The mRNA expression levels of ADAMTS5 and SP1 were determined by reverse transcription‑quantitative PCR, and the interaction between SP1 and ADAMTS5 was analyzed using a dual‑luciferase reporter assay and chromatin immunoprecipitation assay. IL‑1β suppressed proliferation, but promoted apoptosis, extracellular matrix degradation, inflammation and oxidative stress in chondrocytes. ADAMTS5 was upregulated in IL‑1β‑induced chondrocytes, and its knockdown alleviated IL‑1β‑induced chondrocyte injury. SP1 could bind to the ADAMTS5 promoter region to promote its transcription, and SP1 knockdown relieved IL‑1β‑induced chondrocyte injury by reducing ADAMTS5 expression. The SP1/ADAMTS5 axis activated the Wnt/β‑catenin pathway, and the Wnt/β‑catenin pathway agonist, SKL2001, reversed the protective effect of ADAMTS5 knockdown on chondrocyte injury induced by IL‑1β. To the best of our knowledge, the present study was the first to reveal the interaction between SP1 and ADAMTS5 in OA progression and indicated that the SP1/ADAMTS5 axis mediates OA progression by regulating the Wnt/β‑catenin pathway.

Breast cancer (BC) is the most common malignancy in women worldwide. Wnt signaling is involved in tumorigenesis and cancer progression, and is closely associated with the characteristics of BC. Variation in the expression of exosomal microRNAs (miRNAs) modulates key cancer phenotypes, such as cellular proliferation, epithelial‑mesenchymal transition, metastatic potential, immune evasion and treatment resistance. The present review aimed to discuss the importance of Wnt signaling and exosomal miRNAs in regulating the occurrence and development of BC. In addition, the present review determined the crosstalk between Wnt signaling and exosomal miRNAs, and highlighted potential diagnostic biomarkers and therapeutic targets.

The Frizzleds (FZDs) receptors on the cell surface belong to the class F of G protein-coupled receptors (GPCRs) which are the major receptors of WNT protein that mediates the classical WNT signaling pathway and other non-classical pathways. Besides, the FZDs also play a core role in tissue regeneration and tumor occurrence. With the structure and mechanism of FZDs activation becoming clearer, a series of FZDs modulators (inhibitors and agonists) have been developed, with the hope of bringing benefits to the treatment of cancer and degenerative diseases. Most of the FZDs inhibitors (small molecules, antibodies or designed protein inhibitors) block WNT signaling through binding to the cysteine-rich domain (CRD) of FZDs. Several small molecules impede FZDs activation by targeting to the third intracellular domain or the transmembrane domain of FZDs. However, three small molecules (FZM1.8, SAG1.3 and purmorphamine) activate the FZDs through direct interaction with the transmembrane domain. Another type of FZDs agonists are bivalent or tetravalent antibodies which activate the WNT signaling via inducing FZD-LRP5/6 heterodimerization. In this article, we reviewed the FZDs modulators reported in recent years, summarized the critical molecules\' discovery processes and the elucidated relevant structural and pharmacological mechanisms. We believe the summaried molecular mechanisms of the relevant modulators could provide important guidance and reference for the future development of FZD modulators.