The healing of the tendon-bone interface is a complex dynamic process involving the interaction of multiple cellular and molecular signaling pathways. Bone mesenchymal stem cells (BMSCs) have the potential to differentiate into various types of cells, including osteoblasts, chondrocytes and adipocytes, etc., and have the potential to regenerate damaged tissues. They are potential seed cells for promoting tendon-bone healing. How to precisely regulate the proliferation and differentiation of BMSCs to accelerate the process of tendon-bone healing is a current research hotspot. Monomers of traditional Chinese medicine can promote tendon-bone healing by regulating signaling pathways such as Wnt/β-catenin and BMP/Smad to induce osteogenic and chondrogenic differentiation of BMSCs. This article reviews from several aspects such as the regulatory role of related signaling pathways on tendine-bone healing, traditional Chinese medicine monomers and their mechanism of regulating BMSCs to promote tendine-bone healing in order to providing new ideas for promoting tendine-bone healing.
Mesenchymal Stem Cells/cytology* ; Humans ; Animals ; Bone Marrow Cells/cytology* ; Bone and Bones/drug effects* ; Wound Healing/drug effects* ; Medicine, Chinese Traditional ; Tendons/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Signal Transduction/drug effects* ; Cell Differentiation/drug effects*

Objective To investigate the effects of hyaluronic acid and proteoglycan-linked protein 1 (HAPLN1) secreted by synovial fibroblasts (FLS) on the polarization of macrophages (Mϕ) in rheumatoid arthritis (RA). Methods Human monocytic leukemia cells (THP-1) were differentiated into Mϕ, which were subsequently exposed to recombinant HAPLN1 (rHAPLN1). RA-FLS were transfected separately with HAPLN1 overexpression plasmid (HAPLN1^OE) or small interfering RNA targeting HAPLN1 (si-HAPLN1), and then co-cultured with Mϕ to establish a co-culture model. The viability of Mϕ was assessed using the CCK-8 assay, and the proportions of pro-inflammatory M1-type and anti-inflammatory M2-type Mϕ were analyzed by flow cytometry. Additionally, the expression levels of inflammatory markers, including interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), and inducible nitric oxide synthase (iNOS), were quantified using quantitative real-time PCR and Western blot analysis. Results The viability of Mϕ was increased in the rHAPLN1 group compared to the control group. Furthermore, both the M1/Mϕ ratio and inflammatory factor levels were elevated in the rHAPLN1 and HAPLN1^OE groups. In contrast, the si-HAPLN1 group exhibited a decrease in the M1/Mϕ ratio and inflammatory factor expression. Notably, the introduction of rHAPLN1 in rescue experiments further promoted Mϕ polarization towards the M1 phenotype. Conclusion HAPLN1, secreted by RA fibroblast-like synoviocytes (RA-FLS), enhances Mϕ polarization towards the M1 phenotype.
Humans ; Arthritis, Rheumatoid/genetics* ; Macrophages/immunology* ; Fibroblasts/metabolism* ; Phenotype ; Extracellular Matrix Proteins/genetics* ; Proteoglycans/genetics* ; Synovial Membrane/cytology* ; Tumor Necrosis Factor-alpha/genetics* ; Interleukin-1beta/genetics* ; Nitric Oxide Synthase Type II/genetics* ; Cell Differentiation ; Coculture Techniques ; THP-1 Cells

Objective To investigate the effect of gentiopicroside on osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs), and to determine whether its mechanism involves the stromal cell-derived factor 1(SDF-1)/C-X-C chemokine receptor 4 (CXCR4) pathway. Methods BMSCs were divided into six groups: normal culture control group, osteogenic induction model group, low-dose gentiopicroside (L-gentiopicroside, 10 μmol/L) group, medium-dose gentiopicroside (M-gentiopicroside, 20 μmol/L) group, high-dose gentiopicroside (H-gentiopicroside, 40 μmol/L) group, and H-gentiopicroside+SDF-1/CXCR4 pathway inhibitor (AMD3100) group (H-gentiopicroside+AMD3100, 40 μmol/L gentiopicroside+10 μg/mL AMD3100). Cell viability, apoptosis, ALP activity, mineralized nodule formation, and protein levels of the SDF-1/CXCR4 pathway were assessed using the CCK-8 assay, flow cytometry, ALP staining, Alizarin Red S staining, and Western blotting, respectively. Results No mineralized nodules were observed in either the control and model group, although the color of the model group deepened. Compared with the control group, the model group showed significantly increased A value, ALP activity, expression levels of Runt related transcription factor 2 (RUNX2), osteopontin (OPN), SDF-1, CXCR4 proteins, along with a lower apoptosis rate. Compared with the model group, the L-gentiopicroside, M-gentiopicroside and H-gentiopicroside groups showed dose-dependently (Ldifferentiation of BMSCs may be achieved by activating the SDF-1/CXCR4 signaling pathway.
Humans ; Receptors, CXCR4/genetics* ; Mesenchymal Stem Cells/metabolism* ; Chemokine CXCL12/genetics* ; Iridoid Glucosides/pharmacology* ; Osteogenesis/drug effects* ; Cell Differentiation/drug effects* ; Signal Transduction/drug effects* ; Cells, Cultured ; Apoptosis/drug effects* ; Bone Marrow Cells/metabolism*

Objective To investigate how culture duration affects the expression of immune membrane proteins in human monocyte-derived dendritic cells (DCs) and their exosomes (DEXs). Methods Human monocytes were induced with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) to differentiate into DCs and were subsequently matured with tumor necrosis factor α(TNF-α). Exosomes were isolated by ultracentrifugation, and DEXs were identified by transmission electron microscopy and Amnis imaging flow cytometry, which were also used to quantify the expression of immune membrane proteins on DCs and DEXs. Results On the 10th day of culture, DCs displayed high surface expression of CD11c, CD80, CD86, major histocompatibility complex class I (MHC-I), and MHC-II. Expression peaked at day 18(CD11c: 78.66%±20.33%, CD80: 76.41%±10.02%, CD86: 96.43%±0.43%, MHC-I: 84.71%±2.96%, MHC-II: 80.01%±7.03%). After day 24, the overall expression showed a declining trend, with statistically significant differences observed for all markers except CD80 and MHC-II. By day 30, 80% of the DCs still expressed CD80, CD86, and MHC-II. The expression of immune membrane proteins on DEX surfaces also reached its peak on day 18, followed by an overall decline with prolonged culture time, with statistically significant differences observed for all markers except CD80. Correlation analysis revealed a significant positive relationship between the expression levels of immune membrane proteins on DC and DEX surfaces (CD11c: r=0.98; CD80: r=0.65; CD86: r=0.82; MHC-I: r=0.86; MHC-II: r=0.93). Conclusion Human monocyte-derived DCs in vitro express high expression of immune membrane proteins and maintain stable expression over a specific period. The exosomes secreted by these cells similarly demonstrate high surface expression of immune membrane proteins, with temporal trends aligned with those of the parent DCs.
Humans ; Dendritic Cells/immunology* ; Exosomes/immunology* ; Monocytes/metabolism* ; Cells, Cultured ; Time Factors ; B7-1 Antigen/metabolism* ; Membrane Proteins/immunology* ; Cell Culture Techniques/methods* ; B7-2 Antigen/metabolism* ; Cell Differentiation ; CD11c Antigen/metabolism* ; Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology*

Infertility has become one of the most serious diseases worldwide, and 50% of this disease can be attributed to male-related factors. Spermatogenesis, by definition, is a complex process by which spermatogonial stem cells (SSCs) self-renew to maintain stem cell population within the testes and differentiate into mature spermatids. It is of great significance to uncover gene regulation and signaling pathways that are involved in the fate determinations of SSCs with aims to better understand molecular mechanisms underlying human spermatogenesis and identify novel targets for gene therapy of male infertility. Significant achievement has recently been made in demonstrating the signaling molecules and pathways mediating the fate decisions of mammalian SSCs. In this review, we address key gene regulation and crucial signaling transduction pathways in controlling the self-renewal, differentiation, and apoptosis of SSCs, and we illustrate the networks of genes and signaling pathways in SSC fate determinations. We also highlight perspectives and future directions in SSC regulation by genes and their signaling pathways. This review could provide novel insights into the genetic regulation of normal and abnormal spermatogenesis and offer molecular targets to develop new approaches for gene therapy of male infertility.
Humans ; Male ; Signal Transduction/physiology* ; Apoptosis/physiology* ; Spermatogenesis/physiology* ; Cell Differentiation ; Adult Germline Stem Cells/physiology* ; Spermatogonia/cytology* ; Gene Expression Regulation ; Animals ; Infertility, Male/genetics* ; Cell Self Renewal/genetics*

Mouse spermatogenesis entails the maintenance and self-renewal of spermatogonial stem cells (SSCs), which require a complex web-like signaling network transduced by various cytokines. Although brain-derived neurotrophic factor (BDNF) is expressed in Sertoli cells in the testis, and its receptor tropomyosin receptor kinase B (TrkB) is expressed in the spermatogonial population containing SSCs, potential functions of BDNF for spermatogenesis have not been uncovered. Here, we generate BDNF conditional knockout mice and find that BDNF is dispensable for in vivo spermatogenesis and fertility. However, in vitro , we reveal that BDNF -deficient germline stem cells (GSCs) exhibit growth potential not only in the absence of glial cell line-derived neurotrophic factor (GDNF), a master regulator for GSC proliferation, but also in the absence of other factors, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin. GSCs grown without these factors are prone to differentiation, yet they maintain expression of promyelocytic leukemia zinc finger ( Plzf ), an undifferentiated spermatogonial marker. Inhibition of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and Src pathways all interfere with the growth of BDNF-deficient GSCs. Thus, our findings suggest a role for BDNF in maintaining the undifferentiated state of spermatogonia, particularly in situations where there is a shortage of growth factors.
Animals ; Male ; Brain-Derived Neurotrophic Factor/genetics* ; Spermatogonia/cytology* ; Mice ; Spermatogenesis/genetics* ; Mice, Knockout ; Cell Differentiation ; Glial Cell Line-Derived Neurotrophic Factor/genetics* ; Promyelocytic Leukemia Zinc Finger Protein/genetics* ; Cell Survival/physiology* ; Signal Transduction/physiology* ; Cell Proliferation/physiology*

Osteoporosis is a prevalent skeletal condition characterized by reduced bone mass and strength, leading to increased fragility. Buqi-Tongluo (BQTL) decoction, a traditional Chinese medicine (TCM) prescription, has yet to be fully evaluated for its potential in treating bone diseases such as osteoporosis. To investigate the mechanism by which BQTL decoction inhibits osteoclast differentiation in vitro and validate these findings through in vivo experiments. We employed MTS assays to assess the potential proliferative or toxic effects of BQTL on bone marrow macrophages (BMMs) at various concentrations. TRAcP experiments were conducted to examine BQTL's impact on osteoclast differentiation. RT-PCR and Western blot analyses were utilized to evaluate the relative expression levels of osteoclast-specific genes and proteins under BQTL stimulation. Finally, in vivo experiments were performed using an osteoporosis model to further validate the in vitro findings. This study revealed that BQTL suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis and osteoclast resorption activity in vitro in a dose-dependent manner without observable cytotoxicity. The inhibitory effects of BQTL on osteoclast formation and function were attributed to the downregulation of NFATc1 and c-fos activity, primarily through attenuation of the MAPK, NF-κB, and Calcineurin signaling pathways. BQTL's inhibitory capacity was further examined in vivo using an ovariectomized (OVX) rat model, demonstrating a strong protective effect against bone loss. BQTL may serve as an effective therapeutic TCM for the treatment of postmenopausal osteoporosis and the alleviation of bone loss induced by estrogen deficiency and related conditions.
Animals ; NFATC Transcription Factors/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Ovariectomy ; Osteoclasts/metabolism* ; Female ; Osteogenesis/drug effects* ; Rats, Sprague-Dawley ; Rats ; NF-kappa B/genetics* ; Osteoporosis/genetics* ; Signal Transduction/drug effects* ; Bone Resorption/genetics* ; Cell Differentiation/drug effects* ; Humans ; RANK Ligand/metabolism* ; Mitogen-Activated Protein Kinases/genetics* ; Transcription Factors

Andrographolide sulfonate (AS) is a sulfonated derivative of andrographolide extracted from Andrographis paniculata (Burm.f.) Nees, and has been approved for several decades in China. The present study aimed to investigate the novel therapeutic application and possible mechanisms of AS in the treatment of rheumatoid arthritis. Results indicated that administration of AS by injection or gavage significantly reduced the paw swelling, improved body weights, and attenuated pathological changes in joints of rats with adjuvant-induced arthritis. Additionally, the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and IL-1β in the serum and ankle joints were reduced. Bioinformatics analysis, along with the spleen index and measurements of IL-17 and IL-10 levels, suggested a potential relationship between AS and Th17 cells under arthritic conditions. In vitro, AS was shown to block Th17 cell differentiation, as evidenced by the reduced percentages of CD4⁺ IL-17A⁺ T cells and decreased expression levels of RORγt, IL-17A, IL-17F, IL-21, and IL-22, without affecting the cell viability and apoptosis. This effect was attributed to the limited glycolysis, as indicated by metabolomics analysis, reduced glucose uptake, and pH measurements. Further investigation revealed that AS might bind to hexokinase2 (HK2) to down-regulate the protein levels of HK2 but not glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or pyruvate kinase M2 (PKM2), and overexpression of HK2 reversed the inhibition of AS on Th17 cell differentiation. Furthermore, AS impaired the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signals in vivo and in vitro, which was abolished by the addition of lactate. In conclusion, AS significantly improved adjuvant-induced arthritis (AIA) in rats by inhibiting glycolysis-mediated activation of PI3K/AKT to restrain Th17 cell differentiation.
Animals ; Th17 Cells/immunology* ; Diterpenes/pharmacology* ; Arthritis, Rheumatoid/metabolism* ; Proto-Oncogene Proteins c-akt/immunology* ; Glycolysis/drug effects* ; Cell Differentiation/drug effects* ; Phosphatidylinositol 3-Kinases/genetics* ; Rats ; Male ; Rats, Sprague-Dawley ; Humans ; Andrographis paniculata/chemistry* ; Arthritis, Experimental/drug therapy* ; Interleukin-17/immunology* ; Signal Transduction/drug effects*

This study investigated the regulatory potential of salidroside (SAL), a primary active compound in Rhodiola rosea L., on osteoclast differentiation by modulating the hypoxia-inducible factor 1-alpha (HIF-1a) pathway in osteoblasts. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were employed to validate whether the receptor activator of nuclear factor-?B ligand (RANKL) is the downstream target gene of HIF-1a in osteoblasts. The study also utilized lipopolysaccharide (LPS)-induced mouse osteolysis to examine the impact of SAL on osteolysis in vivo. Furthermore, conditioned medium (CM) from SAL-pretreated osteoblasts was used to investigate the paracrine effects on osteoclastogenesis through the HIF-1a pathway. Hypoxic condition-induced overexpression of HIF-1a upregulated RANKL levels by binding to the RANKL promoter and enhancing transcription in osteoblastic cells. In vivo, SAL significantly alleviated bone tissue hypoxia and decreased the expression of HIF-1a by downregulating the expression of RANKL, vascular endothelial growth factor (VEGF), interleukin 6 (IL-6), and angiopoietin-like 4 (ANGPTL4). In the paracrine experiment, conditioned media from SAL-pretreated osteoblasts inhibited differentiation through the HIF-1a/RANKL, VEGF, IL-6, and ANGPTL4 pathways. RANKL emerges as the downstream target gene regulated by HIF-1a in osteoblasts. SAL significantly alleviates bone tissue hypoxia and bone loss in LPS-induced osteolysis through the HIF-1a/RANKL, VEGF, IL-6, and ANGPTL4 pathways. SAL inhibits osteoclast differentiation by regulating osteoblast paracrine secretion.
Animals ; Osteoblasts/cytology* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Glucosides/administration & dosage* ; Cell Differentiation/drug effects* ; Phenols/administration & dosage* ; Mice ; Osteoclasts/metabolism* ; RANK Ligand/genetics* ; Rhodiola/chemistry* ; Osteogenesis/drug effects* ; Signal Transduction/drug effects* ; Interleukin-6/genetics* ; Male ; RAW 264.7 Cells ; Osteolysis/genetics* ; Humans ; Mice, Inbred C57BL

Saponins associated with Panax notoginseng (P. notoginseng) demonstrate significant therapeutic efficacy across multiple diseases. However, certain high-yield saponins face limited clinical applications due to their reduced pharmacological efficacy. This study synthesized and evaluated 36 saponin-1,2,3-triazole derivatives of ginsenosides Rg1/Rb1 and notoginsenoside R1 for anti-adipogenesis activity in vitro. The research revealed that the ginsenosides Rg1-1,2,3-triazole derivative a17 demonstrates superior adipogenesis inhibitory effects. Structure-activity relationships (SARs) analysis indicates that incorporating an amidyl-substituted 1,2,3-triazole into the saponin side chain via Click reaction enhances anti-adipogenesis activity. Additionally, several other derivatives exhibit general adipogenesis inhibition. Compound a17 demonstrated enhanced potency compared to the parent ginsenoside Rg1. Mechanistic investigations revealed that a17 exhibits dose-dependent inhibition of adipogenesis in vitro, accompanied by decreased expression of preadipocytes. Peroxisome proliferator-activated receptor γ (PPARγ), fatty acid synthase (FAS), and fatty acid binding protein 4 (FABP4) adipogenesis regulators. These findings establish the ginsenoside Rg1-1,2,3-triazole derivative a17 as a promising adipocyte differentiation inhibitor and potential therapeutic agent for obesity and associated metabolic disorders. This research provides a foundation for developing effective therapeutic approaches for various metabolic syndromes.
Adipogenesis/drug effects* ; Triazoles/chemical synthesis* ; Ginsenosides/chemical synthesis* ; Saponins/chemical synthesis* ; Animals ; Mice ; Structure-Activity Relationship ; PPAR gamma/genetics* ; 3T3-L1 Cells ; Adipocytes/metabolism* ; Panax notoginseng/chemistry* ; Drug Design ; Molecular Structure ; Humans ; Cell Differentiation/drug effects* ; Fatty Acid-Binding Proteins/genetics*