This study aims to investigate the pharmacological effects and mechanisms of Yougui Yin in treating glucocorticoid-induced osteonecrosis. A rat model of glucocorticoid-associated osteonecrosis of the femoral head(GA-ONFH) was established by intramuscular injection of dexamethasone at 20 mg·kg~(-1) every other day for 8 weeks. Rats were randomly allocated into control, model, and low-and high-dose(1.5 and 3.0 g·kg~(-1), respectively) Yougui Yin groups. After modeling, rats in Yougui Yin groups were administrated with Yougui Yin via gavage, which was followed by femoral specimen collection. Hematoxylin-eosin staining was employed to observe femoral head repair, and immunofluorescence was employed to assess adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs) within the femoral head. Cell experiments were carried out with dexamethasone(1 μmol·L~(-1))-treated BMSCs to evaluate the effects of Yougui Yin-medicated serum on adipogenic differentiation. Animal experiments demonstrated that compared with the model group, Yougui Yin at both high and low doses significantly improved bone mineral density(BMD), bone volume/total volume(BV/TV) ratio, and trabecular thickness(Tb.Th) in the femoral head. Additionally, Yougui Yin alleviated necrosis-like changes and adipocyte infiltration and significantly reduced the expression level of peroxisome proliferator-activated receptor γ(PPARγ) in the femoral head, thereby suppressing the adipogenic differentiation of BMSCs in GA-ONFH rats. The cell experiments revealed that Yougui Yin-medicated serum markedly inhibited dexamethasone-induced adipogenic differentiation of BMSCs and down-regulated the level of PPARγ. The overexpression of PPARγ attenuated the inhibitory effect of Yougui Yin-medicated serum on the adipogenic differentiation of BMSCs, indicating the critical role of PPARγ in Yougui Yin-mediated suppression of adipogenic differentiation of BMSCs. In conclusion, Yougui Yin exerts therapeutic effects on glucocorticoid-induced osteonecrosis by down-regulating PPARγ expression and inhibiting adipogenic differentiation of BMSCs.
Animals ; Mesenchymal Stem Cells/metabolism* ; PPAR gamma/genetics* ; Rats ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Glucocorticoids/adverse effects* ; Rats, Sprague-Dawley ; Adipogenesis/drug effects* ; Osteonecrosis/genetics* ; Cell Differentiation/drug effects* ; Bone Marrow Cells/metabolism* ; Femur Head Necrosis/chemically induced* ; Humans

OBJECTIVE: To investigate the effects and underlying mechanism of ionizing radiation on the adipogenic of mesenchymal stem cells (MSCs). METHODS: Mouse MSCs were cultured in vitro and treated with 2 Gy and 6 Gy radiation with ⁶⁰Co, and the radiation dose rate was 0.98 Gy/min. Bulk RNA-seq was performed on control and irradiated MSCs. The changes of adipogenic differentiation and oxidative stress pathways of MSC were revealed by bioinformatics analysis. Oil Red O staining was used to detect the adipogenic differentiation ability of MSCs in vitro, and real-time fluorescence quantitative PCR (qPCR) was used to detect the expression differences of key regulatory factors Cebpa, Lpl and Pparg after radiation treatment. At the same time, qPCR and Western blot were used to detect the effect of inhibition of Nrf2, a key factor of antioxidant stress pathway, on the expression of key regulatory factors of adipogenesis. Moreover, the species conservation of the irradiation response of human bone marrow MSCs and mouse MSC was determined by qPCR. RESULTS: Bulk RNA-seq suggested that ionizing radiation promotes adipogenic differentiation of MSCs and up-regulation of oxidative stress-related genes and pathways. The results of Oil Red O staining and qPCR showed that ionizing radiation promoted the adipogenesis of MSCs, with high expression of Cebpa, Lpl and Pparg, as well as oxidative stress-related gene Nrf2. Nrf2 pathway inhibitors could further enhance the adipogenesis of MSCs in bone marrow after radiation. Notably, the similar regulation of oxidative pathways and enhanced adipogenesis post irradiation were observed in human bone marrow MSCs. In addition, irradiation exposure led to up-regulated mRNA expression of interleukin-6 and down-regulated mRNA expression of colony stimulating factor 2 in human bone marrow MSCs. CONCLUSION Ionizing radiation promotes adipogenesis of MSCs in mice, and oxidative stress pathway participates in this effect, blocking Nrf2 further promotes the adipogenesis of MSCs. Additionally, irradiation activates oxidative pathways and promotes adipogenic differentiation of human bone marrow MSCs.
Mesenchymal Stem Cells/cytology* ; Oxidative Stress/radiation effects* ; Animals ; Adipogenesis/radiation effects* ; Mice ; Radiation, Ionizing ; Cell Differentiation/radiation effects* ; Humans ; NF-E2-Related Factor 2/metabolism* ; PPAR gamma ; Cells, Cultured

OBJECTIVE: To establish an in vitro cell model simulating acute graft-versus-host disease (aGVHD) bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells (MSCs). METHODS: The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model, respectively. Bone marrow transplantation (BMT) mouse model (n=20) was established by being injected with bone marrow cells (1×10⁷ per mouse) from donor mice within 4-6 hours after receiving a lethal dose (8.0 Gy, 72.76 cGy/min) of γ ray general irradiation. A mouse model of aGVHD (n=20) was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells (1×10⁷ per mouse) and spleen lymphocytes (2×10⁶ per mouse). The blood was removed from the eyeballs and the mouse serum was aspirated on the 7^th day after modeling. Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2%, 5% and 10% BMT mouse serum and aGVHD mouse serum in the medium, respectively. The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining. The expression levels of related proteins PPARγ and CEBPα were detected by Western blot. The expression differences of key adipogenic transcription factors including PPARγ, CEBPα, FABP4 and LPL were determined by real-time quantitative PCR (RT-qPCR). RESULTS: An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established. Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10% compared with BMT serum. Western blot experiments showed that adipogenesis-related proteins PPARγ and CEBPα expressed in MSCs were down-regulated. Further RT-qPCR assay showed that the production of PPARγ, CEBPα, FABP4 and LPL, the key transcription factors for adipogenic differentiation of MSC, were significantly reduced. CONCLUSION The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.
Animals ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Adipogenesis ; Female ; Cell Differentiation ; Graft vs Host Disease/blood* ; Bone Marrow Cells/cytology* ; PPAR gamma/metabolism* ; Disease Models, Animal ; CCAAT-Enhancer-Binding Protein-alpha/metabolism*

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*

OBJECTIVES: Pelvic organ prolapse (POP) is a common condition in postmenopausal women, with an increasing prevalence due to aging. Some women experience POP recurrence after surgical treatment, significantly affecting their physical and mental health. The uterosacral ligament is a critical pelvic support structure. This study aims to investigate the molecular pathological changes in the uterosacral ligament of postmenopausal women with recurrent POP using transcriptomic analysis. METHODS: Transcriptomic data of uterosacral ligament tissues were obtained from the public dataset GSE28660, which includes samples from 4 postmenopausal women with recurrent POP, 4 with primary POP, and 4 without POP. Differentially expressed genes (DEGs) were identified between recurrent POP and both primary and non-POP groups. Further analysis included intersection analysis of DEGs, gene ontology enrichment, protein-protein interaction (PPI) network construction, gene set enrichment analysis (GSEA), single-sample GSEA, and xCell immune cell infiltration analysis to explore molecular pathological changes in recurrent POP. Additionally, histological and molecular differences in the uterosacral ligament were compared between simulated vaginal delivery (SVD) rat models with and without ovariectomy. RESULTS: Compared with primary POP and non-POP groups, recurrent POP exhibited activation of adipogenesis and inflammation-related pathways, while pathways related to muscle proliferation and contraction were downregulated in the uterosacral ligament. Nine key DEGs (ADIPOQ, FABP4, IL-6, LIPE, LPL, PCK1, PLIN1, PPARG, and CD36) were identified, with most enriched in the peroxisome proliferator-activated receptor (PPAR) signaling pathway. These genes were significantly correlated with lipid accumulation, monocyte infiltration, and neutrophil infiltration in the uterosacral ligament. Urodynamic testing revealed that the bladder leak point pressure was significantly higher in ovariectomized SVD rats, both of which had higher values than the sham group. Masson staining showed pronounced adipogenesis in the uterosacral ligament of ovariectomized SVD rats, along with reduced collagen and muscle fibers compared to the sham and non-ovariectomized SVD groups. Furthermore, real-time RT-PCR confirmed significantly elevated expression of key DEGs, including ADIPOQ, IL-6, PCK1, and PLIN1, in the uterosacral ligaments of ovariectomized SVD rats. CONCLUSIONS Adipogenesis and inflammation in the uterosacral ligament may contribute to its reduced supportive function, potentially leading to recurrence POP in postmenopausal women.
Female ; Humans ; Ligaments/pathology* ; Pelvic Organ Prolapse/metabolism* ; Postmenopause ; Animals ; Rats ; Adipogenesis/genetics* ; Recurrence ; Gene Expression Profiling ; Transcriptome ; Middle Aged ; Ovariectomy ; Protein Interaction Maps ; Aged ; Rats, Sprague-Dawley ; Uterus

OBJECTIVE: To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM. METHODS: Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining. The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated, and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells. The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4, FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR. The Western blot was employed to detect the expression levels of IL-6, IL-10, SDF-1α, TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor. RESULTS: The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group, and the BMA content was related to the disease status. The content of BMA decreased in the patients with effective chemotherapy. MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ, DLK1, DGAT1, FABP4 and FASN, but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated. This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts, and the cytokines detected in supernatant changed. PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA. Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal, and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release. The increased BMA and related cytokines effectively promoted the proliferation, migration and drug resistance of MM cells. CONCLUSION The BMA and its associated cytokines are the promoting factors in the survival, proliferation and migration of MM cells. BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.
Humans ; Osteogenesis/genetics* ; Bone Marrow/metabolism* ; Multiple Myeloma/metabolism* ; Drug Resistance, Neoplasm ; Peroxisome Proliferator-Activated Receptors/pharmacology* ; Cell Differentiation ; Adipogenesis ; Cytokines/metabolism* ; Adipocytes/metabolism* ; Bone Marrow Cells/metabolism* ; Cells, Cultured ; PPAR gamma/pharmacology* ; Tumor Microenvironment

OBJECTIVE: To investigate the expression of pyruvate kinase M2 (PKM2) in bone marrow mesenchymal stem cells (BMSCs) in myeloma bone disease (MBD) and its effect on osteogenic and adipogenic differentiation of BMSCs. METHODS: BMSCs were isolated from bone marrow of five patients with multiple myeloma (MM) (MM group) and five with iron deficiency anemia (control group) for culture and identification. The expression of PKM2 protein were compared between the two groups. The differences between osteogenic and adipogenic differentiation of BMSCs were assessed by using alkaline phosphatase (ALP) and oil red O staining, and detecting marker genes of osteogenesis and adipogenesis. The effect of MM cell line (RPMI-8226) and BMSCs co-culture on the expression of PKM2 was explored. Functional analysis was performed to investigate the correlations of PKM2 expression of MM-derived BMSCs with osteogenic and adipogenic differentiation by employing PKM2 activator and inhibitor. The role of orlistat was explored in regulating PKM2 expression, osteogenic and adipogenic differentiation of MM-derived BMSCs. RESULTS: Compared with control, MM-originated BMSCs possessed the ability of increased adipogenic and decreased osteogenic differentiation, and higher level of PKM2 protein. Co-culture of MM cells with BMSCs markedly up-regulated the expression of PKM2 of BMSCs. Up-regulation of PKM2 expression could promote adipogenic differentiation and inhibit osteogenic differentiation of MM-derived BMSCs, while down-regulation of PKM2 showed opposite effect. Orlistat significantly promoted osteogenic differentiation in MM-derived BMSCs via inhibiting the expression of PKM2. CONCLUSION The overexpression of PKM2 can induce the inhibition of osteogenic differentiation of BMSCs in MBD. Orlistat can promote the osteogenic differentiation of BMSCs via inhibiting the expression of PKM2, indicating a potential novel agent of anti-MBD therapy.
Humans ; Adipogenesis ; Bone Diseases/metabolism* ; Bone Marrow Cells ; Cell Differentiation ; Cells, Cultured ; Mesenchymal Stem Cells/physiology* ; Multiple Myeloma/metabolism* ; Orlistat/pharmacology* ; Osteogenesis/genetics*

C-fos is a transcription factor that plays an important role in cell proliferation, differentiation and tumor formation. The aim of this study was to clone the goat c-fos gene, clarify its biological characteristics, and further reveal its regulatory role in the differentiation of goat subcutaneous adipocytes. We cloned the c-fos gene from subcutaneous adipose tissue of Jianzhou big-eared goats by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed its biological characteristics. Using real-time quantitative PCR (qPCR), we detected the expression of c-fos gene in the heart, liver, spleen, lung, kidney, subcutaneous fat, longissimus dorsi and subcutaneous adipocytes of goat upon induced differentiation for 0 h to 120 h. The goat overexpression vector pEGFP-c-fos was constructed and transfected into the subcutaneous preadipocytes for induced differentiation. The morphological changes of lipid droplet accumulation were observed by oil red O staining and bodipy staining. Furthermore, qPCR was used to test the relative mRNA level of the c-fos overexpression on adipogenic differentiation marker genes. The results showed that the cloned goat c-fos gene was 1 477 bp in length, in which the coding sequence was 1 143 bp, encoding a protein of 380 amino acids. Protein structure analysis showed that goat FOS protein has a basic leucine zipper structure, and subcellular localization prediction suggested that it was mainly distributed in the nucleus. The relative expression level of c-fos was higher in the subcutaneous adipose tissue of goats (P < 0.05), and the expression level of c-fos was significantly increased upon induced differentiation of subcutaneous preadipocyte for 48 h (P < 0.01). Overexpression of c-fos significantly inhibited the lipid droplets formation in goat subcutaneous adipocytes, significantly decreased the relative expression levels of the AP2 and C/EBPβ lipogenic marker genes (P < 0.01). Moreover, AP2 and C/EBPβ promoter are predicted to have multiple binding sites. In conclusion, the results indicated that c-fos gene was a negative regulatory factor of subcutaneous adipocyte differentiation in goats, and it might regulate the expression of AP2 and C/EBPβ gene expression.
Animals ; Goats/genetics* ; Cell Differentiation/genetics* ; Adipogenesis/genetics* ; Gene Expression Regulation ; Proteins/genetics* ; Cloning, Molecular

The aim of this study was to clone the goat RPL29 gene and analyze its effect on lipogenesis in intramuscular adipocytes. Using Jianzhou big-eared goats as the object, the goat RPL29 gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR), the gene structure and expressed protein sequence were analyzed by bioinformatics, and the mRNA expression levels of RPL29 in various tissues and different differentiation stages of intramuscular adipocytes of goats were detected by quantitative real-time PCR (qRT-PCR). The RPL29 overexpression vector pEGFP-N1-RPL29 constructed by gene recombination was used to transfect into goat intramuscular preadipocytes and induce differentiation. Subsequently, the effect of overexpression of RPL29 on fat droplet accumulation was revealed morphologically by oil red O and Bodipy staining, and changes in the expression levels of genes related to lipid metabolism were detected by qRT-PCR. The results showed that the length of the goat RPL29 was 507 bp, including a coding sequence (CDS) region of 471 bp which encodes 156 amino acid residues. It is a positively charged and stable hydrophilic protein mainly distributed in the nucleus of cells. Tissue expression profiling showed that the expression level of this gene was much higher in subcutaneous adipose tissue and inter-abdominal adipose tissue of goats than in other tissues (P < 0.05). The temporal expression profile showed that the gene was expressed at the highest level at 84 h of differentiation in goat intramuscular adipocytes, which was highly significantly higher than that in the undifferentiated period (P < 0.01). Overexpression of RPL29 promoted lipid accumulation in intramuscular adipocytes, and the optical density values of oil red O staining were significantly increased (P < 0.05). In addition, overexpression of RPL29 was followed by a highly significant increase in ATGL and ACC gene expression (P < 0.01) and a significant increase in FASN gene expression (P < 0.05). In conclusion, the goat RPL29 may promote intra-muscular adipocyte deposition in goats by up-regulating FASN, ACC and ATGL.
Animals ; Lipogenesis/genetics* ; Adipogenesis/genetics* ; Goats/genetics* ; Adipocytes ; Cell Differentiation/genetics* ; Sequence Analysis ; Cloning, Molecular

This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene. Based on the pre-transcriptome sequencing data obtained previously, the miR-23b-3p, which was differentially expressed in goat intramuscular adipocytes before and after differentiation, was used as an entry point. real-time quantitative-polymerase chain reaction (qPCR) was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes. The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels. The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes. The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes, significantly down-regulated the expression levels of adipogenic marker genes AP2, C/EBPα, FASN, and LPL (P < 0.01). In addition, the expressions of C/EBPβ, DGAT2, GLUT4 and PPARγ were significantly downregulated (P < 0.05). After interfering with the expression of miR-23b-3p, lipid droplet accumulation was increased in goat intramuscular adipocytes. The expression levels of ACC, ATGL, AP2, DGAT2, GLUT4, FASN and SREBP1 were extremely significantly up-regulated (P < 0.01), and the expression levels of C/EBPβ, LPL and PPARγ were significantly up-regulated (P < 0.05). It was predicted that PDE4B might be a target gene of miR-23b-3p. The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p (P < 0.01), and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B (P < 0.05). The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene. MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.
Animals ; MicroRNAs/metabolism* ; Goats/genetics* ; PPAR gamma/metabolism* ; Adipogenesis/genetics* ; Cell Differentiation/genetics* ; Luciferases ; RNA, Messenger