OBJECTIVE: To observe the effects of electroacupuncture (EA) on improving motor function and regulating microglial activation based on Notch receptor 1 (Notch1)/Hes family bHLH transcription factor 1 (Hes1) pathway in mice with Parkinson's disease (PD). METHODS: Thirty-six male C57BL/6 mice were randomly divided into a control group, a model group and an EA group, 12 mice in each group. PD model was established by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 consecutive days in the model group and the EA group. From the 1st day of modeling, EA was applied at "Baihui" (GV20) and bilateral "Shenshu" (BL23) in the EA group, with continuous wave, in frequency of 2 Hz and current of 2 mA, 15 min a time, once a day for 14 days continuously. The behavioral performance was evaluated by gait test, pole climbing test and hanging test, the number of positive cells of tyrosine hydroxylase (TH) and the co-expression positive cells of Notch1/ionized calcium binding adaptor molecule 1 (Iba-1) in the substantia nigra of midbrain was assessed by immunofluorescence, the protein expression of TH, α-synuclein (α-syn), Notch1, Hes1, Iba-1, inducible nitric oxide synthase (iNOS), Arginase-1 (ARG1), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-10 was detected by Western blot, the mRNA expression of Notch1 and Hes1 was detected by real-time PCR. RESULTS: Compared with the control group, in the model group, the stride frequency was accelerated (P<0.001) and the stride length was shortened (P<0.001) for the four limbs, the pole climbing test time was prolonged (P<0.01) and the grip level was reduced (P<0.01); in the substantia nigra of midbrain, the number of positive cells of TH was decreased (P<0.001), the number of co-expression positive cells of Notch1/Iba-1 was increased (P<0.001), the protein expression of α-syn, Notch1, Hes1, Iba-1, iNOS, TNF-α, IL-1βand IL-6 was increased (P<0.01, P<0.05, P<0.001), the protein expression of TH, ARG1 and IL-10 was decreased (P<0.01, P<0.001), the mRNA expression of Notch1 and Hes1 was increased (P<0.01). Compared with the model group, in the EA group, the stride frequency was decelerated (P<0.001) and the stride length was increased (P<0.05, P<0.01, P<0.001) for the four limbs, the pole climbing test time was shortened (P<0.05) and the grip level was increased (P<0.05); in the substantia nigra of midbrain, the number of positive cells of TH was increased (P<0.01), the number of co-expression positive cells of Notch1/Iba-1 was decreased (P<0.001), the protein expression of α-syn, Notch1, Hes1, Iba-1, iNOS, TNF-α, IL-6 and IL-1β was decreased (P<0.05, P<0.01), the protein expression of TH, ARG1 and IL-10 was increased (P<0.05, P<0.001, P<0.01), the mRNA expression of Notch1 and Hes1 was decreased (P<0.05). CONCLUSION EA can improve the behavioral performance and protect the dopaminergic neurons in PD mice, its mechanism may relate to the inhibition of Notch1/Hes1-mediated neuroinflammation, thus inhibiting the microglial activation.
Animals ; Electroacupuncture ; Microglia/metabolism* ; Male ; Receptor, Notch1/metabolism* ; Parkinson Disease/physiopathology* ; Transcription Factor HES-1/metabolism* ; Mice ; Mice, Inbred C57BL ; Humans ; Signal Transduction

Therapeutic resistance in cancer is responsible for numerous cancer deaths in clinical practice. While target mutations are well recognized as the basis of genetic resistance to targeted therapy, nontarget mutation resistance (or nongenetic resistance) remains poorly characterized. Despite its complex and unintegrated mechanisms in the literature, nongenetic resistance is considered from our perspective to be a collective response of innate or acquired resistant subpopulations in heterogeneous tumors to therapy. These subpopulations, e.g., cancer stem-like cells, cancer cells with epithelial-to-mesenchymal transition, and drug-tolerant persisters, are protected by their resistance traits at cellular and molecular levels. This review summarizes recent advances in the research on resistant populations and their resistance traits. NOTCH signaling, as a central regulator of nongenetic resistance, is discussed with a special focus on its canonical maintenance of resistant cancer cells and noncanonical regulation of their resistance traits. This novel view of canonical and noncanonical NOTCH signaling pathways is translated into our proposal of reshaping therapeutic strategies targeting NOTCH signaling in resistant cancer cells. We hope that this review will lead researchers to study the canonical and noncanonical arms of NOTCH signaling as an integrated resistant mechanism, thus promoting the development of innovative therapeutic strategies.
Neoplasms/metabolism* ; Receptors, Notch/metabolism* ; Disease Resistance/physiology* ; Signal Transduction/physiology* ; Humans ; Drug Resistance, Neoplasm/physiology* ; Molecular Targeted Therapy/methods*

OBJECTIVE: To observe the degree of myocardial cell injury and the changes in autophagy level in rats with myocardial ischemia/reperfusion (I/R) injury induced by cardiopulmonary bypass (CPB), and to explore the regulatory role of the long non-coding RNA-urothelial carcinoma antigen 1-microRNA-143-Notch1 axis (lncRNA-UCA1-miR-143-Notch1 axis) in myocardial I/R injury induced by CPB. METHODS: Healthy male Sprague-Dawley (SD) rats were randomly divided into the following groups using the random number method: Sham operation (Sham) group, myocardial I/R injury model group (model group), empty lentivirus group, lncRNA-UCA1 upregulation group, miR-143 downregulation group, and lncRNA-UCA1 upregulation+miR-143 upregulation group, with 9 rats in each group. The rat model of myocardial I/R injury induced by CPB was established by thoracotomy aortic ligation under cardiopulmonary bypass support; in the Sham group, only threading was performed without ligation, and other procedures were the same. Seventy-two hours before modeling, the lncRNA-UCA1 upregulated group was injected with 100 μL of myocardial tissue-specific adeno-associated virus (AAV) overexpression vector of lncRNA-UCA1 via tail vein, the miR-143 downregulated group was injected with 100 μL of AAV short hairpin RNA (shRNA) vector of miR-143 via tail vein, the lncRNA-UCA1 upregulation+miR-143 upregulation group was injected with 100 μL of myocardial tissue-AAV overexpression vector of lncRNA-UCA1 and 100 μL of AAV overexpression vector of miR-143 via tail vein, and the empty vector lentivirus group was injected with 100 μL of AAV empty vector (virus titers were 1×10⁹ TU/mL); the Sham group and the model group were injected with equal amounts of normal saline. The animals were euthanized 24 hours after intervention and cardiac tissue specimens were collected. After hematoxylin eosin (HE) staining, the damage of myocardial cells and the changes of muscle fiber tissue were observed under a light microscope; after dual staining with uranyl acetate and lead citrate, the ultrastructural damage of heart tissue was observed under a transmission electron microscopy; the expression of lncRNA-UCA1, miR-143, and Notch1 mRNA in myocardial tissue was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR); the expression of microtubule 1 light chain 3-II/I (LC3-II/I) and Notch1 protein in myocardial tissue was detected by Western blotting. RESULTS: Compared with the Sham group, the myocardial cells of rats in the model group were enlarged, the intercellular space increased, autophagosomes increased, the arrangement of myocardial fibers was disordered, mitochondrial proliferated and deformed. The expression levels of lncRNA-UCA1 and Notch1 mRNA, as well as the protein expression levels of LC3-II/I and Notch1 were significantly increased, while the expression level of miR-143 was significantly decreased. Compared with the model group, the degree of myocardial cell injury in the lncRNA-UCA1 upregulation group and miR-143 downregulation group was significantly alleviated, the expression levels of Notch1 mRNA, LC3-II/I, and Notch1 protein were significantly increased [Notch1 mRNA (2^-ΔΔCt): 2.66±0.24, 2.03±0.23 vs. 1.45±0.13, LC3-II/I: 2.10±0.21, 1.92±0.19 vs. 1.39±0.14, Notch1 protein (Notch1/GAPDH): 1.72±0.16, 1.57±0.16 vs. 1.34±0.13, all P < 0.05], and the expression level of miR-143 was significantly decreased (2^-ΔΔCt: 0.50±0.06, 0.52±0.06 vs.0.71±0.06, P < 0.05). The expression level of lncRNA-UCA1 in the lncRNA-UCA1 upregulated group was significantly higher than that in the model group (2^-ΔΔCt: 2.47±0.22 vs. 1.43±0.14, P < 0.05), while there was no significant difference in the miR-143 downregulation group compared with the model group (2^-ΔΔCt: 1.50±0.16 vs. 1.43±0.14, P > 0.05). There was no significant difference in the degree of myocardial cell injury in the empty load lentivirus group and the lncRNA-UCA1 upregulation+miR-143 upregulation group compared to the model group. There were no significant differences in the expression of miR-143, Notch1 mRNA, and the autophagy level in these two groups compared to the model group. The expression level of lncRNA-UCA1 in the lncRNA-UCA1 upregulation+miR-143 upregulation group was significantly higher than that in the model group (2^-ΔΔCt: 2.47±0.20 vs. 1.43±0.14, P < 0.05). CONCLUSIONS Autophagy is involved in the pathological process of myocardial I/R injury induced by CPB. The lncRNA-UCA1-microRNA-143-Notch1 axis may regulate the autophagy level to participate in the I/R injury process.
Animals ; MicroRNAs ; Rats, Sprague-Dawley ; RNA, Long Noncoding ; Male ; Myocardial Reperfusion Injury/etiology* ; Rats ; Cardiopulmonary Bypass/adverse effects* ; Receptor, Notch1/metabolism* ; Autophagy

This paper explored the specific mechanism of ginsenoside Rg_1 in regulating mitochondrial fusion through the neurogenic gene Notch homologous protein 1(Notch1) pathway to alleviate hypoxia/reoxygenation(H/R) injury in HL-1 cells. The relative viability of HL-1 cells after six hours of hypoxia and two hours of reoxygenation was detected by cell counting kit-8(CCK-8). The lactate dehydrogenase(LDH) activity in the cell supernatant was detected by the lactate substrate method. The content of adenosine triphosphate(ATP) was detected by the luciferin method. Fluorescence probes were used to detect intracellular reactive oxygen species(Cyto-ROS) levels and mitochondrial membrane potential(ΔΨ_m). Mito-Tracker and Actin were co-imaged to detect the number of mitochondria in cells. Fluorescence quantitative polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels of Notch1, mitochondrial fusion protein 2(Mfn2), and mitochondrial fusion protein 1(Mfn1). The results showed that compared with that of the control group, the cell activity of the model group decreased, and the LDH released into the cell culture supernatant increased. The level of Cyto-ROS increased, and the content of ATP decreased. Compared with that of the model group, the cell activity of the ginsenoside Rg_1 group increased, and the LDH released into the cell culture supernatant decreased. The level of Cyto-ROS decreased, and the ATP content increased. Ginsenoside Rg_1 elevated ΔΨ_m and increased mitochondrial quantity in HL-1 cells with H/R injury and had good protection for mitochondria. After H/R injury, the mRNA and protein expression levels of Notch1 and Mfn1 decreased, while the mRNA and protein expression levels of Mfn2 increased. Ginsenoside Rg_1 increased the mRNA and protein levels of Notch1 and Mfn1, and decreased the mRNA and protein levels of Mfn2. Silencing Notch1 inhibited the action of ginsenoside Rg_1, decreased the mRNA and protein levels of Notch1 and Mfn1, and increased the mRNA and protein levels of Mfn2. In summary, ginsenoside Rg_1 regulated mitochondrial fusion through the Notch1 pathway to alleviate H/R injury in HL-1 cells.
Ginsenosides/pharmacology* ; Receptor, Notch1/genetics* ; Signal Transduction/drug effects* ; Mice ; Animals ; Mitochondrial Dynamics/drug effects* ; Mitochondria/metabolism* ; Cell Line ; Reactive Oxygen Species/metabolism* ; Oxygen/metabolism* ; Cell Hypoxia/drug effects* ; Cell Survival/drug effects* ; Membrane Potential, Mitochondrial/drug effects* ; Humans

OBJECTIVE: To investigate the effectiveness and preliminary mechanisms of icariin (ICA) in enhancing the reparative effects of adipose-derived stem cells (ADSCs) on skin radiation damagies in rats. METHODS: Twelve SPF-grade Sprague Dawley rats [body weight (220±10) g] were subjected to a single dose of 10 Gy X-ray irradiation on a 1.5 cm×1.5 cm area of their dorsal skin, with a dose rate of 200 cGy/min to make skin radiation damage model. After successful modelling, the rats were randomly divided into 4 groups ( n=3), and on day 2, the corresponding cells were injected subcutaneously into the irradiated wounds: group A received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL), group B received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL)+1 μmol/L ICA (0.1 mL), group C received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a hypoxia-inducible factor 2α (HIF-2α) inhibitor+1 μmol/L ICA (0.1 mL), and group D received 0.1 mL of rat ADSCs (1×10 ⁷cells/mL) pretreated with a Notch1 inhibitor+1 μmol/L ICA (0.1 mL). All treatments were administered as single doses. The skin injury in the irradiated areas of the rats was observed continuously from day 1 to day 7 after modelling. On day 28, the rats were sacrificed, and skin tissues from the irradiated areas were harvested for histological examination (HE staining and Masson staining) to assess the repair status and for quantitative collagen content detection. Immunohistochemical staining was performed to detect CD31 expression, while Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to measure the protein and mRNA relative expression levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), fibroblast growth factor 2 (FGF-2), interleukin 10 (IL-10), transforming growth factor β (TGF-β), HIF-2α, and Notch1, 2, and 3. RESULTS: All groups exhibited skin ulcers and redness after irradiation. On day 3, exudation of tissue fluid was observed in all groups. On day 7, group B showed significantly smaller skin injury areas compared to the other 3 groups. On day 28, histological examination revealed that the epidermis was thickened and the dermal fibers were slightly disordered with occasional inflammatory cell aggregation in group A. In group B, the epidermis appeared more normal, the dermal fibers were more orderly, and there was an increase in new blood vessels without significant inflammatory cell aggregation. In contrast, groups C and D showed significantly increased epidermal thickness, disordered and disrupted dermal fibers. Group B had higher collagen fiber content than the other 3 groups, and group D had lower content than group A, with significant differences ( P<0.05). Immunohistochemical staining showed that group B had significantly higher CD31 expression than the other 3 groups, while groups C and D had lower expression than group A, with significant differences ( P<0.05). Western blot and qRT-PCR results indicated that group B had significantly higher relative expression levels of VEGF, PDGF-BB, FGF-2, IL-10, TGF-β, HIF-2α, and Notch1, 2, and 3 proteins and mRNAs compared to the other 3 groups ( P<0.05). CONCLUSION ICA may enhance the reparative effects of ADSCs on rat skin radiation damage by promoting angiogenesis and reducing inflammatory responses through the HIF-2α-VEGF-Notch signaling pathway.
Animals ; Rats, Sprague-Dawley ; Skin/pathology* ; Rats ; Vascular Endothelial Growth Factor A/genetics* ; Basic Helix-Loop-Helix Transcription Factors/genetics* ; Signal Transduction ; Flavonoids/pharmacology* ; Adipose Tissue/cytology* ; Stem Cells/cytology* ; Receptors, Notch/metabolism* ; Radiation Injuries, Experimental/metabolism* ; Wound Healing/drug effects* ; Male

BACKGROUND: Regulatory dendritic cell (DCreg) subset exhibits a unique capacity for inducing immune tolerance among the variety subsets of dendritic cells (DCs) within gut-associated lymphoid tissues (GALTs). Fms-like tyrosine kinase 3 ligand (FLT3L) is involved in the differentiation of DCregs and the subsequent expansion of regulatory T-cells (Tregs) mediated by DCregs, though the precise mechanism remains poorly understood. This study aimed to explore the expansion mechanism of Treg induced by DCreg and the role of FLT3L in this process. METHODS: DCregs were distinguished from other DC subsets isolated from GALTs of BALB/c mice through a mixed lymphocyte reaction assay. The functions and mechanisms by which FLT3L promoted Treg expansion via DCregs were investigated in vitro through co-culture experiments involving DCregs and either CD4 + CD25 - T-cells or CD4 + CD25 + T-cells. Additionally, an in vivo experiment was conducted using a dextran sulfate sodium (DSS)-induced colitis model in mice. RESULTS: CD103 + CD11b + DC exhibited DCreg-like functionality and was identified as DCreg for subsequent investigation. Analysis of Foxp3 + Treg percentages within a co-culture system of CD4 + CD25 - T-cells and DCregs, with or without FLT3L, demonstrated the involvement of the FLT3/FLT3L axis in driving the differentiation of precursor T-cells into Foxp3 + Tregs induced by DCregs. Cell migration and co-culture assays revealed that the FLT3/FLT3L axis enhanced DCreg migration toward Tregs via the Rho pathway. Additionally, it was observed that DCregs could promote Treg proliferation through the Notch pathway, as inhibition of Notch signaling by DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) suppressed Treg expansion within the co-culture system of DCregs and CD4 + T-cells or CD4 + CD25 + T-cells. Furthermore, the FLT3/FLT3L axis influenced JAG1 expression in DCregs, indirectly modulating Treg expansion. In vivo experiments further established that FLT3L promoted DCreg expansion and restored Treg balance in DSS-induced colitis models, thereby ameliorating colitis symptoms in mice. CONCLUSION The FLT3/FLT3L axis is integral to the maintenance of DCreg function in Treg expansion.
Animals ; T-Lymphocytes, Regulatory/immunology* ; Dendritic Cells/immunology* ; Mice ; Mice, Inbred BALB C ; Membrane Proteins/metabolism* ; Receptors, Notch/metabolism* ; Lymphoid Tissue/metabolism* ; Signal Transduction/physiology* ; Coculture Techniques ; Flow Cytometry

Objective To observe the effect of m⁶A methylation regulation on Notch1 pathway on the homing of BMSCs in asthma, and the intervention study of traditional Chinese medicine compound Yanghe Pingchuan Granules. Methods Rat bone mesenchymal stem cells(BMSC)and bronchial epithelial cells were cocultured. The extracted cells were divided into: bronchial epithelial cell group, asthma bronchial epithelial cell+mesenchymal stem cell co-culture group (co-culture group), co-culture cell+normal serum group, coculture cell+serum containing optimal drug group, siRNA FTO+normal serum group, siRNA FTO-NC+normal serum group, and siRNA FTO+serum containing optimal drug group. The vitality and cell cycle changes of co-cultured cells were detected. The level and markers of homing BMSC were detected by immunofluorescence staining. The expression of Notch1 pathway related genes were detected by qRT-PCR. The expression of Notch1 pathway related proteins were detected by Western blot. Results Compared with bronchial epithelial cell group, the co-cultured cell group showed an increase in the homing level of BMSCs and the expression of C-X-C motif chemokine receptor 4 (CXCR4), stromal cell-derived factor 1 (SDF-1), Notch1, transcription factor recombination signal binding protein-J (RBP-J), and hairy enhancer of split 1 (Hes1) proteins. Compared with the co-cultured cell group and co-cultured cell+normal serum group, the co-cultured cell+serum containing optimal drug group showed an increase in the homing level of BMSCs and the expressions of CXCR4 and SDF-1, while the protein and mRNA levels of Notch1 and Hes1 decreased. Compared with the siRNA FTO-NC+normal serum group, the siRNA FTO+normal serum group showed an increase in the levels of Notch1, activated Notch1, RBP-J, Hes1 protein, and cell viability, while the level of homing BMSC decreased. Compared with siRNA FTO+normal serum group, the levels of Notch1, RBP-J mRNA, activated Notch1, and Hes1 protein decreased, while the level of homing BMSCs increased in siRNA FTO+serum containing optimal drug group. The levels of Notch1, RBP-J, and Hes1 mRNA were reduced in the co-cultured cells+serum containing optimal drug group. Compared with siRNA FTO+serum containing optimal drug group, the expressions of Notch1, activated Notch1, RBP-J, Hes1 protein and cell viability decreased, while the level of homing BMSCs increased in the co-cultured cells+serum containing optimal drug group. Conclusion Yanghe Pingchuan Granules may promote the homing of BMSCs in asthma and alleviate asthma inflammation by upregulating the expression of FTO and inhibiting the expression of downstream genes in the Notch1 signaling pathway.
Animals ; Receptor, Notch1/genetics* ; Mesenchymal Stem Cells/cytology* ; Asthma/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Signal Transduction/drug effects* ; Rats ; Coculture Techniques ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics* ; Epithelial Cells/metabolism* ; Rats, Sprague-Dawley ; Cells, Cultured ; Male

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease that threatens the vision of premature infants. Various novel drugs have demonstrated therapeutic potential for ROP by targeting signaling pathways associated with vascular endothelial growth factor (VEGF) [such as PI3K/AKT, hypoxia-inducible factor (HIF)-1α/VEGF], oxidative stress, tumor necrosis factor (TNF)-α, and Notch pathways. Propranolol, insulin-like growth factor-1, and celecoxib attenuate pathological neovascularization via the PI3K/Akt signaling pathway. Tripterine and melatonin inhibit retinal neovascularization by modulating the HIF-1α/VEGF signaling axis. Adiponectin mitigates the damage caused by oxidative stress and preserves endothelial function by enhancing endothelial nitric oxide synthase activity. Omega-3 polyunsaturated fatty acids suppress TNF-α-mediated inflammatory responses, modulate retinal development and angiogenesis, and reduce retinal neovascular lesions. DAPT, a γ-secretase inhibitor, blocks Notch signaling to suppress abnormal vascular proliferation. These agents exhibit synergistic multi-pathway anti-angiogenic effects in preclinical models and early-phase clinical trials, offering critical insights for advancing drug development and clinical translation in ROP management.
Retinopathy of Prematurity/metabolism* ; Humans ; Signal Transduction/drug effects* ; Infant, Newborn ; Vascular Endothelial Growth Factor A/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Oxidative Stress/drug effects* ; Fatty Acids, Omega-3/therapeutic use* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Receptors, Notch/metabolism* ; Angiogenesis Inhibitors/therapeutic use* ; Insulin-Like Growth Factor I/therapeutic use*

Small cell lung cancer (SCLC), a highly aggressive subtype of lung cancer that originates from pulmonary neuroendocrine cells, accounts for 10% to 15% of all lung cancers. It is characterized by a high rate of early metastasis and extremely poor prognosis, often accompanied by challenges such as drug resistance and recurrence. Related researches indicates that the Notch signaling pathway plays a crucial role in the occurrence and development of SCLC by regulating processes such as cell proliferation, differentiation, and apoptosis. In SCLC, abnormal Notch signaling may promote tumor malignancy and the occurrence of drug resistance. Additionally, the Notch pathway is involved in the epithelial-mesenchymal transition of SCLC and influences immune escape mechanisms through interactions with the tumor immune microenvironment. This article reviews the molecular mechanisms of Notch signaling in SCLC, including the roles of its receptors and ligands, signal transduction processes, and its role in tumorigenesis. It also discusses the research progress of Notch signaling as a potential therapeutic target and looks forward to future research directions in this field.
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Humans ; Receptors, Notch/genetics* ; Signal Transduction ; Small Cell Lung Carcinoma/pathology* ; Lung Neoplasms/pathology* ; Animals

OBJECTIVES: The core pathology of osteoporosis lies in bone resorption exceeding bone formation; thus, promoting osteogenesis is a key therapeutic strategy. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) forms the biological basis of bone formation. Astragaloside IV (A-IV), a major active component of Astragalus membranaceus, is known to enhance osteogenesis, but its precise molecular mechanisms remain unclear. This study aims to investigate the effects of A-IV on the proliferation and osteogenic differentiation of BMSCs from osteoporotic rats and to elucidate its molecular mechanism through the regulation of microRNA-21 (miR-21) and Notch2 expression. METHODS: After 1 week of adaptive feeding, mature female SD rats were randomly divided into a sham-operated (Sham) group (n=4) and an ovariectomized (OVX) group (n=8) to establish an osteoporosis model. Twelve weeks after surgery, BMSCs were isolated from femoral bone marrow and cultured. Cells were divided into a S-BMSCs group (from Sham), an O-BMSCs group (from OVX), and an A-BMSCs group (from OVX-derived BMSCs treated with A-IV). S-BMSCs and O-BMSCs were induced for osteogenic differentiation using osteogenic induction medium, whereas A-BMSCs were treated with A-IV before induction. Flow cytometry was used to identify mesenchymal stem cell surface markers (CD29) and hematopoietic stem cell marker (CD34) to confirm BMSC characteristics. Cell proliferation was assessed using the methyl thiazolyl tetrazolium (MTT) assay. Alizarin red staining was performed to quantify calcium nodule formation, and alkaline phosphatase (ALP) activity assays were used to evaluate osteogenic differentiation. Real-time reverse transcription PCR (real-time RT-PCR) was used to detect changes in osteogenic-related genes, runt-related transcription factor 2 (Runx2) and osteopontin (OPN), as well as miR-21 expression. Western blotting was performed to assess Runx2, OPN, and Notch2 protein expression. RESULTS: Flow cytometry confirmed that O-BMSCs retained the phenotypic characteristics of mesenchymal stem cells. A-IV significantly enhanced the proliferation of BMSCs from osteoporotic rats (P<0.05), increased ALP activity, and upregulated the mRNA and protein expression of Runx2 and OPN (P<0.05). Bioinformatic and experimental analyses demonstrated that miR-21 directly targeted Notch2. A-IV treatment increased miR-21 expression while suppressing Notch2 protein expression and inhibiting activation of the Notch signaling pathway (P<0.05). CONCLUSIONS Astragaloside IV promotes the osteogenic differentiation of BMSCs derived from osteoporotic rats by upregulating miR-21 expression and inhibiting the key Notch signaling protein Notch2, thereby relieving the Notch2-mediated suppression of osteogenesis.
Animals ; Triterpenes/pharmacology* ; Saponins/pharmacology* ; Osteogenesis/drug effects* ; MicroRNAs/metabolism* ; Rats, Sprague-Dawley ; Female ; Cell Differentiation/drug effects* ; Mesenchymal Stem Cells/drug effects* ; Signal Transduction/drug effects* ; Osteoporosis/pathology* ; Rats ; Cells, Cultured ; Receptor, Notch2/metabolism* ; Receptors, Notch/metabolism* ; Ovariectomy ; Cell Proliferation/drug effects*