OBJECTIVE: To analyze the clinical characteristics of patients with 11q23 rearrangement acute lymphoblastic leukemia (ALL) with non-KMT2A::AFF1 fusion genes. METHODS: The clinical data of 10 patients with KMT2A fusion gene positive and partner gene non-AFF1 ALL admitted to Henan Cancer Hospital from December 2016 to December 2024 were retrospectively summarized. The immunophenotype, molecular genetic characteristics, clinical manifestations and disease prognosis of these patients were analyzed. This research has been approved by the Medical Ethics Committee of Henan Cancer Hospital (Ethics No.: 2019342). RESULTS: Among the 10 patients, the fusion genes were KMT2A::MLLT1 in 7 cases, KMT2A::MLLT4, KMT2A::MLLT3 and KMT2A::MLLT10 in 1 case each. The European Group for the Immunological Classification of Leukemias (EGIL) classification included 6 cases of T-ALL, 2 cases of pro-B-ALL, 1 case of Common-B-ALL and 1 case of pre-B-ALL. 4 cases of B-ALL all expressed CD19, cCD79a, CD38 and HLA-DR, and some expressed CD34 and CD22, without expression or weak expression of CD10, without expression of CD20. One case was accompanied by myeloid marker CD15 expression. 6 cases of T-ALL all expressed CD34, CD7, most expressed CD38, and some expressed CD3, CD5, CD2, CD4 and CD8, and 1 case expressed CD4 and CD8 together. Chromosomal abnormalities were detected in 3 cases, 5 cases were positive for WT1 fusion gene, and 6 cases had gene alterations. 9 patients achieved the first complete remission (CR1) during chemotherapy, and 1 patient relapsed within 6 months after CR1. At the last follow up, 1 patient (the fusion gene was KMT2A::MLLT4) remained unrelieved. There were 2 cases of KMT2A rearrangement (KMT2A-r) persistent positive (+/+) and 8 cases of KMT2A-r negative (+/-). The overall survival (OS) rate and leukemia-free survival (LFS) rate of patients with KMT2A-r persistent positive were significantly lower than those of patients with negative change, and the differences were statistically significant (P values were all < 0.05). Among the 3 patients who received chemotherapy+allogeneic hematopoietic stem cell transplantation (allo-HSCT), no relapse was observed until the follow up day. The OS rate and LFS rate of patients with KMT2A::MLLT1 and chemotherapy+allo-HSCT were higher than those of non-KMT2A::MLLT1 and single chemotherapy patients, and the differences were not statistically significant (P values were all ≥ 0.05). There was no significant difference in OS rate and LFS rate between T-ALL and B-ALL patients (P values were all ≥ 0.05). The median LFS time of the 10 patients was 32 (0 ~ 100) months, and the median OS time was 36 (1 ~ 101) months. CONCLUSION The 11q23 rearrangement ALL with non-KMT2A::AFF1 transcript is mainly KMT2A::MLLT1, T-ALL is more common, and the rate of chromosomal karyotype detection is relatively low. Persistent positive KMT2A-r is unfavorable for patient survival, and allo-HSCT during the CR1 period may improve patient survival.
Humans ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics* ; Female ; Male ; Myeloid-Lymphoid Leukemia Protein/genetics* ; Histone-Lysine N-Methyltransferase/genetics* ; Adult ; Adolescent ; Chromosomes, Human, Pair 11/genetics* ; Child ; Transcriptional Elongation Factors/genetics* ; Gene Rearrangement ; Oncogene Proteins, Fusion/genetics* ; Retrospective Studies ; Young Adult ; Middle Aged ; Prognosis ; Child, Preschool ; DNA-Binding Proteins/genetics*

OBJECTIVE: To examine the mechanism of action of tanshinone II A (Tan II A) in promoting chemosensitization of osteosarcoma cells to cisplatin (DDP). METHODS: The effects of different concentrations of Tan II A (0-80 µ mol/L) and DDP (0-2 µ mol/L) on the proliferation of osteosarcoma cell lines (U2R, U2OS, 143B, and HOS) at different times were examined using the cell counting kit-8 and colony formation assays. Migration and invasion of U2R and U2OS cells were detected after 24 h treatment with 30 µ mol/L Tan II A, 0.5 µ mol/L DDP alone, and a combination of 10 µ mol/L Tan II A and 0.25 µ mol/L DDP using the transwell assay. After 48 h of treatment of U2R and U2OS cells with predetermined concentrations of each group of drugs, the cell cycle was analyzed using a cell cycle detection kit and flow cytometry. After 48 h treatment, apoptosis of U2R and U2OS cells was detected using annexin V-FITC apoptosis detection kit and flow cytometry. U2R cells were inoculated into the unilateral axilla of nude mice and then the mice were randomly divided into 4 groups of 6 nude mice each. The 4 groups were treated with equal volume of Tan II A (15 mg/kg), DDP (3 mg/kg), Tan II A (7.5 mg/kg) + DDP (1.5 mg/kg), and normal saline, respectively. The body weight of the nude mice was weighed, and the tumor volume and weight were measured. Cell-related gene and signaling pathway expression were detected by RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis. p38 MAPK signaling pathway proteins and apoptotic protein expressions were detected by Western blot. RESULTS: In vitro studies have shown that Tan II A, DDP and the combination of Tan II A and DDP inhibit the proliferation, migration and invasion of osteosarcoma cells. The inhibitory effect was more pronounced in the Tan II A and DDP combined treatment group (P<0.05 or P<0.01). Osteosarcoma cells underwent significantly cell-cycle arrest and cell apoptosis by Tan II A-DDP combination treatment (P<0.05 or P<0.01). In vivo studies demonstrated that the Tan II A-DD combination treatment group significantly inhibited tumor growth compared to the Tan II A and DDP single drug group (P<0.01). Additionally, we found that the combination of Tan II A and DDP treatment enhanced the p38 MAPK signaling pathway. Western blot assays showed higher p-p38, cleaved caspase-3, and Bax and lower caspase-3, and Bcl-2 expressions with the combination of Tan II A and DDP treatment compared to the single drug treatment (P<0.01). CONCLUSION Tan II A synergizes with DDP by activating the p38/MAPK pathway to upregulate cleaved caspase-3 and Bax pro-apoptotic gene expressions, and downregulate caspase-3 and Bcl-2 inhibitory apoptotic gene expressions, thereby enhancing the chemosensitivity of osteosarcoma cells to DDP.
Abietanes/therapeutic use* ; Osteosarcoma/enzymology* ; Cisplatin/therapeutic use* ; Humans ; Cell Line, Tumor ; Animals ; Apoptosis/drug effects* ; Mice, Nude ; Cell Proliferation/drug effects* ; Cell Movement/drug effects* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; MAP Kinase Signaling System/drug effects* ; Bone Neoplasms/enzymology* ; Cell Cycle/drug effects* ; Xenograft Model Antitumor Assays ; Mice ; Drug Resistance, Neoplasm/drug effects* ; Neoplasm Invasiveness ; Mice, Inbred BALB C

OBJECTIVE: To explore the role of the natural compound hesperidin in glycolysis, the key ratelimiting enzyme, in colorectal cancer (CRC) cell lines. METHODS: In vitro, HCT116 and SW620 were treated with different doses of hesperidin (0-500 µmol/L), cell counting kit-8 and colone formation assays were utilized to detected inhibition effect of hesperidin on CRC cell lines. Transwell and wound healing assays were performed to detect the ability of hesperidin (0, 25, 50 and 75 µmol/L) to migrate CRC cells. To confirm the apoptotic-inducing effect of hesperidin, apoptosis and cycle assays were employed. Western blot, glucose uptake, and lactate production determination measurements were applied to determine inhibitory effects of hesperidin (0, 25 and 50 µmol/L) on glycolysis. In vivo, according to the random number table method, nude mice with successful tumor loading were randomly divided into vehicle, low-dose hesperidin (20 mg/kg) and high-dose hesperidin (60 mg/kg) groups, with 6 mice in each group. The body weights and tumor volumes of mice were recorded during 4-week treatment. The expression of key glycolysis rate-limiting enzymes was determined using Western blot, and glucose uptake and lactate production were assessed. Finally, protein interactions were probed with DirectDIA Quantitative Proteomics, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. RESULTS: Hesperidin could inhibit CRC cell line growth (P<0.05 or P<0.01). Moreover, hesperidin presented an inhibitory effect on the migrating abilities of CRC cells. Hesperidin also promoted apoptosis and cell cycle alterations (P<0.05). The immunoblotting results manifested that hesperidin decreased the levels of hexokinase 2, glucose transporter protein 1 (GLUT1), GLUT3, L-lactate dehydrogenase A, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), PFKFB3, and pyruvate kinase isozymes M2 (P<0.01). It remarkably suppressed tumor xenograft growth in nude mice. GO and KEGG analyses showed that hesperidin treatment altered metabolic function. CONCLUSION Hesperidin inhibits glycolysis and is a potential therapeutic choice for CRC treatment.
Hesperidin/therapeutic use* ; Colorectal Neoplasms/metabolism* ; Glycolysis/drug effects* ; Animals ; Humans ; Apoptosis/drug effects* ; Mice, Nude ; Cell Movement/drug effects* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Glucose/metabolism* ; Cell Cycle/drug effects* ; Mice, Inbred BALB C ; Mice ; HCT116 Cells ; Lactic Acid

OBJECTIVE: To investigate the Wnt signaling pathway and miRNAs mechanism of extracts of Plastrum Testudinis (PT) in the treatment of osteoporosis (OP). METHODS: Thirty female Sprague Dawley rats were randomly divided into 5 groups by random number table method, including sham group, ovariectomized group (OVX), ovariectomized groups treated with high-, medium-, and low-dose PT (160, 80, 40 mg/kg per day, respectively), with 6 rats in each group. Except for the sham group, the other rats underwent bilateral ovariectomy to simulate OP and received PT by oral gavage for 10 consecutive weeks. After treatment, bone mineral density was measured by dual-energy X-ray absorptiometry; bone microstructure was analyzed by micro-computed tomography and hematoxylin and eosin staining; and the expressions of osteogenic differentiation-related factors were detected by immunochemistry, Western blot, and quantitative polymerase chain reaction. In addition, Dickkopf-1 (Dkk-1) was used to inhibit the Wnt signaling pathway in bone marrow mesenchymal stem cells (BMSCs) and miRNA overexpression was used to evaluate the effect of miR-214 on the osteogenic differentiation of BMSCs. Subsequently, PT extract was used to rescue the effects of Dkk-1 and miR-214, and its impacts on the osteogenic differentiation-related factors of BMSCs were evaluated. RESULTS: PT-M and PT-L significantly reduced the weight gain in OVX rats (P<0.05). PT also regulated the bone mass and bone microarchitecture of the femur in OVX rats, and increased the expressions of bone formation-related factors including alkaline phosphatase, bone morphogenetic protein type 2, collagen type I alpha 1, and runt-related transcription factor 2 when compared with the OVX group (P<0.05 or P<0.01). Meanwhile, different doses of PT significantly rescued the inhibition of Wnt signaling pathway-related factors in OVX rats, and increased the mRNA or protein expressions of Wnt3a, β-catenin, glycogen synthase kinase-3β, and low-density lipoprotein receptor-related protein 5 (P<0.05 or P<0.01). PT stimulated the osteogenic differentiation of BMSCs inhibited by Dkk-1 and activated the Wnt signaling pathway. In addition, the expression of miR-214 was decreased in OVX rats (P<0.01), and it was negatively correlated with the osteogenic differentiation of BMSCs (P<0.01). MiR-214 mimic inhibited Wnt signaling pathway in BMSCs (P<0.05 or P<0.01). Conversely, PT effectively counteracted the effect of miR-214 mimic, thereby activating the Wnt signaling pathway and stimulating osteogenic differentiation in BMSCs (P<0.05 or P<0.01). CONCLUSION PT stimulates bone formation in OVX rats through β-catenin-mediated Wnt signaling pathway, which may be related to inhibiting miR-214 in BMSCs.
Animals ; MicroRNAs/genetics* ; Female ; Rats, Sprague-Dawley ; Wnt Signaling Pathway/genetics* ; Osteogenesis/genetics* ; Mesenchymal Stem Cells/cytology* ; Cell Differentiation/drug effects* ; Bone Density/drug effects* ; Ovariectomy ; Osteoporosis/drug therapy* ; beta Catenin/metabolism* ; Rats ; Intercellular Signaling Peptides and Proteins/metabolism* ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVE: To elucidate the modulation mechanism of Suanzaoren Decoction (SZRD) on basolateral amygdala (BLA) neuronal activity to alleviate chronic restraint stress (CRS)-related behavioral deficits. METHODS: The male C57BL/6J mice were assigned to 4 groups using the complete randomization method, including control (CON, n=19), CRS (n=19), SZRD (n=21), and fluoxetine (Flu, n=22) groups. Mice were restrained for 6 h per day, over a 21-d period to establish CRS models. The CON group remained in their cages without food or water during the 6-h matching period. SZRD and Flu groups received intragastric administration of SZRD (4.68 g/kg) and Flu (20 mg/kg) daily, respectively, 30 min before restraint for 21 consecutive days. The therapeutic effects of SZRD were evaluated using behavioral tests including the tail suspension test, elevated plus maze test, and forced swimming test. The cellular Fletcher B. Judson murine osteosarcoma proto-oncogene (c-Fos) expression in the BLA was measured using immunofluorescence, while action potential (AP) firing and synaptic transmission in BLA pyramidal neurons were evaluated using whole-cell patch-clamp recordings. RESULTS: SZRD administration significantly increased time spent in the open arms and open-arm entries while reducing immobility time (P<0.05 or P<0.01). It downregulated CRS-induced c-Fos expression and AP firing of pyramidal neurons in the BLA (P<0.01). Additionally, SZRD selectively attenuated excitatory (P<0.01), but not inhibitory, synaptic transmission onto BLA pyramidal neurons. CONCLUSION SZRD alleviated CRS-induced anxiety- and depression-like behaviors in mice by modulating the excitability and synaptic transmission of BLA pyramidal neurons.
Animals ; Drugs, Chinese Herbal/therapeutic use* ; Depression/complications* ; Pyramidal Cells/pathology* ; Male ; Mice, Inbred C57BL ; Basolateral Nuclear Complex/pathology* ; Restraint, Physical ; Anxiety/complications* ; Behavior, Animal/drug effects* ; Stress, Psychological/physiopathology* ; Mice ; Proto-Oncogene Proteins c-fos/metabolism* ; Action Potentials/drug effects* ; Synaptic Transmission/drug effects*

OBJECTIVE: To evaluate the protective effects of resveratrol against acute lung injury (ALI) and investigate the potential mechanisms underlying the reactive oxygen species (ROS)-triggered thioredoxin-interacting protein (TXNIP)/NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) pathway. METHODS: C57BL/6 mice and J774A.1 cells were selected as the research subjects. Thirty Mice were randomly divided into 5 groups of 6 in each group: control with 0.9% saline, 5 mg/kg lipopolysaccharide (LPS) 24 h, 25 mg/kg resveratrol + 5 mg/kg LPS, 100 mg/kg resveratrol + 5 mg/kg LPS, and 4 mg/kg NLRP3 inhibitor CY-09 + 5 mg/kg LPS. For cell stimulation, cells were pretreated with 5 and 20 µmol/L resveratrol for 2 h, and stimulated with or without 1 µg/mL LPS and 3 mmol/L ATP for 2 h. The antioxidant N-acetyl-L-cysteine (NAC, 2 µmol/L) was used as the positive control group. Hematoxylin and eosin staining was used to evaluate the degree of lung LPS-induced tissue damage, and enzyme-linked immunosorbent assay was used to evaluate the contents of interleukin-1 β (IL-1 β) and IL-18 in the serum and cell supernatant. ROS and malondialdehyde (MDA) levels in the lung tissue were detected using the corresponding kits. Western blotting was used to detect the expressions of TXNIP, high-mobility group box 1 (HMGB1), NLRP3, as well as cysteine-aspartic acid protease 1 (caspase-1) and gasdermin D (GSDMD) along with their cleaved forms in lung tissue. Additionally, reverse transcription quantitative polymerase chain reaction was performed to analyze the expression of related inflammatory cytokines. ROS content was detected using flow cytometry and confocal laser microscopy. Mitochondrial morphological changes were observed using transmission electron microscopy, and HMGB1 expression was detected using immunofluorescence. RESULTS: Resveratrol significantly alleviated LPS-induced lung damage with reduced inflammation, interstitial edema, and leukocyte infiltration (P<0.01). It also decreased serum levels of IL-1 β and IL-18 (P<0.05), while downregulating the expressions of NLRP3, IL-6, and other inflammatory markers at both the protein and mRNA levels (P<0.05). Notably, the higher dose (100 mg/kg) demonstrated a better effect than the lower dose (25 mg/kg). In macrophages, resveratrol reduced IL-1 β and IL-18 following LPS and ATP stimulation, suppressed HMGB1 translocation, and inhibited formation and activation of the NLRP3 inflammasome (P<0.05 or P<0.01). These anti-inflammatory effects were mediated through the suppression ROS accumulation (P<0.01) and mitochondrial dysfunction. Transmission electron microscopy revealed that resveratrol preserved mitochondrial structure, preventing the mitochondrial damage seen in LPS-treated groups (P<0.01). The expressions of cleaved caspase-1, cleaved GSDMD, and cytoplasmic HMGB1 were all reduced following resveratrol treatment (P<0.01). Moreover, resveratrol inhibited dissociation of TXNIP from thioredoxin, blocking subsequent activation of NLRP3 and downstream inflammatory cytokines (P<0.01). Similarly, the higher concentration of resveratrol (20 µ mol/L) exhibited superior efficacy in vitro. CONCLUSION Resveratrol can reduce the inflammatory response following ALI and inhibit the activation of NLRP3 inflammasome and the level of HMGB1 in the cytoplasm by inhibiting ROS overproduction.
Acute Lung Injury/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Animals ; Resveratrol/pharmacology* ; Reactive Oxygen Species/metabolism* ; Inflammation/complications* ; Mice, Inbred C57BL ; Carrier Proteins/metabolism* ; Signal Transduction/drug effects* ; Lipopolysaccharides ; Thioredoxins/metabolism* ; Mice ; Lung/drug effects* ; Male ; Cell Line ; Interleukin-1beta/metabolism* ; Cell Cycle Proteins ; Stilbenes/therapeutic use*

OBJECTIVES: Metastasis is the primary cause of death in osteosarcoma, and current clinical treatments remain limited. BRD4, a key epigenetic regulator, has shown therapeutic promise in various cancers through its inhibition. However, the mechanistic role of BRD4 in osteosarcoma remains poorly understood. This study aims to elucidate the molecular mechanisms by which BRD4 regulate osteosarcoma progression and to explore novel therapeutic strategies. METHODS: Immunofluorescence was used to assess BRD4 expression levels in a tissue microarray containing 80 osteosarcoma samples from different patients. The Gene Expression Omnibus (GEO) dataset (GSE42352, containing survival data from 88 osteosarcoma patients) was downloaded to perform Kaplan-Meier survival analysis based on BRD4 gene expression levels. In vivo, an orthotopic intramedullary osteosarcoma model was established using HOS cells in C57 mice, followed by treatment with varying doses of the BRD4 inhibitor (+)-JQ1. Micro-CT, 3D reconstruction of bone tissue, and HE staining were employed to evaluate pathological changes in bone and intestinal lymph nodes. In vitro, cell viability was measured using the methyl thiazolyl tetrazolium (MTT) assay, while colony formation and Transwell assays assessed proliferative and invasive capacities. Chromatin-bound BRD4 was analyzed via co-immunoprecipitation combined with mass spectrometry (Co-IP/MS), and O-GlcNAc glycosylation sites and glycan chains of BRD4 were identified using Co-IP with Nano-LC MS/MS. Real-time PCR and Western blotting were used to analyze the relative mRNA and protein expression levels of target genes, respectively. RESULTS: BRD4 was positively expressed in 61.25% (49/80) of osteosarcoma tissues. Patients with high BRD4 expression exhibited significantly shorter survival times (<i>Pi><0.05). In the orthotopic mouse model, intervention with (+)-JQ1, a potent and commonly used BETi, significantly inhibited tumor growth in vivo and reduced bone destruction (<i>Pi><0.05). (+)-JQ1 treatment significantly suppressed the proliferation (<i>Pi><0.001), invasion (<i>Pi><0.001), and migration (<i>Pi><0.05) of HOS cells. In osteosarcoma cells, BRD4 exhibited O-GlcNAc modifications at both N- and C- C-termini, particularly at Thr73, which is essential for protein stability. This modification also contributed to the activation of the EGFR tyrosine kinase inhibitor resistance pathway (KEGG Pathway: hsa01521). (+)-JQ1 treatment displaced BRD4 from enhancers and downregulated the transcription of pathway-related genes, such as <i>EGFRi> and <i>PDGFCi>, thereby suppressing the malignant behavior of osteosarcoma cells. CONCLUSIONS BRD4 promotes osteosarcoma progression via O-GlcNAc modification at Thr73 and plays a crucial role in tumor growth and metastasis.
Osteosarcoma/drug therapy* ; Humans ; Transcription Factors/metabolism* ; Animals ; Cell Cycle Proteins ; Mice ; Bone Neoplasms/drug therapy* ; Azepines/pharmacology* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Triazoles/pharmacology* ; Mice, Inbred C57BL ; Nuclear Proteins/metabolism* ; Gene Expression Regulation, Neoplastic ; Male ; Bromodomain Containing Proteins

OBJECTIVES: Neurodegenerative diseases are closely associated with myelin loss, and the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) are crucial to remyelination. However, the regulatory mechanisms involved remain incompletely understood. This study aims to investigate how astrocytes (ASTs) regulate the secretion of chitinase-3-like protein 1 (CHI3L1) via connexin 47 (Cx47)-mediated exosome signaling, and its subsequent effect on OPC proliferation. METHODS: Primary cells were isolated from postnatal day 1 Sprague-Dawley (P1SD) rats to establish 3 culture conditions: OPCs alone (Group O), OPCs in direct contact with ASTs (Group C), and OPCs cultured with AST-conditioned medium (Group A). Cellular morphology and proliferation were assessed using optical microscopy, 5-ethynyl-2'- deoxyuridine (EdU) incorporation, and flow cytometry. RNA sequencing (RNA-Seq) and bioinformatics analysis (BA) were conducted to identify differentially expressed genes (DEGs) among groups. Protein expression and cell cycle distribution were analyzed by Western blotting (WB) and flow cytometry. Exosomes were isolated and purified via differential centrifugation, characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), and CHI3L1 expression in exosomes was verified via WB. <i>Cx47i> was silenced using small interfering RNA (siRNA) to evaluate its effect on OPC proliferation and exosome secretion. Artificial exosomes were constructed by encapsulating CHI3L1 in single unilamellar vesicles (SUVs), whose structure and size were validated by NTA and TEM. Following <i>Cx47i> knockdown, artificial exosomes were added back, and OPC proliferation was assessed via flow cytometry and EdU assay. RESULTS: Direct co-cultured with ASTs (Group C) resulted in significantly enhanced OPC proliferation compared to the Group O and Group A (<i>Pi><0.05). RNA-Seq and WB analyses revealed that ASTs promote OPC proliferation and exosome secretion enriched in CHI3L1 through Cx47. <i>Cx47i> knockdown by siRNA led to significant decreases in OPC proliferation and exosome release (<i>Pi><0.05). The inhibitory effect of <i>Cx47i> silencing on OPC proliferation was partially reversed by supplementation with either isolated exosomes or exogenous CHI3L1. CONCLUSIONS This study reveals a novel mechanism by which ASTs regulate OPC proliferation: Through direct contact, ASTs enhance the secretion of CHI3L1-rich exosomes via Cx47, thereby converting intercellular contact signals into secretory signals that promote OPC proliferation. As a key exosomal molecule, CHI3L1 may play an important role in neural function and remyelination and warrants further investigation.
Animals ; Exosomes/metabolism* ; Cell Proliferation ; Rats, Sprague-Dawley ; Rats ; Connexins/genetics* ; Oligodendrocyte Precursor Cells/metabolism* ; Astrocytes/metabolism* ; Chitinase-3-Like Protein 1/metabolism* ; Cells, Cultured ; Cell Differentiation

OBJECTIVES: Acute lung injury (ALI) is an acute respiratory failure syndrome characterized by impaired gas exchange. Due to the lack of effective targeted drugs, it is associated with high mortality and poor prognosis. <i>Tripterygium wilfordiii> (TW) has demonstrated anti-inflammatory activity in the treatment of various diseases. This study aims to investigate the effects and underlying mechanisms of TW on myeloid-derived suppressor cells (MDSCs) in ALI, providing experimental evidence for TW as a potential adjuvant therapy for ALI. METHODS: Eighteen specific pathogen-free (SPF) C57BL/6 mice were randomly divided into normal control (NC; intranasal saline), lipopolysaccharide (LPS; 5 mg/kg intranasally to induce ALI), and LPS+TW (50 mg/kg TW by gavage on the first day of modeling, followed by 5 mg/kg LPS intranasally to induce ALI) groups (<i>ni>=6 each). Lung injury and edema were assessed by histopathological scoring and wet-to-dry weight ratio. Cytokine levels [interleukin (IL)-1β, IL-6, IL-18, tumor necrosis factor-α (TNF-α)] in lung tissue lavage fluid were measured by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to assess the proportions of MDSCs, polymorphonuclear MDSCs (PMN-MDSCs), and monocytic MDSCs (M-MDSCs) in bone marrow, spleen, peripheral blood, and lung tissue, as well as reactive oxygen species (ROS) levels in lung tissues. Messenger RNA (mRNA) expression levels of inducible nitric oxide synthase (<i>iNOSi>) and arginase-1 (<i>ARG-1i>) in lung tissues were determined by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). PMN-MDSCs sorted from the lungs of LPS-treated mice were co-cultured with splenic CD3⁺ T cells and divided into NC, triptolide (TPL)-L, and TPL-H groups, with bovine serum albumin, 25 nmol/L TPL, and 50 nmol/L TPL, respectively. Flow cytometry was used to detect the effect of PMN-MDSCs on T-cell proliferation, and RT-qPCR was used to measure <i>iNOSi> and <i>ARG-1i> mRNA expression. RESULTS: Compared with the NC group, the LPS group showed marked lung pathology with significantly increased histopathological scores and wet-to-dry ratios (both <i>Pi><0.001). TW treatment significantly alleviated lung injury and reduced both indices compared with the LPS group (both <i>Pi><0.05). Cytokine levels were significantly decreased in the LPS+TW group compared with the LPS group (all <i>Pi><0.001). The proportions of MDSCs in CD45⁺ cells from spleen, bone marrow, peripheral blood, and lung, as well as PMN-MDSCs from spleen, peripheral blood, and lung, were significantly reduced in the LPS+TW group compared with the LPS group (all <i>Pi><0.05), accompanied by reduced ROS levels in lung tissues (<i>Pi><0.001). <i>iNOSi> and <i>ARG-1i> mRNA expression in lung tissues was significantly lower in the LPS+TW group than in the LPS group (both <i>Pi><0.001). In vitro, compared with the TPL-L group, the TPL-H group showed significantly increased CD3⁺ T-cell proliferation (<i>Pi><0.001), and decreased <i>iNOSi> and <i>ARG-1i> mRNA expression (all <i>Pi><0.05). CONCLUSIONS TW alleviates the progression of LPS-induced ALI in mice, potentially by reducing the proportion of MDSCs in lung tissues and attenuating the immunosuppressive function of PMN-MDSCs.
Animals ; Acute Lung Injury/chemically induced* ; Myeloid-Derived Suppressor Cells/cytology* ; Tripterygium/chemistry* ; Mice, Inbred C57BL ; Mice ; Cell Differentiation/drug effects* ; Male ; Lipopolysaccharides ; Nitric Oxide Synthase Type II/genetics* ; Cytokines/metabolism* ; Reactive Oxygen Species/metabolism* ; Diterpenes/pharmacology* ; Epoxy Compounds ; Phenanthrenes

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 (<i>ni>=4) and an ovariectomized (OVX) group (<i>ni>=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 (<i>Runx2i>) and osteopontin (<i>OPNi>), 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 (<i>Pi><0.05), increased ALP activity, and upregulated the mRNA and protein expression of <i>Runx2i> and <i>OPNi> (<i>Pi><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 (<i>Pi><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*