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*

OBJECTIVE: To prepare clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with high expression of hematopoietic supporting factors and evaluate their stem cell characteristics. METHODS: Fetal umbilical cord tissues were collected from healthy postpartum women during full-term cesarean section. Wharton's jelly was mechanically separated and hUC-MSCs were obtained by explant culture method and enzyme digestion method in an animal serum-free culture system with addition of human platelet lysate. The phenotypic characteristics of hUC-MSCs obtained by two methods were detected by flow cytometry. The differences in proliferation ability between the two groups of hUC-MSCs were identified through CCK-8 assay and colony forming unit-fibroblast (CFU-F) assay. The differences in multilineage differentiation potential between the two groups of hUC-MSCs were identified through induction of adipogenic, osteogenic, and chondrogenic differentiation. The mRNA expression levels of hematopoietic supporting factors such as SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in the two groups of hUC-MSCs were identified by real-time fluorescence quantiative PCR(RT-qPCR). RESULTS: The results of flow cytometry showed that hUC-MSCs obtained by the two methods both expressed high levels of CD73, CD90 and CD105, while lowly expressed CD31, CD45 and HLA-DR. The results of CCK-8 and CFU-F assay showed that the proliferation ability of hUC-MSCs obtained by explant culture method was better than those obtained by enzyme digestion method. The results of the triple lineage differentiation experiment showed that there was no significant difference in multilineage differentiation potential between the two grous of hUC-MSCs. The results of RT-qPCR showed that the mRNA expression levels of hematopoietic supporting factors SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in hUC-MSCs obtained by explant cultrue method were higher than those obtained by enzyme digestion method. CONCLUSION Clinical-grade hUC-MSCs with high expression levels of hematopoietic supporting factors were successfully cultured in an animal serum-free culture system.
Humans ; Mesenchymal Stem Cells/metabolism* ; Umbilical Cord/cytology* ; Cell Differentiation ; Female ; Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12/metabolism* ; Angiopoietin-1/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Stem Cell Factor/metabolism* ; Flow Cytometry ; Pregnancy

Glutamine provides carbon and nitrogen to support the proliferation of cancer cells. However, the precise reason why cancer cells are particularly dependent on glutamine remains unclear. In this study, we report that glutamine modulates the tumor suppressor F-box and WD repeat domain-containing 7 (FBW7) to promote cancer cell proliferation and survival. Specifically, lysine 604 (K604) in the sixth of the 7 substrate-recruiting WD repeats of FBW7 undergoes glutaminylation (Gln-K604) by glutaminyl tRNA synthetase. Gln-K604 inhibits SCFFBW7-mediated degradation of c-Myc and Mcl-1, enhances glutamine utilization, and stimulates nucleotide and DNA biosynthesis through the activation of c-Myc. Additionally, Gln-K604 promotes resistance to apoptosis by activating Mcl-1. In contrast, SIRT1 deglutaminylates Gln-K604, thereby reversing its effects. Cancer cells lacking Gln-K604 exhibit overexpression of c-Myc and Mcl-1 and display resistance to chemotherapy-induced apoptosis. Silencing both c-MYC and MCL-1 in these cells sensitizes them to chemotherapy. These findings indicate that the glutamine-mediated signal via Gln-K604 is a key driver of cancer progression and suggest potential strategies for targeted cancer therapies based on varying Gln-K604 status.
Glutamine/metabolism* ; Myeloid Cell Leukemia Sequence 1 Protein/genetics* ; Humans ; Proto-Oncogene Proteins c-myc/genetics* ; Cell Proliferation ; Signal Transduction ; Neoplasms/pathology* ; F-Box-WD Repeat-Containing Protein 7/genetics* ; Cell Survival ; Cell Line, Tumor ; Apoptosis

ObjectiveTo explore the therapeutic effect and mechanism of the Danhe granules on hypercholesterolemia rats by observing the changes in the efficacy indicators and the levels of proteins related to the cholesterol metabolism pathway in the rats under the intervention of Danhe granules. MethodSD rats were randomly assigned to either the blank group or the model group based on their body weight. The blank group had normal chow diets， while the model group was fed high-fat diets for seven weeks. One week after the establishment of the model， the content of the serum total cholesterol （TC） in the model rats was detected. According to the TC value， the model group was further randomly divided into a control group， pravastatin sodium tablet group（4.02 mg·kg^-1）， Xuezhikang capsule group（0.12 g·kg^-1）， high-dose， middle-dose， and low-dose groups of Danhe granules（4.536， 2.268， 1.134 g·kg^-1）. After grouping the model groups， each treatment group received continuous oral gavage for six weeks， with weekly measurements of body weight and food intake （the difference between feed intake and feed surplus）. Six weeks later， the levels of serum TC， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， and high-density lipoprotein cholesterol （HDL-C） were measured. The liver pathology and lipid droplet distribution were evaluated by hematoxylin-eosin （HE） staining and oil red O staining， with scoring and calculation conducted. Rat liver tissue was collected， and western blot and immunohistochemistry （IHC） were used to detect the expression levels of cholesterol metabolism-related proteins namely phosphorylated adenosine 5'-monophosphate （AMP）-activated protein kinase （p-AMPK）， AMPK， 3-hydroxy-3-methyl glutaryl coenzyme A reductase （HMGCR）， low-density lipoprotein receptor （LDLR）， cholesterol 7α-hydroxylase （CYP7A1）， Acyl-coenzyme A： cholesterol acyltransferase 2 （ACAT2）， and apolipoprotein B （ApoB） in hypercholesterolemia rats. ResultCompared with the blank group， the model group showed a significantly higher level of serum TC （P<0.01）. The TG level had no significant change， and the HDL-C level was significantly decreased （P<0.05）. The liver index， steatosis score， total score of pathological state， and the positive area ratio of oil red O staining were significantly increased （P<0.01）， and the protein expression levels of p-AMPK， p-AMPK/AMPK， LDLR， and CYP7A1 were significantly decreased （P<0.05， P<0.01）， while the protein expression levels of AMPK， HMGCR， and ACAT2 were significantly increased （P<0.05， P<0.01）. Compared with the model group， the TC level in each dose group of Danhe granules was significantly decreased （P<0.05）， and the positive area ratio of oil red O staining in the pravastatin sodium tablet group and medium-dose group of Danhe granules was significantly decreased （P<0.05）. In each administration group， the protein expression levels of p-AMPK and p-AMPK/AMPK were significantly increased （P<0.05， P<0.01）， and the levels of HMGCR and ACAT2 were significantly decreased （P<0.01）. The ApoB level showed a downward trend. The CYP7A1 level in the pravastatin sodium tablet group and each dose group of Danhe granules was significantly increased （P<0.05， P<0.01）， and the LDLR level in the pravastatin sodium tablet group， Xuezhikang capsule group， and high-dose and medium-dose groups of Danhe granules was significantly increased （P<0.05， P<0.01）. ConclusionDanhe granules can reduce serum TC levels and improve hepatic steatosis. It may activate AMPK， down-regulate the expression of HMGCR， and inhibit cholesterol synthesis. It can also up-regulate the expression of LDLR and CYP7A1， promote cholesterol uptake and excretion， down-regulate the expression of ACAT2 and ApoB， reduce cholesterol absorption and assembly of LDL and other lipoproteins， and thus play a role in the treatment of hypercholesterolemia.

Objective:To establish a mesenchymal stem cell(MSC)-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease(aGVHD).Methods:Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors,and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients.The recipient mouse received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1× 107/mouse)in 6-8 hours post irradiation to establish a bone marrow transplantation(BMT)mouse model(n=20).In addition,the recipient mice received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1 × 107/mouse)and spleen lymphocytes(2 × 106/mouse)in 6-8 hours post irradiation to establish a mouse aGVHD model(n=20).On the day 7 after modeling,the recipient mice were anesthetized and the blood was harvested post eyeball enucleation.The serum was collected by centrifugation.Mouse MSCs were isolated and cultured with the addition of 2％,5％,and 10％recipient serum from BMT group or aGVHD group respectively.The colony-forming unit-fibroblast(CFU-F)experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC.The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining.In addition,the expression of self-renewal-related genes including Oct-4,Sox-2,and Nanog in MSC was detected by real-time fluorescence quantitative PCR(RT-qPCR).Results:We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD.CFU-F assay showed that,on day 7 after the culture,compared with the BMT group,MSC colony formation ability of aGVHD serum concentrations groups of 2％and 5％was significantly reduced(P＜0.05);after the culture,at day 14,compared with the BMT group,MSC colony formation ability in different aGVHD serum concentration was significantly reduced(P＜0.05).The immunofluorescence staining showed that,compared with the BMT group,the proportion of MSC surface molecules CD29+and CD 105+cells was significantly dereased in the aGVHD serum concentration group(P＜0.05),the most significant difference was at a serum concentration of 10％(P＜0.001,P＜0.01).The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4,Sox-2,and Nanog was decreased,the most significant difference was observed at an aGVHD serum concentration of 10％(P＜0.01,P＜0.001,P＜0.001).Conclusion:By co-culturing different concentrations of mouse aGVHD serum and mouse MSC,we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability,which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

Yellowish sweating disease is one of the fluid-retention diseases recorded in Jin Gui Yao Lve(Synopsis of the Golden Cabinet).The symptoms of yellowish sweating disease are complex,involving multiple visceral lesions,which are caused by interior heat and exterior deficiency,together with the concurrent invasion of pathogens of wind and water.Huangqi Shaoyao Guizhi Kujiu Decoction(mainly composed of Astragali Radix,Paeoniae Radix Alba,Cinnamomi Ramulus and vinegar)and Guizhi Plus Huangqi Decoction(mainly composed of Cinnamomi Ramulus and Astragali Radix)are the classical formula for the treatment of yellowish sweating disease.Both of the formulas have the actions of warming defensive qi and dredging yang,removing fluid retention and resolving dampness.Usually suffering heat in the spleen and stomach,together with carelessness in daily living and wind-water pathogens attacking the exterior,contributes to the key pathogenesis of diabetes mellitus.The clinical manifestations,etiology,occurrence and progression,and prognosis of yellowish sweating disease are similar to those of diabetic complications.Therefore,the treatment of diabetes complications such as diabetic kidney disease,diabetic cardiomyopathy,diabetic peripheral neuropathy,diabetes mellitus complicated with liver dysfunction,diabetic foot,and diabetic retinopathy can follow the therapeutic principles of yellowish sweating disease,and can be achieved by the therapies of clearing heat and purging fire,dispelling cold and removing dampness,and nourishing nutritive yin and harmonizing defensive qi with the appropriate formulas.The exploration of the treatment of diabetes mellitus and its complications from the pathogenesis and symptom characteristics of yellowish sweating disease will expand the thoughts for treating diabetic complications with traditional Chinese medicine.

OBJECTIVE: To investigate the mechanism of induction of ferroptosis by brazilin in breast cancer cells. METHODS: Breast cancer 4T1 cells were divided into 6 groups: control, brazilin 1/2 half maximal inhibitory concentration (IC₅₀), IC₅₀, 2×IC₅₀, erastin (10 µg/mL) and capecitabine (10 µg/mL) groups. The effect of brazilin on the proliferation of 4T1 cells was detected by cell counting kit-8 assay, and the treatment dose of brazilin was screened. The effect of brazilin on the mitochondrial morphology of 4T1 cells, and the mitochondrial damage was evaluated under electron microscopy. The levels of Fe²⁺, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase 4 (GPX4) were estimated using various detection kits. The invasion and migration abilities of 4T1 cells were detected by scratch assay and transwell assay. The expressions levels of tumor protein p53, solute carrier family 7 member 11 (SLC7A11), GPX4 and acyl-CoA synthetase long-chain family member 4 (ACSL4) proteins were quantified by Western blot assay. RESULTS: Compared to the control group, the 10 (1/2 IC₅₀), 20 (IC₅₀) and 40 (2×IC₅₀) µg/mL brazilin, erastin, and capecitabine groups showed a significant decrease in the cell survival rate, invasion and migration abilities, GSH, SLC7A11 and GPX4 protein expression levels, and mitochondrial volume and ridge (P<0.05), and a significant increase in the mitochondria membrane density, Fe²⁺, ROS and MDA levels, and p53 and ACSL4 protein expression levels (P<0.05). CONCLUSIONS Brazilin actuated ferroptosis in breast cancer cells, and the underlying mechanism is mainly associated with the p53/SLC7A11/GPX4 signaling pathway.
Ferroptosis/drug effects* ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism* ; Breast Neoplasms/drug therapy* ; Signal Transduction/drug effects* ; Female ; Cell Line, Tumor ; Tumor Suppressor Protein p53/metabolism* ; Amino Acid Transport System y+/metabolism* ; Humans ; Reactive Oxygen Species/metabolism* ; Animals ; Mice ; Cell Proliferation/drug effects* ; Mitochondria/metabolism* ; Coenzyme A Ligases/metabolism* ; Cell Movement/drug effects* ; Benzopyrans

Objective To apply a COMSOL-based finite element analysis method to investigating the electric field effects produced by the human lower limb musculoskeletal system under the synergistic effects of stress field and electromagnetic field.Methods Firstly,a 3D human body model was constructed by Maxon Cinema 4D R21 software,and then imported into COMSOL 6.1 software in STL format.Secondly,an electromagnetic field intervention and stress loading model for the left lower limb of the human body was designed and constructed,in which 15 Hz quasi-pulse group current signals were used for electromagnetic field excitation and the stress field was realized by applying a vibration load with an average compressive force of about 90 N/cm2 to the left foot of the human body.Finally,the electromagnetic properties of human tissue were simulated by numerical simulation,and then the effects of stress field or elecromagnetic field or combined stress field and electromagnetic field on human bioelectric field were compared.Results Simulation results showed that the electric field intensity peaked at the leg joints under both electromagnetic and stress fields acting alone or synergistically,the bioelectric field intensity generated by the human body was related to the distance from the exogenous excitation loading location,and the electric field generated under synergistic action was equivalent to the linear superposition of the bioelectric field in the tissue induced by the electromagnetic field and the stress field acting alone.Conclusion Data supplement is provided for predicting bioelectric field changes within the musculoskeletal tissue,and theoretical foundation is laid for the development and application of multi-physics field synergistic intervention therapy for treating the disorders of the lower limb musculos-keletal system.[Chinese Medical Equipment Journal,2024,45(9):21-26]

Triterpenoids widely exist in nature,displaying a variety of pharmacological activities.Determining triterpenoids in different matrices,especially in biological samples holds great significance.High-performance liquid chromatography(HPLC)has become the predominant method for triterpenoids analysis due to its exceptional analytical performance.However,due to the structural similarities among botanical samples,achieving effective separation of each triterpenoid proves challenging,necessitating significant improvements in analytical methods.Additionally,triterpenoids are characterized by a lack of ultraviolet(UV)absorption groups and chromophores,along with low ionization efficiency in mass spectrometry.Consequently,routine HPLC analysis suffers from poor sensitivity.Chemical derivatization emerges as an indispensable technique in HPLC analysis to enhance its performance.Considering the structural characteristics of triterpenoids,various derivatization reagents such as acid chlorides,rho-damines,isocyanates,sulfonic esters,and amines have been employed for the derivatization analysis of triterpenoids.This review comprehensively summarized the research progress made in derivatization strategies for HPLC detection of triterpenoids.Moreover,the limitations and challenges encountered in previous studies are discussed,and future research directions are proposed to develop more effective derivatization methods.