PURPOSE: To investigate the protective effect of sub-hypothermic mechanical perfusion combined with membrane lung oxygenation on ischemic hypoxic injury of yorkshire brain tissue caused by traumatic blood loss. METHODS: This article performed a random controlled trial. Brain tissue of 7 yorkshire was selected and divided into the sub-low temperature anterograde machine perfusion group (n = 4) and the blank control group (n = 3) using the random number table method. A yorkshire model of brain tissue injury induced by traumatic blood loss was established. Firstly, the perfusion temperature and blood oxygen saturation were monitored in real-time during the perfusion process. The number of red blood cells, hemoglobin content, NA⁺, K⁺, and Ca²⁺ ions concentrations and pH of the perfusate were detected. Following perfusion, we specifically examined the parietal lobe to assess its water content. The prefrontal cortex and hippocampus were then dissected for histological evaluation, allowing us to investigate potential regional differences in tissue injury. The blank control group was sampled directly before perfusion. All statistical analyses and graphs were performed using GraphPad Prism 8.0 Student t-test. All tests were two-sided, and p value of less than 0.05 was considered to indicate statistical significance. RESULTS: The contents of red blood cells and hemoglobin during perfusion were maintained at normal levels but more red blood cells were destroyed 3 h after the perfusion. The blood oxygen saturation of the perfusion group was maintained at 95% - 98%. NA⁺ and K⁺ concentrations were normal most of the time during perfusion but increased significantly at about 4 h. The Ca²⁺ concentration remained within the normal range at each period. Glucose levels were slightly higher than the baseline level. The pH of the perfusion solution was slightly lower at the beginning of perfusion, and then gradually increased to the normal level. The water content of brain tissue in the sub-low and docile perfusion group was 78.95% ± 0.39%, which was significantly higher than that in the control group (75.27% ± 0.55%, t = 10.49, p < 0.001), and the difference was statistically significant. Compared with the blank control group, the structure and morphology of pyramidal neurons in the prefrontal cortex and CA1 region of the hippocampal gyrus were similar, and their integrity was better. The structural integrity of granulosa neurons was destroyed and cell edema increased in the perfusion group compared with the blank control group. Immunofluorescence staining for glail fibrillary acidic protein and Iba1, markers of glial cells, revealed well-preserved cell structures in the perfusion group. While there were indications of abnormal cellular activity, the analysis showed no significant difference in axon thickness or integrity compared to the 1-h blank control group. CONCLUSIONS Mild hypothermic machine perfusion can improve ischemia and hypoxia injury of yorkshire brain tissue caused by traumatic blood loss and delay the necrosis and apoptosis of yorkshire brain tissue by continuous oxygen supply, maintaining ion homeostasis and reducing tissue metabolism level.
Animals ; Perfusion/methods* ; Disease Models, Animal ; Brain Injuries/etiology* ; Swine ; Male ; Hypothermia, Induced/methods*

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

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent inflammation and joint damage, accompanied by the accumulation of plasma cells, which contributes to its pathogenesis. Understanding the genetic alterations occurring during plasma cell differentiation in RA can deepen our comprehension of its pathogenesis and guide the development of targeted therapeutic interventions. Here, our study elucidates the intricate molecular mechanisms underlying plasma cell differentiation by demonstrating that PRDX1 interacts with DOK3 and modulates its degradation by the autophagy-lysosome pathway. This interaction results in the inhibition of plasma cell differentiation, thereby alleviating the progression of collagen-induced arthritis. Additionally, our investigation identifies Salvianolic acid B (SAB) as a potent small molecular glue-like compound that enhances the interaction between PRDX1 and DOK3, consequently impeding the progression of collagen-induced arthritis by inhibiting plasma cell differentiation. Collectively, these findings underscore the therapeutic potential of developing chemical stabilizers for the PRDX1-DOK3 complex in suppressing plasma cell differentiation for RA treatment and establish a theoretical basis for targeting PRDX1-protein interactions as specific therapeutic targets in various diseases.

BACKGROUND:Establishing a nomogram prediction model for postoperative pulmonary infection in hip fractures and taking early intervention measures is crucial for improving patients'quality of life and reducing medical costs. OBJECTIVE:To construct a nomogram risk prediction model of postoperative pulmonary infection in elderly patients with hip fracture,and provide theoretical basis for feasible prevention and early intervention. METHODS:Case data of 305 elderly patients with hip fractures who underwent surgical treatment at Wuxi Traditional Chinese Medicine Hospital Affiliated to Nanjing University of Chinese Medicine between January and October 2020(training set)were retrospectively analyzed.Using univariate and multivariate logistic regression analysis and Hosmer-Lemeshow goodness of fit test,receiver operating characteristic curve was utilized to analyze the diagnostic predictive efficacy of independent risk factors and joint models for postoperative pulmonary infections.Tools glmnet,pROC,and rms in R Studio software were applied to construct a nomogram model for predicting the risk of postoperative pulmonary infection in elderly patients with hip fractures,and calibration curves were further drawn to verify the predictive ability of the nomogram model.Receiver operating characteristic curves,calibration curves,and decision curves were analyzed for 133 elderly patients with hip fractures(validation set)receiving surgery at the same hospital from November 2022 to March 2023 to further predict the predictive ability of the nomogram model. RESULTS AND CONCLUSION:(1)The postoperative pulmonary infection rate in elderly patients with hip fractures in this group was 9.18%(28/305).(2)Single factor and multivariate analysis,as well as forest plots,showed that preoperative hospitalization days,leukocyte count,hypersensitive C-reactive protein,and serum sodium levels were independent risk factors(P<0.05).The Hosmer-Lemeshow goodness of fit test showed good fit(χ2=4.57,P=0.803).Receiver operating characteristic curve analysis was conducted on the independent risk factors and their joint models mentioned above,and the differentiation of each independent risk factor and joint model was good,with statistical significance(P<0.05).(3)The graphical calibration method,C-index,and decision curve were used to validate the nomogram prediction model.The predicted calibration curve was located between the standard curve and the acceptable line,and the predicted risk of the nomogram model was consistent with the actual risk.(4)The validation set used receiver operating characteristic curve,graphic calibration method,and decision curve to validate the prediction model.The results showed good consistency with clinical practice,indicating that the model had a good fit.The nomogram risk prediction model constructed for postoperative pulmonary infection in elderly patients with hip fractures has good predictive performance.The use of the nomogram risk prediction model can screen high-risk populations and provide a theoretical basis for early intervention.

Objective To study the factors influencing the blood concentration of caspofungin(CPFG),construct a prediction model,and validate the predictive effect of the model,so as to provide reference for the individualised dosing of patients with fungal infections in haematology.Methods Seventy-five patients admitted to the Department of Haematology,General Hospital of Tianjin Medical University,who were treated with CPFG for antifungal therapy during the period of March 2021 to June 2022 were selected as the study subjects,and CPFG blood concentration monitoring was carried out to explore the influencing factors of CPFG blood concentration and to construct a prediction model accordingly.Hosmer-Lemeshow(H-L)was used to test the goodness-of-fit of the model,and another 30 patients were selected as the verification group,and the predictive effect of the model was verified by the receiver's operating characteristics(ROC)curve.Results The mean blood concentrations of the patients at 0.5,9 and 24 h were(12.54±4.38),(6.80±2.76),(4.13±2.16)μg·mL-1,and the mean AUC0-24h were(152.05±57.60)μg·mL-1·h.AUC0-24h was lower than the reference value(98 μg·mL-1·h)in two patients.The results of correlation analysis showed that gender showed a correlation with 0.5 h blood concentration(P＜0.05),and there was no correlation with the rest of the two time points blood concentration and AUC0-24h(P＞0.05).Body weight and albumin(Alb)concentration showed correlation with 0.5,9,24 h blood drug concentration and AUC0-24 h(P＜0.05),and the rest of the indicators showed no correlation with blood drug concentration and AUC0_24h at each time point(P＞0.05).The results of multifactorial analysis showed that the factors influencing the patients'0.5 h blood concentration were gender,Alb concentration and body weight,and the factors influencing the 9 and 24 h blood concentration and AUC0-24h were Alb concentration and body weight(P＜0.05).Correlation analysis showed that the daily dose was positively correlated with the plasma concentration of CPFG at 0.5,9 and 24 h and AUC0-24h(P＜0.05).The results of multivariate analysis showed that the daily dose was also one of the influencing factors of the plasma concentration of CPFG(P＜0.05).ROC curve shows that the model has good prediction ability.Conclusion Body weight and Alb are significantly associated with CPFG blood concentrations and area under the drug-time curve,which can be used as a basis for preventive risk avoidance.

Objective This study investigated the impact of occupational mercury(Hg)exposure on human gene transcription and expression,and its potential biological mechanisms. Methods Differentially expressed genes related to Hg exposure were identified and validated using gene expression microarray analysis and extended validation.Hg-exposed cell models and PTEN low-expression models were established in vitro using 293T cells.PTEN gene expression was assessed using qRT-PCR,and Western blotting was used to measure PTEN,AKT,and PI3K protein levels.IL-6 expression was determined by ELISA. Results Combined findings from gene expression microarray analysis,bioinformatics,and population expansion validation indicated significant downregulation of the PTEN gene in the high-concentration Hg exposure group.In the Hg-exposed cell model(25 and 10 μmol/L),a significant decrease in PTEN expression was observed,accompanied by a significant increase in PI3K,AKT,and IL-6 expression.Similarly,a low-expression cell model demonstrated that PTEN gene knockdown led to a significant decrease in PTEN protein expression and a substantial increase in PI3K,AKT,and IL-6 levels. Conclusion This is the first study to report that Hg exposure downregulates the PTEN gene,activates the PI3K/AKT regulatory pathway,and increases the expression of inflammatory factors,ultimately resulting in kidney inflammation.

Objective Mitochondrial reactive oxygen species(mtROS)could cause damage to pancreatic β-cells,rendering them susceptible to oxidative damage.Hence,investigating the potential of the mitochondria-targeted antioxidant(Mito-TEMPO)to protect pancreatic β-cells from ferroptosis by mitigating lipid peroxidation becomes crucial. Methods MIN6 cells were cultured in vitro with 100 μmol/L sodium palmitate(SP)to simulate diabetes.FerroOrange was utilized for the detection of Fe2+fluorescence staining,BODIPY581/591C11 for lipid reactive oxygen species,and MitoSox-Red for mtROS.Alterations in mitophagy levels were assessed through the co-localization of lysosomal and mitochondrial fluorescence.Western blotting was employed to quantify protein levels of Acsl4,GPX4,FSP1,FE,PINK1,Parkin,TOMM20,P62,and LC3.Subsequently,interventions were implemented using Mito-TEMPO and Carbonyl cyanide 3-chlorophenylhydrazone(CCCP)to observe changes in ferroptosis and mitophagy within MIN6 cells. Results We found that SP induced a dose-dependent increase in Fe2+and lipid ROS in MIN6 cells while decreasing the expression levels of GPX4 and FSP1 proteins.Through bioinformatics analysis,it has been uncovered that mitophagy assumes a crucial role within the ferroptosis pathway associated with diabetes.Additionally,SP decreased the expression of mitophagy-related proteins PINK1 and Parkin,leading to mtROS overproduction.Conversely,Mito-TEMPO effectively eliminated mtROS while activating the mitophagy pathways involving PINK1 and Parkin,thereby reducing the occurrence of ferroptosis in MIN6 cells.CCCP also demonstrated efficacy in reducing ferroptosis in MIN6 cells. Conclusion In summary,Mito-TEMPO proved effective in attenuating mtROS production and initiating mitophagy pathways mediated by PINK1 and Parkin in MIN6 cells.Consequently,this decreased iron overload and lipid peroxidation,ultimately safeguarding the cells from ferroptosis.