Background: Advances in intensive care have markedly improved survival after severe brain injury, leading to a growing population of patients with prolonged disorders of consciousness (pDOC). Current management of pDOC remains largely supportive, and evidence-based neuromodulatory interventions are limited; moreover, existing guidelines provide insufficiently explicit recommendations regarding mechanisms of action and objective biomarkers of treatment response. Transcutaneous auricular vagus nerve stimulation (taVNS) has emerged as a potential noninvasive intervention; however, its modulatory effects on brain function in pDOC are not yet well characterized, and the paucity of integrative mechanistic evidence has constrained its translation into routine clinical practice. Objectives: Within a multimodal assessment framework, this study aims to systematically elucidate the neurobiological mechanisms by which taVNS modulates brain function and autonomic activity in patients with pDOC, and to evaluate its clinical potential to enhance levels of consciousness. Methods: In this randomized controlled trial, 60 patients with vegetative state/minimally conscious state will be enrolled and randomly allocated to a taVNS group, a transcutaneous nonauricular vagus nerve stimulation group (sham), or a control group (n = 20 per group) for a 4-week intervention. The primary outcome will be changes in the Coma Recovery Scale-Revised scores from baseline to weeks 1, 2, and 4 of treatment. Secondary outcomes will include functional brain activity assessed by electroencephalography and functional near-infrared spectroscopy, as well as autonomic modulation indexed by heart rate variability. Functional prognosis will be evaluated using the Glasgow Outcome Scale-Extended at the end of treatment and at a 6-month follow-up. Safety will be assessed by continuous monitoring and documentation of adverse events throughout the study period. Results and discussion: By integrating electroencephalography–functional near-infrared spectroscopy with heart rate variability, this study will characterize the effects of taVNS on functional brain networks and consciousness recovery in pDOC across complementary behavioral, electrophysiological, hemodynamic, and autonomic domains, while interrogating potential sources of clinical and neurobiological heterogeneity. The findings are expected to provide a mechanistic and evidence-based foundation for the mechanism-driven clinical implementation of taVNS and the optimization of stimulation protocols in pDOC. Clinical trial registration: International Traditional Medicine Clinical Trial Registry, ITMCTR20250021041, https://itmctr.ccebtcm.org.cn.

Objective:To construct a hierarchical training course system for neonatal nurses based on core competencies, to provide a reference for meeting the training needs of neonatal nurses under the new situation.Methods:Through literature review, questionnaire survey on training needs, and focus group interviews, a preliminary hierarchical training curriculum system for neonatal nurses was developed. Two rounds of Delphi correspondence were conducted with 19 domestic experts to finalize the system.Results:The effective questionnaire recovery rates of the two rounds of expert consultation were 95.00% and 100.00%, and the expert authority coefficient was 0.916, the Kendall harmony coefficient of the first round of expert opinions was 0.351 ( P<0.001), and the Kendall harmony coefficient of the second round of expert opinions was 0.463 ( P<0.001). The hierarchical training course structure and course training content are formed, including N0: 3 first-level items, 9 second-level items, 80 third-level items, N1: 3 first-level items, 9 second-level items, 91 third-level items, N2: 3 first-level items, 9 second-level items, 86 third-level items, N3: 3 first-level items, 10 second-level items, 81 third-level items, N4: 3 first-level items, 10 second-level items, 76 third-level items. Conclusions:The hierarchical training course system for neonatal nurses based on the core competence of nurses is scientific and practical, which can provide a reference for the hierarchical training of neonatal nurses.

BACKGROUND:Programmed cell death protein 1 belongs to the immunoglobulin gene superfamily and can regulate the differentiation of osteoblasts and affect bone homeostasis.However,there are few studies on the regulatory role and mechanism of programmed cell death protein 1 in diabetic osteoporosis.OBJECTIVE:To investigate the regulatory role and mechanism of programmed cell death protein 1 on osteogenic differentiation of rat bone marrow mesenchymal stem cells under high-glucose environment.METHODS:(1)Animal experiment:A total of 12 Sprageu-Dawley rats were randomized into a control group(n=6)and a model group(n=6).The control group was fed routinely,whereas the model group was injected intraperitoneally with streptozotocin to establish a model of type 1 diabetes mellitus,and the high-fat feed was fed for 8 weeks to establish a model of type 1 diabetic osteoporosis.After 8 weeks of feeding,the femurs of rats in the two groups were taken and subjected to hematoxylin-eosin staining and micro-CT assay.The mRNA expression of programmed cell death protein 1 and programmed death ligand 1 was detected.(2)Cell experiment:Passage 3 rat bone marrow mesenchymal stem cells were randomly divided into four groups:normal control group,high-glucose model group cultured in low glucose medium,programmed cell death protein 1-silenced group transfected with programmed cell death protein 1 siRNA,and programmed cell death protein 1-silenced null group transfected with siRNA-NC.After 48 hours of transfection,the normal control group was cultured in a new low-glucose medium,and the other three groups were cultured in a high-glucose medium for another 48 hours of culture followed by osteogenic induction.After 21 days of osteogenic induction,alizarin red staining,and qRT-PCR(programmed cell death protein 1 and RUNX2 mRNA expression)and western blot(β-catenin,GSK-3β,p-GSK-3β and Axin2 protein expression)were performed.RESULTS AND CONCLUSION:In the animal experiment,hematoxylin-eosin staining and micro-CT assay showed successful modeling of type 1 diabetic osteoporosis in the model group.qRT-PCR assay showed that the mRNA expression of programmed cell death protein 1 and programmed cell death ligand 1 was higher in the model group than the control group(P<0.05).In the cell experiment,the results of alizarin red staining showed that the ability of mineralized nodule formation was lower in the high-glucose model group and the programmed cell death protein 1-silenced null group than in the control group and the programmed cell death protein 1-silenced group.Compared with the normal control group,the programmed cell death protein 1 mRNA expression and GSK3β and Axin2 protein expression were elevated in the high-glucose model group and the programmed cell death protein 1-silenced null group(P<0.05),and the RUNX2 mRNA expression and p-GSK3β and β-catenin protein expression were decreased(P<0.05).Compared with the high-glucose model group and the programmed cell death protein 1-silenced null group,programmed cell death protein 1 mRNA expression and GSK3β and Axin2 protein expression were decreased in the programmed cell death protein 1-silenced group(P<0.05),and RUNX2 mRNA expression and p-GSK3β and β-catenin protein expression were elevated(P<0.05).To conclude,programmed cell death protein 1 silencing can activate the Wnt/β-catenin and improve the osteogenic differentiation of rat bone marrow mesenchymal stem cells under high-glucose conditions.

Objective:To construct a hierarchical training course system for neonatal nurses based on core competencies, to provide a reference for meeting the training needs of neonatal nurses under the new situation.Methods:Through literature review, questionnaire survey on training needs, and focus group interviews, a preliminary hierarchical training curriculum system for neonatal nurses was developed. Two rounds of Delphi correspondence were conducted with 19 domestic experts to finalize the system.Results:The effective questionnaire recovery rates of the two rounds of expert consultation were 95.00% and 100.00%, and the expert authority coefficient was 0.916, the Kendall harmony coefficient of the first round of expert opinions was 0.351 ( P<0.001), and the Kendall harmony coefficient of the second round of expert opinions was 0.463 ( P<0.001). The hierarchical training course structure and course training content are formed, including N0: 3 first-level items, 9 second-level items, 80 third-level items, N1: 3 first-level items, 9 second-level items, 91 third-level items, N2: 3 first-level items, 9 second-level items, 86 third-level items, N3: 3 first-level items, 10 second-level items, 81 third-level items, N4: 3 first-level items, 10 second-level items, 76 third-level items. Conclusions:The hierarchical training course system for neonatal nurses based on the core competence of nurses is scientific and practical, which can provide a reference for the hierarchical training of neonatal nurses.

BACKGROUND:Programmed cell death protein 1 belongs to the immunoglobulin gene superfamily and can regulate the differentiation of osteoblasts and affect bone homeostasis.However,there are few studies on the regulatory role and mechanism of programmed cell death protein 1 in diabetic osteoporosis.OBJECTIVE:To investigate the regulatory role and mechanism of programmed cell death protein 1 on osteogenic differentiation of rat bone marrow mesenchymal stem cells under high-glucose environment.METHODS:(1)Animal experiment:A total of 12 Sprageu-Dawley rats were randomized into a control group(n=6)and a model group(n=6).The control group was fed routinely,whereas the model group was injected intraperitoneally with streptozotocin to establish a model of type 1 diabetes mellitus,and the high-fat feed was fed for 8 weeks to establish a model of type 1 diabetic osteoporosis.After 8 weeks of feeding,the femurs of rats in the two groups were taken and subjected to hematoxylin-eosin staining and micro-CT assay.The mRNA expression of programmed cell death protein 1 and programmed death ligand 1 was detected.(2)Cell experiment:Passage 3 rat bone marrow mesenchymal stem cells were randomly divided into four groups:normal control group,high-glucose model group cultured in low glucose medium,programmed cell death protein 1-silenced group transfected with programmed cell death protein 1 siRNA,and programmed cell death protein 1-silenced null group transfected with siRNA-NC.After 48 hours of transfection,the normal control group was cultured in a new low-glucose medium,and the other three groups were cultured in a high-glucose medium for another 48 hours of culture followed by osteogenic induction.After 21 days of osteogenic induction,alizarin red staining,and qRT-PCR(programmed cell death protein 1 and RUNX2 mRNA expression)and western blot(β-catenin,GSK-3β,p-GSK-3β and Axin2 protein expression)were performed.RESULTS AND CONCLUSION:In the animal experiment,hematoxylin-eosin staining and micro-CT assay showed successful modeling of type 1 diabetic osteoporosis in the model group.qRT-PCR assay showed that the mRNA expression of programmed cell death protein 1 and programmed cell death ligand 1 was higher in the model group than the control group(P<0.05).In the cell experiment,the results of alizarin red staining showed that the ability of mineralized nodule formation was lower in the high-glucose model group and the programmed cell death protein 1-silenced null group than in the control group and the programmed cell death protein 1-silenced group.Compared with the normal control group,the programmed cell death protein 1 mRNA expression and GSK3β and Axin2 protein expression were elevated in the high-glucose model group and the programmed cell death protein 1-silenced null group(P<0.05),and the RUNX2 mRNA expression and p-GSK3β and β-catenin protein expression were decreased(P<0.05).Compared with the high-glucose model group and the programmed cell death protein 1-silenced null group,programmed cell death protein 1 mRNA expression and GSK3β and Axin2 protein expression were decreased in the programmed cell death protein 1-silenced group(P<0.05),and RUNX2 mRNA expression and p-GSK3β and β-catenin protein expression were elevated(P<0.05).To conclude,programmed cell death protein 1 silencing can activate the Wnt/β-catenin and improve the osteogenic differentiation of rat bone marrow mesenchymal stem cells under high-glucose conditions.

Objective To identify the potential pathological mechanisms of tuberculous spondylitis(TS).Methods Spinal specimens were collected from 13 TS patients and 13 controls who received treatment at a hospital from March 2021 to March 2023.Specimens were randomly selected from 3 TS patients and 3 controls to perform high-throughput lncRNAs and mRNAs sequencing with Illumina NovaSeq 6000.Differentially expressed lncRNAs(DELncRs)and mRNAs(DEmRs)in TS specimens were identified and analyzed through differential expression,and enrichment analysis was performed.The top 20 DEmRs with high connectivity were identified through protein-protein interaction(PPI)network.Regulatory network of DElncRs and DEmRs was built.Finally,gene expression of the remaining specimens was analyzed using qRT-PCR detection.Results A total of 1 243 DEmRs and 262 DElncRs were identified.Enrichment analysis revealed that muscle contraction,muscle system processes,muscle structural development,PI3K Akt signaling pathway,calcium signaling pathway,and cAMP signaling pathway were activated in TS,while responses to cytokines,cytokine-mediated signaling pathways,regulation of immune system processes,cytokine-cytokine receptor interactions,human T-cell leukemia virus type 1 infection,and phago-somes were inhibited in TS.Three sub-networks were identified in PPI,among which MYL1,TTN,LOC102723407,HLA-A,interleukin(IL)-6,and IL-1β had the highest connectivity and were identified as key DEmRs.MYL1,TTN,and IL-6 were regulated by DElncRs.qRT-PCR validated the differential expression of key DEmRs in TS.Conclusion DEmRs are regulated by lncRNAs and participate in the pathological process of TS,and the immune responses are inhibited in diseases condition.This study reveals key molecules and signaling pathways in TS,providing new insights into the pathological mechanisms of TS,and suggest scientific basis for developing new therapeutic targets.

Objective To identify the potential pathological mechanisms of tuberculous spondylitis(TS).Methods Spinal specimens were collected from 13 TS patients and 13 controls who received treatment at a hospital from March 2021 to March 2023.Specimens were randomly selected from 3 TS patients and 3 controls to perform high-throughput lncRNAs and mRNAs sequencing with Illumina NovaSeq 6000.Differentially expressed lncRNAs(DELncRs)and mRNAs(DEmRs)in TS specimens were identified and analyzed through differential expression,and enrichment analysis was performed.The top 20 DEmRs with high connectivity were identified through protein-protein interaction(PPI)network.Regulatory network of DElncRs and DEmRs was built.Finally,gene expression of the remaining specimens was analyzed using qRT-PCR detection.Results A total of 1 243 DEmRs and 262 DElncRs were identified.Enrichment analysis revealed that muscle contraction,muscle system processes,muscle structural development,PI3K Akt signaling pathway,calcium signaling pathway,and cAMP signaling pathway were activated in TS,while responses to cytokines,cytokine-mediated signaling pathways,regulation of immune system processes,cytokine-cytokine receptor interactions,human T-cell leukemia virus type 1 infection,and phago-somes were inhibited in TS.Three sub-networks were identified in PPI,among which MYL1,TTN,LOC102723407,HLA-A,interleukin(IL)-6,and IL-1β had the highest connectivity and were identified as key DEmRs.MYL1,TTN,and IL-6 were regulated by DElncRs.qRT-PCR validated the differential expression of key DEmRs in TS.Conclusion DEmRs are regulated by lncRNAs and participate in the pathological process of TS,and the immune responses are inhibited in diseases condition.This study reveals key molecules and signaling pathways in TS,providing new insights into the pathological mechanisms of TS,and suggest scientific basis for developing new therapeutic targets.

BACKGROUND:The mechanism of programmed death receptor-1(PD-1)effect on osteogenic differentiation of bone marrow mesenchymal stem cells in high glucose environment remains unclear. OBJECTIVE:To explore the effect of PD-1 on osteogenic differentiation of rat bone marrow mesenchymal stem cells in high glucose environment and its regulatory mechanism. METHODS:Rat bone marrow mesenchymal stem cells were randomly divided into normal glucose group(5.6 mmol/L),high glucose group(30 mmol/L),PD-1 overexpression group,PD-1 overexpression no-load group,PD-1 knockdown group,PD-1 knockdown no-load group,and PI3K/AKT pathway inhibitor group(PD-1 knockdown+5 μmol/L LY294002).Rat bone marrow mesenchymal stem cells were cultured in high glucose to simulate the diabetic environment in vitro.The mRNA expression of PD-1 and ligand PD-L1 and the mRNA expression of osteogenic markers Runx2 and OSX in rat bone marrow mesenchymal stem cells were detected by qRT-PCR.The osteogenic differentiation ability was observed by alkaline phosphatase staining and alizarin red staining.Cell proliferation was detected by CCK-8 assay.The protein expressions of PD-1,PD-L1,p-PI3K,and p-AKT were detected by western blot assay. RESULTS AND CONCLUSION:(1)The levels of PD-1 and PD-L1 were significantly increased in the high glucose environment in vitro,and the osteogenic differentiation ability of bone marrow mesenchymal stem cells was inhibited in the high glucose environment.(2)Knockdown of PD-1 expression could promote osteogenic differentiation of bone marrow mesenchymal stem cells,increase cell proliferation activity,and activate the PI3K/AKT pathway.(3)After addition of PI3K/AKT pathway inhibitor LY294002,the ability of bone marrow mesenchymal stem cells to differentiate into osteoblasts decreased.The results show that PD-1 is dependent on the PI3K/AKT signaling pathway to inhibit osteogenic differentiation of rat bone marrow mesenchymal stem cells under high glucose environment.

OBJECTIVE: In order to enhance the efficacy of anti-breast cancer, paclitaxel nanoparticles (PTX NPs) and polypyrrole nanoparticles (PPy NPs) were combined with photothermal therapy and chemotherapy. At the same time, the two dosage forms of PTX NPs and PTX NPs gel were compared. METHODS: PTX NPs were prepared by self-assembly method, and then the cytotoxicity in vitro was investigated by Methyl thiazolyl tetrazolium (MTT) and other methods, and the efficacy and side effects in vivo were further investigated. RESULTS: The average hydrated diameter, PDI and electric potential of PTX NPs were (210.20 ± 1.57) nm, (0.081 ± 0.003) mV and (15.80 ± 0.35) mV, respectively. MTT results showed that the IC₅₀ value of PTX NPs on 4 T1 cells was 0.490 μg/mL, while that of PTX injection was 1.737 μg/mL. The cell inhibitory effect of PTX NPs was about 3.5 times higher than that of PTX injection. The tumor inhibition rates of PTX NPs and gel were 48.64% and 56.79%, respectively. Together with local photothermal stimulation, the tumor inhibition rate of the PTX NPs reached 91.05%, surpassing that of the gel under the same conditions (48.98%), moreover, the organ index and H&E staining results of PTX NPs showed a decrease in toxicity. CONCLUSION This combination therapy can significantly enhance the effect of anti-breast cancer, and the synergistic effect of chemotherapy and light and heat provides a feasible and effective strategy for the treatment of tumor.

Diabetic kidney disease （DKD） stands as one of the most prevalent microvascular complications of diabetes，noted for its concealed onset and tendency to evolve into end-stage renal disease，profoundly impacting patients' life expectancy and quality of life. Epithelial-to-mesenchymal transition （EMT） is a central pathological process in the initiation and progression of DKD，facilitating disease advancement and renal fibrosis，thus representing a crucial focus of research into the pathological mechanisms of DKD. EMT is driven by the abnormal activation of signaling pathways，including transforming growth factor-β （TGF-β）/Smad，secreted glycoprotein/β-catenin，Notch，tumor necrosis factor-α （TNF-α）/nuclear factor-κB （NF-κB），and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR），leading to renal cellular injury and subsequently accelerating renal fibrosis and the progression of DKD. Traditional Chinese medicine （TCM），characterized by its multi-target and multi-pathway therapeutic approach，demonstrates unique advantages in addressing DKD and EMT. Recent research has shown that active ingredients in TCM，including glycosides，flavonoids，and polyphenols，as well as TCM formulas，can precisely target these relevant signaling pathways，effectively inhibiting cellular injury in DKD and intervening in the EMT process. These findings not only underscore the potential of TCM monomers and formulas in treating DKD and EMT but also pave new directions for research in this field within TCM. This paper systematically reviewed the signaling pathways associated with EMT and provided an in-depth analysis of the research achievements and underlying mechanisms of TCM monomers and formulas in treating DKD and intervening in EMT，aiming to offer new insights and directions for TCM in the treatment of DKD and research on EMT，thereby further promoting the modernization and development of TCM.