Objective To explore the spatial heterogeneity of tumor boundary tissue metabolism in hepatocellular carcinoma (HCC) patients using time-of-flight secondary ion mass spectrometry (TOF-SIMS), and to analyze the characteristics of key metabolites in spatial distribution. Methods After obtaining adjacent non-tumor tissue samples from HCC patients, the samples were snap-frozen and embedded. Serial tissue sections were prepared using frozen section technology, and spatial metabolomic analysis was performed on the tissue sections using TOF-SIMS. The mass spectrometry data were clustered and spatially mapped at the single-cell level using the SEAM algorithm. Results TOF-SIMS combined with the SEAM algorithm successfully achieved high-resolution spatial metabolomic analysis of HCC boundary tissue. The results showed significant metabolic heterogeneity between the intra-and extra-tumoral regions, as well as within the tumor parenchyma, which could be subdivided into two cell subpopulations with different metabolic characteristics. Differential metabolite analysis identified two key phosphate-related ions (m/z=62.97 and m/z=78.96). Quantitative spatial distribution studies revealed that cell populations with high phosphate metabolism (PM+) were significantly enriched in areas close to the tumor boundary. Conclusions There is significant spatial metabolic remodeling at the HCC tumor boundary, and the boundary enrichment characteristics of PM+ cells suggest that the mechanical force environment at the tumor boundary may regulate the metabolic state of cancer cells.
Objective To investigate the changes in peripheral blood and monocyte-derived high mobility group protein B1 (HMGB1) levels in patients with non-puerperal mastitis (NPM) and their correlation with the disease. Methods A total of 83 patients with NPM were divided into the acute group (42 cases) and the chronic group (41 cases) according to their conditions Additionally, 40 healthy subjects were taken as the control group. The levels of HMGB1, C-reactive protein (CRP), and interleukin-6 (IL-6) in the peripheral blood of each group were detected by ELISA. The number of monocytes and the level of HMGB1 were detected by flow cytometry; the expression of HMGB1 in monocytes was detected by qPCR and Western blot; the correlation between the expression of HMGB1 in monocytes and the levels of CRP and IL-6 in peripheral blood was analyzed by the Pearson method. ROC curves were constructed to evaluate the diagnostic value of HMGB1 expression level in monocytes for the condition of NPM. Results The levels of HMGB1, CRP, IL-6 in the peripheral blood of patients with NPM, the number of M1-type monocytes and the HMGB1 mRNA expression in monocytes were all higher than those in the control group. Meanwhile, these levels in the acute group were higher than those in the chronic group (P＜0.05). The level of HMGB1 mRNA in monocytes was positively correlated with CRP (r=0.426) and IL-6 (r=0.569) in peripheral blood (all P＜0.05). The AUC of HMGB1 protein level in M1 monocytes for diagnosing NPM was 0.739 (95% CI: 0.67-0.82), the sensitivity and specificity were 69.8% and 77.2%. The AUC for evaluating the disease condition was 0.828 (95% CI: 0.73-0.91), the sensitivity and specificity were 83.3% and 71.4%. Conclusions The expression of HMGB1 in monocytes of patients with NPM increases and is positively correlated with the levels of peripheral blood CRP and IL-6, which is helpful for the diagnosis and condition assessment of NPM.
Cell membrane engineering is a key technology for endowing cells with novel recognition, response, and regulatory functions, which holds significant value for the advancement of cell therapy and precision medicine. DNA nanotechnology exhibits unique advantages in the precise functionalization of cell membranes owing to its excellent programmability, addressability, and biocompatibility. This article reviews the latest advances in cell membrane engineering based on oligonucleotides, focusing on two major strategies: chemical covalent modification and noncovalent binding. The modification mechanisms, technical characteristics, and application status of these methods in cell therapy are systematically elaborated. Finally, representative strategies for DNA-mediated cell membrane modification are summarized, and the development prospects in the fields of cell engineering and precision medicine are discussed.
Objective To investigate the implementation status of the 2022 edition of the Standardized Residency Training Content and Standards in emergency medicine residency training bases and to identify existing challenges, in order to provide evidence for future optimization of the standards. Methods A cross-sectional questionnaire survey was conducted among teaching administrators and residents from emergency medicine residency training bases nationwide. The questionnaire covered perceptions of standard implementation and practical challenges, implementation and effectiveness of teaching measures, as well as evaluation and reform suggestions regarding the assessment system. Results A total of 156 valid responses were collected. Most respondents (97.44%) agreed that clinical competency should serve as the core training objective. Approximately 38.46% supported allowing moderate regional flexibility within a unified national framework. During implementation, insufficient teaching incentives and heavy clinical and research workload was identified as major challenges (62.18%). Most teaching activities were routinely implemented, and practice-oriented activities were more frequently rated as effective compared with some theory-based activities. Regarding assessment, 83.33% recommended that process evaluations be tailored by individual training bases, and 78.21% supported increasing the proportion and difficulty of case-based analysis. Conclusions The overall philosophy of the emergency medicine residency training standards has been widely recognized. However, further improvements are needed in institutional support, teaching optimization, and competency-oriented assessment reform. The findings of this study provide practical evidence for future revision of the standards and continuous quality improvement in residency training.
Objective To investigate the expression pattern of platelet-derived growth factor receptor alpha (PDGFRA) in bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) from patients with adolescent idiopathic scoliosis (AIS), and to evaluate its effects on osteogenic differentiation and proliferation of BM-MSCs. Methods Previously generated BM-MSC transcriptomic sequencing data from an established cohort were analyzed to identify differentially expressed genes (AIS, n=12; controls, n=5), and the findings were validated by qRT-PCR(AIS, n=12; controls, n=9). PDGFRA was knocked down by siRNA in normal BM-MSCs. After osteogenic induction, osteogenesis-related parameters were assessed by ALP staining/activity assay, Alizarin Red S staining, qRT-PCR, and Western blot. Cell proliferation was evaluated using CCK-8 assays. Results Compared with healthy controls, PDGFRA was significantly downregulated in BM-MSCs from patients with AIS. During the early stage of osteogenic induction, PDGFRA expression increased significantly on day 6 in healthy BM-MSCs. PDGFRA knockdown suppressed both early osteogenic activity and late mineralization in BM-MSCs, and reduced the expression of osteogenesis-related markers, including RUNX2, ALP, OPN, COL1A1, OSX, and IBSP. In addition, PDGFRA knockdown impaired the proliferation capacity of BM-MSCs. Conclusions PDGFRA is involved in the proliferation and osteogenic differentiation of BM-MSCs from healthy participants. Reduced PDGFRA expression in AIS BM-MSCs may contribute to impaired osteogenesis and low bone mass in AIS.
Objective To investigate the impacts of baculovirus IAP repeat containing protein 2 (BIRC2) on mesangial cell injury and autophagy in human diabetic nephropathy (DN) in vitro. Methods Human glomerular mesangial cells HGMCs were cultured routinely and randomly divided into control group, model group, sh NC group, BIRC2 shRNA group, and BIRC2 shRNA+YC-1 group(HIF-1α inhibitor YC-1, 25 μmol/L). CCK-8 method was applied to detect the activity of HGMCs. Flow cytometry was applied to detect cell apoptosis. ELISA method was applied to detect the levels of interleukin(IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) of cells in each group. Transmission electron microscopy was applied to observe the number of autophagosomes of cells in each group. Western blot was applied to detect the expression of BIRC2, HIF-1α/BNIP3/mTOR pathway related proteins and autophagy related protein Beclin-1, and microtubule-associated protein 1 light chain 3 (LC3) of cells in each group. Results Compared with the control group, the absorbance (A) value (48 h, 72 h), IL-1β, IL-6, TNF-α levels, BIRC2 protein expression, and mTOR phosphorylation levels of HGMCs in the model group were significantly increased(P＜0.05), the apoptosis rate of HGMCs, the number of autophagosomes, and the expression levels of HIF-1α, BNIP3, Beclin-1, and LC3 Ⅱ/Ⅰ proteins were significantly reduced (P＜0.05). Compared with the model group and sh NC group, the changes in the corresponding indicators of HGMCs in the BIRC2 shRNA group were opposite (P＜0.05). Conclusions Downregulation of BIRC2 expression alleviates the damage of mesangial cells in human diabetic nephropathy and promotes autophagy in vitro.
Objective To explore the function of centrosomal protein 89 (CEP89) in neuroblastoma (NB). Methods The relationship between the expression of CEP89 mRNA and the prognosis of NB patients was clarified through the analysis of three independent primary NB cohorts via Genomics Analysis and Visualization Platform R2. CEP89 protein was examined by immunohistochemical staining in tumors and adjacent tissues from NB patients. CEP89 mRNA in NB cell lines BE(2)-M17,SK-N-BE(2),SH-SY5Y and normal cell lines MCF10A,IMR-90 and hTERT RPE-1 was analyzed by RT-qPCR. Cell lines were transfected with CEP89-targeting siRNA or shRNA. Cell proliferation was detected by crystal violet staining,real time cell analysis (RTCA) and colony formation assays. Cell apoptosis and cell cycle were detected by flow cytometry and cell-cycle analysis. Wound-healing assay was performed to measure cell migration in vitro. Ki67 was analyzed by Western blot and immunofluorescence assay. Results Analysis of R2 database revealed that higher CEP89 expression was associated with poorer survival in NB patients. CEP89 protein expression level was significantly higher in NB patients tumors compared with adjacent tissues. CEP89 showed significantly increased expression at mRNA level in NB cells compared with non-NB normal cells. CEP89 knockdown reduced NB cell viability more significantly compared with normal cells as demonstrated by crystal violet staining,RTCA and colony formation assays. CEP89 knockdown significantly induced apoptosis,limited cell migration,and decreased the expression of Ki67 in NB cells. Conclusions CEP89 expression is significantly increased in NB tumors and NB cells. CEP89 promotes the NB cell proliferation in vitro.
Objective To establish a mouse model of combined pulmonary fibrosis and emphysema(CPFE)in-duced by intratracheal instillation of bleomycin(BLM)combined with porcine pancreatic elastase(PPE).Methods Male C57BL/6 mice were randomly divided into a control group and an experimental group.Mice in the experimental group received intratracheal instillation of a suspension of BLM(2.5 mg/kg)and PPE(1.5 U/100 g).The control group received instillation of phosphate-buffered saline(PBS).After four weeks,lung function was assessed.Histopathological changes in lung tissue were observed using hematoxylin-eosin staining,and the mean linear intercept(MLI)was measured.Fibrosis severity was evaluated using Masson's trichrome staining,and the subepithelial collagen deposition thickness(SEc/Pbm)in the airways was determined.Additionally,hydroxypro-line content in lung tissue was measured.Results Compared with the control group,the experimental group showed an increase in functional residual capacity(FRC),a decrease in FEV50(forced expiratory volume at 50 ms)/FVC and maximum mid-expiratory flow(MMEF),a trend toward decreased dynamic pulmonary compliance(Cdyn),no significant change in inspiratory capacity(IC),and no significant change in chord compli-ance(Cchord).Histologically,the experimental group exhibited enlarged alveolar septa and alveolar wall disruption in some lung regions,along with significant collagen deposition in the airways and parts of the lung pa-renchyma.Furthermore,MLI,SEc/Pbm,and hydroxyproline content were all significantly increased.Conclusions Intratracheal instillation of BLM combined with PPE successfully establishes a CPFE model in C57BL/6 mice within four weeks,characterized by both emphysema-like damage and fibrotic changes.This model provides an ex-perimental basis for future research on the mechanisms underlying the development and progression of CPFE and for evaluating intervention strategies.
Objective To establish a mouse model with specific knockout of dopamine D2 receptor (D2R) in the enteric nervous system (ENS) and elucidate its mechanism in promoting intestinal motility. Methods The Cre-LoxP system was employed to generate ENS-specific D2R knockout mice (D2RNmu-CKO) and control mice (D2Rflox/flox). Expression levels of D2R and choline acetyltransferase (ChAT) were detected using immunofluorescence staining, DAB staining, RT-qPCR, and Western blot. Acetylcholine (ACh) content was measured by ELISA. Intestinal transit was assessed in vivo using X-ray imaging in live small animals. Results Immunofluo- rescence staining confirmed the co-localization of D2R with the neuronal marker PGP 9.5 as well as the cholinergic neuronal marker ChAT in the duodenal myenteric plexus (MP) of wild-type mice, indicating that D2R is expressed in cholinergic neurons of the duodenal ENS. Genotyping confirmed successful establishment of D2RNmu-CKO mice (genotypes: Nmu-Cre+/+D2R-flox+/+ or Nmu-Cre+/-D2R-flox+/+) and control D2Rflox/flox mice (genotype: Nmu-Cre-/- D2R-flox+/+). Compared with control mice, D2RNmu-CKO mice exhibited significantly reduced mRNA(n=6, P＜0.001) and protein (n=8, P＜0.01) expression of D2R in the duodenal muscle layer, while mRNA(n=6, P＜0.001) and protein (n=6, P＜0.05) expression of ChAT were markedly increased. The content of ACh was also significantly elevated (n=8, P＜0.01). DAB staining further revealed a significant increase in the number of ChAT-positive neurons in the duodenal MP of D2RNmu-CKO mice (n=6, P＜0.01). Intestinal transit assays demonstrated accelerated intestinal transit and significantly shortened whole-gut transit time in D2RNmu-CKO mice (n=5, P＜0.05). Conclusions This study successfully established an ENS-specific D2R knockout mouse model and revealed that loss of ENS D2R promotes intestinal transit by enhancing ACh release. These findings provide a theoretical basis for novel strategies targeting ENS D2R to treat gastrointestinal motility disorders such as functional dyspepsia and slow-transit constipation.
Objective To investigate aberrant alternative splicing and splicing factor-mediated regulation in chronic lymphocytic leukemia (CLL). Methods High-throughput transcriptome sequencing was performed on peripheral blood mononuclear cells from 3 healthy controls (HC), and public data from 2 CLL patients and 6 patients treated with voruciclib (PT) were included. The rMATS software was used to quantify alternative splicing events (ASEs) and identify differential alternative splicing events (DASEs). The expression changes and self-splicing alterations of splicing factors (SFs)were analyzed. Results A total of 12 933, 32 908, and 32 862 ASEs were identified in HC, CLL, and PT samples, respectively, with skipped exons being the predominant type. 4 134 DASEs were detected in CLL vs. HC, among which 69.5% corresponded to decrease in percent spliced-in (PSI). Complex interactions were observed between PSI changes and gene expression alterations. Splicing factors exhibited alterations at both expression and PSI levels in CLL. In total, 330 DASEs displayed opposite ΔPSI trends between disease progression and treatment response including 276 events with a “PSI decrease in disease-PSI increase after treatment” pattern enriched in the cell cycle and DNA repair processes, and 54 events with a “PSI increase in disease-PSI decrease after treatment” pattern enriched in autophagy and phosphorylation pathways. Conclusions Aberrant ASEs exist in CLL, and the altered expression and splicing levels of SFs may contribute to this dysregulation.
Objective To investigate the time-dependent transcriptomic alterations during liver organoid formation from HepaRG cells under three-dimensional culture from 0 to 120 h, and to characterize molecular features associated with the programmatic transition from proliferation-related processes to hepatic functional differentiation. Methods HepaRG cells were mixed with Matrigel (1∶1, v/v) and seeded into ultra-low-attachment 96-well plates. Samples collected at 0 h, 14 h, 24 h, 72 h, and 120 h were subjected to RNA sequencing (RNA-seq), followed by principal component analysis (PCA)/correlation assessment, DESeq2-based differential expression, time-course clustering with enrichment analysis, and protein-protein interaction (PPI) network analysis. Results HepaRG cells self-assembled into spheroid organoids under three-dimensional culture, accompanied by increased albumin (ALB) expression. The number of differentially expressed genes (DEGs) peaked at 72 h and 120 h. Six temporal clusters revealed an early-to-late transition from proliferation/cell-cycle programs to hepatic functional programs (lipid metabolism, lipoprotein remodeling, and the complement pathway). Conclusions HepaRG organoid formation involves dynamic transcriptomic remodeling. The 72-120 h interval constitutes a critical stage with enhanced hepatic function-related programs, offering transcriptomic support for maturation evaluation and mechanistic studies.
Objective To investigate the effects of Mcart1 gene knockout on macrophage metabolism and phenotype. Methods Two types of bone marrow-derived macrophages (BMDMs) were obtained from Mcart1flox/flox and Mcart1Lyz2-cre mice. Basal respiration of both cell types was measured using a Seahorse analyzer. The activity of α-ketoglutarate dehydrogenase (OGDH) was assessed in Mcart1 stably knockout RAW264.7 cells (Mcart1-/--RAW264.7) and wild-type RAW264.7 cells (RAW264.7). The two types of BMDMs were induced into M1 macrophages using LPS, and whole-cell reactive oxygen species (ROS) levels were measured by flow cytometry, while dynamic expression of inflammatory mediators was monitored by qPCR. The two types of BMDMs were also induced into M2 macrophages using IL-4, and expression of M2 anti-inflammatory and tissue repair mediators was measured by qPCR, while protein levels of classical M2 activation markers were detected using Western blot. Results Compared with Mcart1flox/flox BMDMs, Mcart1 knockout significantly reduced basal respiration in BMDMs, and Mcart1 stable knockout decreased OGDH activity in macrophages(P＜0.01). Mcart1 knockout significantly enhanced ROS production in M1 macrophages and upregulated early inflammatory mediators Il1b, Tnf, and Nos2(P＜0.05), while having no significant effect on the expression of M2 anti-inflammatory and tissue repair mediators Arg1, Mrc1, Retnla, Cd163, Fn1, or classical activation proteins p-Stat6 and p-Stat3 in M2 macrophages. Conclusions Mcart1 knockout significantly suppresses basal respiration in macrophages, inhibits OGDH enzyme activity, increases oxidative stress and inflammatory mediator levels in M1 macrophages, and does not significantly affect functional mediator levels in M2 macrophages.
Objective To systematically analyze the expression characteristics of peripheral blood lymphocyte subsets in patients with newly diagnosed lung cancer at different disease stages, and to explore their correlations with clinicopathological features, gender, age and other factors. Methods A total of 183 patients with newly diagnosed untreated lung cancer (106 early-stage, 77 late-stage) from January 2019 to December 2020 were enrolled. Flow cytometry was used to detect 28 peripheral blood lymphocyte subsets and activation markers. Comparisons between groups were performed using t-test, Welch t-test, or Mann-Whitney U test, and subgroup analyses were conducted based on age, gender, pathological type, and metastatic site. Results Early-stage patients exhibited higher levels of lymphocytes, B cells, CD4+ T cells, and CD28 (P＜0.05), while late-stage patients showed significant increase in white blood cell count, NK cell proportion, and CD8+ T cell activation markers (P＜0.001). Subgroup analyses revealed that in the early-stage elderly group, NK cell proportion was higher than that in the younger group (P＜0.001), while B cell proportion was lower (P＜0.05); in early-stage lung squamous cell carcinoma, white blood cell count was higher than that in adenocarcinoma (P＜0.05), while adenocarcinoma exhibited higher lymphocyte and B cell proportions (P＜0.05); in late-stage squamous cell carcinoma, lymphocyte, NK cell, and CD8+CD38+ activation levels were lower than those in adenocarcinoma (P＜0.05); patients with pulmonary metastasis showed lower expression of CD8+HLA-DR+ and CD8+CD38+ compared with those without pulmonary metastasis (P＜0.05); patients with multiple metastases showed lower expression of CD4+CD28+ percentage and CD8+CD28+ compared with those with solitary metastasis (P＜0.05). Conclusions There are systematic differences in peripheral immune characteristics between patients with early-and late-stage lung cancer, which are influenced by various clinical factors including age, gender, pathological type, and metastatic site, providing a basis for disease staging assessment and individualized treatment strategies based on peripheral immune phenotypes.
Objective To evaluate the effectiveness of a progressive multimodal teaching approach “self-directed theoretical learning → clinical observation → lecture-based learning (LBL) → case-based learning (CBL)” in transabdominal bowel ultrasound training. Methods Thirteen visiting ultrasound radiologists without prior experience in intestinal ultrasound who were trained at the Department of Ultrasound, Peking Union Medical College Hospital between September 2024 and March 2025 were enrolled. All participants completed the four-stage training curriculum. Test scores were collected immediately after the self-learning phase and again after the concentrated LBL+CBL block to evaluate learning outcomes. Examinations consisted of three modules: basic principles (30 points), image interpretation (40 points), and intestinal disease (30 points). Results After self-learning phase, the mean total score was 62.3±18.8. The disease-knowledge module showed a score percentage of 33.3% (33.3%,66.6%), significantly lower than both the basic-principle (74.4%±24.2 %, P＜0.01) and image-interpretation (67.3%±23.7 %, P＜0.05) modules.Following LBL+CBL block, the mean total score rose to 93.9±7.7 (P＜0.001). All three sub-scores improved significantly [basic knowledge: 20(20,30) vs. 30(30,30), P＜0.05; image interpretation: 30(20,35) vs. 40(35,40), P＜0.001; disease knowledge: 10(10,20) vs. 30(25,30), P＜0.001]. The greatest improvement was observed in disease-related knowledge. In post-course surveys, 84.6 % of participants rated the LBL+CBL component as “considerably” or “markedly” helpful beyond self-study alone. Conclusions The progressive multimodal teaching of “self-directed theoretical learning → clinical observation → LBL → CBL” is effective for training inexperienced ultrasound physicians and shows promise in strengthening disease-related knowledge systems. This study offers a useful reference for establishing standardized intestinal ultrasound training programs in China.
Objective To analyze the clinical characteristics of patients with hemorrhagic cerebrovascular disease in late pregnancy and summarize the anesthetic management experience for cesarean delivery. Methods Medical records of patients who underwent lower uterine segment cesarean section for hemorrhagic cerebrovascular disease in late pregnancy at Beijing Tiantan Hospital, Capital Medical University, from January 1, 2020, to January 1, 2025, were retrospectively reviewed. Clinical data, multidisciplinary treatment, anesthetic management, and the outcomes were analyzed. Results Among the 12 patients, 4 (33.3%) underwent emergency cesarean delivery for pregnancy termination, while 8 (66.7%) underwent elective cesarean delivery after expectant management. Neuraxial anesthesia was administered in 10 cases (83.3%), and general anesthesia in 2 cases (16.7%). Except for one case of recurrent cerebral hemorrhage due to delayed neurosurgical intervention, maternal and fetal outcomes were favorable. Conclusions Multidisciplinary collaboration is essential for management of late-pregnancy patients with hemorrhagic cerebrovascular disease. Anesthetic management should extend beyond the intraoperative period of cesarean delivery, emphasizing comprehensive monitoring throughout the perinatal and postpartum periods.
Severe interstitial lung disease (SILD) is a large group of complex and rapidly progressing diseases with an extremely high mortality rate. Early diagnosis, assessment and treatment of patients with SILD are of critical importance. The application of quantitative computed tomography (QCT), positron emission tomography/computed tomography (PET/CT) and bronchoalveolar lavage fluid metagenomic next-generation sequencing (BALF-mNGS) in the diagnosis of SILD holds promise for improving diagnostic rates and accuracy. Glucocorticoids/immunosuppressants serve as the cornerstone of treatment, while tofacitinib, antifibrotic drugs and hypoxia management play significant roles in improving patient prognosis. However, continuous research and the development of individualized treatment strategy are still necessary to provide more solid evidence and more diverse options for clinical practice.
Objective To investigate the function of NAT10 in the mouse myeloid progenitor cell line 32D. Methods The NAT10 gene was knocked out in 32D cells using CRISPR/Cas9 technology, and the knockout efficiency was verified by RT-qPCR and Western blot. Flow cytometry was employed to assess cell cycle distribution and apoptosis, and the experimental results were statistically analyzed. Results A NAT10 knockout 32D cell model was successfully established. Compared with the control group, NAT10 knockout resulted in a marked reduction in the percentage of cells in the G1 phase accompanied by a significant increase in the percentage of cells in the S phase. Meanwhile, the proportion of early and late apoptotic cells was significantly reduced (P＜0.05). Conclusions NAT10 plays an important role in the proliferation and survival of 32D cells. Its loss can lead to cell cycle activation and inhibition of apoptosis, suggesting that NAT10 may be involved in the regulation of myeloid progenitor cell growth and in limiting their excessive proliferation.
Exosomes can be secreted by different cells in post-stroke cognitive impairment (PSCI) and participate in the occurrence and development of PSCI by affecting the neuroinflammatory response, the blood-brain barrier, repair of damaged neurons, and reconstruction of neurovascular unit. Compared with cognitive assessment scales and traditional treatment drugs, exosomes have the advantages of early diagnosis and treatment. Exosomes can be used as new diagnostic and therapeutic targets for PSCI, providing new ideas for the diagnosis and treatment of PSCI.
Objective To explore the intrinsic relationship between microglia in Alzheimer's disease(AD)and natural aging,and their role in mediating microenvironmental proteostasis.Methods Nearly 1.69 million brain single-cell and single-nucleus transcriptomes from 18 public datasets were integrated and annotated using deep gen-erative models.A"transcriptomic aging score"was constructed to compare the transcriptional remodeling of micro-glia between natural aging and AD models.Multiplex immunofluorescence staining on brain sections was performed to verify the in situ expression of key molecules for proteostasis and lysosomes.A Trem2-deficient model was intro-duced to explore the regulatory mechanisms.Results AD pathology significantly accelerated the age-dependent accumulation of late-stage disease-associated microglia(DAM-Late).The AD transcriptional profile showed a highly significant positive correlation with natural aging(R=0.481),manifesting as a"pathological amplification"of aging-mediated lipid metabolism and lysosomal programs.Metabolic stress led to the collapse of microglial pro-teostasis,with a significant increase in the area and number of αB-CRYSTALLIN,CD68,and UBC puncta carried by single cells in the hypothalamus of AD mice.TREM2 acted as a key switch initiating the compensatory program,and its deficiency led to phenotypic decoupling and functional compensation failure.Conclusions The pathological activation of AD microglia significantly amplifies endogenous aging characteristics.Under continuous pathological stress,this adaptive compensation may ultimately mediate severe impairment of proteostasis in the microenvironment and adjacent cells.
Objective To investigate the effect of carboxyamidotriazole (CAI) on TNF-α-induced activation of fibroblast-like synoviocyte(FLS) derived from patients with rheumatoid arthritis (RA). Methods FLS were isolated and cultured from synovial tissues of RA patients and identified by immunofluorescence. The cells were stimulated with TNF-α in the presence or absence of various concentrations of CAI. Cell proliferation was assessed by CCK-8 assay. The contents of IL-6, IL-8 and MMP-3 in the culture supernatant were measured by ELISA. NF-κB p65 DNA-binding activity in the nuclear extracts was detected using TransAM kit. Results TNF-α stimulation significantly promoted the proliferation of RA-FLS, upregulated the secretion of IL-6, IL-8 and MMP-3, and enhanced the DNA-binding activity of NF-κB p65. Treatment with 10 μmol/L CAI inhibited TNF-α-induced FLS proliferation (P＜0.05), IL-6 and MMP-3 secretion (P＜0.01), as well as NF-κB p65 activation (P＜0.01). Furthermore, 20, 30 and 40 μmol/L CAI significantly suppressed all the TNF-α-induced elevations in the detected parameters (P＜0.01). Conclusions CAI may attenuate TNF-α-induced proliferation and production of IL-6, IL-8 and MMP-3 in RA-FLS through suppressing NF-κB activation, suggesting that CAI has the potential to be developed as a therapeutic agent for RA.
