ObjectiveA middle cerebral artery occlusion (MCAO) mouse model is established by electrocoagulation of the middle cerebral artery. The study examines the mechanism by which exosomes (EXO) derived from human amniotic mesenchymal stem cells (hAMSCs) improve ischemic stroke and regulate neural ferroptosis-related injury. MethodsThirty-two SPF-grade male C57BL/6J mice aged 6 - 8 weeks were randomly divided into four groups (n=8 per group): sham group (Sham), model group (MCAO), MCAO plus normal saline group (MCAO+NaCl), and MCAO plus exosome group (MCAO+EXO). The mouse MCAO model was established by electrocoagulation of the middle cerebral artery. Mice in the Sham group underwent exposure of the middle cerebral artery without electrocoagulation. Twenty-four hours before MCAO induction, mice in the MCAO+EXO group received a tail vein injection of 100 μL of exosomes derived from the culture supernatant of hAMSCs at a concentration of 9.5×10¹¹ particles/mL. Mice in the MCAO+NaCl group were injected with an equal volume of normal saline via the tail vein. Twenty-four hours after model establishment, neurological deficits were evaluated using the Longa neurological deficit scoring system. Cerebral infarct volume was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Hematoxylin and eosin (HE) staining was performed to evaluate morphological changes of neurons in the ischemic brain regions. The contents of ferrous iron (Fe²⁺), malondialdehyde (MDA), total glutathione (total GSH), oxidized glutathione (GSSG), and reduced glutathione (GSH) in the infarct core and peri-infarct regions were determined using microcolorimetric assays to evaluate differences among groups. The mRNA expression levels of ferroptosis-related factors, including nuclear factor erythroid 2-related factor 2 (NRF2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) in the infarct core and peri-infarct regions were measured by real-time quantitative PCR. Protein expression levels of NRF2, SLC7A11, and GPX4 in the infarct and peri-infarct regions of each group were analyzed by Western blotting. ResultsCompared with the MCAO group, the Longa neurological deficit score was significantly reduced in the MCAO+EXO group (P<0.01). Prominent cerebral infarction was observed in the MCAO group, whereas the infarct volume ratio was markedly decreased in the MCAO+EXO group compared with the MCAO group (P<0.001). Histopathological analysis revealed that mice in the MCAO group exhibited obvious neuronal damage, including cytoplasmic vacuolar degeneration, nuclear pyknosis and fragmentation, unclear nuclear structure, and disorganized neuronal arrangement, compared with the Sham group. In contrast, neurons in the MCAO+EXO group showed relatively preserved morphology, with intact cellular structures and large, regular nuclei located centrally within the cells. Biochemical analysis demonstrated that Fe²⁺ and MDA levels in the infarct core and peri-infarct regions were significantly increased in the MCAO group compared with the Sham group (P<0.001). These levels were significantly reduced in the MCAO+EXO group compared with the MCAO group (P<0.01). In addition, total glutathione (total GSH), oxidized glutathione (GSSG), and reduced glutathione (GSH) levels were markedly decreased in the MCAO group relative to the Sham group (P<0.01). Compared with the MCAO group, the MCAO+EXO group exhibited significantly increased levels of total GSH and GSH (P<0.001), while no significant change was observed in GSSG levels (P>0.05). Furthermore, both mRNA and protein expression levels of nuclear factor erythroid 2-related factor 2 (NRF2), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) were significantly downregulated in the MCAO group compared with the Sham group (P<0.01, P<0.001). In contrast, both mRNA and protein expression levels of NRF2, SLC7A11, and GPX4 were significantly upregulated in the MCAO+EXO group compared with the MCAO group (P<0.05). ConclusionIn the mouse MCAO model, tail vein injection of exosomes derived from hAMSCs can improve motor function, reduce infarct area, protect neuronal cell morphology, and reduce the degree of nerve injury. Exosomes may exert a protective effect by activating the NRF2/SLC7A11/GPX4 pathway and reducing ferroptosis in neuronal cells of MCAO model mice.

ObjectiveTo compare female C57BL/6J and BALB/c mice models of ulcerative colitis (UC) induced by dextran sulfate sodium salt (DSS), providing a reference for selecting female animal models in studies of innovative drugs for UC treatment. MethodsA total of 48 female mice aged 6–8 weeks, including 24 C57BL/6J and 24 BALB/c mice, were divided into four groups: control group, UC-3d group (2.5% DSS drinking for 3 days), UC-7d group (2.5% DSS drinking for 7 days), and UC-withdrawal group (2.5% DSS drinking for 7 days followed by 3 days of pure water replacement), with 6 mice in each group. Body weight, fecal occult blood, and stool characteristics were recorded daily, and disease activity index (DAI) scores were calculated. At the end of DSS induction, mice were gavaged with fluorescein isothiocyanate (FITC)-labeled dextran solution before tissue collection. Four hours later, under deep anesthesia induced by inhalation of 4% isoflurane, blood samples were collected from the retro-orbital venous plexus, and euthanasia was performed by cervical dislocation. The colon was then separated immediately, and its length from the anus to the ileocecal junction was measured. Colon tissues were fixed, embedded, and sectioned, and they were evaluated using hematoxylin-eosin (HE) staining and alcian blue-periodic acid-Schiff (AB-PAS) staining to comprehensively assess the degree of colonic inflammation. The spleen was also collected and weighed, and the spleen coefficient was calculated. Serum FITC fluorescence intensity and D-lactic acid concentration were measured to evaluate intestinal permeability and barrier injury. The grooming duration, the number of buried marbles, the time spent in the center of the open field, and the total distance traveled were measured to evaluate anxiety/depression-like behaviors in mice. ResultsFecal occult blood appeared in both C57BL/6J and BALB/c mice after 3 days of ad libitum access to DSS. Compared with the control group, there was no significant change in the weight loss rate of female BALB/c mice in the UC-7d and UC-withdrawal groups (P > 0.05), but the DAI score increased (P < 0.01). Conversely, both the weight loss rate and DAI score increased for female C57BL/6J mice in the UC-7d and UC-withdrawal groups (P < 0.01). In female C57BL/6J mice, both the UC-7d and UC-withdrawal groups had higher spleen coefficients than the control group (P < 0.05). Additionally, in these groups, colon length was significantly reduced (P < 0.01), FITC fluorescence intensity in serum was markedly increased (P < 0.01), obvious inflammatory reaction areas appeared in the colon, and the goblet cells and acidic mucus layer were damaged. In contrast, in female BALB/c mice, no significant differences were observed in colon length, serum FITC fluorescence intensity, or D-lactic acid concentration between the UC-7d and UC-withdrawal groups compared with the control group (P > 0.05). Only a few inflammatory cells and damaged goblet cells were observed in the colonic mucosal layer, and no other obvious pathological changes were detected. In addition, female C57BL/6J mice in the UC-7d group showed a significantly reduced total distance in the open field and fewer buried marbles (P < 0.05), indicating depression-like behavior, as well as prolonged grooming duration (P < 0.05), indicating anxiety-like behavior. ConclusionFemale BALB/c mice are not sensitive to 2.5% DSS and do not show typical colonic pathological changes of UC. In contrast, ad libitum access to 2.5% DSS for 7 days successfully induces typical UC pathological symptoms and colonic pathological changes in female C57BL/6J mice. These changes persist for 3 days after DSS withdrawal, and the mice also exhibit symptoms of anxiety/depression. The results suggest that this model can be used for research on the pathogenesis and therapeutic drugs of UC.

Objective:This study aimed to identify potential prognostic and biotherapeutic targets for oral squamous cell carcinoma(OSCC)by investigating the regulatory effects of M1/M2 macrophages on OSCC progression and their underlying mechanisms.Methods:The ef-fects of M1/M2 macrophages on the proliferation,migration,invasion,and apoptosis of SCC-15 cells were examined.Immunohistochemical staining was used to detect the expression of CD68 and M2 macrophage marker proteins in human OSCC tissue microarrays,and their cor-relations with clinicopathological features were analyzed.Results:M1 macrophages promoted apoptosis of SCC-15 cells while inhibiting their proliferation,migration,and invasion.In contrast,M2 macrophages exhibited the opposite effects.The infiltration density of tumor-associ-ated macrophages(TAMs)in OSCC tissues was significantly positively correlated(P<0.05)with lymph node metastasis.Conclusions:M1 mac-rophages suppress OSCC progression,whereas M2 macrophages promote it.

Objective To investigate the relationship between serum ferritin(SF)and metabolic associated fatty liver disease(MAFLD)in adolescents.Methods This study utilized data from 2017 to 2020 national health and nutrition examination survey database,ultimately including 559 adolescents aged 12～18 years,divided into MAFLD group(119 cases)and non-MAFLD group(440 cases).Data on demographic characteristics,serum iron metabolism indicators,liver function,lipid profiles,glucose levels,and other relevant parameters were collected.Liver steatosis and fibrosis were assessed using vibration-controlled transient elastography.Pearson correlation and Logistic regression analyses were performed to evaluate the relationship between SF and MAFLD.Results SF levels were significantly higher in MAFLD group compared to non-MAFLD group(P<0.05).Pearson correlation analysis indicated that SF was positively correlated with the controlled attenuation parameter,liver stiffness measurement,body mass index(BMI),alanine aminotransferase(ALT),γ-glutamyl transferase,triglycerides,and uric acid levels(P<0.05),but negatively correlated with high-density lipoprotein cholesterol levels(P<0.05).Adolescents were divided into 4 groups according to the quartiles of SF levels(Q1 goup:SF 1.9-25.6ng/ml;Q2 goup:SF 25.7-41.7ng/ml;Q3 goup:SF 41.8-70.4ng/ml;Q4 goup:SF 70.5-445.0ng/ml).The results of Logistic regression analysis showed that when the variables were not adjusted,the risk of MAFLD in Q4 group adolescents was significantly increased,this association disappeared after adjusting for gender,age,race,BMI,and other factors.BMI(OR=1.250,95％CI:1.176-1.329),globulin(OR=2.991,95％CI:1.218-7.343),ALT(OR=1.052,95％CI:1.003-1.104),and white blood cell count(OR=1.225,95％CI:1.041-1.441)were identified as independent risk factors for MAFLD.Conclusion SF levels are significantly elevated in adolescents with MAFLD but are not independently associated with the occurrence of MAFLD.BMI,globulin,ALT,and white blood cell count are important risk factors for adolescent MAFLD.

Objective:To investigate immunological non-responders (INRs) and immunological responders (IRs) during long-term antiretroviral therapy (ART), and study the dynamics of HIV reservoir and α4β7 cells in INRs and IRs and their correlation.Methods:Twenty-six patients with chronic HIV infection who received ART for 5 years were included. They were divided into INRs (CD4 + T cell counts≤350 cells/μl, n=9) and IRs (CD4 + T cell counts≥500 cells/μl, n=17) based on immune reconstitution outcomes. The percentages and numbers of α4β7 cells in both groups at baseline, ART 1, 3, and 5 years were detected by flow cytometry, and the levels of HIV DNA and cell-associated HIV RNA were quantified by real-time fluorescent quantitative PCR during the same periods. HIV viral decay, α4β7 cells dynamics, and their correlations with T cells were compared at baseline, ART 1, 3 and 5 years between the two groups. Results:Over 5 years of ART, INRs exhibited higher HIV reservoir levels compared to IRs, but the decline trend was not slow. The counts of α4β7 cell were lower and the growth trend was slow in INRs ( P<0.05). α4β7 cell counts were strongly positively correlated with CD4 + T cell counts at all timepoints (Year 1: r=0.887; Year 3: r=0.878; Year 5: r=0.887; P all <0.001), while showing significantly negative correlations with activated CD38 + HLA-DR + CD4 + T cells (Year 1: r=-0.619, P=0.001), CD38 + HLA-DR + CD8 + T cells (Year 1: r=-0.517; Year 5: r=-0.532; P all <0.01), and PD-1 + CD4 + T cells (Year 1: r=-0.476, Year 5: r=-0.390, P all <0.05). Conclusions:During long-term ART, INRs maintained higher HIV reservoir and lower α4β7 cell counts compared with IRs, and decreased α4β7 cells may be associated with disease progression.

Oral cancer is a common inflammation-associated malignant tumor of the head and neck.In recent years,the role of the tumor microenvironment in the progression of oral cancer has received increasing attention.In the microenvironment of solid tumors,including or-al cancer,tumor-associated macrophages(TAMs)are among the most common type of immune cell and are closely associated with the poor prognosis of more than 80%of human malignant tumors.An increasing body of evidence indicates that TAMs play important roles in the oc-currence and progression of oral cancer.On the basis of the most recent research progress,both domestic and international,we systematic-ally reviewed the phenotypic characteristics,clinicopathological correlations,and molecular regulatory mechanisms of TAMs in the microen-vironment of oral squamous cell carcinoma(OSCC).In particular,we focus on the key signaling pathways promoting tumorigenesis and de-velopment and immune escape mechanisms,with the aim of constructing a systematic theoretical framework for developing therapeutic strategies targeting TAMs and providing a scientific basis for clinical translational research.

Objective To investigate the effects of auricular point seed-pressing and earpoint scraping on sleep quality,sleep structure index and neurotransmitter in patients with sleep disorders in neurology department.Methods A total of 110 patients with sleep disorders treated in the Department of Neurology at Nanjing Hospital Affiliated to Nanjing Medical University from June 2022 to June 2024 were selected as the study subjects.The patients were randomly divided into an observation group and a control group using a random number table,with 55 cases in each group.The control group was treated with Estazolam Tablets,while the observation group received auricular point seed-pressing therapy combined with ear scraping therapy.The treatment course lasted for 4 weeks.After treatment,the clinical efficacy of the two groups was evaluated.Changes in the Pittsburgh Sleep Quality Index(PSQI)scores were observed before and after treatment,as well as sleep structure indicators,including total sleep time(TST),sleep latency(SL),arousal index(AI),non-rapid eye movement sleep time(NREM)[light sleep stage(S1),light to moderate sleep stage(S2),moderate sleep stage(S3),deep sleep stage(S4)],actual sleep time(AST),sleep efficiency(SE),and rapid eye movement sleep time(REM).The serum levels of norepinephrine(NE),5-hydroxytryptamine(5-HT),and gamma-aminobutyric acid(GABA)were compared before and after treatment,as well as changes in the Quality of Life Scale(SF-36)scores.The safety and incidence of adverse reactions in the two groups were also evaluated.Results(1)The total effective rate in the observation group was 96.61%(53/55),while it was 81.82%(45/55)in the control group.The efficacy of the observation group was superior to that of the control group,with a statistically significant difference(P＜0.05).(2)After treatment,the PSQI scores of both groups were significantly improved(P＜0.05),and the observation group showed significantly better improvement in PSQI scores compared to the control group,with a statistically significant difference(P＜0.05).(3)After treatment,the sleep structure indicators in the observation group,including TST,SL,AI,S1,S2,S3,S4,AST,SE,and REM,were significantly improved,and the differences were statistically significant compared to the control group(P＜0.05).(4)After treatment,the serum levels of NE,5-HT,and GABA in both groups were significantly improved,and the observation group showed significantly better improvement in these levels compared to the control group,with a statistically significant difference(P＜0.05).(5)After treatment,the SF-36 scores of both groups were significantly improved,and the observation group showed significantly better improvement in SF-36 scores compared to the control group,with a statistically significant difference(P＜0.05).(6)The total incidence of adverse reactions in the observation group was 0.00%(0/55),while it was 10.91%(6/55)in the control group.The incidence of adverse reactions in the observation group was significantly lower than that in the control group,with a statistically significant difference(P＜0.05).Conclusion Auricular point seed-pressing therapy combined with ear scraping therapy for the treatment of sleep disorders at the Department of Neurology can significantly improve patients'sleep quality,enhance sleep structure-related indicators,regulate neurotransmitter levels,thereby improving patients'quality of life with high safety and efficacy.

In order to explore the relationship between ubiquitin proteasome system(UPS)and au-tophagy after macrophage was infected with Brucella.On the one hand,the levels of LC3 Ⅱ、P62 and 20S proteasomes in cell supernatant and peritoneal fluid were determined respectively after RAW264.7 cells were infected and BALB/c mice were intraperitoneal inoculated with Brucella suis(B.suis).The results displayed that UPS was activated firstly,followed by autophagy after B.suis infection.On the other hand,we prepared the UPS-inhibited-cells and ALP-inhibited-cells by lacta-cystin and 3-methy ladenine(3-MA),and then the cells were infected by B.suis and the levels of LC3 Ⅱ、P62 与 20S proteasomes in cell supernatant were determined.The results showed that the ALP function was significantly improved when the effect of M

Objective:To explore the distribution and clearance of 99mTc labeled diethylenetriamine pentaacetic acid(99mTc-DTPA)in different brain regions of adult rats after administration through brain extracellular space(ECS)pathway.Methods:After the injection of a volume of 2 μL and radioactive activity of about 3.7 MBq(100 μCi)of 99mTc-DTPA into the caudate nucleus and thalamus of SD rats through stereotactic positioning of rat brain,the single photon emission computed tomography/computed tomography(SPECT/CT)for small animals was used for imaging at different time points,and the dyna-mic distribution and clearance of the tracer in the whole body were observed continuously.The SD rats were injected with 99mTc-DTPA into thalamus and caudate nucleus respectively for biological distribution in vivo.They were put to death 4 h later.Their blood and urine were collected.The brain,cerebellum,heart,liver,spleen,lung,and kidney were taken and weighed by γ counter to measure its radioactivity.Results:SPECT/CT imaging results showed that after 99mTc-DTPA was administered through brain ECS,the radioactivity was concentrated in the brain,kidney and bladder.The tracer administered to the left caudate nucleus was preferentially drained to the right cerebellum,while the tracer administered to the right caudate nucleus was preferentially drained to the left cerebellum.There was a phenomenon of"con-tralateral cerebellar dominant drainage"in the caudate nucleus.The thalamic area preferentially drained to the ipsilateral cerebellum after administration.Four hours after administration via ECS,high radioac-tive uptake appeared in urine,cerebellum and brain,followed by blood and kidney.The radioactive up-take values of heart,liver,spleen and lung were low,which were mainly excreted through urinary sys-tem.Conclusion:Intracerebral ECS administration is a promising method of administration,but there are significant differences in distribution and clearance in different brain regions.This study further ex-pands the content and significance of"ECS regions",and also provides an important theoretical founda-tion for the treatment of encephalopathy and the research of new drugs through brain ECS in the future.

Objective:To evaluate the efficacy of preoperative prediction of regional lymph node (RLN) metastasis in renal cell carcinoma (RCC) using a machine learning model based on habitat imaging radiomics from renal MRI.Methods:This cross-sectional study retrospectively analyzed 220 patients with RCC who underwent nephrectomy and RLN dissection at four medical centers of Chinese PLA General Hospital from January 2010 to August 2023. The cohort included 65 patients with RLN metastasis and 155 without. A stratified random sampling method was used to divide 175 patients from the first medical center into a training set ( n=140) and an internal test set ( n=35) in an 8∶2 ratio, while 45 patients from the third, fourth, and fifth medical centers constituted the external test set. The primary RCC lesions were categorized into 15 habitat subregions based on corticomedullary-phase enhancement and T 2WI signal intensity on MRI, and the volume fractions of different subregions were analyzed. In the training cohort, radiomics features derived from the habitat subregions were used to construct a radiomics model employing various machine learning algorithms, including extremely random trees (ET), gradient boosting decision trees (GBDT), random forest (RF), and support vector machine (SVM). The optimal model was selected and combined with RLN short-axis diameter to develop a combined model. The efficacy of each model in predicting RLN metastasis was evaluated using the receiver operating characteristic (ROC) curve. Results:The volume fraction of hyper-enhanced hyper-intense regions in the non-metastatic group was significantly higher than that in the metastatic group (0.05±0.09 vs. 0.02±0.03; t=3.00, P=0.003). Among the machine learning models constructed using 15 optimal habitat radiomics features, the SVM model demonstrated the best performance, with area under the ROC curve (AUC) values of 0.85 (95% CI 0.72-0.98) in the internal test set and 0.82 (95% CI 0.67-0.98) in the external test set, surpassing those of the ET, GBDT, and RF models. The combined model, integrating the SVM model with RLN short-axis diameter, achieved AUC values of 0.94 (95% CI 0.85-1.00) in the internal test set and 0.89 (95% CI 0.78-1.00) in the external test set, with RLN short-axis diameter contributing AUC values of 0.81 (95% CI 0.66-0.96) and 0.81 (95% CI 0.68-0.94), respectively. The diagnostic sensitivity of the combined model was 91.7% in the internal test set and 85.7% in the external test set, with specificities of 78.3% and 67.7%, respectively. Conclusion:The combined model based on MRI habitat imaging radiomics and RLN short-axis diameter demonstrates excellent preoperative assessment capability for RLN metastasis in RCC.