Objective: To explore the metabolic molecular mechanism of regulatory T cells (Tregs) resisting ferroptosis under a hypoxic stress microenvironment, and to provide scientific support for finding intervention targets to improve the immune microenvironment of oral squamous cell carcinoma (OSCC). Methods: This study was approved by the Institutional Medical Ethics Committee. Multiplex immunofluorescence was used to detect the distribution of Tregs and the localization of hypoxic regions in clinical pathological specimens from patients with OSCC tissues. Animal experiments were approved by the Laboratory Animal Ethics Committee. A tumor-bearing model was established in C57BL/6 mice. The submandibular, subscapular, inguinal, and popliteal lymph nodes were collected along with the spleen, and were homogenized to prepare single cell suspensions. C57BL/6 mouse naive CD4⁺ T cells were induced to differentiate into Tregs in vitro, and a 1% hypoxic culture model was established. Normoxia and hypoxia groups were established, and Treg viability under hypoxic conditions was assessed by flow cytometry. Transcriptomics and metabolomics were applied to analyze differentially expressed genes, metabolic pathways, and changes in cholesterol metabolites. Western blot was performed to detect the expression of emopamil-binding protein (EBP), glutathione peroxidase 4 (GPX4), ferroptosis suppressor protein 1 (FSP1), and acyl-CoA synthetase long-chain family member 4 (ACSL4). The safe concentration of the EBP inhibitor TASIN-30 was determined by the CCK-8 assay. On this basis, four groups were established: normoxia control, hypoxia, hypoxia+TASIN-30, and hypoxia+TASIN-30+7-dehydrocholesterol (7-DHC). Treg viability in each group was measured by flow cytometry; intracellular Fe2+ content, lipid peroxidation level, and reactive oxygen species (ROS) accumulation were detected by cellular immunofluorescence and flow cytometry; the reduced/oxidized glutathione (GSH/GSSG) ratio was measured using a microplate reader; and mitochondrial morphology was observed by transmission electron microscopy. In vivo tumor-inhibition experiments in C3H/HeJ mice were performed to validate the in vivo effects of EBP inhibition, and clinical prognostic survival analysis based on the TCGA-HNSC cohort was performed to evaluate the clinical prognostic value of ferroptosis resistance in Tregs. Results: Tregs were highly enriched in OSCC tissue, preferentially accumulated in hypoxic areas, and could maintain cell survival under hypoxia. The cholesterol metabolism pathway was upregulated in the hypoxic group, with increased EBP mRNA expression (P < 0.001) and 7-DHC accumulation. Compared with the hypoxic group alone, the hypoxic + TASIN-30 group showed decreased GPX4 and FSP1 expression (P < 0.001, P = 0.003); increased ACSL4 expression (P < 0.001); a decreased viable cell ratio (P < 0.001); increased Fe2+, lipid peroxidation, and ROS levels (P < 0.001); a decreased GSH/GSSG ratio (P < 0.001); and mitochondrial shrinkage. Exogenous supplementation of 7-DHC reversed the effects of EBP inhibition. In vivo inhibition of EBP significantly inhibited tumor growth (P < 0.001). Conclusion Tregs upregulate EBP and promote 7-DHC accumulation in hypoxic microenvironments, thereby inhibiting Treg ferroptosis and maintaining their survival and immunosuppressive function. Inhibition of EBP can weaken the resistance of Tregs to ferroptosis under hypoxia.

ObjectiveTo investigate the effects of Liuwei Dihuangwan on mammalian target of rapamycin （mTOR）-mediated synaptic plasticity in the prefrontal cortex-hippocampus circuit in the rat model of valproic acid （VPA）-induced autism spectrum disorder （ASD） and to explore its potential mechanism for reducing behavioral abnormalities in ASD rats. MethodsThirteen SD pregnant rats were randomized into the VPA group （10 rats） and the control group （3 rats）. On gestational day 13， the VPA group was intraperitoneally injected with VPA solution （600 mg·kg^-1）， while the control group was injected with an equal volume of normal saline. Male pups of 21 days old from both groups underwent behavioral tests to screen the ASD model rats （32 rats）. These rats were then randomly assigned to the model （distilled water）， vitamin D （1 480 U·kg^-1）， low-dose （0.75 g·kg^-1） Liuwei Dihuangwan， and high-dose （1.5 g·kg^-1） Liuwei Dihuangwan groups， and normal male pups were selected as the blank group （distilled water）， with 8 rats in each group. Each group was treated with the corresponding drug or an equal volume of distilled water via gavage daily. After drug administration， behavioral tests were conducted again， followed by anesthesia and tissue collection. Nissl staining was carried out to observe the morphological changes of neurons in the prefrontal cortex and hippocampus. Transmission electron microscopy was used to observe changes in hippocampal synaptic structure. Immunohistochemistry and Western blot were employed to determine the expression of mTOR， phosphatidylinositol 3-kinase （PI3K）， and protein kinase B （Akt） in the prefrontal cortex， as well as synaptophysin-1 （SYP-1）， postsynaptic density 95 （PSD95）， and N-methyl-D-aspartate receptors （NMDARs） in the hippocampus. ResultsCompared with the control group， the model group exhibited severe growth retardation and developmental deformities， with abnormal behavioral performance and pathological damage to neurons and synapses in the prefrontal cortex and hippocampus. Compared with the model group， all treatment groups showed reduced behavioral abnormalities and pathological damage to neurons and synapses in the prefrontal cortex and hippocampus， with the most significant effects in the high-dose Liuwei Dihuangwan group. Immunohistochemistry and Western blotting results showed that compared with the control group， the model group exhibited significantly increased protein levels and phosphorylation ratios of p-mTOR， p-Akt， and p-PI3K in the prefrontal cortex （P<0.05， P<0.01）， and decreased protein levels of SYP-1 and PSD95 and increased protein level of NMDARs in the hippocampus （P<0.01）. Compared with those in the model group， the protein levels of p-mTOR， p-Akt， and p-PI3K in the prefrontal cortex decreased in the vitamin D and high-dose Liuwei Dihuangwan groups （P<0.05）， and the p-mTOR/mTOR， p-Akt/Akt， and p-PI3K/PI3K ratios in the prefrontal cortex declined in the vitamin D group （P<0.05）. The high-dose Liuwei Dihuangwan group showed a reduction in p-PI3K/PI3K ratio （P<0.05）. The protein level of SYP-1 and PSD95 in the hippocampus increased in the vitamin D， low-dose Liuwei Dihuangwan， and high-dose Liuwei Dihuangwan groups （P<0.05， P<0.01）， while the protein level of NMDARs decreased in the vitamin D and high-dose Liuwei Dihuangwan groups （P<0.01）. ConclusionLiuwei Dihuangwan significantly reduces VPA-induced ASD-like behaviors and enhances the regeneration and repair of neurons in the prefrontal cortex and synaptic structures in the hippocampus by downregulating of PI3K/Akt/mTOR expression in the prefrontal cortex， upregulating SYP-1 and PSD95 expression in the hippocampus， and inhibiting NMDARs expression.

OBJECTIVE: To identify and validate novel biomarkers for the early diagnosis of sepsis-associated acute kidney injury (SA-AKI) and precise continuous renal replacement therapy (CRRT) using proteomics. METHODS: A prospective multicenter cohort study was conducted. Patients with sepsis admitted to five hospitals in Taizhou City of Zhejiang Province from April 2019 to December 2021 were continuously enrolled, based on the occurrence of acute kidney injury (AKI). Sepsis patients were divided into SA-AKI group and non-SA-AKI group, and healthy individuals who underwent physical examinations during the same period were used as control (NC group). Peripheral blood samples from participants were collected for protein mass spectrometry analysis. Differentially expressed proteins were identified, and functional enrichment analysis was conducted on these proteins. The levels of target proteins were detected by enzyme linked immunosorbent assay (ELISA), and the predictive value of target protein for SA-AKI were evaluated by receiver operator characteristic curve (ROC curve). Additionally, sepsis patients and healthy individuals were selected from one hospital to externally verify the expression level of the target protein and its predictive value for SA-AKI, as well as the accuracy of CRRT treatment. RESULTS: A total of 37 patients with sepsis (including 19 with AKI and 18 without AKI) and 31 healthy individuals were enrolled for proteomic analysis. Seven proteins were identified with significantly differential expression between the SA-AKI group and non-SA-AKI group: namely cystatin C (CST3), β ₂-microglobulin (β ₂M), insulin-like growth factor-binding protein 4 (IGFBP4), complement factor I (CFI), complement factor D (CFD), CD59, and glycoprotein prostaglandin D2 synthase (PTGDS). Functional enrichment analysis revealed that these proteins were involved in immune response, complement activation, coagulation cascade, and neutrophil degranulation. ELISA results demonstrated specific expression of each target protein in the SA-AKI group. Additionally, 65 patients with sepsis (38 with AKI and 27 without AKI) and 20 healthy individuals were selected for external validation of the 7 target proteins. ELISA results showed that there were statistically significant differences in the expression levels of CST3, β ₂M, IGFBP4, CFD, and CD59 between the SA-AKI group and non-SA-AKI group. ROC curve analysis indicated that the area under the curve (AUC) values of CST3, β ₂M, IGFBP4, CFD, and CD59 for predicting SA-AKI were 0.788, 0.723, 0.723, 0.795, and 0.836, respectively, all exceeding 0.7. Further analysis of patients who underwent CRRT or not revealed that IGFBP4 had a good predictive value, with an AUC of 0.84. CONCLUSIONS Based on proteomic analysis, CST3, β ₂M, IGFBP4, CFD, and CD59 may serve as potential biomarkers for the diagnosis of SA-AKI, among which IGFBP4 might be a potential biomarker for predicting the need for CRRT in SA-AKI patients. However, further clinical validation is required.
Humans ; Sepsis/complications* ; Acute Kidney Injury/blood* ; Proteomics ; Prospective Studies ; Biomarkers/blood* ; Male ; Female ; beta 2-Microglobulin/blood* ; Middle Aged ; Cystatin C/blood* ; Aged

Objective To investigate the expression characteristics of granzyme B(GZMB)and C-X-C motif chemokine ligand 9(CXCL9)in gastric cancer(GC)tissues,explore their association with the tumor immune microenvironment(TME),and evaluate their clinical prognostic significance.Methods Bioinformatic analyses were conducted using public databases(TIMER,GEPIA,UALCAN)to validate the expression levels of GZMB and CXCL9 across various cancer types,with a focus on GC.Paired tumor and adjacent normal tissue samples were collected from 89 GC patients who underwent radical gastrectomy at Nanyang First People's Hospital between 2018 and 2019.The mRNA expression levels of GZMB and CXCL9 were measured using quantitative real-time PCR(qRT-PCR),and their associations with clinicopathological characteristics were statistically analyzed.Immune cell infiltration levels were estimated using the TIMER and GSCA databases.Diagnostic performance was evaluated through receiver operating characteristic(ROC)curve analysis,while survival outcomes were assessed using data from the Kaplan-Meier Plotter database and patient follow-up records.Results Both database analysis and qRT-PCR results demonstrated that GZMB and CXCL9 expression levels were significantly elevated in GC tissues compared to adjacent non-tumor tissues(P<0.05).Clinical correlation analysis revealed no significant associations between mRNA expression levels of GZMB and CXCL9 and clinicopathological parameters,including gender,age,pathological type,tumor differentiation,TNM stage,tumor size,or lymph node metastasis(P>0.05).Immune infiltration analysis indicated that both genes were significantly positively correlated with CD8+T cell and dendritic cell infiltration(P<0.05),while showing a negative correlation with B cell infiltration(P<0.05).ROC curve analysis showed that the combined detection of GZMB and CXCL9 yielded an AUC of 0.890(95%CI:0.843～0.936),which was higher than that of GZMB alone(AUC=0.832,95%CI:0.772～0.891)or CXCL9 alone(AUC=0.782,95%CI:0.715～0.850).Survival analysis further revealed that patients with high expression of GZMB and CXCL9 had significantly improved overall survival compared to those with low expression(P<0.05).Conclusion GZMB and CXCL9 are highly expressed in GC and are strongly associated with the infiltration of anti-tumor immune cells.These molecules represent promising diagnostic biomarkers for GC,and their elevated expression is correlated with a more favorable prognosis in patients.

Objective To investigate the expression characteristics of granzyme B(GZMB)and C-X-C motif chemokine ligand 9(CXCL9)in gastric cancer(GC)tissues,explore their association with the tumor immune microenvironment(TME),and evaluate their clinical prognostic significance.Methods Bioinformatic analyses were conducted using public databases(TIMER,GEPIA,UALCAN)to validate the expression levels of GZMB and CXCL9 across various cancer types,with a focus on GC.Paired tumor and adjacent normal tissue samples were collected from 89 GC patients who underwent radical gastrectomy at Nanyang First People's Hospital between 2018 and 2019.The mRNA expression levels of GZMB and CXCL9 were measured using quantitative real-time PCR(qRT-PCR),and their associations with clinicopathological characteristics were statistically analyzed.Immune cell infiltration levels were estimated using the TIMER and GSCA databases.Diagnostic performance was evaluated through receiver operating characteristic(ROC)curve analysis,while survival outcomes were assessed using data from the Kaplan-Meier Plotter database and patient follow-up records.Results Both database analysis and qRT-PCR results demonstrated that GZMB and CXCL9 expression levels were significantly elevated in GC tissues compared to adjacent non-tumor tissues(P<0.05).Clinical correlation analysis revealed no significant associations between mRNA expression levels of GZMB and CXCL9 and clinicopathological parameters,including gender,age,pathological type,tumor differentiation,TNM stage,tumor size,or lymph node metastasis(P>0.05).Immune infiltration analysis indicated that both genes were significantly positively correlated with CD8+T cell and dendritic cell infiltration(P<0.05),while showing a negative correlation with B cell infiltration(P<0.05).ROC curve analysis showed that the combined detection of GZMB and CXCL9 yielded an AUC of 0.890(95%CI:0.843～0.936),which was higher than that of GZMB alone(AUC=0.832,95%CI:0.772～0.891)or CXCL9 alone(AUC=0.782,95%CI:0.715～0.850).Survival analysis further revealed that patients with high expression of GZMB and CXCL9 had significantly improved overall survival compared to those with low expression(P<0.05).Conclusion GZMB and CXCL9 are highly expressed in GC and are strongly associated with the infiltration of anti-tumor immune cells.These molecules represent promising diagnostic biomarkers for GC,and their elevated expression is correlated with a more favorable prognosis in patients.

Objective:To explore the impact of sleep duration, physical exercise, and their interactions on the risk of dyslipidemia in older adults aged ≥80 (the oldest old) in China.Methods:The study subjects were the oldest old from four rounds of Healthy Aging and Biomarkers Cohort Study (2008-2009, 2011-2012, 2014 and 2017-2018). The information about their demographic characteristics, lifestyles, physical examination results and others were collected, and fasting venous blood samples were collected from them for blood lipid testing. Competing risk model was used to analyze the causal associations of sleep duration and physical exercise with the risk for dyslipidemia. Restricted cubic spline (RCS) function was used to explore the dose-response relationship between sleep duration and the risk for dyslipidemia. Additive and multiplicative interaction model were used to explore the interaction of sleep duration and physical exercise on the risk for dyslipidemia.Results:The average age of 1 809 subjects was (93.1±7.7) years, 65.1% of them were women. The average sleep duration of the subjects was (8.0±2.5) hours/day, 28.1% of them had sleep duration for less than 7 hours/day, and 27.2% had sleep for duration more than 9 hours/day at baseline survey. During the 9-year cumulative follow-up of 6 150.6 person years (follow-up of average 3.4 years for one person), there were 304 new cases of dyslipidemia, with an incidence density of 4 942.6/100 000 person years. The results of competitive risk model analysis showed that compared with those who slept for 7-9 hours/day, the risk for dyslipidemia in oldest old with sleep duration >9 hours/day increased by 22% ( HR=1.22, 95% CI: 1.07-1.39). Compared with the oldest old having no physical exercise, the risk for dyslipidemia in the oldest old having physical exercise decreased by 33% ( HR=0.67, 95% CI: 0.57-0.78). The RCS function showed a linear positive dose-response relationship between sleep duration and the risk for hyperlipidemia. The interaction analysis showed that physical exercise and sleep duration had an antagonistic effect on the risk for hyperlipidemia. Conclusion:Physical exercise could reduce the adverse effects of prolonged sleep on blood lipids in the oldest old.

Objective To analyze the characteristics of platelet changes and their influencing factors during postoperative hospitalization in patients who underwent transcatheter aortic valve implantation (TAVI). Methods The patients who underwent TAVI at Beijing Anzhen Hospital Valve Surgery Center between March 2017 and October 2021 were retrospectively selected. The patients were divided into a self-limiting group and a non-self-limiting group according to the characteristics of postoperative platelet decline. In addition, the general preoperative data, preoperative and postoperative ultrasound data, intraoperative data, and the use of anticoagulant drugs during the postoperative stay in the hospital were compared between the two groups. Results A total of 249 patients were enrolled in this study. There were 175 (70.3%) patients in the self-limiting group, including 100 males and 75 females, and there were 74 (29.7%) patients in the non-self-limiting group, including 43 males and 31 females, with no statistical difference between the two groups (P=0.863). The mean age of patients was 73.11±8.88 years in the self-limiting group and 71.54±10.39 years in the non-self-limiting group (P=0.231). The decline of platelets in the self-limiting group generally occurred on the postoperative day 2 and reached the lowest count on the postoperative day 4, and returned to the baseline level on the postoperative day 5-7, while the platelets in the non-self-limiting group changed by simple rise, fall or irregular fluctuation. Patients in the self-limiting group had severer preoperative aortic stenosis (P<0.001) and used more extracorporeal circulation assistance during surgery (P<0.001). Postoperatively, patients in the self-limiting group were more likely to have periaortic valve leakage than those in the non-self-limiting group (P=0.013). Conclusion Platelet changes in most patients after TAVI show a self-limiting decline, which may be related to the severity of patients’ preoperative aortic stenosis, intraoperative extracorporeal circulation device use, and postoperative perivalvular leakage.

Osteoporosis can be induced by various factors including prolonged glucocorticoid usage, diminished estrogen levels, secondary hyperparathyroidism, and alterations in the microenvironment of bone tissue. The bone metabolism imbalance(osteogenic-lipogenic imbalance) plays a crucial role in the development of osteoporosis. This imbalance is primarily driven by an increase in the differentiation of bone marrow mesenchymal stem cells into adipocytes and a decrease in their differentiation into osteoblasts, thus forming the core of the osteogenic-lipogenic imbalance observed in osteoporosis. The bone morphogenesis protein(BMP) plays a crucial role in the regulation of the osteogenic-lipid balance in osteoporosis. This regulatory function is accomplished through both the Smad-dependent and Smad-independent pathways. This review centers on the Smad-dependent and Smad-independent pathways facilitated by BMP, offering a comprehensive overview of the potential mechanisms through which BMP-2, 4, 6, 7, and 9 contribute to the regulation of osteogenesis and lipid metabolism in osteoporosis via these pathways. In order to present novel insights for the identification of efficacious targets for clinical anti-osteoporosis medications.

Objective:To investigate the distribution characteristics of skeletal muscle mass and strength in the older adults over 65 years old in 18 longevity areas in China.Methods:The subjects were selected from the Healthy Aging and Biomarkers Cohort Study conducted in 18 longevity areas of China. A total of 4 662 older adults over 65 years old from a cross- sectional survey in 2021 were included in the study. The information about their sociodemographic characteristics, lifestyle, nutrient intake and other factors were collected through questionnaire surveys and physical examinations. Grip strength was measured by using professional electronic grip dynamometer. Total skeletal muscle mass (TSM) was measured using bioelectrical impedance analysis, and TSM was adjusted by height squared and BMI to obtain TSM Ht2 and TSM BMI. The proportion of individuals with low muscle mass and strength was determined according to the recommended method by the Asian Working Group for Sarcopenia (AWGS). Descriptive analysis was conducted on the population and regional distribution characteristics of people with different muscle mass and grip strength. A generalized additive model was used to analyze the age-related trends of muscle mass and grip strength. Results:The age of 4 662 study subjects was (82.69±10.54) years, men accounted for 46.85% (2 184 cases) and Han Chinese accounted for 96.27% (4 488 cases). The M( Q1, Q3) of TSM, TSM Ht2 and TSM BMI in men were 23.30 (20.50, 26.20) kg, 9.02 (8.13, 9.89) kg/m 2, and 1.01 (0.90, 1.13) kg·(kg/m 2) -1, respectively, which were all higher than those in women [TSM: 18.20 (15.70, 20.70) kg, TSM Ht2: 8.18 (7.42, 9.07) kg/m 2 and TSM BMI: 0.79 (0.69, 0.90) kg·(kg/m 2) -1], the differences were significant (all P<0.001). The grip strength of men [ M( Q1, Q3): 24.50 (17.80, 30.80) kg] was higher than that of women [ M( Q1, Q3): 15.60 (11.10, 19.90) kg], the difference was significant ( P<0.001). Southern elderly men had lower TSM and TSM Ht2 compared with northern elderly men (all P<0.001), while there was no significant regional difference in TSM BMI ( P>0.05). Southern elderly women had higher TSM Ht2 and TSM BMI compared with northern elderly women (all P<0.001), while there was no significant regional difference in TSM ( P>0.05). Furthermore, according to the method recommended by AWGS, the elderly with low muscle mass and grip strength were characterized by older age, illiteracy, being unmarried/divorced/widowed, poor chewing ability, impaired activity of daily living and living in southern region. Conclusion:There were population and regional differences in muscle mass and grip strength in the older adults over 65 years in 18 longevity areas of China, and these differences showed decreasing trends with age.