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.

Objective To explore the influence of cardiac electrophysiological(EP)mode on image quality and radiation dose in performing hysterosalpingography(HSG)with digital subtraction angiography(DSA).Methods The clinical data of 97 infertility patients,who were admitted to the Jinhua Municipal People's Hospital of China from October to November of 2024,were prospectively collected.Among them,50 patients who received treatment in October of 2024 by using DSA abdominal acquisition mode were collected as the control group,and other 47 patients who received treatment in November of 2024 by using EP acquisition mode were collected as the study group.The image quality and radiation dose of the two groups were analyzed.Results The patients' image quality of both groups met the diagnostic requirements,and the diagnostic conclusions made by two physicians were consistent.No statistically significant differences in the positive rate,number of spot shot films,radiation dose of spot shot films,and continuous exposure time existed between the two groups(P＞0.05).Compared with the control group,in the study group the number of images increased by 8.3 times,the continuous exposure radiation dose was decreased by 90.0％,and the total radiation dose was decreased by 83.0％.The differences in the above indexes between the two groups were statistically significant(P＜0.05).Conclusion In performing HSG with DSA,the use of EP mode for image acquisition can meet the diagnostic requirements,besides it can effectively reduce the radiation dose while increasing the number of image frames,therefore,this technique is worthy of clinical promotion.

Objective:To investigate the curative effect and mechanism of hyperbaric oxygen therapy on nonalcoholic fatty liver disease in mice.Methods:Twenty-one 8-week-old male C57BL/6J mice were divided into three groups: control group (normal diet), model group (high-fat and high-cholesterol diet), and hyperbaric oxygen group (high-fat and high-cholesterol diet + hyperbaric oxygen therapy), with seven mice in each group. The changes in body weight, serum liver enzymes, and blood lipids were compared after treatment between the three groups. Hematoxylin-eosin staining, Oil Red O staining, Sirius red staining, and F4/80 immunohistochemical staining were used to observe the pathological changes in liver tissues. RT-qPCR and Western blot methods were used to detect the expression levels of oxidative stress and inflammatory factors. One-way analysis of variance was used for comparison among the groups.Results:Mice in the hyperbaric oxygen group had significantly improved liver histopathology. The serological levels of alanine aminotransferase, aspartate aminotransferase, and cholesterol were (77.50±13.59) U/L, (156.06±23.68) U/L, and (4.80±0.53) mmol/L, which were significantly lower than those in the model group [(109.43±16.88) U/L, (216.62±18.79) U/L, and (5.86±0.53) mmol/L, P<0.05], and accompanied by lower levels of lipid deposition, macrophage infiltration, and fibrosis. In addition, compared with the model group, the expression of antioxidant stress protein nuclear transcription factor erythroid 2-related factor 2 [(0.30±0.06) and (2.16±1.21), P<0.05] and heme oxygenase-1 [(0.48±0.19) and (1.01±0.18), P<0.05] in liver tissue showed an upward trend following hyperbaric oxygen treatment, which was also validated at the transcriptional level ( P<0.05). Simultaneously, compared with the model group, the mRNA expressions of tumor necrosis factor-α [(2.60±0.71) and (0.66±0.15), P<0.05], interleukin-1β [(2.41±1.01) and (0.78±0.23), P<0.05], and interleukin-6 [(3.61±2.17) and (0.94±0.25), P<0.05] in the liver tissue of mice in the hyperbaric oxygen group were decreased. The tumor necrosis factor-α protein level [(7.50±4.73) and (1.05±0.58), P<0.05] and interleukin-1β [(1.65±0.35) and (1.02±0.02), P<0.05] was reduced following hyperbaric oxygen treatment compared with those in the model group. Conclusion:Hyperbaric oxygen therapy can slow down the progression of nonalcoholic fatty liver disease by regulating the levels of oxidative stress and inflammation in the mice.

AIM:To observe the neuroprotective effects of total saponins of Trillium tschonoskii Maxim(TST)on rats with vascular dementia(VD)and explore the drug's impact on astrocytes and the Sonic Hedgehog(Shh)pathway as well as its mechanisms of action.METHODS:A rat model of vascular dementia was established by bilateral common carotid artery occlusion.Subsequently,the rats were randomly divided into four groups:donepezil hydrochloride group,TST group,model group,and sham group,with 18 rats in each group.After 5 weeks of gavage,samples were collected.Cognitive function was evaluated by the water maze test.Histopathological changes in brain tissue were examined with HE and Nissl staining.The ultrastructure of astrocytes was analyzed by transmission electron microscopy.The localization and expression of the neuronal nuclear antigen(NeuN),glial fibrillary acidic protein(GFAP),Shh,and glioma-associated oncogene homolog 1(Gli1)were identified using immunohistochemistry.Immunofluorescence was also employed to exam-ine the expression of NeuN and GFAP.Finally,Western blot analysis was used to measure the protein levels of NeuN,GFAP,Shh,Patched 1(Ptch1),Gli1,Bax,Bcl-2,tumor necrosis factor-α(TNF-α),and interleukin-10(IL-10)in the hippocampus.RESULTS:Compared to the sham group,the model rats demonstrated prolonged escape latency,disorga-nized and loosened neuronal arrangement in the hippocampus and cortex,reduced Nissl bodies,and significant neuronal damage.Astrocytes displayed chromatin aggregation,swollen mitochondria with disrupted cristae structures,and swollen endoplasmic reticulum regions.The expression of NeuN,Shh,and Gli1 positive cells significantly decreased,while GFAP positive cells significantly increased.Additionally,the protein levels of NeuN,Shh,Ptch1,Gli1,IL-10,and Bcl-2 in the hippocampus were reduced(P＜0.05),whereas those of GFAP,Bax,and TNF-α were elevated(P＜0.01).Com-pared to the model group,the donepezil hydrochloride and TST groups effectively improved the aforementioned indicators.CONCLUSION:Total saponins of trillium tschonoskii maxim can effectively alleviate pathological damage to neurons in VD rats,thereby improving learning and memory abilities.The underlying mechanisms may involve inhibiting the overacti-vation of astrocytes,activate the Shh/Ptch1/Gli1 signaling pathway,suppress neuroinflammation,and reduce neuronal apoptosis.

Selenium is an essential trace element for human body.The primary functional form of selenium in the organism is selenoproteins(Sep),which plays a crucial role in maintaining normal physiological functions,influencing immune responses,and is also implicated in tumorigenesis and related metabolic disorders.Recent studies have shown that Sep is involved in the pathophysiological process of inflammatory bowel disease(IBD).This article reviewed the mechanisms of Sep in IBD,aiming to explore the pathogenic mechanisms of IBD and provide insights for the diagnosis and treatment of the disease.

Objective To investigate the key targets and pathways of Juhongtai formula granules in improving acute lung injury(ALI).Methods Juhongtai formula granules and their drug-containing serum components were analyzed by UPLC-QTOF-MS/MS,and the active ingredients were screened based on the"Lipinski Rule of Five".The action targets of the above active ingredients were obtained through the TCMSP,Swiss Target Prediction and SuperPred databases,and the ALI-related targets were obtained from the GeneCards,OMIM,PharmGKB,CTD databases and GEO chip.The target protein-protein interaction network was constructed using the String database and Cytoscape to analyze the key targets.The DAVID database was used for GO functional annotation and KEGG pathway enrichment analysis,and the CB-dock2 platform was used for molecular docking verification.After one week of adaptive feeding,the experimental SD rats were randomly divided into the blank group,the model group,the positive control group,the negative control group and the Juhongtai formula granules group,with 6 rats in each group.The rat model of ALI was replicated by tracheal infusion of lipopolysaccharide.The pathological morphology of lung tissues in each group of rats was observed by HE staining,and the mRNA expressions of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1 β(IL-1β)and nitric oxide were detected by reverse transcription quantitative polymerase chain reaction(RT-qPCR).Results In total,44 components of the Juhongtai formula granules were identified,including 26 drug-containing serum components,and 21 key compounds were screened.A total of 22 intersection targets were obtained.The pathway enrichment results indicated the action of these targets on the advanced glycation end product receptor(AGE-RAGE)and TNF signaling pathways.The docking results showed that limonin,kaempferol and naringenin could be matched with prostaglandin peroxidase 2,nitric oxide synthase 2 and TNF.Animal experiments confirmed that the Juhongtai formula granules can alleviate inflammatory infiltration in lung tissue and reduce the expressions of TNF-α,IL6,IL-1β mRNA and nitric oxide.Conclusion Juhongtai formula granules can inhibit ALI-related inflammatory targets through the synergistic effects of multiple components such as limonin and kaempferol,regulate signaling pathways such as AGE-RAGE and TNF,reduce the production of inflammatory factors and nitric oxide,and improve ALI.

OBJECTIVE: To explore the clinical features of the sepsis in immunocompromised hosts and establish an early warning equation. METHODS: A retrospective study was conducted on sepsis patients admitted to the intensive care unit (ICU) of Binzhou Medical University Hospital from October 2011 to October 2022. General information, infection site, etiology results and drug susceptibility, clinical symptoms, inflammatory indicators, acute physiology and chronic health status evaluation II (APACHE II), sequential organ failure assessment (SOFA), incidence of immune paralysis, and outcome during hospitalization were collected. Based on whether they met the diagnostic criteria for immunocompromised hosts, patients were divided into immunocompromised group and immune normal group. The clinical information of the two groups were compared. Multivariate Logistic regression was used to analyze the risk factors of patients with immunocompromised sepsis and the regression equation model was initially established. Omnibus test and Hosmer-Lemeshow test were used to evaluate the model. RESULTS: A total of 169 patients with sepsis were included, including 61 in the immunocompromised group and 108 in the normal immune group. The top 3 infection sites in the immunocompromised group were bloodstream infection, pulmonary infection and abdominal infection. The top 3 infection sites in the normal immune group were pulmonary infection, bloodstream infection and abdominal infection. The infection rate of Gram-negative bacteria in the immunocompromised group was significantly lower than that in the normal group [49.2% (30/61) vs. 64.8% (70/108), P < 0.05]. The infection rate of Gram-positive bacteria [27.9% (17/61) vs. 13.9% (15/108)] and multidrug-resistant bacteria [54.1% (33/61) vs. 29.6% (32/108)] were significantly higher than those in normal immune group (both P < 0.05). In terms of clinical symptoms, the proportion of fever in the immunocompromised group was significantly lower than that in the immune normal group [49.2% (30/61) vs. 66.7% (72/108), P < 0.05]. Neutrophil count (NEU) and neutrophil percentage (NEU%) in the immunocompromised group were significantly lower than those in the normal immune group. Lymphocyte percentage (LYM%), neutrophil/lymphocyte ratio (NLR), C-reactive protein (CRP), procalcitonin (PCT), APACHE II score, combined shock rate, incidence of immune paralysis, and mortality during hospitalization in the immunocompromised group were significantly higher than those in the normal immune group. Logistic regression analysis showed that NLR, CRP and PCT were risk factors for patients with immunocompromised sepsis (all P < 0.05). The above indicators were used as covariables to construct a Logistic regression equation, that was, Logit (P) = 0.025X₁+0.010X₂+0.013X₃-2.945, where X₁, X₂ and X₃ represent NLR, CRP and PCT respectively. Omnibus test and Hosmer-Lemeshow test show that the model fits well and has certain early warning value. CONCLUSIONS Patients with immunocompromised sepsis have more intense inflammatory response, with Gram-negative bacteria being the predominant pathogen, and a higher incidence of Gram-positive bacterial infections and multi-drug resistant infections. The severity of the disease, in-hospital mortality, the incidence of shock and the incidence of immune paralysis after sepsis were significantly higher. NLR, CRP and PCT were independent risk factors for sepsis in immunocompromised hosts. The regression equation constructed based on this may have early warning significance for patients with immunocompromised sepsis.
Humans ; Sepsis/immunology* ; Immunocompromised Host ; Retrospective Studies ; Risk Factors ; Intensive Care Units ; Logistic Models ; Male ; APACHE ; Female ; Middle Aged ; Aged

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

BACKGROUND:Acute exercise tends to cause skeletal muscle tissue damage and lipid metabolism disorders in vivo,but the mechanism by which acute exercise combined with electroacupuncture modulates metabolic and autophagic pathways in vivo is unclear. OBJECTIVE:To observe the changes in metabolic enzymes and autophagy levels in skeletal muscle of rats subjected to acute exercise by electroacupuncture at the acupoints of"Zusanli"and"Huantiao." METHODS:Fifty male Sprague-Dawley rats were randomly divided into three groups:quiet control group(n=10),model group(n=20),and reverse electroacupuncture group(n=20).The latter two groups were set up with two time points,i.e.immediate and 3 hours after exercise groups(n=10 per time point).The model group and the reverse electroacupuncture group underwent acute exercise training after adaptive treadmill training.The rats in the reverse electroacupuncture group underwent electroacupuncture treatment(parameters:electroacupuncture on both sides of the rats at the acupoints of"Zusanli"and"Huantiao,"continuous wave,frequency of 2 Hz,intensity of 2 mA,leaving the needle in the body for 30 minutes,once a day for 7 consecutive days)before treadmill training.Bilateral gastrocnemius muscle tissues were taken under anesthesia immediately after exercise and 3 hours after exercise,and hematoxylin-eosin staining was used to observe the histopathological changes of rat skeletal muscle.ELISA kit was used to detect the activities of hepatic lipase,fatty acid synthase,hormone-sensitive lipase,and carnitine palmitoyltransferase 1 in rat skeletal muscle tissues.Immunohistochemistry and western blot were used to detect the changes in the expression of autophagy genes. RESULTS AND CONCLUSION:After hematoxylin-eosin staining,the arrangement of gastrocnemius muscle fibers in the model group was disturbed,swollen and ruptured immediately after exercise and 3 hours after exercise.In the reverse electroacupuncture group,gastrocnemius muscle fibers were tightly arranged and the number of swollen and ruptured cells was greatly reduced immediately after exercise and 3 hours after exercise,and there was no significant difference when compared with the quiet control group.Compared with the quiet control group,the activities of hepatic lipase and fatty acid synthase were lower while the activities of lipoprotein lipase,hormone-sensitive lipase,and carnitine palmitoyltransferase 1 were higher in the model group and the reverse electroacupuncture group 3 hours after exercise(P＜0.05 or P＜0.01).Compared with the model group,the activities of lipoprotein lipase and carnitine palmitoyltransferase 1 were higher in the reverse electroacupuncture group immediately after exercise(P＜0.05),while the activity of lipoprotein lipase was higher and the activity of hormone-sensitive lipase was lower in the reverse electroacupuncture group 3 hours after exercise(P＜0.01).Immunohistochemical results showed that compared with the quiet control group,the expression of P62,autophagy-related gene 5 and autophagy-related gene 7 was higher in the model group immediately and 3 hours after exercise,as well as in the reverse electroacupuncture group immediately after exercise(P＜0.05 or P＜0.01);compared with the model group,the expression of P62 and autophagy-related gene 7 was lower in the reverse electroacupuncture group immediately and 3 hours after exercise(P＜0.05).Western blot results showed that the protein expression of P62 and autophagy-related gene 7 in the reverse electroacupuncture group was lower than that in the model group immediately after exercise(P＜0.05);the protein expression of Parkin in the model group was higher than that in the quiet control group immediately and 3 hours after exercise(P＜0.05);and the protein expression of Parkin in the reverse electroacupuncture group was lower than that in the model group immediately and 3 hours after exercise(P＜0.05).To conclude,acute exercise induces disorders,swelling and rupture of gastrocnemius muscle fibers in rats and electroacupuncture on both sides of the acupoints of"Zusanli"and"Huantiao"can improve the level of lipid metabolism and regulate autophagy cells in rat skeletal muscle,preventing the disorders of lipid metabolism and damage of gastrocnemius muscle tissues caused by acute exercise.The mechanism may be closely related to the regulation of autophagy-related factor P62,autophagy-related gene 5,autophagy-related gene 7,and Parkin protein expression to promote the occurrence of autophagy or regulate the autophagy pathway in rat skeletal muscle cells.

Objective To investigate the key targets and pathways of Juhongtai formula granules in improving acute lung injury(ALI).Methods Juhongtai formula granules and their drug-containing serum components were analyzed by UPLC-QTOF-MS/MS,and the active ingredients were screened based on the"Lipinski Rule of Five".The action targets of the above active ingredients were obtained through the TCMSP,Swiss Target Prediction and SuperPred databases,and the ALI-related targets were obtained from the GeneCards,OMIM,PharmGKB,CTD databases and GEO chip.The target protein-protein interaction network was constructed using the String database and Cytoscape to analyze the key targets.The DAVID database was used for GO functional annotation and KEGG pathway enrichment analysis,and the CB-dock2 platform was used for molecular docking verification.After one week of adaptive feeding,the experimental SD rats were randomly divided into the blank group,the model group,the positive control group,the negative control group and the Juhongtai formula granules group,with 6 rats in each group.The rat model of ALI was replicated by tracheal infusion of lipopolysaccharide.The pathological morphology of lung tissues in each group of rats was observed by HE staining,and the mRNA expressions of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-1 β(IL-1β)and nitric oxide were detected by reverse transcription quantitative polymerase chain reaction(RT-qPCR).Results In total,44 components of the Juhongtai formula granules were identified,including 26 drug-containing serum components,and 21 key compounds were screened.A total of 22 intersection targets were obtained.The pathway enrichment results indicated the action of these targets on the advanced glycation end product receptor(AGE-RAGE)and TNF signaling pathways.The docking results showed that limonin,kaempferol and naringenin could be matched with prostaglandin peroxidase 2,nitric oxide synthase 2 and TNF.Animal experiments confirmed that the Juhongtai formula granules can alleviate inflammatory infiltration in lung tissue and reduce the expressions of TNF-α,IL6,IL-1β mRNA and nitric oxide.Conclusion Juhongtai formula granules can inhibit ALI-related inflammatory targets through the synergistic effects of multiple components such as limonin and kaempferol,regulate signaling pathways such as AGE-RAGE and TNF,reduce the production of inflammatory factors and nitric oxide,and improve ALI.