Objective: To investigate the causal relationship between neutrophil extracellular traps (NETs) and head and neck squamous cell carcinoma (HNSCC) using Mendelian randomization (MR) methods, and to explore the clinical significance of NETs in the occurrence and development of HNSCC. Methods: Data related to NET biomarker myeloperoxidase-DNA complex (MPO-DNA) complex and HNSCC were obtained from the pooled statistical data of the Genome-Wide Association Study database (GWAS). This study was reviewed and approved by the Medical Ethics Committee, and informed consent was obtained from patients. Patients with HNSCC admitted to Department of Oral Maxillofacial, the First Affiliated Hospital of Harbin Medical University were included as the research group, and volunteers matched for age and gender were randomly selected from the Clinical Examination Center as the control group. The levels of MPO-DNA and citrullinated histone H3 (CitH3), two markers of NETs, as well as the levels of soluble adhesion factor CD44 variant 6 (CD44v6) and leukocyte differentiation antigen CD109, markers of lymph node metastasis, were measured in all subjects. Blood coagulation indicators, including plasma prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), D-dimer (DD), and fibrinogen levels (FIB), were recorded to analyze the relationship and potential mechanisms between NETs and HNSCC. Results: MR results indicated a possible causal relationship between NETs and HNSCC. The inverse variance weighted P values for the four datasets were P1 = 0.037, P2 = 0.017, P3 = 0.004, and P4 = 0.023. Ultimately, 52 patients with head and neck squamous cell carcinoma and 20 healthy individuals were included. Compared with the control group, the expression levels of NETs markers MPO-DNA, CitH3, lymph node metastasis markers CD44v6 and CD109, and coagulation indicators FIB and DD were significantly elevated in the group with head and neck squamous cell carcinoma, with statistically significant differences (all P<0.001). In correlation studies between NETs markers and lymph node metastasis markers, as well as coagulation indicators, the Pearson correlation coefficient was 0.686, 0.531, 0.7, and 0.5 for MPO-DNA and DD, FIB, CD44v6, and CD109, respectively, and the Pearson correlation coefficient was 0.456, 0.503, 0.525, and 0.603 for CitH3 and DD, FIB, CD44v6, and CD109, respectively (P<0.05). In terms of diagnostic efficacy, the area under the curve (AUC) for MPO-DNA, CitH3, and MPO-DNA + CitH3 in patients with head and neck squamous cell carcinoma was 0.863, 0.892, and 0.905, respectively, with an increasing AUC of the receiver operating characteristic curve (ROC) in the order mentioned. Levels of MPO-DNA and CitH3 in patients with early-stage head and neck squamous cell carcinoma were (132.4 ± 16.4) ng/mL and (21.3 ± 2.9) ng/mL, respectively, which were lower than those in patients with advanced head and neck squamous cell carcinoma, who had MPO-DNA and CitH3 levels of (199.3 ± 33.1) ng/mL and (26.6 ± 3.7) ng/mL, respectively. The serum concentrations of FIB, DD, CD44v6, and CD109 in patients with high MPO-DNA expression were significantly higher than those in patients with low MPO-DNA expression (all P<0.05). The serum concentrations of FIB, CD44v6, and CD109 in patients with high CitH3 expression were significantly higher than those in patients with low CitH3 expression (all P<0.05). Conclusion The study indicates a potential causal relationship between NETs and HNSCC. NETs-related markers may serve as potential biomarkers for HNSCC, as they correlate with the hypercoagulable state of the cancer. NETs-related markers have potential diagnostic utility for HNSCC and are associated with tumor progression.

Objective: To investigate the prognostic significance and biological functions of neutrophil extracellular traps (NETs) related genes in oral squamous cell carcinoma (OSCC). Methods: A total of 333 transcriptome datasets and 6 single-cell sequencing datasets of OSCC were retrieved from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Based on 69 NETs related gene sets, univariate Cox and Lasso-Cox regression were used to construct a prognostic risk model for OSCC. The model's efficacy was evaluated through Kaplan-Meier analysis and receiver operating characteristic (ROC) curves, and risk scoring and nomogram analysis were conducted. Further, the relationship between NETs risk scores and angiogenesis, epithelial-mesenchymal transition (EMT), and cell cycle was explored. Enrichment analysis was performed to annotate the functional characteristics of relevant pathways. Kaplan-Meier analysis was employed to screen for prognostic key genes. Candidate targets were validated through drug prediction and molecular docking assays. Single-cell RNA sequencing was utilized to characterize the expression profile of the key gene cathepsin G (CTSG) within the tumor microenvironment (TME). Using pan-cancer and OSCC related data retrieved from the TCGA database, we analyzed the differences in CTSG expression between tumor tissues and normal tissues. Subsequently, immunohistochemical staining experiments were performed on tissue microarrays to validate its expression at the protein level. Results: A prognostic risk model based on six NETs related genes (F3, AKT1, CTSG, VNN3, MPO, and IL17A) was successfully established. Patients in the high-risk group exhibited significantly shorter overall survival (OS) (P < 0.000 1). The area under the ROC curve (AUC) of the established model for predicting 1-, 3-, and 5-year overall survival (OS) rates was 0.718, 0.820, and 0.805, respectively. The NETs related risk score was identified as an independent prognostic factor (P < 0.001), with the constructed nomogram demonstrating good calibration. The NETs related risk score correlated with angiogenesis (r = ˗0.20,, P < 0.001), EMT (r = 0.17, P < 0.01), G1/S phase transition (r = 0.11, P < 0.05), and G2/M phase transition (r = 0.17, P < 0.01). GSEA（gene set enrichment analysis）revealed that the high-risk group was significantly enriched in pathways including basal cell carcinoma, whereas the low-risk group exhibited significant enrichment in pathways such as alpha-linolenic acid metabolism (P < 0.05). Kaplan-Meier analysis revealed that patients with low expression of CTSG had a poorer prognosis (P < 0.001). Molecular docking assays demonstrated a stable binding interaction between CTSG and glutathione (binding energy: -7.4 kcal/mol). Single-cell RNA sequencing analysis further showed that CTSG was highly expressed in mast cell subsets but weakly expressed in malignant cells (P < 0.001). TCGA pan-cancer analysis revealed that CTSG is underexpressed in multiple cancer tissues, including OSCC (P < 0.05). Immunohistochemical staining confirmed that CTSG protein expression was lower in tumor tissues than in paracancerous tissues (P < 0.01). Conclusion The NETs related prognostic model established in this study exhibits robust predictive performance. CTSG was identified as a key prognostic gene, thereby providing a novel biomarker and potential therapeutic target for prognostic evaluation and targeted therapy of OSCC.

Objective : To construct a molecular classification system for head and neck squamous cell carcinoma (HNSCC) utilizing hypoxia-related gene (HAG) expression profiles, and to comprehensively examine the clinicopathological significance and biological functions of the hypoxia gene stanniocalcin 2 (STC2) in HNSCC. Methods : Transcriptomic data and clinical information of 546 HNSCC samples were obtained from The Cancer Genome Atlas (TCGA) database, and based on the expression profiles of 200 HRGs, HNSCC was classified subclasses using non-negative matrix factorization (NMF). HNSCC was classified into three subclasses (C1, C2, and C3), and the molecular characteristics and prognostic differences of the subclasses were assessed by comparing the tumor mutation load, functional enrichment analysis, drug sensitivity, and clinical features among the subclasses. LASSO-Cox regression was used to screen prognosis-related genes and construct prognostic models. Using oral squamous cell carcinoma (OSCC)-related data in the TCGA database, we analyzed the expression differences of STC2 in OSCC and control samples, and detected the mRNA and protein expression of STC2 in oral squamous carcinoma samples using qRT-PCR and immunohistochemistry. We knocked down STC2 in CAL-27 cells and verified the knockdown efficiency by qRT-PCR and Western blot. CCK-8 assay and cell scratch assay were used to assess the effect of STC2 on cell proliferation and migration ability. Results: Based on HRGs expression profiles, HNSCC was categorized into three subclasses (C1, C2, and C3). Subclass C1 had moderate hypoxic activity and good prognosis; subclass C2 had the highest hypoxic activity, poor prognosis, and poor sensitivity to CTLA-4 inhibitors (P<0.05); subclass C3 had the lowest hypoxic activity and moderate prognosis, and STC2 belonged to subclass C3. The frequency of cyclin-dependent kinase inhibitor 2A (CDKN2A), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), and tumor protein p53 (TP 53) mutations was higher in HNSCC. C1 genomic gain and deletion burden were significantly higher than C3 subclass (P<0.05) and C2 genomic gain than C3 subclass (P<0.05). The C2 subclass was significantly enriched in hypoxia-associated pathways, such as glycine metabolism and base excision repair (P<0.05). The C1, C2, and C3 subclasses were significantly positively correlated in terms of sex (male) (Cramer’s V=0.15), radiation exposure (Cramer’s V=0.12), medication (Cramer’s V=0.18), and pathological grading (G1/G2) (Cramer’s V=0.25) (P<0.05). Nine prognosis-related genes were screened by LASSO-Cox regression, among which high expression of STC2 was positively correlated with poorer overall survival (OS) in HNSCC patients (P<0.01). Bioinformatics analysis showed that STC2 mRNA expression was higher in OSCC than in normal controls (P<0.05). qRT-PCR and immunohistochemistry confirmed that both mRNA and protein expression of STC2 were significantly upregulated in OSCC tissues and cells (P<0.01). In vitro experiments showed that STC2 expression was knocked down to approximately 80% in CAL-27 cells (P<0.001), and the STC2 knockdown group had a reduced value-added rate (P<0.001) and a reduced percentage of scratch closure (P<0.05) compared with the control group. Conclusion We successfully constructed a molecular typing system for HNSCC based on the expression profiles of HRGs and categorized HNSCC into three subclasses with significant prognostic differences, among which the C2 subclass had the highest hypoxic activity and the poorest prognosis. STC2 was highly expressed in HNSCC and suggested a poor prognosis, demonstrating that it may be a potential target for HNSCC treatment.

Objective To explore the risk factors of frozen shoulder,and to provide the basis for the prevention of frozen shoulder.Methods A total of 114 patients with frozen shoulder who were hospitalized in the First Affiliated Hospital of Zhejiang Chinese Medical University from January 2020 to August 2022 were included in case group,and 114 physical examination patients with no history of frozen shoulder were included in control group.The clinical data of two groups were collected and the risk factors of frozen shoulder were analyzed by multivariate Logistic regression.Results There were statistically significant differences in cervical radiculopathy,type 2 diabetes mellitus,hepatitis,chronic non-atrophic gastritis,lumbar disc herniation,osteoporosis and thyroid sarcoidosis between two groups(P<0.05).Multivariate Logistic analysis showed that cervical radiculopathy(OR=6.114,95%CI:1.458-25.642)and type 2 diabetes mellitus(OR=24.069,95%CI:4.023-144.007)were independent risk factors for the onset of frozen shoulder.Conclusion Patients with type 2 diabetes mellitus and cervical radiculopathy have a higher risk of frozen shoulder,and should pay attention to early prevention.

Neutrophil extracellular traps(NETs)are fibrous web-like structures composed of decondensed chromatin and granular proteins released by neutrophils,with the ability to capture and kill bacteria.Pathogens,such as bacteria and viruses,can trigger the formation of NETs via NETosis,a type of programmed cell death that has two distinct forms:suicidal NETosis and vital NETosis.Numerous studies have found that NETs interact with immune cells in the tumor microenvironment,where they activate macrophages,promote immunosuppressive effects of myeloid-derived suppressor cells,and coat the tumor surface to prevent cytotoxic effects of CD8+T cells and natural killer cells.Recent research has identified a substantial presence of NETs in oral squamous cell carcinoma(OSCC)tissues,indicating a complex re-lationship between NETs and OSCC development.Depending on the phenotype of neutrophils,NETs may exhibit pro-tu-mor or anti-tumor effects.For instance,NETs derived from N1-type neutrophils may exert anti-tumor effects,while TGF-β-induced NETs derived from N2-type neutrophils may exert pro-carcinogenic activity,thereby contributing to the devel-opment of oral squamous metaplasia.Furthermore,NETs likely play a role in OSCC metastasis by capturing circulating tumor cells and inducing a hypercoagulable state,thereby facilitating tumor-related thrombus formation and hematoge-nous metastasis.The involvement of NETs in the occurrence and progression of OSCC opens new avenues for anti-tumor therapy and prognostication.Inhibiting NET formation can significantly suppress the development of chemotherapy-in-duced drug resistance and reduce the risk of thrombosis in OSCC patients,thereby inhibiting tumor metastasis.Current-ly,multiple prognostic models based on NET-related genes have been constructed and validated for head and neck squa-mous cell carcinoma,indicating the potential clinical value of NETs.However,the association between NETs and OSCC treatment is still unclear,necessitating further research on its underlying mechanisms and feasibility.This article attempts to review the relationship between NETs and OSCC,aiming to provide novel perspectives for OSCC treatment.

Ulcerative colitis （UC） is a commonly seen digestive system disease with unclear pathogenesis. The condition is complex and variable， often chronic， and has a long treatment period with no specific cure. Currently， the treatment of UC often involves the use of corticosteroids， aminosalicylates， and biologics in western medicine， which provide fast-acting and definite efficacy in the short term. However， with prolonged medication， some patients may develop drug resistance and worsening of the disease， leading to the occurrence of colon cancer. Research has found that oxidative stress is one of the important pathogenic factors in UC and influences its onset and development. Oxidative stress is a state of imbalance between oxidative products and the antioxidant system in the body， characterized by overexpression of oxidative products such as malondialdehyde （MDA）， reactive oxygen species （ROS）， nitric oxide （NO）， or deficiency of antioxidant enzymes such as superoxide dismutase （SOD）， catalase （CAT）， and glutathione （GSH）. It is worth noting that traditional Chinese medicine （TCM）， as a unique characteristic medicine of China， has achieved significant efficacy in the treatment of UC. Studies have shown that TCM effectively inhibits the occurrence of UC by suppressing the accumulation of metabolites and antagonizes the development of UC by enhancing the antioxidant system. Therefore， using TCM to regulate the oxidative balance as a diagnostic and therapeutic approach may be a new method and direction for the treatment of UC in the future. Based on the above research， this article summarized the mechanisms of key pathogenic proteins in oxidative stress and the occurrence and development of UC， and compiled the effective ingredients of Chinese medicine， single drugs， prescriptions， and acupuncture and moxibustion in regulating upstream and downstream target proteins of oxidative stress. These interventions can reduce pathological damage to the intestinal mucosa， lower the colon injury index， enrich the intestinal microbiota， increase colon length， and improve clinical symptoms of UC. The article is expected to expand the application of TCM in the treatment of UC and provide a reliable scientific theoretical basis.

Objective:To establish the biology reference interval (RI) of peripheral blood procalcitonin (PCT) for children between 3 days and 6 years old in China.Methods:Totally 3 353 reference individuals with apparent health or no specific diseases were recruited in 18 hospitals throughout the country during October 2020 to May 2021. Reference individuals were divided into four groups: 3-28 days, 29 days - 1 year, 1-3 years and 4-6 years. Vein blood or capillary blood were collected by percutaneous puncture from every reference individual. The PCT level in serum and the capillary whole blood were assayed by Roche Cobas e601 and Norman NRM411-S7 immunoanalyzer. Outliers were deleted and 95th percentiles of every group were provided as RIs. Man-Whitney U test or Kruskal-Wallis test were used performed to assess the difference among different gender, age or method groups. Results:The difference of PCT distribution between male and female is not statistically significant, but the difference between serum and capillary whole blood is statistically significant. The differences between age groups are significant too. For Roche e601, serum PCT RI of 3-28 days group is <0.23 μg/L, 29 days - 6 years are <0.11 μg/L. For NRM411, Serum PCT RI of 3-28 days group is <0.21 μg/L, 29 days - 1 year: <0.09 μg/L, 1 - 6 years: <0.10 μg/L. For whole blood PCT, RI of 3-28 days group is <0.26 μg/L, 29 days - 6 years is <0.15 μg/L.Conclusions:Serum and capillary whole blood PCT have different RIs, however, capillary whole blood PCT testing is valuable in pediatric application. Children in 3-28 days show higher PCT levels than other age group. To establish the RIs and understand the differences among different groups are essential for the interpretation and clinical application of peripheral blood PCT testing results.

Objective:To explore independent predictors for poor outcome at 3 months in elderly patients with acute cerebral infarction(ACI)treated with intravenous thrombolysis(IVT), and to develop a nomogram-based predictive model.Methods:This was a retrospective cohort study.Clinical, laboratory and imaging data of 346 elderly patients with ACI treated with IVT from January 2016 to April 2021 in our hospital were collected.Poor outcome was defined as a modified Rankin Scale(mRS)score >2 at 3 months after the stroke.Logistic regression analysis was used to screen for independent factors predicting poor outcome in elderly ACI patients treated with IVT, and a corresponding nomogram model was developed using the R software.The ROC curve, calibration plots and decision curve analysis were used to evaluate discrimination, calibration and clinical application value of the nomogram model.Results:Among 346 candidates, 109 developed a poor outcome, representing a rate of 31.5%.Logistic regression analysis showed that symptomatic hemorrhagic transformation( OR=15.647, 95% CI: 8.913-27.454), stroke severity(moderate stroke, OR=3.322, 95% CI: 1.414-7.811; moderate-severe stroke, OR=8.169, 95% CI: 4.102-16.258; severe stroke, OR=9.653, 95% CI: 5.440-17.121), stroke-associated pneumonia( OR=2.239, 95% CI: 1.134-4.420), and heart failure( OR=2.758, 95% CI: 1.424-5.336)were independent predictors for poor outcome at 3 months in elderly ACI patients treated with intravenous thrombolysis(all P<0.05). With the area under curve(AUC-ROC)value at 0.85(95% CI: 0.80-0.89), the nomogram model, which was composed of the above four predictors, demonstrated good discrimination.On the calibration plot, the mean absolute error was 0.020, indicating that the model had good calibration.Decision curve analysis revealed that the model had good clinical application value. Conclusions:The nomogram model composed of symptomatic hemorrhagic transformation, stroke severity, stroke-associated pneumonia and heart failure may predict poor outcome at 3 months in elderly ACI patients treated with IVT, with high prediction accuracy and high clinical application value.

Autophagy is a crucial physiological process for cellular self-renewing and homeostasis,which plays a dual role in both tumorigenesis and anti-tumor treatment.It plays an antineoplastic role by maintaining genomic stability,as well as a protective role in tumorgenesis and progression.Autophagy improves the effectiveness of anti-tumor therapy,meanwhile,it involves in resistance of tumor cells to chemotherapy and irradiation.Using relevant autophagy regulators in different backgrounds is expected to become a new strategy for improving sensitivity of tumor cells to anti-tumor treatment.

Resveratrol is a natural polyphenolic substance with a variety of bioactivities,such as anti-oxidation,anti-tumor and apoptosis induction.This paper summarized the recent research progress of resveratrol induced cancerous cell apoptosis,including human cervical cancer HeLa cells,leukemia cells,hepatoma cells and gastric cancer cells,and analyzed the molecular mechanism behind resveratrol-induced cell apoptosis in tumor.In addition,resveratrol was investigated by blocking the cell cycle,regulating the expressions of related genes and proteins and mitochondria-induced apoptosis pathways as a potential application of cancer drugs to clinical researches.