Objective: To analyze the expression of Niemann-Pick C1 like-1 (NPC1L1) mRNA in colon cancer and its clinical significance based on bioinformatics methods. Methods: NPC1L1 mRNA expression data, clinicopathological characteristics, and survival data of colon cancer patients were collected from public databases including TIMER2.0, TCGA and GEO. Differences in NPC1L1 mRNA expression between tumor and adjacent non-tumor tissues were analyzed using R software. Patients were stratified into high- and low-expression groups based on the optimal cutoff value of NPC1L1 mRNA data. The correlation between NPC1L1 expression and clinicopathological features was assessed. Kaplan-Meier survival curves and Log-rank tests were used to compare 5-year survival rates between groups. Cox regression analysis was performed to identify prognostic factors for colon cancer. Gene Set Enrichment Analysis (GSEA) was performed using the Cluster Profiler package in R. Two human colon cancer cell lines, HCT116 and SW620, were cultured and passaged. Cells were divided into negative control, si-NPC1L1-1, si-NPC1L1-2, si-NPC1L1-3, and si-NPC1L1-4 groups, transiently transfected with corresponding small interfering RNAs (siRNAs). Protein levels were verified by Western blotting, and the most effective siRNA sequence was selected for further experiments. HCT116 and SW620 cells were then divided into siNC (control) and si-NPC1L1 (knockdown) groups, and colony formation assays were performed to assess clonogenic potential. Results: NPC1L1 mRNA expression was significantly higher in colon cancer tissues than that in adjacent normal tissues (P<0.001). High NPC1L1 mRNA expression was significantly associated with lymph node metastasis in colorectal cancer patients (χ2=2.126, P=0.034). The area under the ROC curve was 0.785 (95%CI: 0.728-0.839). The 5year survival rate in the high-expression of NPC1L1 mRNA group was significantly lower than that in the low-expression group (P=0.003). Univariate and multivariate Cox regression analyses indicated that NPC1L1 mRNA may be an independent prognostic factor for colorectal cancer patients (P<0.05). GSEA revealed that the highexpression group was enriched in 10 signaling pathways, including the Wnt pathway (P<0.05). Cell experiments showed that the si-NPC1L1-3 sequence achieved the highest knockdown efficiency (>65%) in both HCT116 and SW620 cells. Colony formation assays demonstrated that the number of colonies formed by si-NPC1L1-treated HCT116 and SW620 cells was significantly lower than those in the negative control group (P<0.001). Conclusion: NPC1L1 mRNA is significantly overexpressed in colon cancer and negatively correlated with patient prognosis, suggesting its potential as a biomarker for diagnosis and treatment.

Objective: To investigate the expression of hsa_circ_0076987 in gastric cancer cells and plasma exosomes, and to study its effect on the biological function of gastric cancer cells. Methods: The expression of circRNAs in plasma exosomes of six pairs of gastric cancer patients and healthy individuals was detected by circRNA microarray technology. qRT-PCR was used to detect the expression of hsa_circ_0076987 in gastric cancer cells and plasma exosomes, and its relationship with clinicopathological characteristics was analyzed. The diagnostic value of hsa_circ_0076987 in gastric cancer was analyzed by receiver operating characteristic (ROC) curve. Overexpression plasmid was used to overexpress hsa_circ_0076987 in gastric cancer cells. The changes in the cell proliferation, migration and invasion ability of gastric cancer cells were detected by CCK-8 assay, cell clone formation assay, Transwell migration and invasion assay, respectively. Results: The results of qRT-PCR showed that hsa_circ_0076987 was lowly expressed in gastric cancer cells and gastric cancer plasma exosomes (both P<0.01) and correlated with tumor neural infiltration (P<0.05). The ROC curve showed that the area under the curve (AUC) of the hsa_circ_0076987 was 0.709, the sensitivity was 0.606, the specificity was 0.697, 95%CI: 0.585～0.833 (P<0.05). Overexpression of hsa_circ_0076987 inhibited the proliferation, migration and invasion ability of gastric cancer cells. Conclusion: hsa_circ_0076987 is lowly expressed in gastric cancer cells and plasma exosomes. It′s expected to be a novel marker and therapeutic target for gastric cancer diagnosis.

Objective: To investigate the role of potassium voltage-gated channel subfamily D member 1 (KCND1) in pancreatic cancer radioresistance and its underlying mechanisms. Methods: Human pancreatic cancer cell line PANC1 was cultured in vitro and divided into control group (no irradiation) and X-ray irradiation group (8 Gy). After 48 hours of irradiation, mRNA expression levels of potassium channel-related genes were analyzed by RNA sequencing (RNA-seq) and real-time quantitative PCR; KCND1 protein expression was detected by immunofluorescence. CRISPR/Cas9 technology was used to knockout KCND1 in PANC1 cells, which were then divided into four groups: sgControl, sgKCND1, sgControl + X-ray (8 Gy), and sgKCND1 + X-ray (8 Gy). After 48 hours of irradiation, caspase3 and Bax protein expression were assessed by Western blotting; apoptosis and reactive oxygen species (ROS) levels were measured by flow cytometry; E-cadherin and N-cadherin expression levels were evaluated using Western blotting and immunofluorescence; and cell migration ability was determined by scratch wound healing assay. Twenty-four 6-8-week-old C57BL/6 mice were subcutaneously inoculated with PANC02 cells on the right back. Two weeks later, mice were randomly assigned to four groups: control group, X-ray irradiation (5 Gy) group, imipramine (20 mg/kg) group, and X-ray irradiation (5 Gy) + imipramine (20 mg/kg) group, with six mice per group. Tumor volume and weight were measured and compared. Expression levels of anti-apoptotic and pro-apoptotic proteins, as well as E-cadherin and N-cadherin, were analyzed in subcutaneous tumor tissues by Western blotting. Results: Compared with the control group, mRNA expression levels of potassium channels including KCNN4, KCND1, KCNA7, KCNA5, KCNMA1, and KCNJ14 were significantly upregulated in the X-ray irradiation group (P<0.05 or P<0.01), among which KCND1 showed the most prominent upregulation. Compared with the X-ray irradiation group, the sgKCND1 + X-ray group showed significantly higher expression of caspase-3 and Bax proteins (P<0.05 or P<0.01), significantly reduced N-cadherin expression (P<0.01), markedly elevated E-cadherin expression (P<0.05), and a significantly lower scratch wound healing rate (P<0.01). Compared with the X-ray irradiation group, the X-ray+imipramine group exhibited significantly smaller tumor volumes and reduced tumor weights (P<0.05). Western blotting results showed that, compared with the X-ray group, the X-ray+imipramine group had significantly lower expression of the anti-apoptotic protein Bcl-2 (P<0.05), while pro-apoptotic proteins cleaved caspase-3 and Bax were significantly upregulated (P<0.05); additionally, E-cadherin expression was significantly increased (P<0.01), while N-cadherin expression was significantly decreased (P<0.05). Conclusion: The potassium channel KCND1 may contribute to radioresistance in pancreatic cancer by promoting epithelial-mesenchymal transition and enhancing apoptosis resistance in pancreatic cancer cells.

Objective: To investigate the damaging effects of triptolide on the blood-testis barrier and its underlying molecular mechanisms. Methods: Nine healthy male C57BL/6 mice (20-25 g) were randomly divided into control group, low-dose triptolide group (0.2 mg/kg), and high-dose triptolide group (0.5 mg/kg), with 3 animals per group. After continuous administration for 28 days, body weight, testicular volume, and testicular histomorphology were observed in mice exposed to different doses of triptolide. Intergroup comparisons of testis-to-body weight ratio, germ cell apoptosis level, expression of blood-testis barrier-related proteins and barrier leakage were performed among the three groups. Results: Compared with the control group, mice in triptolide-treated groups exhibited markedly reduced body weight gain, testicular weight and testis-to-body weight ratio (all P<0.01). HE staining revealed massive loss of germ cells and prominent vacuolization in the seminiferous tubular lumen of triptolideexposed mice. The sperm count in epididymal tissues was decreased upon triptolide administration, and this reduction was further aggravated in the high-dose group, accompanied by abundant necrotic germ cells. TUNEL assay revealed markedly elevated apoptosis in seminiferous tubules of triptolidetreated mice (all P<0.01). Western blotting indicated that the expression levels of blood-testis barrier-related proteins (E-cadherin, occludin, β-catenin, VE-cadherin, Arp3, and Claudin 11) were significantly downregulated in triptolide-treated groups (all P<0.01). Biotin tracer staining demonstrated varying degrees of blood-testis barrier injury in triptolidetreated mice, and the severity of barrier leakage increased in a dose-dependent manner. Conclusion: Triptolide treatment can be used to stably establish a mouse model of blood-testis barrier injury. Its mechanism is closely related to the synergistic regulation of multiple pathways, including Sertoli cell apoptosis, downregulated expression of blood-testis barrier-related proteins and oxidative stress response.

Objective: To investigate the effect of nebulized human umbilical cord mesenchymal stem cells-derived extracellular vesicles (hUCMSC-EVs) on house dust mite (HDM)- and lipopolysaccharide (LPS)-induced neutrophilic inflammation in asthmatic mice and its potential mechanism. Methods: HUCMSC-EVs were isolated using ultra-high-speed centrifugation. The morphology of hUCMSC-EVs was observed via transmission electron microscopy. Western blotting was performed to detect the expression of cluster of differentiation 81 (CD81), heat shock protein 70 (HSP70), tumor susceptibility gene 101 (TSG101) and Calnexin in the EVs. A mouse model of neutrophilic asthma induced by HDM-LPS was established and treated with nebulized hUCMSCEVs. Fifteen 6-8-week-old female BALB/c mice were randomly divided into three groups: control group, HDM-LPS group, and HDM-LPS+EVs group (5 mice per group). Hematoxylin-eosin (HE) staining was used to observe inflammatory cell infiltration around the pulmonary airways, while periodic acid-Schiff (PAS) staining was applied to detect goblet cell metaplasia in the lungs. A hemocytometer was used to count the total number of inflammatory cells in bronchoalveolar lavage fluid (BALF), and Wright-Giemsa staining was employed to count the total number of neutrophils in BALF. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to measure the mRNA expression levels of C-X-C motif chemokine ligand 15 (Cxcl15) and Cxcl5 in mouse lung tissues. Relevant kits were used to detect the contents of ferrous irons and malondialdehyde (MDA) in lung tissues, and Western blotting was used to determine the expression of glutathione peroxidase 4 (GPX4). In vitro, HBE135-cells were treated with HDM-LPS or HDM-LPS+EVs; laser confocal microscopy was used to observe the uptake of CM-Dil-labeled hUCMSC-EVs by the huaman bronchial epithelial cell line HBE135-E6E7 cells. In vitro, HBE135-E6E7 cells were treated with HDM-LPS or HDM-LPS+EVs. IL-8 and Cxcl5 mRNA expression was analyzed by qRT-PCR, while ferrous ion, MDA, and GPX4 levels were determined using kits and Western blotting, respectively. Results: HUCMSC-EVs exhibited cuplike membranous structure and expressed CD81, HSP70 and TSG101. Compared with the control group, the HDM-LPS group exhibited obvious inflammatory cell infiltration around airways and airway epithelial goblet cell metaplasia, with significantly increased inflammation score and PAS staining scores (all P<0.01). The total number of cells and neutrophils in the BALF was significantly increased (all P<0.01). In lung tissues, the levels of Cxcl15 mRNA, Cxcl5 mRNA, ferrous ions, and MDA were significantly upregulated, while GPX4 expression was significantly downregulated (P<0.01 or P<0.05). Compared with the HDM-LPS group, the HDM-LPS+EVs group showed significantly reduced airway inflammation and PAS scores in mice, along with a marked decrease in total cell and neutrophil counts in BALF (P<0.01). In lung tissues, the levels of Cxcl15 mRNA, Cxcl5 mRNA, ferrous ions, and MDA were reduced, whereas GPX4 expression was increased (P<0.01 or P<0.05). In vitro experiments revealed that hUCMSC-EVs could be endocytosed by HBE135-E6E7 cells; and compared with the HDM-LPS group, the HDM-LPS+EVs group showed significantly reduced levels of ferrous ions, MDA, and the expression of IL-8 mRNA and Cxcl5 mRNA, while GPX4 expression was markedly upregulated (P<0.01 or P<0.05). Conclusion: Nebulized hUCMSC-EVs could significantly alleviate airway inflammation in mice with neutrophilic asthma induced by HDM-LPS, likely through inhibition of ferroptosis in bronchial epithelial cells, thereby exerting anti-inflammatory effects.

Objective: To investigate the relationship between age at menarche, age at menopause or reproductive lifespan and cognitive function in postmenopausal women. Methods: Based on data from the 2018 China Health and Retirement Longitudinal Study (CHARLS), a total of 1 829 postmenopausal women with complete key data were selected and divided into non-cognitive impairment group (n=1 230) and cognitive impairment group (n=599) according to the Mini-Mental State Examination (MMSE), and their basic characteristics were compared. Logistic regression analysis was used to examine the relationship between reproductive factors and cognitive function, and restricted cubic spline (RCS) curves were further plotted to examine potential nonlinear associations between the above factors and cognitive function. Results: There were statistically significant differences between the cognitive impairment group and the non-cognitive impairment group in marital status, place of residence, education level, smoking status, hypertension, depressive symptoms, sleep duration, age at menopause, reproductive span and MMSE scores (all P<005). After adjusting for confounding factors, Logistic regression analysis showed that compared to women with menopause age between 51-55 years, those with menopause age ≤45 years had a higher risk of cognitive impairment (OR=1.59, 95%CI: 1.15-2.10, P=0.014); compared to women with a reproductive span of 36-40 years, those with a reproductive span ≤30 years had a higher prevalence of cognitive impairment (OR=1.68, 95%CI: 1.26-2.25, P=0.008). No association was found between age at menarche and cognitive function. Smooth curve fitting did not reveal any nonlinear relationship between reproductive factors and cognitive function. Conclusion: Women with earlier menopause (≤45 years) and shorter reproductive lifespan (≤30 years) have a higher risk of cognitive impairment, while no significant association was observed between age at menarche and cognitive impairment.

Objective: To investigate the effect of vitamin D on B cell activation and total immunoglobulin E (IgE) secretion in children with house dust mite allergy and the underlying molecular mechanisms. Methods: A total of 126 children with house dust mite allergy (allergy group) and 81 healthy children (control group) were enrolled. Serum levels of 25-hydroxyvitamin D ［25-(OH)D］, total IgE, dermatophagoides pteronyssinus peptidase 1(Derp1)-specific IgE, and soluble CD23 (sCD23) were measured. Pearson correlation analysis was used to assess the relationships between serum 25-(OH)D levels and total IgE, Derp1-specific IgE, sCD23 levels, and the proportion of CD19⁺CD23⁺B cells in peripheral blood. The diagnostic value of serum 25-(OH)D for house dust mite allergy was assessed using receiver operating characteristic (ROC) curve analysis. CD19⁺B cells isolated from allergic children were divided into negative control group (routine culture), positive control group (1 μg/mL lipopolysaccharide), and and three treatment groups with 10, 50, and 100 nmol/L 1,25-(OH)2D3, respectively; cells were co-cultured for 48 hours. Flow cytometry was used to detect membrane expression of CD23, while Western blotting assessed levels of signal transducer and activator of transcription 3 (STAT3), STAT5, p-STAT3, and p-STAT5. ELISA was employed to measure IgE secretion. Time-course experiments were conducted to observe dynamic changes in STAT3 phosphorylation and IgE secretion at different time points after 100 nmol/L 1,25-(OH)2D3 treatment. CD19+B cells were further divided into four groups: negative control group (routine culture), 1,25-(OH)2D3 group ［100 nmol/L 1,25-(OH)2D3］, IL-6 group (20 ng/mL IL-6), and 1,25-(OH)2D3+IL-6 group ［100 nmol/L 1,25-(OH)2D3 + 20 ng/mL IL-6］. Membrane expression of CD23 and IgE secretion were analyzed in each group. Results: Serum 25-(OH)D level in the allergy group was significantly lower than that in the control group (P<0.001) and showed a weak negative correlation with sCD23 levels (r=-0.248). ROC curve analysis indicated that serum 25-(OH)D had moderate diagnostic value for house dust mite allergy in children (AUC=0.769). In vitro experiments demonstrated that compared with the negative control group, CD23 membrane expression, p-STAT3 expression and IgE secretion were significantly reduced in the 1,25-(OH)2D3 groups in a concentration-dependent manner (all P<0.01). Time-course experiments revealed that 100 nmol/L 1,25-(OH)2D3 significantly inhibited p-STAT3 expression within 30 minutes and markedly reduced IgE secretion after 48 hours (P<0.01). Compared with the 1,25-(OH)2D3 group, the 1,25-(OH)2D3+IL-6 group showed significantly increased CD23 membrane expression and IgE secretion (P<0.05). Conclusion: Vitamin D may downregulate CD23 expression by inhibiting STAT3 phosphorylation, thereby reducing IgE production from B cells in house dust mite allergy.

Objective: To develop a predictive model for bone metastasis in patients with prostate cancer based on the SEER database, so as to provide a quantitative assessment tool for early identification of high-risk patients in clinical practice. Methods: A total of 15 055 patients diagnosed with prostate cancer complicated by bone metastasis between January 1, 2018 and December 31, 2021 were extracted from the SEER database. Stratified random sampling was performed to divide the cohort into a training set (n=10 538) and an internal validation set (n=4 517) at a ratio of 7∶3. Cox proportional hazards regression models were used to screen independent prognostic risk factors associated with overall survival (OS) and cancer-specific survival (CSS) of prostate cancer patients with bone metastasis. Nomogram models for OS and CSS were separately constructed based onthe identified independent prognostic factors. The concordance C-index, receiver operating characteristic (ROC) curves, calibration curves and decision curve analysis (DCA) were adopted to comprehensively evaluate the model performance in terms of discrimination, calibration and clinical net benefit. Results: Multivariate Cox regression analysis revealed that age ≥80 years, race, marital status, Gleason score of 7-9, T4/TX stage, brain metastasis, liver metastasis, radical resection, radiotherapy and chemotherapy were independent risk factors for OS in patients with prostate cancer (all P<0.05). Age 50-59 years, age ≥80 years, race, marital status, Gleason score of 7-9, T4/TX stage, brain metastasis, liver metastasis and lung metastasis were independent risk factors for CSS (all P<0.05). The C-index of the OS nomogram was 0.710 (95%CI: 0.701-0.718) in the training cohort and 0.710 (95%CI: 0.697-0.723) in the validation cohort. For the CSS nomogram, the C-index was 0.705 (95%CI: 0.695-0.715) in the training cohort and 0.697 (95%CI: 0.682-0.712) in the validation cohort. ROC curves of the OS nomogram yielded AUC values of 0.75, 0.72 and 0.72 for survival at 1, 2 and 3 years in the training dataset, with corresponding values of 0.74, 0.71 and 0.70 in the validation cohort. For the CSS nomogram, the AUC at 1, 2 and 3 years were 0.74, 0.71 and 0.71 in the training cohort, and 0.72, 0.69 and 0.69 in the validation cohort. Calibration curves of the OS nomogram demonstrated favorable consistency between predicted survival probabilities and actual observed survival probabilities. Similarly, calibration curves of the CSS nomogram showed strong agreement between predicted cancer-specific survival probabilities and real-world observations. DCA indicated that both the OS and CSS nomograms achieved higher clinical benefit. Conclusion: The constructed nomogram model was used to predict the 1, 2 and 3 year OS and CSS in patients with bone metastatic prostate cancer. The model exhibited favorable discrimination capacity and possessed prominent clinical application value.

Objective: To investigate the expression levels and prognostic value of peripheral blood Bcell activating factor of TNF family (BAFF), high-mobility group box-1 (HMGB1) and miR-219a-5p in children with epilepsy. Methods: A total of 84 children with epilepsy admitted to Qingdao Municipal Hospital from April 2023 to March 2024 were selected as the epilepsy group, and 84 healthy children receiving physical examinations during the same period were set as the control group. According to the Glasgow Outcome Scale, the epilepsy group was subdivided into a good prognosis subgroup (n=61) and a poor prognosis subgroup (n=23). Baseline data of all subjects were collected. The expression levels of peripheral blood BAFF, HMGB1 and miR-219a-5p were detected by ELISA, fluorescence flow cytometry and qRT-PCR, respectively. The differences in clinical indicators and molecular expression were compared between the two groups and different prognosis subgroups. Multivariate Logistic regression analysis was performed to screen independent risk factors for poor epilepsy prognosis. Spearman correlation analysis was used to analyze the correlations among the three molecular markers, and receiver operating characteristic (ROC) curves were plotted to evaluate the efficacy of combined detection of the three indicators in predicting poor prognosis. Results: The expression levels of BAFF, HMGB1 and miR-219a-5p in the epilepsy group were significantly higher than those in the control group (P＜0.01). The pretreatment seizure frequency, seizure duration and levels of the three molecular markers in the poor prognosis subgroup were all higher than those in the good prognosis subgroup (all P＜0.01). Logistic regression analysis showed that high pre-treatment seizure frequency, prolonged seizure duration and overexpression of peripheral BAFF, HMGB1 and miR-219a-5p were independent risk factors for poor prognosis of pediatric epilepsy (P＜0.05). Spearman analysis indicated that BAFF, HMGB1 and miR-219a-5p were positively correlated with each other, with correlation coefficients r of 0.468, 0.432 and 0.547, respectively (P＜0.05). ROC curve analysis revealed that the area under the curve of the combined three indicators was 0.851, with a sensitivity of 0.87 and a specificity of 0.81, and the predictive efficacy was superior to that of single marker detection (P＜0.05). Conclusion: BAFF, HMGB1 and miR-219a-5p are highly expressed in the peripheral blood of children with epilepsy, and all may serve as independent risk factors for poor prognosis. Combined detection of the three indicators can significantly improve the predictive efficiency of adverse prognosis in pediatric epilepsy.

Objective: To evaluate the effect of steady-state ilaprazole on the pharmacokinetics of clopidogrel in healthy subjects. Methods: A randomized, open-label, crossover, self-controlled trial was conducted. A total of 40 healthy subjects were randomly assigned to two groups (Group A and Group B, n=20 each). Group A received clopidogrel 75 mg once daily for 7 consecutive days; Group B received clopidogrel 75 mg plus ilaprazole 10 mg once daily for 7 days. After a 10-day washout period, the two groups crossed over to the alternative regimen. Plasma concentrations of clopidogrel, its active metabolite, and clopidogrel carboxylic acid were determined for pharmacokinetic analysis. Results: The 90%CI of the geometric mean ratios for systemic exposure of clopidogrel active metabolite in the all subjects and CYP2C19 extensive metabolizers were within the range of 0.80 to 1.25. Conclusion: No clinically significant pharmacokinetic interaction was observed between ilaprazole and clopidogrel in healthy subjects, nor was there obvious clinical relevance in CYP2C19 extensive metabolizers.