Objective: To prepare shikonin liposomes (SHK-L) and preliminarily evaluate their anti-aging effects. Methods: The SHK-L prescription was optimized through single factor experiments to investigate its in vitro release and oral bioavailability in rats. The in vivo safety was evaluated through zebrafish toxicity experiments, and the anti-aging effect was examined by staining the β-galactosidase in aging zebrafish. Results: The optimal prescription was SHK 6 mg, lecithin 70 mg, cholesterol 10 mg, vitamin E polyethylene glycol succinate (TPGS) 30 mg, and sodium dodecyl sulphate (SDS) 1.5 mg, with a particle size of (206.96±0.75) nm, polydispersity coefficient (0.186±0.030), encapsulation rate of (96.88±2.19)% and drug loading was (5.36±0.01)%. SHK-L improved the drug release rate compared to SHK. According to in vivo pharmacokinetic data, SHK-L had a relative bioavailability of 413.37% of SHK. At 96 hours post-administration, LC₅₀ of shikonin and SHK-L in zebrafish were (0.112±0.007) μg/mL and (0.141±0.012) μg/mL, respectively. Conclusion: SHK-L can enhance the solubility and oral bioavailability of insoluble drugs, as well as their anti-aging effect and safety.

Objective: To construct a bioaffinity chromatography of TrkA-lipid rafts@CNBr-Sepharose 4B gels and apply it to the assessment of the efficacy of the affinity adsorption of TrkA targeted drugs. Methods: TrkA-lipid rafts@CNBr-Sepharose 4B gels stationary phase was prepared and characterized through the coupling reaction of cyanogen bromide immobilized ligand. The stability was assessed by repeating the up-sampling experiment over a period of 30 days. Affinity adsorption experiments were conducted to investigate the affinity adsorption behavior of TrkA-targeted drugs on both blank agarose gels columns and TrkA-lipid rafts@CNBr-Sepharose 4B gels columns. Results: The Western blotting results revealed that the lipid rafts contained Flotillin-1, Caveolin-1, and TrkA proteins. Following the coupling of U251 cell lipid rafts coupled Sepharose 4B gels, it was observed that the lipid rafts maintained a stable solid loading on the column bed even after rinsing with multiple column volumes of buffer. Adsorption experiments confirmed the specific affinity adsorption of the TrkA-lipid rafts@CNBr-Sepharose 4B gels stationary phase. Conclusion: The bioaffinity chromatography of TrkA-lipid rafts@CNBr-Sepharose 4B gels was successfully constructed, thus preliminarily validating its feasibility and stability.

Objective: To develop a wound dressing incorporating Clostridium butyricum (C. butyricum, anaerobic probiotic), which is capable of producing antibacterial substances and exerting hydrogen-producing properties under normoxic conditions, and investigate the effects of its selective bacteria inhibitionand reactive oxygen species (ROS) scavenging. Methods: C. butyricum and Bacillus subtilis (B. subtilis, aerobic probiotic) were simultaneously encapsulated in hydrogel to prepare a dual-probiotic hydrogel (BC-gel). B. subtilis was used to consume oxygen within the hydrogel. This creates an anaerobic microenvironment for C. butyricum to maintain its viability. The selective bacteria inhibition of BC-gel was evaluated by plate counting method, while the selective scavenging of ROS was evaluated through flow cytometry. Results: BC-gel can effectively inhibit the proliferation of Escherichia coli and methicillin-resistant Staphylococcus aureus. While maintaining the normal growth of Bifidobacterium and Lactobacillus, BC-gel has the ability to scavenge ·OH and ONOO- free radicals, but has no significant decreasing effect on O-2· and NO·. Conclusion: BC-gel exhibits favorable selective bacteria inhibition and ROS scavenging properties.

Objective: To investigate the changes in gene expression in pancreatic cancer during radiotherapy combined with immunotherapy and to screen and validate key genes involved in mice. Methods: Twenty C57BL/6J male mice aged 6-8 weeks were randomly divided into 4 groups: control group, radiotherapy group, immunotherapy group and radiotherapy combined with immunotherapy group, with 5 mice in each group. The tumor-bearing mouse model was established by subcutaneous injection of pancreatic cancer KPC cells, and the radiotherapy group was irradiated with 8 Gy radiation, the immunotherapy group received a single intraperitoneal injection of PD-1 monoclonal antibody (200 μg/mouse), the radiotherapy combined with immunotherapy group received a single dose of 8 Gy radiation followed by intraperitoneal injection of PD-1 monoclonal antibody (200 μg/mouse), and the control group did not receive any treatment. The tumor tissues were harvested 7 days later, and the transcriptome sequencing was performed. The differentially expressed genes (DEGs) with significant expression were analyzed by GO, KEGG and Reactome functional annotation to explore the related signaling pathways. Venn analysis was performed to identify the intersection of DEGs, and survival analysis was used to identify key genes. The expression levels of these key genes were analyzed, and their correlation with immune cell infiltration and immune checkpoint expression was examined. A proteinprotein interaction (PPI) network was constructed to analyze the interactions of these key genes. Two subcutaneous tumorbearing models of male mice with pancreatic cancer KPC and PANC02 cell lines C57BL/6J were constructed. The combined radiotherapy and immunotherapy treatment was administered on the following day and maintained for 3 days. Quantitative reverse transcription PCR (qRT-PCR) was used to assess the relative mRNA expression of key genes in tumor tissues. Results: The results of transcriptome sequencing analysis showed that compared with the radiotherapy group and the immunotherapy group, the radiotherapy combined with immunotherapy group had the richest DEGs and mainly participated in the signal pathways related to metabolism, the immune system and signal transduction. Venn analysis of upregulated genes in the radiotherapy, immunotherapy, and combined radiotherapy and immunotherapy groups identified 11 common genes. Survival analysis revealed that myxovirus resistance 1 (MX1) was a key gene, which was highly expressed in pancreatic cancer and significantly negatively correlated with the prognosis of pancreatic cancer patients (P<0.05). The MX1 mRNA expression in pancreatic cancer was positively correlated with the infiltration levels of neutrophils and dendritic cells (r=0.413, 0.333, both P<0.05), and was also positively correlated with the expression of immune checkpoints CD80, CD274, and CD86 (all P<0.05). The gene cluster interacting with MX1 in pancreatic cancer included OASL1, RSAD2, IFIT1, IFIT2 and IFIT3. qRT-PCR results showed that in the tumor-bearing mouse models of pancreatic cancer KPC and PANC02 cells, compared with the control group, the relative expression level of MX1 mRNA in the radiotherapy combined with immunotherapy group increased significantly (P<0.05). Conclusion: The relative expression of MX1 mRNA significantly increased in radiotherapy combined with immunotherapy for pancreatic cancer, and it may be one of the key genes.

Objective: To investigate the therapeutic effects of human umbilical cord mesenchymal stem cell derived exosomes (hucMSC-Exo) on acute myocardial infarction (AMI) and to elucidate the underlying mechanisms. Methods: Human umbilical cord mesenchymal stem cells (hucMSCs) were isolated and cultured. Exosomes were extracted from the culture media. AMI model was constructed by ligating the anterior descending branch of the coronary artery in mice. The mice were randomly divided into Sham, AMI, Exo and Fer-1 groups, and PBS, PBS, hucMSC-Exo and Ferrostatin-1 were injected into the marginal zone of the ischemic myocardium of each group, respectively. The levels of the antioxidants glutathione (GSH) content and malondialdehyde (MDA) content in mouse myocardial tissues measured by corresponding assay kits, and reactive oxygen species (ROS) levels detected by DHE probe were used to assess the severity of ferroptosis in mouse myocardial tissue; Cardiac ultrasound and Masson staining were used to evaluate the cardiac function and fibrosis level in mice; HL-1 cells were treated with DMSO, Erastin and hucMSC-Exo and subjected to RNA sequencing, and the potential mechanism of the therapeutic effect of hucMSC-Exo was evaluated by KEGG enrichment analysis and clustering heat map analysis. The viability of HL-1 cells after hucMSC-Exo or hucMSC-Exo+BI-6C9 co-treatment was detected by CCK-8 assay. BH3 interacting domain death agonist (Bid) expression levels in infarcted myocardial tissues of mice were detected by qRT-PCR. Results: Compared with the AMI group, the Exo and Fer-1 groups of mice showed a significant decrease in MDA and ROS levels in the infarcted region of the myocardium, an increase in GSH content, a reduction in myocardial fibrosis, and an improvement in cardiac function; among the mice in the Exo group, the treatment effect was more pronounced compared to the Fer-1 group on the 28th day after AMI. KEGG enrichment analysis showed that the reversed genes by hucMSC-Exo were mainly enriched in the p53 signaling pathway; clustering heatmap analysis of p53-related genes revealed that Bid gene expression was significantly higher in HL-1 cells in the Erastin group compared with the control group, and the level of Bid expression in HL-1 cells in the Exo group was significantly lower than that in the Erastin group. The therapeutic effect of hucMSC-Exo in HL-1 cells was not significantly increased after treatment with the Bid inhibitor BI-6C9. Bid mRNA level in the heart tissues of mice in the AMI group was substantially increased compared with that in the control group, and was significantly decreased in the Exo group compared with that in the AMI group. Conclusion: hucMSC-Exo inhibits Bid expression to mitigate ferroptosis after AMI, thereby alleviate myocardial injury.

Objective: To explore the regulatory role and mechanism of high mobility group box 1 (HMGB1) in the radiosensitivity of triple-negative breast cancer (TNBC) cells. Methods: The expression level of HMGB1 in breast cancer subtypes and its relationship with the survival prognosis of breast cancer patients were analyzed by using The Cancer Genome Atlas (TCGA). Breast epithelial cells MCF-10A, non-TNBC cells MCF-7, SK-BR-3, and TNBC cells MDA-MB-231, MDA-MB-436, MDA-MB-468 were cultured in vitro. The mRNA and protein expression levels of HMGB1 were detected by qRT-PCR and Western blotting. Cell proliferation was assessed at different time points (0, 24, 48, 72 and 96 h) after 4 Gy X-ray irradiation, and the radiosensitivity of the cells was evaluated after different doses (0, 2, 4, 6, 8 Gy) of irradiation. HMGB1 expression was knocked down or overexpressed in MDA-MB-231 cells using plasmids or lentivirus, respectively, and cell proliferation, clone formation ability and apoptosis level after 4 Gy X-ray irradiation were detected by CCK-8 assay, colony formation assay, and flow cytometry, respectively. Changes in nuclear and intracellular HMGB1 in MDA-MB-231 cells after 0, 4, and 8 Gy irradiation were detected by immunofluorescence assay. MDA-MB-231 cells were divided into shControl, shHMGB1-2, Vector, and Flag-HMGB1 groups, irradiated with 4 Gy for 72 h, and changes in nuclear, HMGB1, and γ-H2AX were detected by immunofluorescence assay. Protein expression of p-AKT, AKT, p-GSK-3β, GSK-3β, Caspase 9, p-H2AX, and HMGB1 were detected by Western blotting in MDA-MB-231 cells after 0, 4, 8 Gy irradiated, and in shControl, shHMGB1-2, Vector, Flag-HMGB1 groups cells after 4 Gy irradiation for 72 h. The proliferation inhibition rate was assessed by CCK-8 assay in control, radiotherapy, AKT inhibitor, and AKT inhibitor + radiotherapy groups. Cell proliferation activity was examined by CCK-8 assay, and protein expression of p-AKT/AKT, Caspase 9, and p-H2AX were detected by Western blotting in Vector, Flag-HMGB1, Vector+AKT inhibitor, and Flag-HMGB1+AKT inhibitor groups after 4 Gy irradiation. Results: HMGB1 was highly expressed in TNBC, and its high expression was associated with poor prognosis. The mRNA and protein expression levels of HMGB1 was higher in MDA-MB-231/436/468 cells than in MCF-7 and SK-BR-3 cells, with the former having lower radiosensitivity. Knockdown of HMGB1 significantly increased the radiosensitivity of MDA-MB-231 cells, while overexpression of HMGB1 significantly decreased it. Knockdown of HMGB1 resulted in a significant decrease in p-AKT expression levels and a significant increase in p-GSK-3β and p-H2AX expression levels post 4 Gy irradiation. Overexpression of HMGB1 resulted in a significant increase in p-AKT expression levels and a significant decrease in p-GSK-3β and pH2AX expression levels post 4 Gy irradiation. The resistance-promoting effect of HMGB1 on radiotherapy was abolished by AKT inhibitor AKTi-1/2 treatment. Conclusion: HMGB1 is highly expressed in TNBC cells and regulates the radio-sensitivity of cells through the PI3K/AKT pathway. Thus, it may be implicated that HMGB1 may potentially serve as an important molecular target for radio-resistance in TNBC.

Objective: To construct an end-to-end deep learning model for the multi-class classification task of T staging in advanced gastric cancer using CT images. Methods: This retrospective study included enhanced venous phase images from 423 gastric cancer patients, which were randomly divided into the training set and the test set at an 8∶2 ratio. We employed a deep learning automatic segmentation model based on the 3D-nnUNet for segmenting tumors. Simultaneously, a multi-class classification model based on the SmallFocusNet was developed for classification of T staging of advanced gastric cancer. Finally, these models were integrated to construct an end-to-end deep learning model for CT-T staging diagnosis of advanced gastric cancer. The performance of the segmentation model was evaluated using the Dice similarity coefficient (DSC), Intersection over Union (IoU), and 95% Hausdorff Distance (HD_95). The prediction efficacy of the deep learning model was assessed using area under the ROC curve (AUC) values, sensitivity, and specificity. Additionally, the diagnostic performance of the deep learning model for classification of T staging of advanced gastric cancer was compared with that of radiologists. Results: In the test set, the DSC and IoU of the automatic segmentation model were 0.869±0.095 and 0.779±0.137, respectively. The macro-average AUC value of the deep learning model was 0.882 (95%CI: 0.812-0.926). The AUC values for distinguishing T2, T3 and T4a stage tumors were 0.960 (95%CI: 0.915-0.990), 0.739 (95%CI: 0.616-0.849) and 0.917 (95%CI: 0.812-0.926), respectively. The average sensitivity was 0.769 (95%CI: 0.676-0.853), with sensitivities for distinguishing T2, T3 and T4a stage tumors of 0.808 (95%CI: 0.654-0.923), 0.750 (95%CI: 0.571-0.893) and 0.750 (95%CI: 0.594-0.906), respectively. Furthermore, the deep learning model outperformed radiologists in the diagnostic performance of T staging for advanced gastric cancer. Conclusion: The end-to-end deep learning model, which integrates multi-channel and attention mechanisms based on enhanced CT images, demonstrates high accuracy and consistency in preoperative T staging diagnosis of advanced gastric cancer.

Objective: To investigate the risk factors for diuretic resistance in patients with acute exacerbation of chronic heart failure and to construct a Nomogram model, aiming to provide a reference for the early identification of patients with diuretic resistance. Methods: A retrospective study was designed by including 215 patients with acute exacerbation of chronic heart failure who were admitted to the Affiliated People′s Hospital of Jiangsu University from January 2021 to August 2024. Patients were grouped into two groups, one is resistance group, the other non-resistance based on the criteria for diagnosing diuretic resistance, and baseline data were collected for both groups. Using the least absolute shrinkage and selection operator (LASSO) regression for univariate screening, followed by multifactorial Logistic regression for further screening, a nomogram model was constructed and subjected to internal validation. Results: LASSO regression selected 6 variables, and further analysis using multiple Logistic regression revealed that an elevated Nterminal pro B-type natriuretic peptide (NT-proBNP) level was an independent risk factor for diuretic resistance ［OR (95%CI): 2.342 (1.087-5.043), P<0.05］, while higher levels of estimated glomerular filtration rate (eGFR), hemoglobin, serum sodium, and left ventricular ejection fraction (LVEF) were identified as protective factors ［OR (95%CI): 0.978 (0.965-0.990), 0.973 (0.958-0.989), 0.893 (0.822-0.971), 0.944 (0.918-0.971), all P<0.05］. A nomogram model was constructed based on the above risk factors. The area under the receiver operating characteristic (ROC) curve was 0.846 (95%CI: 0.795-0.897). The calibration curve and Hosmer-Lemeshow test indicated that the model fit well (P=0.159). The clinical decision curve analysis demonstrated that the model provided a favorable net clinical benefit when the threshold probability ranged from 0% to 87.0%. Conclusion: The Nomogram model, incorporating NT-proBNP, hemoglobin, blood sodium, eGFR, and LVEF, effectively assesses the risk of diuretic resistance in patients with acute exacerbation of chronic heart failure.

Objective: To investigate the association of single nucleotide polymorphisms (SNP) of programmed death receptor 1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) with postoperative prognosis of patients with hepatocellular carcinoma (HCC). Methods: A total of 395 HCC patients who underwent partial hepatectomy from January 2002 to December 2012 were included. The SNP loci of PD-1 and CTLA-4 were identified by genotyping, and the survival status of the patients was evaluated by the Kaplan-Meier survival curve, the survival rate was compared by using the Log-Rank test; multivariate analysis of the factors influencing the prognosis of patients was further conducted through the Cox proportional hazards model. Results: A total of 7 SNP loci were detected, including 4 SNP of the PD-1 gene(rs10204525, rs36084323, rs2227982, rs7421861) and 3 SNP of the CTLA-4 gene(rs16840252, rs733618, rs231775). Among them, rs10204525, rs36084323 and rs16840252 were significantly correlated with the postoperative prognosis of HCC patients(P<0.05), while no significant correlation was shown at other loci(P>0.05). The results of Cox regression analysis showed that rs10204525, rs36084323 and rs16840252 were independent influencing factors for the postoperative prognosis of HCC patients(P<0.05). Conclusion: The SNP rs10204525 and rs36084323 of the PD-1 gene and the SNP rs16840252 of the CTLA-4 gene were significantly associated with the postoperative prognosis of HCC patients and were independent influencing factors.