Objective: To investigate the impact of lipotoxic hepatocytederived exosome (LTH-ex) on ferroptosis and activation of hepatic stellate cell (HSC). Methods: Lipotoxic damage was induced in the human liver cell line LO2 using oleic acid and palmitic acid. The supernatants of the lipotoxic damaged liver cells were collected and LTH-ex was extracted through ultracentrifugation. The characterization of LTH-ex was identified using transmission electron microscopy and Nanosight nanoparticle analysis system, including assessing morphology, particle size and concentration, and LTH-ex surface markers through Western blotting. The human HSC cell line LX-2 was cocultured with LTH-ex or the ferroptosis activator Erastin for 48 h, and then all cells were divided into three groups: the PBS control group, the LTH-ex treatment group, and the co-treatment group consisting of LTH-ex and the ferroptosis inducer Erastin. Cell viability and mitochondrial membrane potential of LX-2 were evaluated using FDA and JC-1 staining. The iron ion concentration kit was used to detect the iron level in LX-2, the expression of mRNA and protein of death suppressor genes GPX4, xCT, and HSC activation gene α-SMA, were measured through qRT-PCR and Western blotting. Additionally, a mouse model of nonalcoholic fatty liver disease was established by feeding a high-fat diet, followed by injection of LTH-ex through the tail vein. Liver tissue structure was observed using HE staining, while GPX4 and α-SMA protein expression was assessed through immunohistochemistry staining. Sirius red staining was employed to detect collagen deposition in the liver tissue. Results: The LTH-ex particles were approximately 110 nm in size, displaying the typical lipid bilayer structure of exosomes and containing exosome marker proteins TSG101, Alix, and CD63. In comparison to the PBS control group, the LTH-ex treatment group demonstrated enhanced viability of LX-2 cells, an increased mitochondrial membrane potential, decreased iron levels, and upregulation of the ferroptosis-inhibiting genes GPX4 and xCT, as well as HSC activation gene α-SMA. Conversely, when compared to the LTH-ex treatment group, the co-treatment group of LTH-ex and the ferroptosis inducer Erastin exhibited reduced LX-2 cell viability and mitochondrial membrane potential, alongside downregulation of GPX4, xCT, and α-SMA. HE staining, immunohistochemistry, and Sirius red staining demonstrated that LTH-ex could exacerbate vacuolar degeneration of hepatocytes in the liver tissue of mice with nonalcoholic fatty liver disease. Additionally, LTH-ex promoted the expression of GPX4 and αSMA proteins and increased collagen deposition in the liver tissue. expression. Conclusion: LTH-ex increase the cell viability of hepatic stellate cells and the expression of ferroptosis inhibitory genes GPX4 and xCT, and it may be implicated that LTH-ex may promote hepatic stellate cell activation by inhibiting ferroptosis, and exacerbate collagen deposition in the liver tissue of non-alcoholic fatty liver disease mice.

Objective: To investigate the role and mechanism of celastrol in the treatment of relapsed/refractory diffuse large B-cell lymphoma (DLBCL) by inducing ferroptosis. Methods: The rituximab-resistant lymphoma cell line SU-DHL-2-R was established by the concentration gradient method. The CCK-8 assay was used to determine cell viability after treatment with different concentrations of celastrol. SU-DHL-2-R cells were divided into control group, celastrol group, ferroptosis inhibitor (Fer-1) group, and celastrol+Fer1 group. qRT-PCR and Western blotting were used to detect the expression levels of ferroptosis-related genes SLC7A11 and GPX4. GSH and Fe2+ levels were detected using commercial kits, and reactive oxygen species (ROS) levels were detected using the DCFH-DA probe method. The protein expressions of PI3K, p-PI3K, Akt and pAkt were detected by Western blotting. SU-DHL-2-R cells were injected subcutaneously into SCID mice to establish lymphoma tumor bearing mouse models. The tumor bearing mice were divided into control and celastrol treatment groups. The survival status of the mice was monitored and tumor volume was measured. The expressions of SLC7A11 and GPX4 in the lymphoma tissues of the mice were detected by immunohistochemistry. Results: The SU-DHL-2-R cell model was successfully established, and the drug resistance index was 12. Celastrol significantly inhibited the proliferation of SU-DHL-2 and SU-DHL-2-R cells. Compared with the control group, the mRNA and protein expressions of SLC7A11 and GPX4 were downregulated, the level of GSH was decreased, ROS accumulation was induced, Fe2+ levels were increased, and the protein expression levels of pPI3K and p-Akt were significantly downregulated in the celastrol group. However compared with the celastrol group, the above changes were attenuated in the celastrol+Fer-1 group. The tumor-bearing mouse model of lymphoma was established. Compared with the control group, the tumor volume of mice treated with celastrol was reduced, and the expression of SLC7A11 and GPX4 was significantly decreased. Conclusion: Celastrol may inhibit the progression of relapsed/refractory DLBCL by inducing ferroptosis through targeting the PI3K/Akt pathway.

Objective: To figure out the possible mechanisms of ferroptosis and explore potential ferroptosisrelated genes (FRGs) biomarkers and pharmacological compounds for atherosclerosis (AS). Methods: The AS transcriptome dataset GSE100927 was downloaded from the gene expression omnibus (GEO) database. FRGs were downloaded from FerrDb database. To screen the ferroptosisrelated hub genes in AS (FRG-hubs) by weighted gene coexpression network analysis, differential gene analysis, LASSO regression and random forest algorithm. Singlesample gene set enrichment (ssGSEA) analysis was used to evaluate the immunological landscape. Furthermore, transcription factors and miRNAs regulatory networks were constructed by the NetworkAnalyst database, and candidate drugs were searched from DSigDB database. The expression of FRGhubs was verified by AS model mice. Results: zinc finger E-Box binding homeobox 1 (ZEB1), mitogenactivated protein kinase kinase kinase 11 (MAP3K11), and cyclin dependent kinase inhibitor 2A (CDKN2A) were identified as FRG-hubs, and the nomogram model based on them demonstrated high reliability and effectiveness. In addition, FRG-hubs and immune cell infiltration showed a significant association. AS patients could be classified into 2 ferroptosis-related cluster with different immune cell infiltration. Predictions were made for 13 transcription factors, 8 miRNAs, and 10 drugs targeting FRGhubs. The results of qRT-PCR and Western blotting show that compared with the control group, the mRNA and protein expression levels of Map3k11 and Cdkn2a are significantly elevated in the AS model mice, while the expression level of the Zeb1 is significantly decreased. Conclusion: ZEB1, MAP3K11 and CDKN2A may be involved in the occurrence and development of AS by regulating immune-related pathways through ferroptosis.

Objective: To explore the effect of neural stem cell secretome (NSC-S) on ferroptosis human neuroblastoma SH-SY5Y cells and its underlying mechanism. Methods: Human neuroblastoma SH-SY5Y cells were used to establish neuronal ferroptosis model in vitro. And the cells were divided into three groups: control group, Erastin group and Erastin+NSC-S group; cells were treated with DMEM, DMEM containing 10 μmol/L Erastin, and NSC-S containing 10 μmol/L Erastin, respectively. The activities of lactate dehydrogenase (LDH), intracellular reactive oxygen species and malondialdehyde in the supernatants of cells in each group were detected by commercial kits, and the contents of free ferrous ions were detected by fluorescent probes. The expression level of glutathione peroxidase 4 (GPX4) protein in cells was determined by Western blotting. The components of NSC-S were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis and screening were conducted. In addition, SH-SY5Y cells were divided into four groups, which were control group, Erastin group, Erastin+NSC-S group, and Erastin+Parkinson′s disease protein 7 (PARK7) group. And cells were treated with DMEM, DMEM containing 10 μmol/L Erastin, NSC-S containing 10 μmol/L Erastin, and DMEM containing 200 μg/L PARK7+10 μmol/L Erastin, respectively. The viability of SH-SY5Y cells was detected by the CCK-8 assay, and the contents of reactive oxygen species, malondialdehyde, ferrous ions in the cells as well as the expression level of GPX4 protein were also detected. Results: Compared with the Erastin group, the LDH activity of SH-SY5Y cells supernatant in the Erastin+NSC-S group was significantly decreased (P<0.001), the contents of intracellular reactive oxygen species, malondialdehyde and ferrous ions were significantly reduced (P<0.001 or P<0.05), and the relative expression level of GPX4 protein was greatly increased (P<0.05). LC-MS/MS analysis revealed that NSC-S contained PARK7 protein. Compared with the Erastin group, the viability of SH-SY5Y cells in the Erastin+PARK7 group was significantly enhanced (P<0.001), the contents of reactive oxygen species, malondialdehyde and ferrous ions were significantly decreased (P<0.001), and the relative expression level of GPX4 protein was significantly increased (P<0.05). Conclusion: NSC-S could inhibit ferroptosis induced by Erastin in human neuroblastoma SH-SY5Y cells, which may be related to the mediation of PARK7 in NSC-S.

Objective: To investigate the effect of 3,3′-diindolylmethane (DIM) on paclitaxelresistant nonsmall cell lung cancer A549/T cells and explore its underlying mechanism. Methods: Western blotting was used to detect transferrin receptor (TFRC), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4) in human normal bronchial epithelial BEAS-2b cells, human NSCLC A549 cells, paclitaxel-resistant human NSCLC A549/T cells, and human large cell lung cancer H460 cells to select the optimal cell line. A549 and A549/T cells were treated with various concentrations of DIM (0, 20, 40, 80, 160 μmol/L), and cell proliferation was assessed by CCK-8 assay; and the expression of TFRC, SLC7A11 and GPX4 proteins in A549/T cells were determined by Western blotting to screen out the optimal DIM concentration. Additionally, A549/T cells were divided into the control group, which was cultured in highglucose medium containing 10% fetal bovine serum for 24 h; the Erastin group, treated with 10 μmol/L Erastin for 12 h alone; the DIM group, treated only with 80 μmol/L DIM for 24 h; the DIM+Erastin group, cells were pre-treated with 10 μmol/L Erastin for 12 h, then treated with 80 μmol/L DIM in the following 24 h. Cell viability was detected by CCK8 assay, and the expression of TFRC, SLC7A11 and GPX4 proteins was assessed by Western blotting. Results: Compared with the BEAS-2b group, TFRC and SLC7A11 expression was significantly increased in A549 cells (P<0.05), TFRC, SLC7A11 and GPX4 expression was significantly increased in A549/T cells (P<0.05). No significant differences in TFRC, SLC7A11 and GPX4 expression were observed in H460 cells (P>005). In A549/T cells, compared with the 0 μmol/L group, the expression of TFRC, SLC7A11 and GPX4 proteins showed significant alterations in the 80 μmol/L DIM group (all P<0.05). Compared with 0 μmol/L DIM group, in A549 cells, cell viability was significantly decreased in 20, 40, 80 and 160 μmol/L DIM groups (all P<0.05); in A549/T cells, cell proliferation was significantly decreased in 80 and 160 μmol/L DIM groups (all P<0.05). In A549/T cells, compared with the control group, DIM group, Erastin group and DIM+Erastin group showed a significant increase in TFRC expression (all P<0.05), while the expression of SLC7A11 and GPX4 proteins significantly decreased (all P<0.05), and cell proliferation significantly decreased (all P<0.05). Conclusion: DIM may inhibit the proliferation of paclitaxel-resistant non-small cell lung cancer A549/T cells by inducing ferroptosis, and ferroptosis inducers could be used to enhance the anti-cancer effect of DIM.

Objective: To investigate the expression of SET domain containing lysine methyltransferase 7 (SETD7) in ovarian cancer and its effect on epithelial-mesenchymal transition (EMT), as well as the proliferation and migration of ovarian cancer cells. Methods: The expression level of SETD7 in ovarian cancer microarrays was detected by immunohistochemical staining, and the correlation between SETD7 expression and clinicopathological features and prognosis of ovarian cancer patients was analyzed. Both stable knockdown SETD7 and overexpression SETD7 cell lines were constructed by using lentiviral expression system; cell counting, CCK-8, Transwell and scratch assay were used to evaluate the proliferation and migration ability of ovarian cancer cells, while Western blotting was used to determine the expression levels of EMT-related proteins. Results: Compared with normal ovarian tissues, the expression level of SETD7 protein in human ovarian cancer tissues was significantly up-regulated (P<0.01). High expression of SETD7 was greatly correlated with M stage, FIGO stage and progression-free survival in ovarian cancer patients (P<0.05). Knockdown of SETD7 significantly inhibited the proliferation and migration of ovarian cancer A2780 cells (P<0.05), while overexpression was the opposite in SKOV3 cells. Overexpression of SETD7 up-regulated the expression of N-cadherin and Snail, and down-regulated the expression of E-cadherin, while its knockdown exerted the opposite in A2780 cells. Conclusion: SETD7 is up-regulated in human ovarian cancer tissues and cells, which promotes the migration, proliferation and EMT process of ovarian cancer cells.

Objective: To analyze the expression levels of gap junction protein beta 5 (GJB5) in pancreatic cancer and its relationship with patient prognosis, and to preliminarily explore its effect on the malignant biological behavior of pancreatic cancer cells. Methods: Using data from the UCSC XENA database, the relative expression level of GJB5 mRNA in human pan-cancer tissues and their correlation with patient prognosis and various clinicopathological characteristics were analyzed. Gene ontology (GO) and Kyoto encyclopedia of gene and genomes (KEGG) analysis in over representation analysis were used to explore the potential biological functions of GJB5. Gene set enrichment analysis (GSEA) was employed to predict the signaling pathways regulated by GJB5related differentially expressed genes. qRTPCR was used to detect the expression levels of GJB5 mRNA in human pancreatic ductal epithelial cells (HPNE) and five human pancreatic cancer cell lines (CFPAC1, PaTu 8988T, BxPC3, MIA PaCa2 and PANC1), and the optimal pancreatic cancer cell lines were selected for transfection. In vitro experiments, lentiviral transfection was used to establish GJB5 knockdown and overexpression models in human pancreatic cancer PaTu 8988-T and BxPC-3 cells, and to assess the effects of GJB5 on the growth, proliferation, migration, and invasion of pancreatic cancer cells. Results: Bioinformatics analysis showed that the relative expression of GJB5 mRNA was significantly upregulated in pancreatic cancer tissues compared to adjacent normal tissues (P<0.05). High GJB5 mRNA expression levels were significantly correlated with the poor prognosis of pancreatic cancer patients (P<0.05). The expression of GJB5 mRNA was significantly correlated with histological grade of pancreatic cancer patients (P<005)，and there was no significant correlation with gender, smoking history, alcohol history, T stage and N stage (P>005). GO/KEGG analysis indicated that GJB5 plays an important role in various biological processes, including signal release, synaptic signaling regulation, chemical synapse transmission regulation, membrane potential regulation, and hormone transport. GSEA results suggested that GJB5-related differentially expressed genes might be involved in regulating multiple signaling pathways, such as KRAS, apoptosis and protein ubiquitination. Compared with HPNE cells, the relative expression of GJB5 mRNA was significantly increased in the five human pancreatic cancer cell lines (all P<0.05). Knockdown of GJB5 mRNA significantly inhibited the growth, proliferation of PaTu 8988-T and BxPC-3 cells, migration and invasion capabilities of PaTu 8988T cells; on the contrary, overexpression of GJB5 mRNA promoted the growth, proliferation of PaTu 8988-T and BxPC-3 cells, migration and invasion capabilities of PaTu 8988-T cells. Conclusion: GJB5 is significantly overexpressed in pancreatic cancer tissues and pancreatic cancer cell lines, and GJB5 overexpression is related to the poor prognosis of pancreatic cancer patients, and promotes the malignant growth of pancreatic cancer cells lines PaTu 8988-T and BxPC-3.

Objective: To investigate the origin and distribution of IL-9 derived cells in patients with different types of thyroid cancer. Methods: Patients with papillary thyroid carcinoma (22 cases) and medullary thyroid carcinoma (8 cases) were recruited from March 2021 to December 2023, along with 30 healthy individuals undergoing physical examinations as controls. Blood samples were collected preoperatively and within 7 days postoperatively from the thyroid cancer patients and the controls. The expression of IL-9 in peripheral blood mononuclear cells (PBMCs) was measured using flow cytometry. The levels of cytokines IL-13, IL-4, IL-33, IL-25, IL-1β, IL-9, and IL-21 in serum were determined through enzymelinked immunosorbent assay (ELISA). The mRNA levels of PU-1, SMAD2, TGFβ, and STAT3 in blood cells were measured via quantitative realtime PCR (qRTPCR). During surgeries, thyroid cancer tissue and adjacent normal tissue samples were obtained. The mRNA expression levels of IL-25, IL-33, IL-1β, IL-13, RORα, and IL-9 in tissues were detected by qRT-PCR. Immunofluorescence staining was applied to analyze IL-9 expression in cancerous and adjacent normal tissues. Results: Flow cytometry showed that the distribution frequency of IL-9-producing T cells (Th9 cells) was higher in papillary thyroid carcinoma patients than those in medullary thyroid carcinoma patients, and higher in medullary thyroid carcinoma patients than those in the control group. After surgery, the distribution frequency of Th9 cells increased significantly in both papillary thyroid carcinoma and medullary thyroid carcinoma groups. However, the distribution of type 2 innate lymphoid cells (ILC2s) producing IL9 showed no significant difference among the groups. Preoperatively, papillary thyroid carcinoma and medullary thyroid carcinoma patients had significantly higher serum levels of IL-9, IL-33, IL-25, IL-1β, IL-4, and IL-21 than the control group. Postoperatively, IL-9 and IL-4 levels increased further, while IL-33 and IL-25 levels decreased significantly. qRT-PCR revealed that the mRNA expression of Th9related transcription factors PU-1, SMAD2, TGFβ, and STAT3 was lowest in the control group and increased significantly after surgery in thyroid cancer patients. In cancer tissue, qRT-PCR showed that IL-25 and IL-33 mRNA expression in medullary thyroid carcinoma and papillary thyroid carcinoma were significantly higher than those in adjacent tissues, and the medullary thyroid carcinoma was significantly higher than that in papillary thyroid carcinoma tissues. Contrastedly IL-9 and IL-1β mRNA expression were decreased. Immunofluorescence staining showed that IL-9 expression was lower in cancer tissue than in adjacent normal tissue and was mainly located in CD4⁺ T cells. Conclusion: IL-9 derived from Th9 plays an antitumor role in patients with thyroid cancer. The presence and distribution of Th9 cells correlate with the malignancy of thyroid cancer. IL-9 and Th9 cell may be considered as promising new targets for the diagnosis and therapeutic monitoring of thyroid cancer.

Objective: To develop and validate a nomogram model utilizing CT radiomics features for predicting the efficacy of radiotherapy in esophageal squamous cancer. Methods: A retrospective analysis was conducted for 201 patients diagnosed with esophageal squamous cancer at Xuzhou Cancer Hospital and Xuzhou Mining Group General Hospital between January 2019 and January 2024. The dataset was partitioned into the training set (n=79), the testing set (n=34), and the external validation set (n=88). Radiomics features were extracted from pre-radiotherapy CT images of patients, followed by dimensionality reduction. Univariate and multivariate Logistic regression analyses were employed to identify the significant clinical features of the constructed model. The support vector machine learning algorithm was utilized to construct three predictive models: the clinical model, the radiomics model, and the nomogram model. The nomogram model was visualized using a nomogram. The predictive performance of the constructed model was assessed through external validation. The model′s clinical applicability were measured by dicision curve analysis (DCA). Results: A total of 1 834 CT radiomics features were extracted. Following feature screening and dimensionality reduction, eleven optimal radiomics features were selected for subsequent model construction. The area under curve (AUC) for the nomogram model in the training set was 0980, which exceeded that of both the clinical model (AUC=0.859) and the radiomics model (AUC=0.926). In the testing set, the AUC for the nomogram model was 0.834, surpassing the clinical model (AUC=0.638) and the radiomics model (AUC=0.818). Furthermore, in the external validation set, the AUC for the nomogram model was 0883, outperforming the clinical model (AUC=0.652) and the radiomics model (AUC=0.765). DCA results demonstrated that the nomogram model had the highest clinical net benefit in comparison to the alternative models. Conclusion: The developed nomogram model, which integrates CT radiomics and clinical features, may be applied for predicting the efficacy of radiotherapy for esophageal squamous cancer.

Objective: To assess the predictive value of systemic inflammatory markers such as C-reactive protein (CRP), and total bilirubin (TBil) combined with carbohydrate antigen 199 (CA199) for malignant biliary stricture (MBS). Methods: A total of 241 patients diagnosed with biliary strictures by endoscopic retrograde cholangiopancreatography in the Affiliated People′s Hospital of Jiangsu University from January 2019 to October 2022 were selected. According to the clinical diagnosis, the patients were divided into MBS group and benign biliary strictures (BBS) group. The relevant laboratory examination indicators of the two group patients were collected; and the clinicopathological characteristics of the two groups were compared. The risk factors of MBS were determined through binary Logistic regression analysis, and the predictive effects of each index were evaluated by using the receiver operating characteristic (ROC) curve. Results: Compared with the BBS group, patients in the MBS group were significantly older (P<0.05), and the levels of TBil, CA199, and platelet-lymphocyte ratio were greatly increased (P<0.05), while the levels of CRP and lymphocyte-monocyte ratio were significantly decreased (P<0.05). The results of multivariate Logistic regression analysis showed that advanced age, high CA199, high TBil and low CRP were all independent risk factors of MBS (P<0.05). The results of the ROC curve showed that the areas under the curve (AUC) of age, TBil, CA199 and CRP for predicting the occurrence of MBS were 0.574, 0.731, 0.750 and 0.523, respectively, and the AUC of age+CA199+TBil+CRP for predicting MBS was 0.807. Conclusion: Age, CRP and TBil combined with CA199 may be used to predict for MBS.

Objective: To integrate the two elements of selenium and magnesium into nanoparticles to prepare selenium-magnesium nanoparticles, and explore their antibacterial effect and antibacterial mechanism. Methods: The morphology and particle size distribution of selenium-magnesium nanoparticles were characterized by transmission electron microscopy and nanoparticle size analyzer. The antibacterial effect of selenium-magnesium nanoparticles was evaluated by plate counting method, disk diffusion method, and bacterial live/dead staining method. The content of reactive oxygen species in bacteria after exposure to nanoparticles was detected by DCFH-DA fluorescence probe to explore its potential antibacterial mechanism. Results: Selenium-magnesium nanoparticles are irregular spherical under transmission electron microscopy, and the particle size is mainly concentrated in the range of 50-70 nm. They exhibit excellent antibacterial properties, especially against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. The potential antibacterial mechanism involves induction of reactive oxygen species production in bacteria. Conclusion: The prepared selenium-magnesium nanoparticles exhibit good antibacterial properties, it has been preliminarily demonstrated that bacterial killing is achieved by inducing the production of reactive oxygen species within bacterial.