Abstract:
Objectivec To investigate the therapeutic effect of wall-broken Ganoderma lucidum spore powder (GLS) on a mouse model of hepatocellular carcinoma and to explore its underlying mechanism. Methods Fifty 14-day-old C57BL/6J mice were divided into a control group, a model group and low, medium and high-dose GLS groups(0.25, 0.5, 1.0 g/kg ), with 10 mice in each group. A DEN/CCL4 mouse hepatocellular carcinoma model was established, and GLS was administered by gavage starting from the 8th week, once a day for 14 weeks. At the end of the final administration, the number of liver nodules was observed , and the serum alpha-fetoprotein (AFP) was detected using enzyme-linked immunosorbent assay. The content of catalase (CAT) in serum and liver tissue was determined by ammonium molybdate method. The content of reduced glutathione (GSH) in serum and liver tissue was measured by microplate assay. The content of glutathione peroxidase (GSH-PX) in serum and liver tissue was detected by colorimetric method. Immunohistochemistry (IHC-P) was employed to detect the protein expression of proliferating cell nuclear antigen (PCNA). R eal-time fluorescence quantitative polymerase chain reaction was utilized to detect the relative expression of nuclear factor E2-related factor 2 (Nrf2), Kelch-like epichlorohydrin-associated protein 1 (Keap1),NAD(P)H quinone oxidoreductase 1 (Nqo1), heme oxygenase-1 ( Ho-1 ) and glutamate cysteine ligase catalytic subunit (Gclc) mRNA. Results Compared with the control group, the content of serum AFP were significantly higher in the model group (P<0.01), while the contents of CAT, GSH and GSH-PX in liver and serum were significantly lower in the model group (P<0.01). The relative expression of Keap1 and Nqo1 mRNA was significantly higher (P<0.01), the relative expression of Nrf2, Ho-1, Gclc mRNA relative expression was significantly lower (P<0.01), and the positive expression rate of PCNA was significantly higher (P<0.01). Compared with the model group, the total number of liver nodules and content of serum AFP in the administered group were significantly lower (P<0.01), and the contents of CAT, GSH, and GSH-PX in the liver and serum of mice significantly increased (P<0.05 or P<0.01). The expression of Keap1 and Nqo1 mRNA significantly decreased (P<0.05). The expression of Nrf2, Ho-1 and Gclc mRNA significantly increased (P<0.05), and positive expression rate of PCNA significantly decreased (P<0.01). Conclusion GLS has a significant protective effect on DEN/CCL4 -induced mouse hepatocellular carcinoma model, and the mechanism of action is related to the regulation of oxidative stress in mouse liver.

Abstract:
Objective To explore the ameliorative effect of the endoplasmic reticulum stress inhibitor 4-phenylbutyric acid (4-PBA) on bile acid metabolism disorders induced by bacterial lipopolysaccharide (LPS) in late-pregnant mice, and to clarify the regulatory mechanism of endoplasmic reticulum stress (ERS) in intrahepatic cholestasis of pregnancy (ICP). Methods Twenty-four pregnant mice were divided into control group, LPS group, 4-PBA group, and LPS+4-PBA group. On the 16th day of pregnancy, the LPS group received intraperitoneal injection of LPS (200 μg/kg) and the 4-PBA group received injection of 4-PBA (150 mg/kg). In the LPS+4-PBA group, 4-PBA was intraperitoneally injected first, followed by LPS administration 1 h later. Serum total bile acid (TBA), liver injury markers, bile acid profiles, and the expression of key genes and proteins were detected 6 h after LPS treatment. Results LPS exposure increased serum TBA levels in pregnant mice(P<0.05), activated ERS markers and inflammatory factors, and downregulated bile acid transporters and the expression of the rate limiting enzyme CYP7A1(all P<0.05). 4-PBA intervention effectively inhibited ERS, reduced TBA levels(P<0.05), and upregulated Ntcp, Bsep, Mdr3, and Mrp3 gene expression(all P<0.05). Conclusion ERS plays an important role in LPS interference with the homeostasis of bile acid metabolism during pregnancy. Inhibiting ERS can improve bile stasis by regulating bile acid transporters, providing a theoretical basis for targeted therapy of ICP.

Abstract:
Objective To humanize the variable region of the IL9-2G1 rabbit monoclonal antibody (IL9 2G1 mAb) and to evaluate its biological activity. Methods The humanized sequences of IL9-2G1 mAb were designed. The heavy chain variable region (VH) and light chain variable region (VL) of the antibody were humanized and cloned into vector backbones containing the constant region frameworks of human IgG1 and Ig Kappa subtypes. Protein purification was carried out using the Chinese hamster ovary cell (CHO) expression system. Enzyme-linked immunosorbent assay (ELISA) was used to qualitatively analyze the antigen-binding activity ofthe antibodies. After screening the optimal humanized antibody candidate, surface plasmon resonance (SPR) was performed to detect molecular affinity. Western blot was used to examine the effects of the rIL-9 group and the rIL-9 + hIL-9 mAb group on the phosphorylation of Janus kinase 1/signal transducer and activator of transcription 3 (JAK1/STAT3), while ELISA was employed to assess the impact of the Th9 group and the Th9 + hIL-9 mAb group on interleukin-9 (IL-9) secretion. Results Purification and ELISA results showed that the heavy chain OH and light chain FL exhibited good compatibility and expression performance. SPR results indicated that the equilibrium dissociation constant (KD) of M501042-OHFL against the analyte M30903 was 1.33×10_7 M, which was superior to the parental 2G1 antibody. The Western blot results showed that the levels of phosphorylated JAK1 (p-JAK1) and phosphorylated STAT3 (p-STAT3) were significantly decreased in the rIL-9 + hIL-9 mAb group compared with the rIL-9 group (P<0.01). The ELISA results indicated that the secretion of IL-9 was significantly reduced in the Th9 + hIL-9 mAb group compared with the Th9 group (P<0.01). Conclusion Humanization modification is performed on the variable region of IL9-2G1 rabbit monoclonal antibody, and the humanized antibody hIL-9 mAb OHFL with high affinity and strong binding stability is successfully constructed, which provides an experimental basis for subsequent research.

Abstract:
Objective This study aims to construct and evaluate various machine learning models for predicting the risk of chronic obstructive pulmonary disease (COPD) in individuals, thereby providing data support for early screening and intervention.Methods A total of 823 subjects were selected for this study, comprising l42 individuals in the high-risk group for COPD and 68l individuals in the low-risk group. Data collected included demographic characteristics, smoking history, symptoms (such as cough and shortness of breath), and scores from the Chronic obstructive pulmonary disease screening questionnaire. Four machine learning algorithms—Logistic Regression, Random Forest, Support Vector Machine, and XGBoost—were utilized to construct risk prediction models. The performance of these models was assessed using 5-fold cross-validation, with evaluation metrics including accuracy, precision, recall, Fl-score, area under the receiver operating characteristic curve (AUC), and average precision (AP). Furthermore, a feature importance analysis was performed.Results The Logistic Regression model exhibited superior performance, achieving an AUC of 0.982 and an AP of 0.939. This was closely followed by the Random Forest model, which recorded an AUC of 0.975 and an AP of 0.890. Feature importance analysis revealed that smoking history, symptoms of shortness of breath, and body weight were significant predictors. All models demonstrated robust performance in identifying low-risk populations, with precision values exceeding 0.93; however, variations were observed in their efficacy in identifying high-risk populations.Conclusion Machine learning models have proven effective in identifying individuals at high risk for COPD. Among these, the logistic regression model exhibits the best overall performance, efficiently identifying high-risk populations and serving as a valuable clinical auxiliary screening tool. Various models, each with distinct performance characteristics, are suited to different clinical screening scenarios, thereby offering targeted decision-making support for the establishment of a hierarchical and intelligent COPD screening pathway.

Abstract:
Objective To explore the structural characteristics, biocompatibility, anti-inflammatory and antioxidant properties of hyaluronic acid (HA)/silk fibroin (SF) composite hydrogel scaffolds loaded with epigallocatechin gallate (EGCG), and to provide a new material basis for the treatment of periodontitis. Methods The composite hydrogel scaffolds were constructed by cross-linking HA and SF and loaded with EGCG. The water absorption rate, degradation property and material characteristics were detected by scanning electron microscopy. The cytocompatibility and anti-inflammatory and antioxidant properties were evaluated by in vitro experiments. Results The EGCG-loaded scaffolds exhibited a porous network structure and desirable swelling properties. In vitro experiments demonstrated that the scaffolds possessed good biocompatibility, along with the ability to inhibit the expression of inflammatory factors and scavenge reactive oxygen species. Conclusion The HA/SF-EGCG composite scaffolds have excellent biocompatibility and dual functional activities of anti-inflammatory and antioxidant, providing a new strategy for improving the clinical treatment effect of periodontitis.

Abstract:
Objective To prepare photosensitive nanodiscs loaded with rapamycin and evaluate their in vitro antitumor activity against ovarian cancer. Methods Nanodiscs were prepared by the thin-film hydration method， and their particle size， potential and morphology were characterized by dynamic light scattering and transmission electron microscopy. High performance liquid chromatography was established for the determination of rapamycin content， and the specificity， linearity， precision， repeatability， stability， and recovery rate of the method were verified. The singlet oxygen generation and light-controlled drug release properties of nanodiscs under near-infrared light were evaluated respectively by singlet oxygen detection probe and reverse dialysis method. The cytotoxicity of the nanodiscs against human ovarian cancer cells (OVCAR-8) was evaluated by detecting cell metabolic activity. Confocal microscopy and flow cytometry were used to investigate the cellular uptake and endocytosis mechanisms of the nanodiscs. Results The nanodiscs exhibited a hydrodynamic diameter of (63.66±0.27) nm， a zeta potential of (-18.2 ± 2.4) mV， and demonstrated good stability. The results from transmission electron microscopy revealed that the nanodiscs exhibited spherical and short rod-like morphologies. The peak area of rapamycin showed good linearity in the concentration range of 0.2～40 μg/mL (R=0.999 9)， and the relative standard deviations (RSDs) of precision， repeatability and recovery were all less than 5%. Under near-infrared light irradiation， the nanodiscs could effectively produce singlet oxygen. At the same time， the singlet oxygen generated could induce lipid membrane oxidation， which significantly promoted the responsive release of rapamycin (P<0.001). Nanodiscs had no obvious cytotoxicity， but the cytotoxicity to ovarian cancer cells was significantly enhanced after near-infrared irradiation. Cellular uptake experiments confirmed that the nanodiscs mainly entered cells through clathrin-mediated， energy-dependent endocytosis (P<0.001)， with drug release triggered by light exposure. Conclusion Rapamycin photosensitive nanodiscs were successfully prepared， which showed significant light-controlled release properties and enhanced anti-ovarian cancer activity in vitro.

Abstract:
Objective To investigate the protective effect of liver-targeted fibroblast growth factor 21 (FGF21) overexpression against CCl₄-induced acute liver injury (ALI) and its association with silent information regulator 1/glutathione peroxidase 4 (SIRT1/GPX4) expression and ferroptosis-related markers. Methods Female ICR mice (8 weeks old) were randomly divided into Control, CCl₄, adeno- associated virus (AAV)-FGF21, and AAV-FGF21 + CCl4 groups. Liver-targeted FGF21 overexpression was achieved via tail vein injection of AAV8-FGF21 virus with green fluorescent protein (GFP) labeling. ALI was induced by intraperitoneal injection of CCl₄ (0.3 mL/kg), with sample collection at 12 hours. Body weight and liver weight were recorded to calculate the liver weight/body weight. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using a biochemical analyzer. FGF21 expression in liver tissues was detected by immunofluorescence. Hepatic histopathology was evaluated by HE staining. RT-qPCR was performed to assess mRNA expression of ferroptosis and iron metabolism-related genes (ACSL4, PTGS2, HJV, FPN, GPX4), FGF21, and SIRT1. Protein expression ofFGF21, SIRT1, and GPX4 were determined by Western blot. Iron content, malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) activity in liver homogenate were quantified using assay kits. Results Compared with the Control group, the FGF21 mRNA and protein expression elevated in CCl4 group;serum ALT/AST levels and liver weight/body weigh increased. The HE staining results showed that the liver cell membranes were ruptured. The mRNA levels of ACSL4 and PTGS2, the iron content and MDA levels all increased (P<0.01). The mRNA levels of SIRT1, GPX4, HJV, and FPN, the GSH level and SOD activity all decreased (P<0.05), and the protein levels ofSIRT1 and GPX4 both decreased (P<0.05). Compared with the CCl4 group, the serum levels of ALT and AST in the AAV- FGF21 + CCl4 group were decreased (P<0.01), and the liver/body ratio of mice decreased (P<0.01). The results of HE staining showed that the degree of liver injury was alleviated. The mRNA levels of ACSL4 and PTGS2, iron content and MDA level all decreased (P<0.01), while the mRNA levels ofSIRT1, GPX4, HJV, FPN, GSH level and SOD activity all increased (P<0.05). The protein levels of SIRT1 and GPX4 both increased (P<0.05). Conclusion FGF21 overexpression is associated with enhanced SIRT1/GPX4 expression and suppression of ferroptosis features, thereby contributing to the alleviation of CCl4-induced acute liver injury.

Abstract:
Objective To investigate the effect of Semaphorin 3A (Sema3A) on the progression of pathological cardiac hypertrophy. Methods Wild-type C57BL 6J mice, Sema3A global overexpressing (Sema3A/Cre-ERTM) mice, and their littermate controls (Cre-ERTM) were used, with 6 mice per group. A pressure-overload induced cardiac hypertrophy model was established via transverse aortic constriction (TAC), with a sham-operated group (Sham) as the control. Cardiac hypertrophy was assessed by gross morphology, heart weight/body weight ratio (HW/BW), heart weight/tibia length ratio (HW/HL), and heart weight/lung weight ratio (HW/PW). Cardiac function was evaluated by measuring ejection fraction (EF) and fractional shortening (FS) via echocardiography. In heart tissues, the mRNA level of atrial natriuretic peptide (ANP) and Sema3A were detected by RT-qPCR, Sema3A protein level was detected by western blot, morphological structure was detected by hematoxylin and eosin (HE) staining, level of fibrosis was detected by Masson’s trichrome staining, and angiogenesis was detected by immunohistochemical detection of CD31.Forin vitroexperiments, rat H9C2 cardiomyocytes were treated with angiotensin II (Ang II) to induce hypertrophy, with PBS-treated cells as the control. After 48 hours, the mRNA expression of ANP, B-type natriuretic peptide (BNP) and Sema3A were measured by RT-qPCR, and Sema3A protein level was assessed by western blot.Results At the animal level, compared with the sham group, mice subjected to TAC showed significantly increased mRNA levels of ANP in cardiac tissue, as well as markedly elevated mRNA and protein levels of Sema3A (P<0.05). Compared with the Cre-ERTM + TAC group, the Sema3A/Cre-ERTM + TAC group exhibited more pronounced cardiac hypertrophy, greater ventricular wall thickening, higher HW/BW, HW/HL, and HW/PW ratios, more significant reductions in EF and FS, more severe cardiac fibrosis, and a significant decrease in cardiac CD31 expression levels (P<0.05).At the cellular level, compared with the control group, H9C2 cells treated with Ang II displayed significantly increased mRNA levels ofANP and BNP, along with markedly elevated mRNA and protein levels of Sema3A (P<0.05).ConclusionSema3A promotes the progression of pressure overload-induced cardiac hypertrophy by inhibiting angiogenesis.

Abstract:
Objective To explore the risk factors for liver injury in sepsis, so as to provide a reference for the diagnosis and treatment of sepsis patients with liver injury. Methods A retrospective analysis was conducted on the clinical data of 402 patients with sepsis. According to the occurrence of acute liver injury during ICU stay, the patients were divided into a sepsis with liver injury group and a sepsis without liver injury group. Univariate analysis was used to screen clinical indicators potentially associated with liver injury, and a multivariate Logistic regression model was then established to identify the independent risk factors for acute liver injury in sepsis patients. Results A total of 229 sepsis patients who met the inclusion criteria were enrolled, among whom 110 cases (48.03%) had liver injury, and 52 patients (47.27%) in the liver injury group died. There were statistically significant differences between the two groups in APACHE II score, SOFA score, platelet count, lactate level, fibrinogen content, diabetes mellitus, abdominal infection, vasoactive drug therapy, CRRT therapy, and mechanical ventilation therapy (P < 0.05). Multivariate Logistic regression analysis showed that SOFA score (OR = 1.19, 95%CI: 1.06 ~ 1.33), diabetes mellitus (OR = 3.27, 95%CI: 1.65 ~ 6.48), abdominal infection (OR = 2.74, 95%CI: 1.33 ~ 5.62), and mechanical ventilation therapy (OR = 5.24, 95%CI: 1.58 ~ 17.41) were independent risk factors. Conclusion Diabetes, mellitus, abdominal infection, SOFA score, and mechanical ventilation therapy are independent risk factors for acute liver injury in sepsis patients.

Abstract:
Objective To focused on patients with unexplained repeated implantation failure (URIF) and compare the clinical efficacy of different administration routes of granulocyte colony- stimulating factor (G-CSF) in patients undergoing frozen-thawed embryo transfer cycles.Methods A retrospective case-control study was conducted. A total of 187 patients with unexplained recurrent implantation failure (URIF) who received medical treatment were enrolled in this study. According to whether G-CSF was used on the day of endometrial transformation and its administration route in frozen-thawed embryo transfer cycles, the patients were divided into three groups: control group (without G-CSF, n = 71), subcutaneous injection group (subcutaneous G- CSF, n = 72), and intrauterine infusion group (intrauterine G-CSF infusion, n = 44). The pregnancy outcomes among the three groups were compared, and Logistic regression analysis was performed. Results There were significant differences in biochemical pregnancy rates among the three groups (P < 0.05), indicating statistical significance in the overall comparison. In terms of numerical trend, the biochemical pregnancy rate was in the order: subcutaneous injection group > intrauterine infusion group > control group. Further pairwise comparison showed that only the difference between the subcutaneous injection group and the control group was statistically significant (P < 0.017), while no significant differences were found between the other groups (P > 0.017). The overall comparison of early miscarriage rates among the three groups also showed a significant difference (P < 0.05), indicating a statistically significant difference in the distribution among the groups. The numerical trend was: intrauterine perfusion group > subcutaneous injection group > control group. Pairwise comparison results showed that only the difference between the intrauterine perfusion group and the control group was statistically significant (P < 0.017), while there was no statistically significant difference between any other two groups (P > 0.017).Multivariate Logistic regression analysis showed that endometrial thickness on the day of transformation, number of transferred embryos, and subcutaneous G-CSF injection were independent factors associated with an increased biochemical pregnancy rate, where as age was an independent factor for a decreased biochemical pregnancy rate. For clinical pregnancy outcome, endometrial thickness on the day of transformation, number of transferred embryos, and subcutaneous G-CSF injection were also independent factors for an increased clinical pregnancy rate, while BMI was an independent factor for a decreased clinical pregnancy rate. Conclusion Subcutaneous injection of G-CSF only improved the biochemical pregnancy rate in URIF patients. Multivariate Logistic regression analysis showed that subcutaneous G-CSF injection was a favorable factor for clinical pregnancy rate in URIF patients; however, no significant improvement in clinical pregnancy rate was observed among the three groups. Both the comparison of pregnancy outcomes and the results of multivariate logistic regression analysis indicates that intrauterine perfusion of G-CSF has no significant therapeutic effect in the treatment of patients with URIF.

Abstract:
Objective To construct a chimeric antigen receptor macrophage (CAR-M) targeting interleukin-13 receptor α (IL-13Rα) and intracellular domain integration, which can double activate the signal adaptor protein OX40 L of macrophages and T cells. Methods The CAR molecule targeting IL- 13Rα (IL-13Rα-OX40L-CAR) with OX40L as the intracellular signal domain was designed and synthesized, and it was cloned into the adenovirus expression vector. After THP-1 cells were induced to differentiate into macrophages (THP-1-M) by phorbol ester, the cells were infected with recombinant adenovirus to construct THP-1 macrophages stably expressing green fluorescent protein (GFP) labeled anti-IL- 13RαCAR. The expression efficiency of CAR molecules was monitored by flow cytometry. The empty vector group was used as the negative control, the CD3ζ group was used as the positive control, and the CD3ζ-OX40L group was used as the experimental group. The phagocytic function of macrophages in each group on IL- 13Rα high expression glioma cells (U251) and low expression glioma cells (T98G) was detected by in vitro co-culture experiments. Results CAR adenovirus was efficiently transfected into THP-1-M to obtain anti-IL- 13RαCAR-M. After co-cultured with glioma cells with different expression levels of IL- 13Rα, the results of flow cytometry showed that there was no significant difference among the three groups for T98G cells with low expression of IL- 13Rα . After co-culture with U251 cells with high expression of IL- 13Rα, the CD3ζ group showed enhanced phagocytosis compared with the empty vector group (P<0.05). Compared with the CD3ζ group, the phagocytosis ability of the CD3ζ-OX40L group was further enhanced (P<0.05). Conclusion Anti-IL13Rα-OX40L-CAR-M is successfully constructed by constructing IL- 13Rα-OX40L-CAR adenovirus and transfecting PMA-induced differentiated THP-1-M. It can engulf glioma cells with high expression of IL- 13Rα by specific targeting, and shows activation of intracellular domain superior to traditional CD3ζ .

Abstract:
Objective To evaluate the efficacy of immediate postoperative oral administration of 2 mg estradiol/estradiol dydrogesterone tablets in reducing secondary surgery for retained products of conception(RPOC), preventing intrauterine adhesions(IUA), and promoting endometrial repair following induced abortion. Methods A retrospective analysis was conducted on 494 patients who underwent induced abortion. The control group (n=214) received conventional postoperative treatment with antibiotics and Yimucao granules, while the observation group (n=280) received immediate administration of estradiol/dydrogesterone tablets (each tablet contains 2 mg of estradiol),combined with antibiotics and Yimucao granules. The two groups were compared for the secondary surgery rate for RPOC, incidence of IUA, endometrial thickness at 2 weeks postoperatively, duration of postoperative vaginal bleeding, time to menstrual resumption, incidence of reduced menstrual volume after resumption, and postoperative complications (pelvic infection, intrauterine fluid accumulation). Results The observation group showed a lower rate of secondary surgery for RPOC compared to the control group (0.4% vs 2.3%), but the difference was not statistically significant (P=0.13, Fisher’s exact test). The observation group also had a significantly lower incidence of IUA (P<0.05), greater postoperative endometrial thickness at 2 weeks (P<0.05), and significantly shorter duration of postoperative vaginal bleeding and time to menstrual resumption. The incidence of reduced menstrual volume postoperatively and complications (pelvic infection, intrauterine fluid accumulation) were also significantly lower in the observation group (all P<0.05). Conclusion Immediate oral administration of estradiol/dydrogesterone tablets after induced abortion significantly prevents intrauterine adhesions, shortens postoperative vaginal bleeding duration, promotes endometrial repair, improves menstrual recovery, and potentially reduces the risk of secondary surgery for retained products of conception.

Abstract:
Objective To investigate the effect of hypoxia on pemetrexed (PE)-mediated apoptosis in non-small cell lung cancer cells. Methods A549 cells were cultured under 21% O₂ (normoxia) or 1% O₂ (hypoxia), and treated with 8 μmol/L PE for 48 h. The cells were divided into normoxic control group, normoxic vehicle group, normoxic drug-treated group, hypoxic control group, hypoxic vehicle group and hypoxic drug-treated group. Trypan blue staining was used to observe cell death after PE intervention under normoxia and hypoxia. Flow cytometry was adopted to detect cell apoptosis rate. Wound healing assay was used to assess cell migration. Western blot was performed to detect the protein levels of MRP-1, Cleaved Caspase-3, E-Cadherin and N-Cadherin. Results Trypan blue staining results showed that the cell death rate was reduced in PE-treated cells under hypoxia compared with the normoxic PE group (P<0.001). Flow cytometry results indicated that PE induced cell apoptosis under both normoxic and hypoxic conditions.However, the apoptosis rate of the normoxic PE group was significantly higher than that of the hypoxic PE group (P<0.000 1). Wound healing assay showed that the migration distance of A549 cells decreased after PE treatment for 24 h and 48 h under normoxia and hypoxia when compared with the control group (P<0.05), while no significant difference was found between the normoxic PE group and the hypoxic PE group. Western blot results revealed that MRP-1 expression was markedly higher in hypoxic PE-treated cells than in normoxic PE-treated cells (P<0.000 1). Compared with the control group, Cleaved Caspase-3 expression was upregulated in A549 cells following PE intervention under normoxia (P<0.05), whereas no obvious alteration in Cleaved Caspase-3 expression was detected in hypoxic PE-treated cells. Under both normoxic and hypoxic conditions, PE treatment downregulated the expression of E-Cadherin (P<0.000 1 ，P<0.001) and N-Cadherin (P<0.05 ，P<0.000 1). In addition, the expression levels of E-Cadherin and N-Cadherin in the hypoxic PE group were significantly lower than those in the normoxic PE group (P<0.000 1). Conclusion Hypoxia significantly attenuates the pro-apoptotic effect of PE on A549 cells and blocks Cleaved Caspase-3 activation. This mechanism may be related to hypoxia-induced MRP-1 overexpression and reduced drug accumulation. Interfering with MRP-1 or restoring the activity of the apoptosis pathway may serve as a novel strategy to overcome hypoxia-induced drug resistance.

Abstract:
Objective. , To evaluate the mechanical properties and biocompatibility of a 3 mol% yttria-stabilized tetragonal zirconia polycrystal (3Y-TZP)-polymethyl methacrylate (PMMA) biomimetic layered composite and to explore the feasibility of using this composite for ceramic dental crowns. Methods The micro-morphology of 3Y-TZP powder was observed using scanning electron microscopy and metallographic microscopy. The ball-milled 3Y-TZP powder was stirred together with bacterial cellulose to obtain a homogeneous ceramic slurry. Uniform ceramic films were prepared by tape casting. After stacking the ceramic films, the composite was finally obtained through pressure-less sintering, silane coupling, and organic matter infiltration. The microstructure of the composite was observed using scanning electron microscopy. The mechanical properties of the composite were measured using a universal testing machine, and the energy dissipation rate and impact toughness of the samples were evaluated through ball drop and falling weight impact tests. The biocompatibility of the composite was investigated using CCK-8 assay, live/dead cell staining, cell adhesion assay, and Transwell cell migration assay. Results The composite exhibited a distinct layered structure. As the thickness of the ceramic layer decreased, the bending and compressive strengths of the material gradually decreased, while its impact resistance was significantly improved. The maximum energy dissipation rate and impact toughness can reach 75.8% and 44.1 kJ/m²respectively. No significant differences were observed in the CCK-8 assay, live/dead cell staining, or cell migration assay results between the blank group and the material group, and cells were able to adhere well to the composite. Conclusion This study successfully fabricates 3Y-TZP-PMMA composite materials, which possess both excellent mechanical properties and biocompatibility, demonstrating promising prospects for clinical application.

Abstract:
Objective To optimize the flow cytometric sorting workflow for myeloid-derived suppressor cells (MDSCs) from murine Lewis lung carcinoma (LLC) subcutaneous tumor tissues, improving single-cell suspension preparation efficiency and sorting purity, thereby providing high-quality cells for subsequent functional studies. Methods Subcutaneous LLC tumor tissues were harvested from C57BL/6 mice, and two enzymatic digestion protocols were first compared to evaluate their effects on single-cell suspension preparation. After staining with a viability dye (Live/Dead) and fluorochrome-conjugated anti-mouse antibodies against CD45, CD11b, and Gr-1, cells were loaded onto a Beckman CytoFLEX cell sorter. Next, conventional and optimized gating strategies were applied. In the optimized strategy, the CD45+ population was used as the initial gate to directly define an MDSC-enriched region, followed by selection of CD11b+Gr-1+ cells. Finally, purity was assessed by flow cytometry, viability was determined by trypan blue staining, and RT-qPCR was performed to measure the expression of MDSC signature genes, including Arg1, Nos2, IL-10, and S100A8/A9. Results The optimized enzymatic digestion protocol significantly increased the yield and viability of single cells. After optimization of the gating strategy, the post-sort positivity rate (purity) was markedly improved. Arg1, Nos2, IL-10and S100A8/A9 were highly expressed in the sorted cells, indicating that the isolated cells retained characteristic MDSC features and functional signatures. Conclusion By optimizing both the tissue digestion protocol and the flow cytometric gating strategy, we establish an efficient workflow for isolating MDSCs from tumor tissues. This method improves the yield and purity of single-cell preparations while preserving antigen integrity, providing a reliable methodological foundation for subsequent studies on MDSC function and metabolism.

Abstract:
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent synovial inflammation and progressive joint destruction. Recent studies have shown that lactate metabolic reprogramming plays a key role in the pathological process of RA, and the accumulation of lactate in the synovial microenvironment triggers a cascade of immune regulatory effects. These metabolic changes create a self-sustaining cycle of inflammation and tissue damage, leading to progressively worsening disease. Therefore, lactate metabolism has been identified as a therapeutic target. Therapeutic strategies targeting lactate metabolism, such as LDHA inhibitors, targeting PKM2, MCT inhibitors, GPR81 antagonists, and lactylation modification regulators, have shown great potential in restoring immune balance and protecting joint integrity. This review summarizes the mechanisms of lactate metabolism in RA and its therapeutic applications, providing a theoretical basis for the development of novel metabolic intervention therapies.

Abstract:
Objective To investigate the relationship between Type ⅠHelicobacter pylori (H. pylori) infection and the risk of colorectal neoplasms (CRN) in patients attending gastroenterology clinics, and to evaluate its predictive value for CRN in this population. Methods We included 2,148 patients presenting to the Department of Gastroenterology, with 677 assigned to the CRN group and 1,471 to the non-CRN group.H.pylori antibody typing was performed using Western blot, with type Ⅰ defined as positivity for CagA and/or VacA antibodies, and type II as positivity only for UreA/B antibodies. Statistical analyses included univariate screening, multivariate logistic regression, and receiver operating characteristic (ROC) curve evaluation. Results Type ⅠH.pylori seropositivity was significantly more prevalent in the CRN group than in the non-CRN group (54.51% vs 45.62%, P<0.0167). No significant differences were observed for type II H.pylorior seronegative subjects. The CRN group had higher proportions of males, hypertension, diabetes, smoking, and alcohol use, as well as elevated body mass index, fasting plasma glucose, triglycerides, and uric acid, along with lower high-density lipoprotein levels (all P<0.05). Multivariate analysis confirmed type Ⅰ H.pylori (OR=1.303), alcohol consumption (OR=2.315), male sex (OR=1.592), and age (OR=1.048) as independent risk factors for CRN. Although type I H.pylori alone showed limited predictive value (AUC=0.544), a combined model incorporating alcohol use, age, and sex demonstrated significantly improved discriminative ability (AUC=0.681, sensitivity 59.1%, specificity 68.2%). Conclusion Type ⅠH. pylori infection predicts the risk of CRN in gastroenterology patients. Assessing it can help to optimize the identification of high-risk individuals and inform targeted screening strategies.

Abstract:
Objective To investigate the distribution characteristics and correlation of paired oral and esophageal microbiota in patients with esophageal squamous cell carcinoma (ESCC). Methods A total of 46 pathologically confirmed ESCC patients were enrolled, and oral swabs and cancerous tissue samples were collected. The microbial composition was analyzed using 16S rRNA gene sequencing, and bioinformatics methods were employed to compare the microbiota diversity, compositional differences, and correlations between the two sites. Results The Alpha diversity of the oral microbiota in ESCC patients was significantly higher than that of their esophageal microbiota, though the overall community structure was similar between the two sites. LEfSe analysis revealed that Spirochaetes, Prevotella, Catonella, Enterococcus, Fusobacterium periodonticum, Veillonella parvula, Capnocytophaga sputigena, and Neisseria subflava were significantly enriched in ESCC tumor tissues. In contrast, Actinobacteria, Proteobacteria, Streptococcus, Neisseria, Haemophilus, Streptococcus mitis, Haemophilus parainfluenzae, Porphyromonas gingivalis, Haemophilus haemolyticus, and Prevotella tannerae were significantly enriched in the oral cavity. Correlation analysis demonstrated significant associations between the two sites at taxonomic levels such as Spirochaetes, Treponema, Granulicatella elegans, Neisseria macacae, Treponema amylovorum, Fusobacterium periodonticum, and Neisseria meningitidis (P<0.05). Conclusion The oral and esophageal microbiota in ESCC patients exhibit both distinct differences and similarities. Oral microbiota may influence the esophageal microenvironment through migration or local immune modulation.

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Objective To construct B cell conditional knockout Spi1 gene mice and analysis their genotypes to provide an animal model basis for disease pathogenesis and drug target research. Methods Mb1-cre transgenic mice were crossbred with Spi1flox/flox mice,. The genotypes of the offspring mice were identified using polymerase chain reaction (PCR) and agarose gel electrophoresis. The mice with the genotype of Mb1-cre×Spi1flox/flox that were screened out will be the homozygous mice with B cell-specific conditional knockout of the Spi1 gene. Results PCR identification results showed that when using flox primers for detection, only a 220 bp band was amplified, indicating a genotype of Spi1flox/flox in the mice. When using Mb1-Cre primers for detection, a 383 bp band was amplified, indicating a genotype of Mb1-cre×Spi1flox/flox in the mice; Flow cytometry results showed that compared to Spi1flox/flox mice, Mb1-cre×Spi1flox/flox mice exhibited significantly reduced PU.1 expression levels in bone marrow-derived mononuclear cells, peripheral blood mononuclear cells, and spleen-derived B cells. Moreover, PU.1 exhibited normal expression in other immune cells, such as CD4+ T cells and macrophages. Conclusion By utilizing the Cre/LoxP system and CRISPR/Cas9 technology, this study successfully generates mice with B cell-specific conditional knockout of the Spi1 gene, providing a reliable animal model for in-depth exploration of the specific role of PU.1 in B cell-related diseases.

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Objective To investigate the protective effect of magnolol ester derivative YW against cisplatin (Cis)-induced acute kidney injury (AKI) and to explore its effect and preliminary mechanisms on Cis-induced AKI. Methods The research group designed and synthesized the magnolol ester derivative YW using magnolol as the parent core. Molecular docking was employed to predict the binding interaction between YW and lysine acetyltransferase 7 (KAT7). ACis-induced AKI model was established in C57BL/6J mice. Mice were divided into the normal control group, model group, YW low/medium/high dose groups (17.5, 35, 70 mg/kg), and the positive control amifostine group. Renal function was assessed by measuring serum creatinine (Scr) and blood urea nitrogen (BUN) levels. Pathological changes were evaluated via H&E staining and scoring of kidney tissues. Protein expression levels of kidney injury molecule 1 (KIM-1), lysine acetyltransferase 7 (KAT7), and cyclin-dependent kinase inhibitor 1A (p21) in kidney tissues were detected by Western blot (WB). HK-2 cells were treated with different concentrations of YW; cell viability was measured by CCK-8 assay. WB was used to detect the expression levels of the renal injury marker KIM-1, KAT7, and its downstream protein p21. Cellular senescence was assessed by senescence-associated β-galactosidase (SA-β-Gal) staining. Results YW could stably bind to KAT7. In vivo experiments showed that, compared with the model group, all YW dose groups,especially the medium and high doses, significantly reduced the elevated levels of Scr and BUN in AKI mice (P<0.001) and effectively ameliorated renal histopathological damage. YW significantly downregulated the protein expression levels of KIM-1, KAT7, and p21 in kidney tissues (P<0.05).Concentrations of YW ≤10 μmol/L had no significant effect on HK-2 cell viability. YW inhibited the Cis-induced decline in HK-2 cell viability (P<0.05), suppressed the Cis-induced upregulation of KIM-1, KAT7, and p21 proteins (P<0.05), and significantly decreased the percentage of SA-β-Gal-positive cells (P<0.01). Conclusion YW suppresses the expression of KAT7 and p21 proteins and inhibits the decline in HK-2 cell viability in cisplatin-induced AKI, significantly mitigates HK-2 cell senescence, and improves renal function, providing preliminary experimental evidence for developing AKI treatment strategies based on structural optimization of natural products.

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Objective To investigate the therapeutic mechanism of artemether in diabetic sarcopenia（DS）using network pharmacology and animal experiments. Methods Potential active components and therapeutic targets were screened using artemisinin as the parent compound. The predicted targets were intersected with DS-related targets, followed by construction of a protein-protein interaction (PPI) network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to identify the key biological processes and signaling pathways. Molecular docking was further used to evaluate the binding affinity between artemether and core targets. The DS mouse model was established using db/db mice, followed by artemether intervention. Fasting blood glucose, body weight, diabetes-related symptoms, body composition, grip strength, serum and skeletal muscle triglyceride levels, and muscle fiber cross-sectional area (CSA) were assessed. The expression levels of forkhead box O1 (FoxO1), Atrogin-1, muscle RING finger protein 1 (MuRF1), acyl-CoA synthetase short-chain family member 2 (ACSS2), carnitine palmitoyltransferase 2 (CPT2), and fatty acid-binding protein 3 (FABP3) in skeletal muscle were detected by qRT-PCR, Western blot, immunofluorescence, and immunohistochemistry. In addition, metabolomics was performed to analyze changes in acylcarnitine metabolites in skeletal muscle. Results A total of 68 overlapping targets between artemether and diabetic sarcopenia (DS) were identified. The core targets included AKT1, FoxO1, NFKB1, FBXO32, and TRIM63, which were mainly enriched in the phosphoinositide 3-kinase/protein kinase B (PI3K-Akt) signaling pathway, forkhead box O (FoxO) signaling pathway, tumor necrosis factor (TNF) signaling pathway, and insulin resistance-related pathways. Molecular docking analysis showed that artemether exhibited favorable binding affinity with the core targets. Animal experiments demonstrated that artemether reduced fasting blood glucose, ameliorated metabolic symptoms, increased lean mass and grip strength, decreased serum and skeletal muscle triglyceride levels, and increased muscle fiber cross-sectional area (CSA) in DS mice. In addition, artemether downregulated the mRNA and protein expression levels of FoxO1, FBXO32/Atrogin-1, TRIM63/MuRF1, ACSS2, CPT2, and FABP3, and improved the disturbance of acylcarnitine metabolism in skeletal muscle. Conclusion Artemether improves metabolic and skeletal muscle phenotypes in DS mice. Its effects may be associated with the amelioration of lipid metabolic disorders and the downregulation of FoxO1 and its downstream ubiquitin-proteasome pathway-related factors Atrogin-1 and MuRF1 in skeletal muscle.

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Objective To investigate whether the expression of Piezo1 in bulbar conjunctival fibroblasts contributes to the development of conjunctivochalasis (CCH). Methods Primary bulbar conjunctival fibroblasts from CCH patients and normal controls (NC) were obtained by tissue block applanation, purified and then subjected to fibroblast identification. Immunohistochemistry was used to detect the expression of Piezo1 in the NC and CCH groups. Immunofluorescence was used to detect the expression of Piezo1 in bulbar conjunctival fibroblasts in the CCH and NC groups, as well as the expression of MMP-9 after the intervention of the Piezo1 agonist, Yoda1. qRT-PCR was used to detect the expression of mRNA of Piezo1 and MMP-9 in the CCH and NC groups, as well as the expression of mRNA of MMP-9 after the intervention of Yoda1. In addition, confocal microscopy was used to detect Ca2+influx in bulbar conjunctival fibroblasts in the NC group after Yoda1 intervention. Membrane clamp was used to detect the current amplitude of NC bulbar conjunctival fibroblasts after Yoda1 intervention to verify the functional expression of Piezo1. Results Primary bulbar conjunctival fibroblasts were successfully cultured. Immunohistochemistry revealed that Piezo1 protein expression was elevated in the CCH group (P<0.01). qRT-PCR assay revealed that mRNA expression of Piezo1 was elevated in the CCH group (P<0.01), mRNA expression of MMP-9 was elevated in the CCH group (P<0.01), and mRNA expression of MMP-9 was elevated in the CCH+Yoda1 group (P<0.01). Immunofluorescence detection of Piezo1 revealed enhanced fluorescence in the CCH group relative to the NC group (P<0.05), and the fluorescence of MMP-9 was enhanced by the addition of Yoda1 stimulation compared with that of the CCH group (P<0.05). Confocal microscopy of NC group bulbar conjunctival fibroblasts after the addition of Yoda1 showed enhanced fluorescence of Piezo1 (P<0.05). The addition of Yoda1 activated enhanced current amplitude, showed by membrane clamp detection of NC group bulbar conjunctival fibroblasts (P<0.000 1). Conclusion Mechanical damage produced by conjunctival tissues in conjunctivochalasis upregulates Piezo1 expression, and Piezo1 activation may amplify the mechanical damage caused by CCH.

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Objective To investigate the association between insulin resistance (IR) and cardiovascular metabolic indicators, including blood pressure, blood lipids, and serum uric acid, in a middle-aged population. Methods Based on the baseline survey of the Hong Sifang cohort of China National Salt Industry Group, a total of 575 participants were included. According to the homeostasis model assessment of insulin resistance, the participants were divided into an IR group (n = 116) and a non-IR group (n = 459). Differences between the two groups were compared using the t-test and chi-square test. Multivariate logistic regression analysis was performed to evaluate the associations between IR and abnormalities in metabolic indicators. Results A total of 575 participants were included, including 390 men and 185 women, with a mean age of (48.58 ± 5.14) years and a mean body mass index (BMI) of (24.16 ± 2.97) kg/m². Blood pressure, serum uric acid, and blood lipid levels were significantly higher in the IR group than in the non-IR group (P<0.001). After adjusting for potential confounders including age, sex, and BMI, IR was independently associated with hypertension, dyslipidemia, and hyperuricemia, with odds ratios (OR) and 95% confidence intervals (CI) of 2.57 (1.61-4.09), 3.81 (2.36-6. 17), and 2.68 (1.53-4.71), respectively (all P<0.001). Conclusion Insulin resistance is significantly associated with multiple cardiovascular metabolic risk factors in the middle-aged population. Monitoring IR may contribute to the early prevention and control of cardiovascular diseases.

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Objective To investigate the effects of Hand-Arm Bimanual Intensive Training (HABIT) based on the FITT-CORRECT principles on upper limb function and functional independence in children with spastic hemiplegic cerebral palsy. Methods A total of 87 children with spastic hemiplegia were enrolled and randomly divided into an observation group (n=44), which received HABIT based on the FITT-CORRECT principles, and a control group (n=43), which received conventional HABIT. Both groups received intervention for 6 weeks. The Quality of Upper Extremity Skills Test (QUEST), Peabody Developmental Motor Scales–Fine Motor (PDMS-FM), and the Functional Independence Measure for Children (WeeFIM) were administered before and after treatment. The root mean square (RMS) values of the biceps and triceps brachii during maximum isometric contraction of elbow flexion and extension on the affected side were also measured. Results Before treatment, there were no significant differences between the two groups in QUEST, PDMS-FM, WeeFIM scores, or RMS values of the biceps and triceps during maximum isometric contraction of the affected elbow (P>0.05). After 6 weeks of treatment, no significant change was observed in the RMS values of the triceps during elbow flexion in either group (P>0.05). All other outcome measures improved significantly in both groups (P<0.05). The observation group showed significantly greater improvements in dissociated movement and grasp scores of QUEST, grasp and visual-motor integration scores of PDMS-FM, WeeFIM scores, as well as RMS values of the biceps during elbow flexion and both the biceps and triceps during elbow extension on the affected side (P<0.05). Conclusion Compared with conventional HABIT, HABIT based on the FITT-CORRECT principles is more effective in improving upper limb function and functional independence in children with spastic hemiplegia.

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Macropinocytosis (MP) is an actin-dependent process by which cells internalize large volumes of extracellular fluid. This engulfed material is subsequently digested and degraded within lysosomes, providing essential nutrients that enable cell survival and growth under hypoxic and nutrient-scarce microenvironments. In the pathogenesis of hepatocellular carcinoma (HCC), sustained liver inflammation plays a critical role. Chronic liver inflammation not only creates favorable conditions for MP activation but also triggers relevant signaling pathways, thereby establishing a persistent positive feedback loop. This review aims to systematically elucidate the mechanistic basis of MP, summarize recent advances in its regulation via relevant signaling pathways during HCC progression, and discuss the potential value of targeting MP as a therapeutic strategy for HCC, thereby providing insights for future clinical research.

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Thalamic hemorrhage, a severe form of spontaneous intracerebral hemorrhage (ICH), accounts for 6% to 25% of all ICH cases. It is associated with high mortality, imposing a substantial burden on both patients and society. Current research on the surgical management ofthalamic hemorrhage primarily focuses on the indications, technical nuances, clinical outcomes, and complications of various surgical approaches. Stereotactic minimally invasive surgery for small-volume thalamic hemorrhage has been proven to improve patient prognosis, particularly for localized hematomas with a volume less than 15mL. However, a systematic and standardized evaluation of the efficacy across different surgical methods is still lacking. This review analyzes existing literatures to explore different surgical approaches for thalamic hemorrhage, including craniotomy, stereotactic minimally invasive evacuation, and neuroendoscopic evacuation, assessing their respective advantages and limitations. The analysis reveals that traditional craniotomy is being used less frequently in the treatment of deep-seated thalamic hemorrhage due to its significant invasiveness. Stereotactic minimally invasive surgery demonstrates advantages in effectively improving neurological outcomes and reducing mortality for small to moderate hematomas, while neuroendoscopic surgery offers unique value in evacuating intraventricular hematomas and reducing shunt dependency. Additionally, the timing of surgery has a significant impact on patient prognosis, although the optimal timing remains to be further clarified. Current evidence is largely derived from retrospective studies, highlighting the need for high-quality prospective research to provide a more robust foundation for clinical decision-making. This review aims to provide scientific evidence for clinical practice and to guide future research directions in this field.

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Objective To investigate the differential expression of LEFTY1 in peritoneal dialysis (PD)-associated peritoneal fibrosis (PF) and its clinical value. Methods Levels of LEFTY1, transforming growth factor-β (TGF-β), and fibronectin (FN) in peritoneal dialysis effluent were measured in 72 PD patients, and clinical data were collected. Differences in these indicators were compared among groups with different dialysis durations, and the correlation between LEFTY1 and clinical parameters was analyzed to evaluate its predictive value for ultrafiltration insufficiency. Results LEFTY1 levels decreased progressively with longer dialysis duration: short-term group [(2 262.49±270.47) ng/mL] > mid-term group [(1 853.52±226.06) ng/mL] > long-term group [(1 767.24±264.57) ng/mL], with significant differences among groups (P<0.001). TGF-β and FN levels increased with prolonged dialysis duration (P<0.05). Correlation analysis revealed that LEFTY1 was significantly negatively correlated with FN (r=-0.327, P=0.004), dialysis duration (r=-0.496,P<0.001), peritoneal Kt/V (r=-0.333, P=0.004), peritoneal creatinine clearance (r=-0.239, P=0.043), and serum calcium (r=-0.410, P<0.001), while positively correlated with total creatinine clearance (r=0.283, P=0.016). The area under the ROC curve for LEFTY1 combined with peritoneal Kt/V and peritoneal Ccr in predicting ultrafiltration insufficiency was 0.739. Multivariate regression analysis identified dialysis duration as an independent influencing factor for LEFTY1 expression (P=0.001). Conclusion The expression of LEFTY1 in the peritoneal dialysis effluent of PD patients decreases with prolonged dialysis duration and is negatively correlated with pro-fibrotic indicators, suggesting that it may play a potential anti-fibrotic role in the progression of PF. The combined detection of LEFTY1 and solute removal indicators holds value for the early identification of ultrafiltration dysfunction.

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Objective To develop an early - warning approach for influenza in Anhui Province using the Moving Epidemic Method (MEM), offering a scientific basis for influenza prevention and control in the province. Methods Based on the surveillance data reported weekly by influenza sentinel hospitals and network laboratories in the “China Influenza Surveillance Information System”, the weekly influenza virus detection positive rate (positive rate, PR) across the province was analyzed. The MEM was used to estimate the epidemic threshold and classification intensity threshold of influenza in Anhui Province before and after the COVID-19 pandemic, respectively. The early warning effect of the MEM was evaluated using cross-validation. Results Epidemic and graded intensity thresholds of influenza were derived using PR and MEM. Prior to the COVID - 19 pandemic, the starting, medium - intensity, high - intensity, extremely - high - intensity, and ending thresholds of influenza epidemics were 17.08%, 38.47%, 53.20%, 59.71%, and 12.34% respectively. At this point, the model exhibited sensitivity of 0.98, specificity of 0.86, Matthews Correlation Coefficient of 0.84, and Youden Index of 0.84. After the COVID - 19 pandemic, these thresholds were 25.61%, 43.81%, 63.29%, 74.46%, and 17.82% respectively. At this point, the model demonstrates sensitivity of 0.86, specificity of 0.92, Matthews Correlation Coefficient of 0.78, and Youden Index of 0.77. Conclusion MEM shows good applicability in estimating the epidemic and graded intensity thresholds of influenza during the winter and spring in Anhui Province. Both the influenza epidemic threshold and graded intensity thresholds after the COVID- 19 pandemic are higher than those before the pandemic.

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Objective To investigate the association between single and combined exposure to arsenic (As), mercury (Hg), and cadmium (Cd) in urine and hearing loss in adults. Methods A total of 400 adult patients were recruited for the study, comprising 200 participants in the normal hearing group and 200 in the hearing loss group. General demographic information of the study subjects was collected by a self-administered questionnaire, morning urine samples were tested for As, Hg, and Cd concentrations, and hearing status was assessed by Pure Tone Audiometry. A binary logistic regression model was used to explore the association between the concentrations ofthe three metals in urine and hearing loss, and a Bayesian kernel-machine regression (BKMR) model was used to further explore the association between the concentrations of the three metal mixtures and hearing loss. Results After correction for urinary creatinine, As, Hg, and Cd concentrations were higher in the hearing loss group than those in the normal hearing group (P<0.05). Binary logistic regression indicated that concentrations of As, Hg, and Cd were positively associated with hearing loss, and the risk of hearing loss was significantly higher in the second quartile of Cd concentration compared to the first quartile (OR=2.000, 95% CI: 1.009-3.964). BKMR modeling analysis showed that the combined exposure levels of As, Hg, and Cd significantly increased the risk of hearing loss, with Cd being the most important contributor (posterior inclusion probability=0.412), and that there were potential interactions between As and Cd, and Cd and Hg. Conclusion Combined exposure to As, Hg, and Cd in urine is significantly associated with adult hearing loss, with Cd being the primary contributor in the exposure combination. The interactions between As and Cd, as well as Cd and Hg, may have adverse effects on hearing loss.

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Objective To investigate the mechanism by which activation of α7nicotinic acetylcholine receptors (α7nAChR) optimizes brown adipose tissue (BAT) thermogenesis in obese mice. Methods Obesity was induced in C57BL/6J mice via a high-fat diet. Fifty 8-week-old mice were randomly assigned to five groups: low-fat diet (Control), high-fat diet (HFD), high-fat diet plus a β3-adrenergic receptor agonist (HFD+β3), high-fat diet plus a β3-adrenergic receptor agonist and an α7nAChR selective agonist (HFD+β3+GTS-21), and high-fat diet plus a β3- adrenergic receptor agonist and an α7nAChR selective antagonist (HFD+β3+α-BGT). Haematoxylin and eosin (HE) staining was used to evaluate BAT morphology. Transmission electron microscopy was performed to assess mitochondrial number and lipid droplet morphology in adipocytes. ELISA was used to measure levels of tumour necrosis factor-α (TNF-α), interleukin-lβ(IL-lβ), interleukin-10(IL-10), transforming growth factor-β (TGF-β), cyclic adenosine monophosphate (cAMP), and norepinephrine (NE). RT-qPCR was conducted to determine mRNA expression levels of vascular endothelial growth factor A (VEGF-A), nitric oxide synthase 2 (NOS2), arginase 1 (Arg1), uncoupling protein 1 (UCP1), PR domain-containing protein 16 (PRDM16), and peroxisome proliferator- activated receptor γ coactivator-lα (PGC-1α) in BAT. Immunohistochemistry was used to detect macrophage markers (CD3l, CD86, and CD206). Protein expression levels of UCPl, α7nAChR, nuclear factor-κB p65(NF-κB p65), phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) were analyzed by Western blot. Results Compared with the Control group, the HFD group showed enlarged lipid droplets and reduced mitochondrial numbers in adipocytes, accompanied by increased TNF-α and IL-lβ and decreased IL-10, TGF-β, Argl, and VEGF-A (all P<0.0l). Compared with the HFD+β3 group, the HFD+β3+GTS-21 group showed a smaller lipid droplet area and a higher mitochondrial number, along with reduced levels of pro-inflammatory factors, NOS2, and NF-κB p65, and increased levels of anti-inflammatory factors, Arg1, and phosphorylation of JAK2 and STAT3 (all P<0.01). Conclusion Activation of α7nAChR in combination with a β3-adrenergic receptor agonist effectively enhances thermogenesis, alleviates local adipose tissue hypoxia, and shifts the local inflammatory phenotype toward an M2-like state, thereby reducing inflammation in brown adipose tissue and improving its thermogenic capacity.

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Objective To investigate the expression patterns of key steroidogenic enzymes (CYP11A1, HSD3B1, and CYP19A1) in placental tissues from preeclampsia (PE) patients and evaluate their potential as biomarkers for PE. Methods A case-control study was conducted using placental tissues from PE patients (PE group, n=3) and normotensive pregnant women (control group, n=3). RNA sequencing was performed to identify differentially expressed genes (DEGs), followed by validation of target gene expression using Western blot, qRT-PCR, and immunohistochemistry. Steroid hormone levels in placental tissues and serum were measured by ELISA. Spearman correlation analysis was used to assess associations between gene expression and clinical parameters, and receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive value of target genes for PE. Results The results showed that 501 DEGs (P<0.05 and |Log2fold change| >1) were identified in PE placental tissues, with KEGG enrichment analysis revealing significant involvement in steroid hormone biosynthesis pathways. Compared with the control group, PE placental tissues exhibited significantly decreased protein and mRNA expression of HSD3B1 and CYP19A1 (P< 0.01), while CYP11A1 expression was upregulated (P<0.05). Serum estradiol (E2) and progesterone (P4) levels were significantly reduced in the PE group (P<0.01), whereas testosterone (T) levels were elevated (P<0.05). Correlation analysis demonstrated that CYP11A1 expression was positively associated with systolic blood pressure (SBP), diastolic blood pressure (DBP), and soluble fms-like tyrosine kinase-1 (sFlt-1) (P<0.01), but negatively correlated with placental growth factor (PLGF). Conversely, HSD3B1 and CYP19A1 expression showed inverse correlations with these clinical parameters. ROC analysis revealed that CYP11A1, HSD3B1, and CYP19A1 each had an area under the curve (AUC) of 0.778 for predicting PE, with 95%CI ranging from 0.614 to 0.942 (P=0.004). The sensitivity was 94.4%, and the specificity was 66.7%. Conclusion These findings suggest that dysregulated expression of steroidogenic enzymes in PE placental tissues contributes to hormonal imbalance, thereby playing a role in PE pathogenesis. Furthermore, CYP11A1, HSD3B1, and CYP19A1 may serve as potential diagnostic biomarkers for PE.

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Objective To investigate the metabolic mechanisms of Limosilactobacillus reuteri (L.reuteri) in alleviating chemotherapy-induced intestinal mucosal injury (CIMI). Methods C57BL/6 mice were randomly divided into the Control group, Model group, and L. reuteri treatment group (n = 8 per group). Mice in the L. reuteri group were intragastrically administered L. reuteri (1 × 109 CFU/mL) for 10 consecutive days. From day 5, mice in the Model and L. reuteri groups were intraperitoneally injected with 5-FU (65 mg/kg) for 6 consecutive days to establish a CIMI model.Body weight changes, colon length, and related indicators were recorded. Intestinal histopathological injury was assessed by H&E staining with measurement of villus height and crypt depth. The expression levels of zonula occludens-1 (ZO-1), Occludin, Claudin-4, Muc1, and Muc2 in ileal tissues were determined by RT-qPCR and immunohistochemistry to evaluate intestinal barrier function. In addition, untargeted metabolomics was performed on cecal contents from each group to analyze metabolic profiles and enrichment of differential metabolic pathways. Results L.reuteri intervention significantly alleviated body weight loss (P<0.001) and colon shortening (P<0.05) in CIMI mice, effectively reducing ileal tissue pathological damage, and upregulating the expression of ZO-1, Occludin, Claudin4, Muc1, and Muc2 (P<0.05). Untargeted metabolomics analysis showed that L. reuteri treatment reversed the abnormal metabolic profile in the cecal contents of CIMI mice, bringing it closer to normal. Various differential metabolites, such as acylcarnitines and polyunsaturated fatty acids, might play an important role in L. reuteri-mediated alleviation of CIMI. These differential metabolites were primarily enriched in key pathways such as linoleic acid metabolism, tryptophan metabolism, and unsaturated fatty acid biosynthesis. Conclusion This study reveals that L. reuteri improves intestinal barrier function and alleviates CIMI by regulating the intestinal metabolic profile, particularly lipid and amino acid metabolism. It provides a novel potential strategy for the prevention and treatment ofCIMI.

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Objective To investigate the role of Disco Interacting Protein 2 Homolog B (DIP2B) in the proliferation, migration, and invasion processes of gastric cancer cells. Methods Bioinformatic analysis was performed using public datasets, including The Cancer Genome Atlas (TCGA), GEPIA and TIMER2.0, to assess DIP2B expression and its clinical prognostic value in gastric cancer. DIP2B expression levels in gastric cancer cell lines were validated by Western blot and RT-qPCR. ADIP2B knockdown cell model was established via siRNA mediated gene silencing, and the knockdown efficiency was confirmed. Cell proliferation was assessed using real-time cellular analysis (RTCA) and colony formation assays. Cell migration and invasion were evaluated by Transwell assays, while flow cytometry was employed to detect changes in apoptosis and cell cycle distribution. Results DIP2B was significantly upregulated in gastric cancer cells and correlated with poor prognosis in patients (P<0.05). The experimental results revealed that DIP2B knockdown markedly suppressed the proliferation, migration, and invasion of gastric cancer cells, while promoting apoptosis and inducing cell cycle arrest (P<0.05). Conclusion DIP2B is highly expressed in gastric cancer and associated with unfavorable prognosis. Knockdown of DIP2B inhibits the proliferation, migration, and invasion of gastric cancer cells and promotes apoptosis, suggesting that DIP2B may serve as a potential prognostic biomarker and therapeutic target for gastric cancer.

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Objective To construct an Il-9 (Il9) gene knockout mouse model on a C57BL/6N background, establish a reliable genotyping method, and provide a standardized animal model for investigating Il9-related immune mechanisms and diseases. Methods Il9 knockout mouse strain (C57BL/6N-Il9em1Cya) was generated using conventional gene knockout technology. The method for DNA extraction from mouse tails was optimized to ensure template quality. Two sets of specific primers (F1/R1 and F1/R2) were designed to amplify target fragments by PCR, and mouse genotypes were determined by agarose gel electrophoresis. Specifically, primer pair F1/R1 targeted the knockout sequence with an expected product of 465 bp, while F1/R2 targeted the wild-type sequence with an expected product of 703 bp. Subsequently, Western blot was performed to verify the knockout efficiency of Il9 in primary immune cells and key organs of the offspring mice, and flow cytometry was used to assess the effects of Il9 deficiency on the function of the immune system in major immune cell populations. Results Il9 knockout mice were successfully bred and genotyped. PCR identification showed that homozygous mice (-/-) exhibited only a 465 bp band, wild-type mice (+/+) displayed only a 703 bp band, and heterozygous mice (+/-) showed both bands simultaneously. Meanwhile, Western blot analysis revealed significantly decreased Il9 expression in various organs of the offspring mice (P<0.001). Flow cytometric results demonstrated that Il9 deficiency exerted no obvious effects on the proportions of peripheral blood lymphocytes, splenic lymphocytes, and peritoneal macrophages. Conclusion An Il9 gene knockout mouse model on a C57BL/6N background is successfully constructed. The established PCR genotyping system is highly efficient and accurate, which can effectively distinguish mice of different genotypes. This model provides a stable and reliable experimental basis for subsequent functional studies of Il9 and exploration of the mechanisms underlying Il9-related diseases.

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Objective To investigate whether the CD151 YXXφ motif point mutant affects vascular permeability by regulating vesicle internalization and recycling. Methods CD151 knockout (KO) and wild-type (WT) mice were used. A modified Miles assay was performed to compare vascular permeability in the dorsal and postauricular skin of the two groups of mice under vascular endothelial growth factor A (VEGF-A) stimulation. The CD151 YXXφ motif point mutant YALA was constructed and transfected into human endothelial cells (EA.hy926) using an adenoviral vector. Multiple experimental groups were set up to detect the permeability of endothelial cell monolayers to FITC-dextran under baseline conditions and after VEGF-A stimulation. Western blot (WB), RT-qPCR, and immunofluorescence were used to examine the expression and distribution of VE-cadherin. The internalization rate of VE-cadherin was quantitatively compared using a cell surface biotinylation assay. Phalloidin was used to label F-actin to observe changes in cytoskeletal tension. Results The Miles assay showed that under VEGF-A stimulation, dye extravasation in the dorsal and postauricular skin of KO mice was significantly higher than that in WT mice (all P<0.05). At 60 min and 120 min after VEGF-A stimulation, the permeability of endothelial cell monolayers overexpressing the YALA mutant to FITC-dextran significantly increased (P<0.05). WB and RT-qPCR results indicated that the YALA mutant did not affect the total protein or mRNA expression levels of VE-cadherin. Immunofluorescence revealed that the YALA mutant disrupted the continuous distribution of VE-cadherin on the cell membrane and its perinuclear co-localization with CD151 under VEGF-A stimulation. The cell surface biotinylation assay confirmed that the YALA mutant significantly reduced the internalization rate of VE-cadherin (P<0.05). Cytoskeletal staining showed that the YALA mutant caused the formation of thick tension fibers in the actin cytoskeleton of endothelial cells, indicating cell contraction. Conclusion The CD151 YXXφ motif point mutation affects vesicle internalization and recycling in endothelial cells, influences the expression and internalization of VE-cadherin, and consequently affects vascular permeability.

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Objective To explore the expression levels of long non-coding RNA (lncRNA) H19 and microRNA-22-3p (miR-22-3p) in the serum of patients with acute myocardial infarction (AMI) and their diagnostic value for AMI. Methods A total of 176 AMI patients from the Department of Cardiology were selected as the experimental group and were divided into ST-segment elevation myocardial infarction (STEMI) group and non-ST-segment elevation myocardial infarction (NSTEMI) group based on their medical history and electrocardiogram. Meanwhile,156 patients with negative angiography during the same period were selected as the control group (CON group). The relative expression levels of lncRNA H19 and miR-22-3p in the serum of the two groups were detected by real-time fluorescence quantitative polymerase chain reaction. The diagnostic value of miR-22-3p and lncRNA H19 in AMI was evaluated by receiver operating characteristic curve (ROC) analysis. The levels of serum cardiac troponin I (cTnI), creatine kinase (CK), creatine kinase MB isoenzyme (CK-MB), and high-sensitivity C-reactive protein (CRP) were detected in all study subjects. Spearman correlation analyses were used to evaluate the correlations between lncRNA H19 and miR-22-3p and cTnI, CK, CK-MB and CRP. Results Compared with the control group, the expression level of lncRNA H19 was upregulated and the expression level of miR-22-3p was downregulated in the AMI group (all P < 0.05); compared with the NSTEMI group, the expression level of lncRNA H19 was upregulated and the expression level of miR-22-3p was downregulated in the STEMI group (P < 0.05). The areas under the ROC curves (AUC) for the diagnosis of AMI by miR-22-3p, lncRNA H19 and their combination were 0.555, 0.977, and 0.983, respectively. lncRNA H19 was positively correlated with cTnI, CK,CK-MB and CRP (P < 0.05), and negatively correlated with LVEF (P < 0.05); miR-22-3p was negatively correlated with cTnI, CK and CRP, and positively correlated with LVEF (P < 0.05).Conclusion The expression level of lncRNA H19 is elevated and the expression level of miR-22-3p decreased in AMI patients, and their levels may have potential value as an auxiliary biomarker for AMI diagnosis and cardiac function assessment.

Abstract:
Objective To investigate the molecular mechanisms by which kaempferol induces apoptosis in hepatic stellate cells (HSC). Methods HSC were divided into five groups: blank control, transforming growth factor-β1(TGF-β1) (10 ng/mL) group, and groups treated with different concentrations of kaempferol (8, 16, 32 µmol/L). Cell counting kit-8(CCK-8) assay was used to evaluate chrysin's effect on HSC proliferation. Transwell assay was used to assess chrysin's impact on HSC migration. Flow cytometry was used to detect apoptosis, reactive oxygen species (ROS) expression, and mitochondrial permeability transition pore (mPTP) opening across groups. 5,5,,6,6,-Tetrachloro-1,1,,3,3,-tetraethylimidacarbocyanine iodide(JC-1) assay was used to test mitochondrial membrane potential. Western blot was used to detect apoptosis-related mitochondrial proteins and extracellular matrix protein expression. Transmission electron microscopy was used to examine mitochondrial structural alterations. Molecular docking and kinetic simulations was used to analyze the targeted interaction between kaempferol and NAD-dependent deacetylase sirtuin-3 (SIRT3). Results Kaempferol significantly inhibited the proliferation and migration of HSC while promoting their apoptosis. It markedly increased ROS expression and opened the mPTP in these cells, concurrently reducing their mitochondrial membrane potential. It increased the expression of Bcl-2 associated X protein (Bax), Cleaved-Caspase3, and Cleaved-Caspase9 while decreasing the expression of B lymphocytoma-2 (Bcl2) and SIRT3. Kaempferol promoted mitochondrial fission in HSC. Molecular docking and kinetic simulations revealed that kaempferol exhibits favorable binding affinity and stability with SIRT3. Conclusion Kaempferol induces HSC apoptosis by targeting SIRT3 to mediate ROS regulation of the mitochondrial apoptosis pathway, thereby reducing extracellular matrix deposition. This demonstrates kaempferol's potential anti-fibrotic effects in liver.

Abstract:
Objective To investigate morphological and functional neural network alterations in the primary visual cortex (V1) during early-stage diabetes. Methods 32 SPF male C57BL/6J mice were randomly divided into control and diabetic groups. Type 1 diabetes was induced by intraperitoneal streptozotocin (STZ) injection, with model validation via oral glucose tolerance test (OGTT) at week 4. Nissl staining was used for structural analysis. Immunohistochemistry was used for assessing CaMKII and SST expression, and SST signal density on CaMKII⁺ neurons in V1. In vivo electrophysiology was used for studying neuronal firing and functional connectivity. Results Compared with the control mice, nissl staining showed diabetic mice exhibited significant V1 thinning (P=0.02), reduced neuronal density (P=0.01); immunohistochemistry found decreased SST integral optical density (P=0.02) and elevated mean fluorescence intensity (P=0.0l), and lower SST density on CaMKⅡ+ neurons; In vivo electrophysiology studies indicated reduced action potential peak-to-trough ratio and the absolute values of slope (P<0.01), as well as impaired functional connectivity, including conditionalfiring, mutualinformation, and Granger causality (P<0.000 1). Conclusion Early diabetes induces V1 structural atrophy accompanied by SSTergic system disruption and multi-level functional degradation from single neurons to neural networks.

Abstract:
Objective To investigate the inhibitory effect of gingerol, an active component of ginger, on Staphylococcus aureus (S. aureus) infection, and to preliminarily explore its mechanism related to extracellular vesicles (EVs). Methods S. aureus infection models were established in HUVECs, Vero E6 cells, and C57BL/6 mice. Experimental groups included control, infection, and gingerol-treated groups. Bacterial load and VE-cadherin protein expression were detected using immunofluorescence and Western blot. EVs were isolated by size exclusion chromatography and characterized by transmission electron microscopy and nanoparticle tracking analysis. miRNA sequencing was performed on EVs. Results Gingerol treatment significantly reduced the bacterial load in both in vitro and in vivo infection models and upregulated VE-cadherin expression. miRNA sequencing of EVs revealed that S. aureus infection upregulated the expression of hsa-miR-320c, while gingerol treatment reversed this abnormal expression. Bioinformatic analysis further predicted that the target genes of hsa-miR-320c were significantly enriched in cell junction-related pathways. Conclusion Gingerol exhibits clear antibacterial and host-protective effects, by regulating hsa-miR-320c in EVs to maintain endothelial barrier integrity.

Abstract:
Objective To investigate whether intestinal deficiency of lanosterol synthase (LSS), a key enzyme in cholesterol synthesis, influences the progression of metabolic dysfunction-associated steatotic liver disease (MASLD) by regulating intestinal cholesterol absorption and immune response. Methods LSS heterozygous knockout (LSS+/-) mice and wild-type (WT) controls were generated using CRISPR/Cas9 technology and fed either a high-fat diet (HFD) or regular chow (CHOW). The model was validated by genotyping. Hepatic steatosis was assessed by H&E and Oil Red O staining. Immunohistochemistry was used to detect the localization and expression of NPC1L1 and CD36 proteins in the intestine. Western blot analysis was performed to measure JNK phosphorylation and TLR4 protein levels in intestinal tissues. Real-time quantitative polymerase chain reaction (qPCR) was employed to examine the mRNA expression of TLR4 and IL-6. Results LSS+/- mice were successfully validated by genotyping and reduced intestinal LSS protein expression. H&E and Oil Red O staining of liver sections showed that, compared with WT mice fed a CHOW diet, WT mice fed a HFD exhibited a marked increase in hepatic lipid vacuoles. In contrast, compared with HFD-fed WT mice, HFD-fed LSS+/- mice displayed significantly attenuated hepatic lipid deposition and reduced serum ALT levels (P<0.05). Immunohistochemical analysis revealed that, compared with WT mice, the expression of the cholesterol absorption protein NPC1L1 in the intestinal villi of LSS+/- mice was downregulated under both CHOW and HFD conditions (P<0.001 for HFD group). Conversely, the expression of the fatty acid transporter CD36 was upregulated in the intestines of LSS+/- mice (P < 0.05 for CHOW group, P<0.01 for HFD group). Western blot analysis demonstrated that, compared with WT mice, TLR4 protein expression in the intestines of LSS+/- mice significantly increased under both CHOW and HFD conditions (P < 0.05 for CHOW group, P < 0.05 for HFD group). JNK phosphorylation level was significantly elevated in LSS+/- mice under CHOW condition (P < 0.05). Under HFD condition, total JNK protein expression increased, but its phosphorylation level showed no significant change. qPCR analysis showed that, compared with WT mice, the mRNA levels of TLR4 (P<0.01 for CHOW group, P<0.000 1 for HFD group) and IL-6 (P<0.001 for CHOW group, P<0.01 for HFD group) were significantly upregulated in the intestines of LSS+/-mice. Conclusion LSS deficiency counteracts hepatic lipid deposition by orchestrating a synergistic reprogramming involving restricted intestinal cholesterol absorption, enhanced fatty acid utilization, and activation of immune pathways, suggesting intestinal LSS as a potential therapeutic target of MASLD.

Abstract:
Objective To investigate the short-term clinical outcomes and long-term prognostic value of preserving the superior rectal artery (SRA) versus high ligation of the inferior mesenteric artery (IMA) in laparoscopic sigmoid colon cancer resection (LSCER) for sigmoid colon cancer (SCC). Methods A retrospective comparative analysis was performed of 316 SCC patients who underwent LSCER. Patients were divided into the SRA preservation group (Group A,132 cases) and IMA high ligation group (Group B,184 cases) according to the vascular management strategy. Short-term clinical indicators and long-term follow-up outcomes were compared between the two groups. Results There were no significant differences in baseline data between the two groups (P > 0.05). The mean operation time of Group A was (190 ± 61.9) minutes, which was significantly longer than that of Group B [(165 ± 49.1) minutes, P < 0.001]. However, Group A demonstrated significantly shorter time to drain removal, time to first flatus, and postoperative hospital stay (P < 0.001). No significant differences were observed in intraoperative blood loss, number of lymph nodes dissected, or incidence of anastomotic leakage (AL) between the two groups. Long-term follow-up results showed that there were no significant differences in 3-year overall survival rate and 3-year disease-free survival rate between the two groups. Conclusion Laparoscopic sigmoid colon cancer resection with SRA preservation is safe and feasible for the treatment of SCC. Although the operation time is slightly prolonged, it can effectively promote short-term postoperative recovery without affecting the curative effect of tumor resection or long-term prognosis, and thus has important clinical promotion value.