Animal experiments are widely used in biomedical research for safety assessment, toxicological analysis, efficacy evaluation, and mechanism exploration. In recent years, the ethical review system has become more stringent, and awareness of animal welfare has continuously increased. To promote more efficient and cost-effective drug research and development, the United States passed the Food and Drug Administration (FDA) Modernization Act 2.0 in September 2022, which removed the federal mandate requiring animal testing in preclinical drug research. In April 2025, the FDA further proposed to adopt a series of "new alternative methods" in the research and development of drugs such as monoclonal antibodies, which included artificial intelligence computing models, organoid toxicity tests, and 3D micro-physiological systems, thereby gradually phasing out traditional animal experiment models. Among these cutting-edge technologies, 3D bioprinting models are a significant alternative and complement to animal models, owing to their high biomimetic properties, reproducibility, and scalability. This review provides a comprehensive overview of advancements and applications of 3D bioprinting technology in the fields of biomedical and pharmaceutical research. It starts by detailing the essential elements of 3D bioprinting, including the selection and functional design of biomaterials, along with an explanation of the principles and characteristics of various printing strategies, highlighting the advantages in constructing complex multicellular spatial structures, regulating microenvironments, and guiding cell fate. It then discusses the typical applications of 3D bioprinting in drug research and development,including high-throughput screening of drug efficacy by constructing disease models such as tumors, infectious diseases, and rare diseases, as well as conducting drug toxicology research by building organ-specific models such as those of liver and heart. Additionally,the review examines the role of 3D bioprinting in tissue engineering, discussing its contributions to the construction of functional tissues such as bone, cartilage, skin, and blood vessels, as well as the latest progress in regeneration and replacement. Furthermore, this review analyzes the complementary advantages of 3D bioprinting models and animal models in the research of disease progression, drug mechanisms, precision medicine, drug development, and tissue regeneration, and discusses the potential and challenges of their integration in improving model accuracy and physiological relevance. In conclusion, as a cutting-edge in vitro modeling and manufacturing technology, 3D bioprinting is gradually establishing a comprehensive application system covering disease modeling, drug screening, toxicity prediction, and tissue regeneration.

Animal experiments are widely used in biomedical research for safety assessment, toxicological analysis, efficacy evaluation, and mechanism exploration. In recent years, the ethical review system has become more stringent, and awareness of animal welfare has continuously increased. To promote more efficient and cost-effective drug research and development, the United States passed the Food and Drug Administration (FDA) Modernization Act 2.0 in September 2022, which removed the federal mandate requiring animal testing in preclinical drug research. In April 2025, the FDA further proposed to adopt a series of "new alternative methods" in the research and development of drugs such as monoclonal antibodies, which included artificial intelligence computing models, organoid toxicity tests, and 3D micro-physiological systems, thereby gradually phasing out traditional animal experiment models. Among these cutting-edge technologies, 3D bioprinting models are a significant alternative and complement to animal models, owing to their high biomimetic properties, reproducibility, and scalability. This review provides a comprehensive overview of advancements and applications of 3D bioprinting technology in the fields of biomedical and pharmaceutical research. It starts by detailing the essential elements of 3D bioprinting, including the selection and functional design of biomaterials, along with an explanation of the principles and characteristics of various printing strategies, highlighting the advantages in constructing complex multicellular spatial structures, regulating microenvironments, and guiding cell fate. It then discusses the typical applications of 3D bioprinting in drug research and development,including high-throughput screening of drug efficacy by constructing disease models such as tumors, infectious diseases, and rare diseases, as well as conducting drug toxicology research by building organ-specific models such as those of liver and heart. Additionally,the review examines the role of 3D bioprinting in tissue engineering, discussing its contributions to the construction of functional tissues such as bone, cartilage, skin, and blood vessels, as well as the latest progress in regeneration and replacement. Furthermore, this review analyzes the complementary advantages of 3D bioprinting models and animal models in the research of disease progression, drug mechanisms, precision medicine, drug development, and tissue regeneration, and discusses the potential and challenges of their integration in improving model accuracy and physiological relevance. In conclusion, as a cutting-edge in vitro modeling and manufacturing technology, 3D bioprinting is gradually establishing a comprehensive application system covering disease modeling, drug screening, toxicity prediction, and tissue regeneration.

In the context of global climate change, the impact of ambient temperature change on cardiovascular health has become increasingly significant. Epidemiological studies have shown that both high and low temperature can lead to the occurrence of adverse cardiovascular outcomes. For example, exposure to low temperature and high temperature, and diurnal temperature difference can increase the risk of cardiovascular diseases (CVD) mortality. Exposure to cold and heat, heatwaves and cold spells may induce myocardial infarction. Low temperature, high temperature and variations in temperature can increase the risk of heart failure. There are complex interactions among the effects of temperature and humidity, wind speed, radiation, precipitation and air pollution on adverse cardiovascular outcomes. Valnerable groups such as the elderly, patients with chronic diseases, and outdoor workers are particularly susceptible to temperature-related CVD risks. The mechanisms by which temperature affects adverse cardiovascular outcomes involves hemodynamic changes, autonomic nervous system dysregulation, blood rheology alterations, as well as inflammation and metabolic abnormalities, but many research gaps remain. Future studies should conduct in-depth research on mechanisms, individual differences, climate change impacts, and public health interventions to provide more effective policies and interventions to reduce the burden of CVD.

Objective To investigate the relationship between serum levels of plasminogen activator inhibi-tor-1(PAI-1)and annexin A2(ANXA2)and the carotid atherosclerosis stability(CAS)plaque in patients with transient ischemic attack(TIA).Methods A total of 131 patients with TIA admitted to the hospital from January 2021 to January 2023 were selected as the TIA group,and 46 healthy people in the same period were selected as the control group.According to the CAS plaque stability of TIA patients,they were divided into unstable plaque group(64 cases)and stable plaque/no plaque group(67 cases).The serum levels of PAI-1 and ANXA2 were detected by enzyme-linked immunosorbent assay and chemiluminescence immunoassay,respectively.Multivariate Logistic regression analysis was used to analyze the factors of CAS plaque stability in TIA patients.Receiver operating characteristic curve was used to analyze the predictive value of serum PAI-1 and ANXA2 levels for CAS instability in TIA patients.Results Compared with the control group,the levels of PAI-1 and ANXA2 in TIA group were significantly increased(P<0.05).Compared with the stable plaque/no plaque group,the unstable plaque group was significant increaseed in the serum levels of PAI-1 and ANXA2(P<0.05).Smoking,high risk of TIA,elevated PAI-1 and ANXA2 were independent risk factors for CAS plaque stability in patients with TIA(P<0.05).The area under the curve predicted by serum PAI-1 and ANXA2 levels combined was 0.879,which was larger than 0.788 and 0.783 predicted by serum PAI-1 and ANXA2 levels alone(P<0.05).Conclusion The increased levels of serum PAI-1 and ANXA2 are closely re-lated to CAS plaque instability in patients with TIA.The combination of serum PAI-1 and ANXA2 levels has a higher value in predicting CAS plaque instability in patients with TIA.

ObjectiveTo evaluate the clinical effectiveness and safety of Bairui Granules （百蕊颗粒） in the treatment of acute pharyngitis with wind-heat syndrome. MethodsA multicenter， double-blind， double-simulation， randomised controlled trial was conducted， in which 162 patients with acute pharyngitis and wind-heat syndrome from 7 centers were recruited， and each center was divided into trial group and control group on the ratio of 2∶1. In the trial group， 108 cases were orally administered with Bairui Granules plus Reyanning Granules （热炎宁颗粒） simulant， and in the control group， 54 cases were orally administered with Reyanning Granules plus Bairui Granules simulant for 5 days， with a follow-up visit on the 6th day. Full analysis set （FAS） analysis and per protocol set （PPS） were used for analysis， respectively. The primary efficacy index was the disappearance rate of sore throat after 5-day treatment； the secondary efficacy indexes were the disappearance rate of sore throat after 3-day treatment， as well as the visual analogue score （VAS） of sore throat before treatment， every day during the treatment， and follow-up on day 6， and the traditional Chinese medicine （TCM） syndrome score was performed before treatment and at the follow-up on day 6. The effectiveness on TCM syndrome was evaluated at the follow-up on day 6， and the changes of vital signs， blood routine， urine routine， liver functions， kidney function， the adverse events before and after the treatment were recorded， and safety analysis set （SS） was analysed. Results162 patients entered the FAS and SS analyses， and 158 cases （105 cases in the trial group and 53 cases in the control group） entered the PPS analysis. FAS analysis showed that the disappearance rate of sore throat after 5-day treatment was 80.56% （87/108） in the trial group and 64.81% （35/54） in the control group， and the difference between groups was statistically significant （χ² = 5.10， P = 0.0239）. PPS analysis showed that the disappearance rate of sore throat after 5-day treatment was 80.00% （84/105） in the trial group and 64.15% （34/53） in the control group， and the difference between groups was statistically significant （χ² =4.85， P = 0.0277）. FAS and SS analyses both showed that the difference in disappearance rate of sore throat between groups on 3-day treatment was not statistically significant （P＞0.05）. Compared with those before treatment， the VAS scores of sore throat were lower in both groups during treatment on day 2， 3， 4， 5， and follow-up on day 6 （P＜0.01）， but the difference between groups at each time point was not statistically significant （P＞0.05）. TCM syndrome scores of both groups at the follow-up were lower than that before treatment， and those of the trial group were lower than those of the control group （P＜0.01）. The cure rate and effective rate of TCM syndrome of the trial group were significantly higher than those of the control group （P＜0.01）. There was no significant difference in blood routine， urine routine， liver function， kidney function between groups before and after treatment （P＞0.05）， and no serious adverse events occured in both groups. ConclusionBairui Granules showed clinical effectiveness in the treatment of acute pharyngitis of wind-heat syndrome， and it could significantly improve the clinical symptoms， accelerate the disappearance time of sore throat with good safety.

Objective     To explore the correlation between the quantitative and qualitative features of CT images and the invasiveness of pulmonary ground-glass nodules, providing reference value for preoperative planning of patients with ground-glass nodules. Methods    The patients with ground-glass nodules who underwent surgical treatment and were diagnosed with pulmonary adenocarcinoma from September 2020 to July 2022 at the Third Affiliated Hospital of Kunming Medical University were collected. Based on the pathological diagnosis results, they were divided into two groups: a non-invasive adenocarcinoma group with in situ and minimally invasive adenocarcinoma, and an invasive adenocarcinoma group. Imaging features were collected, and a univariate logistic regression analysis was conducted on the clinical and imaging data of the patients. Variables with statistical difference were selected for multivariate logistic regression analysis to establish a predictive model of invasive adenocarcinoma based on independent risk factors. Finally, the sensitivity and specificity were calculated based on the Youden index. Results     A total of 555 patients were collected. The were 310 patients in the non-invasive adenocarcinoma group, including 235 females and 75 males, with a meadian age of 49 (43, 58) years, and 245 patients in the invasive adenocarcinoma group, including 163 females and 82 males, with a meadian age of 53 (46, 61) years. The binary logistic regression analysis showed that the maximum diameter (OR=4.707, 95%CI 2.060 to 10.758), consolidation/tumor ratio (CTR, OR=1.027, 95%CI 1.011 to 1.043), maximum CT value (OR=1.025, 95%CI 1.004 to 1.047), mean CT value (OR=1.035, 95%CI 1.008 to 1.063), spiculation sign (OR=2.055, 95%CI 1.148 to 3.679), and vascular convergence sign (OR=2.508, 95%CI 1.345 to 4.676) were independent risk factors for the occurrence of invasive adenocarcinoma (P<0.05). Based on the independent predictive factors, a predictive model of invasive adenocarcinoma was constructed. The formula for the model prediction was: Logit(P)=–1.293+1.549×maximum diameter of lesion+0.026×CTR+0.025×maximum CT value+0.034×mean CT value+0.72×spiculation sign+0.919×vascular convergence sign. The area under the receiver operating characteristic curve of the model was 0.910 (95%CI 0.885 to 0.934), indicating that the model had good discrimination ability. The calibration curve showed that the predictive model had good calibration, and the decision analysis curve showed that the model had good clinical utility. Conclusion     The predictive model combining quantitative and qualitative features of CT has a good predictive ability for the invasiveness of ground-glass nodules. Its predictive performance is higher than any single indicator.

Oxalate is an organic dicarboxylic acid that is a common component of plant foods.The kidneys are essential organs for oxalate excretion,but excessive oxalates may induce kidney stones.Jupiter micro-tubule associated homolog 2(JPT2)is a critical molecule in Ca2+mobilization,and its intrinsic mecha-nism in oxalate exposure and kidney stones remains unclear.This study aimed to reveal the mechanism of JPT2 in oxalate exposure and kidney stones.Genetic approaches were used to control JPT2 expression in cells and mice,and theJPT2 mechanism of action was analyzed using transcriptomics and untargeted metabolomics.The results showed that oxalate exposure triggered the upregulation of JPT2,which is involved in nicotinic acid adenine dinucleotide phosphate(NAADP)-mediated Ca2+mobilization.Tran-scriptomic analysis revealed that cell adhesion and macrophage inflammatory polarization were inhibited by JPT2 knockdown,and these were dominated by phosphatidylinositol 3-kinase(PI3K)/AKT signaling,respectively.Untargeted metabolomics indicated that JPT2 knockdown inhibited the produc-tion of succinic acid semialdehyde(SSA)in macrophages.Furthermore,JPT2 deficiency in mice inhibited kidney stones mineralization.In conclusion,this study demonstrates that oxalate exposure facilitates kidney stones by promoting crystal-cell adhesion,and modulating macrophage metabolism and in-flammatory polarization via JPT2/PI3K/AKT signaling.

Objective To investigate the overall and subgroup efficacy,subgroup differences,predictors of efficacy and safety of roxadustat in the treatment of refractory non-severe aplastic anemia(NSAA)and low-risk myelodysplastic syndromes(LR-MDS).Methods Patients with refractory NSAA and LR-MDS who were admitted to the Department of Hematology,Peking Union Medical College Hospital from August 2020 to December 2022 were enrolled.All patients received first-line standard treatment,including recombinant human erythropoietin(rhEPO)for at least 3 months before roxadustat treatment.All patients received roxadustat 2.5 mg/kg every other day for at least 3 months,and were followed up for at least 8 months.The clinical characteristics of patients,roxadustat efficacy,predictors of efficacy,adverse effects,relapse and disease clonal evolution were analyzed.Results A total of 40 patients including 24 refractory NSAA and 16 LR-MDS were included.median age was 56(18～81)years and 40%were males.65%of the patients were transfusion dependent.Median follow-up was 21(9～34)months.22.5%,25.0%,47.5%,55.0%,57.5%,60.0%and 50.0%of the patients achieved haemato-logical improvement-erythroid(HI-E)at months 1,2,3,4,5,6,and end of the follow-up period,respectively,and no factors affecting HI-E were identified.The hemoglobin change from baseline was statistically different between the two groups at the end of the follow-up period.50%of patients were relieved from transfusion dependence.Adverse reactions were reported in 22.5%of patients.28.5%of patients relapsed after achieving HI-E,with a median time to relapse of 7(4～12)months.No clonal evolution was observed at the end of the follow-up period.Conclusions Our preliminary findings suggested that Roxadustat may be effective for patients with NSAA or LR-MDS refractory to conventional therapies and rhEPO,with mild adverse effects and low relapse rate.The degree of hemoglobin improvement may be better in the refractory NSAA patients.

Objective To explore the mechanism of ferroptosis induced by endoplasmic reticulum stress(ERs)in acute respiratory distress syndrome(ARDS).Methods In order to determine the effects of LPS on oxidative stress and Fe2+level of mouse capillary alveolar epithelial cells(MLE12 cells),the cells were treated with LPS(0,1,2,5 μg/ml)for 24 h.To verify the role of ferroptosis in lipopolysaccharide(LPS)-induced cell death,MLE12 cells were divided into control(Con)group,iron removal inhibitor(Fer-1)group,LPS group and LPS+Fer-1 group.LPS+Fer-1 group was pretreated with 10 μmol/L Fer-1 for 6 h,then the cells were exposed to 5 μg/ml LPS for 24 h.Con group was treated with solvent DMSO for 24 h.Fer-1 group was pretreated with 10 μmol/L Fer-1 for 6 h,and then treated with DMSO for 24 h.The cells in LPS group were exposed to 5 μg/ml LPS for 24 h.The MLE12 cells were divided into three groups:Con+Vector group,Con+sequence similarity family 134 mem-ber B(FAM134B)group,LPS+Vector group and LPS+FAM134B group.After transfected with vector or FAM134B overexpression plasmid for 48 h,the cells were exposed or not exposed to 5 μg/ml LPS for 24 h.Cell vi-ability was measured by CCK-8.The levels of malondialdehyde(MDA),glutathione and iron,the protein levels of ferroptosis markers[cyclooxygenase 2(PTGS2),glutathione peroxidase 4(GPX4)]and ERs markers[glucose reg-ulatory protein 78(GRP78),activated transcription factor 4(ATF4)and C/EBP homologous protein(CHOP)]were measured in different groups.In order to further confirm the results of in vitro cell experiments,40 mice were randomly divided into Con+Vector group,Con+FAM134B group,LPS+Vector group and LPS+FAM134B group,with 10 mice in each group.LPS-induced sepsis models were established in LPS+Vector group and LPS+FAM134B group,and the levels of GPX4 and ERs in lung tissue were evaluated by immunofluorescence staining and protein blot.Results LPS treatment increased the levels of PTGS2 and MDA,and decreased the levels of GPX4 and GSH in MLE12 cells in a dose-dependent manner.Compared with LPS group,the cell viability,GPX4 and GSH levels in LPS+Fer-1 group increased significantly(P<0.05),while the PTGS2 protein level and MDA level decreased significantly(P<0.05).Compared with LPS+Vector group,LPS+FAM134B group significantly increased cell viability(P<0.05),decreased PTGS2 protein level(P<0.05)and increased GPX4 level(P<0.05).At the same time,the level of MDA in LPS+FAM134B group was lower than that in LPS+Vector group(P<0.05),and the level of GSH was higher than that in LPS+Vector group(P<0.05).In animal experiment,compared with LPS+Vector group,the expression levels of 4-HNE,ATF4 and CHOP in lung tissue of LPS+FAM134B group decreased significantly(P<0.05),and the expression levels of GPX4,FAM134B group in-creased significantly(P<0.05).Conclusion LPS induces ferroptosis and ERs in MLE12 cells in a dose-depend-ent manner.Activating the endoplasmic reticulum autophagy associated FAM134B receptor helps to inhibit ERs and alleviate cell ferroptosis.

Background and purpose:Abnormal DNA methylation is closely associated with the onset and progression of tumors.This study aimed to investigate the expression of intermediate filament family orphan 1(IFFO1),a methylation-driven gene(MDG)in pancreatic adenocarcinoma(PAAD),along with its effects on the invasion and metastasis of PAAD cells,as well as its potential as a diagnostic and prognostic biomarker.Methods:mRNA expression data(TCGA-PAAD-mRNA),DNA methylation data(TCGA-PAAD-meth,GSE53051,PACA-AU)of PAAD and adjacent normal tissues,as well as DNA methylation data of healthy individuals'blood(GSE69270),were obtained from the The Cancer Genome Atlas(TCGA),International Cancer Genome Consortium(ICGC)and Gene Expression Omnibus(GEO)databases.By performing differential expression analysis combined with differential methylation analysis,we screened for MDG in PAAD.In the TCGA database,Pearson correlation tests were employed to verify the relationship between IFFO1 promoter methylation level and its expression level.Additionally,Kaplan-Meier survival analysis was conducted to evaluate the relationship among IFFO1 promoter methylation level,expression level,and the prognosis of PAAD.Pathological sections of cancer tissues and corresponding adjacent tissues from 27 PAAD patients were obtained from Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine.All samples involved in this study were approved by the human ethics committee of Shanghai General Hospital,Shanghai Jiao Tong University School of Medicine(ethics number:hospital ethics review[2017]No.53).Immunohistochemistry staining(IHC)was utilized to detect the expression of IFFO1 in cancer tissues and corresponding adjacent tissues from 27 PAAD patients.Based on the median expression level of IFFO1,patients in the TCGA database were classified into high-expression and low-expression groups.Subsequently,differential analysis,gene ontology(GO)enrichment analysis and gene set enrichment analysis(GSEA)were performed.Western blot and real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)were employed to assess the expression variations of IFFO1 between the normal pancreatic ductal epithelial cell line H6C7 and the PAAD cell lines MIA PaCa2,BxPC-3,AsPC-1 and Capan-2.The impact of IFFO1 overexpression on the migration and invasion capacities of PAAD cell lines AsPC-1 and Capan-2 was evaluated using scratch and invasion assays.Additionally,receiver operating characteristic(ROC)curves and Kaplan-Meier survival analysis were utilized to assess the diagnostic and prognostic significance of IFFO1 methylation levels in the TCGA pan-cancer cohort.Results:Through the cross-screening of five datasets,41 MDG in PAAD were identified.Among these,IFFO1 was found to be the gene most closely associated with the prognosis of PAAD[hazard ratio(HR)=0.28,P＜0.001].IFFO1 exhibited high methylation and low expression levels in PAAD.Moreover,a significant negative correlation was observed between the methylation level of its promoter and its expression level(r=-0.55,P＜0.001).IHC results indicated that IFFO1 expression was significantly lower in PAAD tissues than in adjacent non-tumor tissues(P＜0.05).TCGA survival analysis demonstrated that patients with high methylation or low expression of IFFO1 had poorer overall survival(P＜0.05).Both GO and GSEA analyses indicated that the pathway"Negative regulation of cell migration"was enriched in patients with high IFFO1 expression.Western blot and RTFQ-PCR results demonstrated that IFFO1 expression in normal pancreatic ductal epithelial cells H6C7 was significantly higher compared with PAAD cell lines MIA PaCa2,BxPC-3,AsPC-1,and Capan-2.Overexpression of IFFO1 significantly inhibited the migration and invasion of the PAAD cell lines AsPC-1 and Capan-2.Additionally,pan-cancer analysis revealed that IFFO1 exhibited abnormal promoter methylation and low expression across various cancer types,with its methylation levels demonstrating significant diagnostic and prognostic prediction value among different tumors.Conclusion:Promoter hypermethylation results in decreased expression of IFFO1 in PAAD.IFFO1 may suppress the invasion and migration abilities of PAAD cells.Furthermore,IFFO1 methylation holds great promise as a novel biomarker for the diagnosis and prognosis of PAAD.