ObjectiveThis paper aims to observe the role of Danlou tablet in treating coronary heart disease （CHD） with phlegm-stasis intermingling syndrome in minipigs by improving platelet function and explore the potential pharmacological mechanism of Danlou tablet in regulating platelet function by using proteomics technology. MethodsThirty Bama minipigs were randomly divided into a normal control group （6 pigs） and a high-fat diet group （24 pigs）. After 2 weeks of high-fat diet feeding， the high-fat diet group was randomly subdivided into a model group， an atorvastatin group （1 mg·kg^-1）， and Danlou tablet groups （0.6 g·kg^-1 and 0.3 g·kg^-1）. All groups continued to receive a high-fat diet for 8 weeks after the procedure. The normal control group was given a regular diet， underwent only coronary angiography， and did not receive an interventional injury procedure. The model group and each administration group were fed a high-fat diet. Two weeks later， they underwent a coronary angiography injury procedure. After the procedure， drugs were mixed into the feed every morning for 8 consecutive weeks， with the minipigs maintained on a continuous high-fat diet during this period. Quantitative proteomics technology was further used to study platelet proteins， and differential proteins were obtained by screening. Bioinformatics analysis was performed to analyze key regulatory proteins and biological pathways involved in the therapeutic effect of Danlou tablet on CHD with phlegm-stasis intermingling syndrome. ResultsCompared with the normal control group， the model group showed a significant increase in total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C）， and low-density lipoprotein cholesterol （LDL-C） of minipigs' serum （P<0.01）， a significant shortening in prothrombin time of （PT） （P<0.01）， a coagulation function index， and an increase in whole blood viscosity （P<0.01） and platelet aggregation rate （P<0.01）. Moreover， the platelet morphology was altered， and the contents of endothelin-1 （ET-1） and nitric oxide （NO） were significantly increased （P<0.01）. Hemodynamic parameters were obviously abnormal， including significantly decreased systolic blood pressure （SBP）， diastolic blood pressure （DBP）， mean arterial pressure （MAP）， left ventricular systolic pressure （LVSP）， and left ventricular maximal positive dp/dt （LV+dp/dt_max） （P<0.01）. Left ventricular maximal negative dp/dt （LV-dp/dt_max） was significantly increased （P<0.01）. Besides， there were myocardial cell hypertrophy， obvious edematous degeneration， massive interstitial inflammatory cell infiltration， high degree of fibrosis， and coronary endothelial atherosclerosis. TC and TG levels in minipigs' serum were significantly reduced in Danlou tablet groups with 0.6 g·kg^-1 and 0.3 g·kg^-1 （P<0.05， P<0.01）， compared with those in the model group. LDL-C was decreased in the Danlou tablet group with 0.6 g·kg^-1 （P<0.05）. The whole blood viscosity under low and high shear conditions was significantly reduced in the Danlou tablet group with 0.6 g·kg^-1 （P<0.05）. In groups with all doses of Danlou tablet， maximum aggregation rate （MAR） and average aggregation rate （AAR） were significantly decreased （P<0.05， P<0.01）， and platelets' morphological changes such as pseudopodia extension were reduced. ET-1 levels in the serum were significantly reduced. In the Danlou tablet group with 0.6 g·kg^-1， NO level in the serum was reduced （P<0.05）. In groups with all doses of Danlou tablet， DBP and MAP were significantly increased （P<0.05）. In the Danlou tablet group with 0.6 g·kg^-1， LVSP and LV+dp/dt_max were significantly increased （P<0.05， P<0.01）， and LV-dp/dt_max was significantly decreased （P<0.05）. In groups with all doses of Danlou tablet， edematous degeneration in myocardial tissue was milder， and coronary artery lesion degree was significantly alleviated. Compared with the normal control group， there were 94 differentially expressed proteins in the model group， including 81 up-regulated and 13 down-regulated proteins. Compared with the model group， the Danlou tablet group with 0.6 g·kg^-1 showed 174 differentially expressed proteins， including 100 up-regulated and 74 down-regulated proteins. A total of 30 proteins were reversed after Danlou tablet intervention. Bioinformatics analysis revealed that its pharmacological mechanism may exert anti-platelet activation， aggregation， and adhesion effects through biological pathways such as regulation of actin cytoskeleton， platelet activation pathway， Fcγ receptor-mediated phagocytosis， as well as proteins such as growth factor receptor-bound protein 2 （GRB2）， Ras-related C3 botulinum toxin substrate 2 （RAC2）， RAC1， and heat shock protein 90 alpha family class A member 1 （HSP90AA1）. ConclusionDanlou tablet can effectively reduce platelet activation and aggregation， exerting a good therapeutic effect on CHD with phlegm-stasis intermingling syndrome in minipigs. Its pharmacological mechanism may involve regulating biological pathways such as actin cytoskeleton and platelet activation pathway， as well as proteins like GRB2， RAC2， RAC1， and HSP90AA1， thereby exerting a pharmacological effect in anti-platelet activation， aggregation， and adhesion.

Iron overload is strongly associated with heart disease. Ferroptosis is a new form of regulated cell death indicated in cardiac ischemia-reperfusion (I/R) injury. However, the specific molecular mechanism of myocardial injury caused by iron overload in the heart is still unclear, and the involvement of ferroptosis in iron overload-induced myocardial injury is not fully understood. In this study, we observed that ferroptosis participated in developing of iron overload and I/R-induced cardiomyopathy. Mechanistically, we discovered that Parkin inhibited iron overload-induced ferroptosis in cardiomyocytes by promoting the ubiquitination of long-chain acyl-CoA synthetase 4 (ACSL4), a crucial protein involved in ferroptosis-related lipid metabolism pathways. Additionally, we identified p53 as a transcription factor that transcriptionally suppressed Parkin expression in iron-overloaded cardiomyocytes, thereby regulating iron overload-induced ferroptosis. In animal studies, cardiac-specific Parkin knockout mice (Myh6-CreER ^T2 /Parkin ^fl/fl ) fed a high-iron diet presented more severe myocardial damage, and the high iron levels exacerbated myocardial I/R injury. However, the ferroptosis inhibitor Fer-1 significantly suppressed iron overload-induced ferroptosis and myocardial I/R injury. Moreover, Parkin effectively protected against impaired mitochondrial function and prevented iron overload-induced mitochondrial lipid peroxidation. These findings unveil a novel regulatory pathway involving p53-Parkin-ACSL4 in heart disease by inhibiting of ferroptosis.

Vein graft (VG) failure (VGF) is associated with VG intimal hyperplasia, which is characterized by abnormal accumulation of vascular smooth muscle cells (VSMCs). Most neointimal VSMCs are derived from pre-existing VSMCs via a process of VSMC phenotypic transition, also known as dedifferentiation. There is increasing evidence to suggest that ginger or its bioactive ingredients may block VSMC dedifferentiation, exerting vasoprotective functions; however, the precise mechanisms have not been fully characterized. Therefore, we investigated the effect of ginger on VSMC phenotypic transition in VG remodeling after transplantation. Ginger significantly inhibited neointimal hyperplasia and promoted lumen (L) opening in a 3-month VG, which was primarily achieved by reducing ferroptotic stress. Ferroptotic stress is a pro-ferroptotic state. Contractile VSMCs did not die but instead gained a proliferative capacity and switched to the secretory type, forming neointima (NI) after vein transplantation. Ginger and its two main vasoprotective ingredients (6-gingerol and 6-shogaol) inhibit VSMC dedifferentiation by reducing ferroptotic stress. Network pharmacology analysis revealed that 6-gingerol inhibits ferroptotic stress by targeting P53, while 6-shogaol inhibits ferroptotic stress by targeting 5-lipoxygenase (Alox5), both promoting ferroptosis. Furthermore, both ingredients co-target peroxisome proliferator-activated receptor gamma (PPARγ), decreasing PPARγ-mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) expression. Nox1 promotes intracellular reactive oxygen species (ROS) production and directly induces VSMC dedifferentiation. In addition, Nox1 is a ferroptosis-promoting gene that encourages ferroptotic stress production, indirectly leading to VSMC dedifferentiation. Ginger, a natural multi-targeted ferroptotic stress inhibitor, finely and effectively prevents VSMC phenotypic transition and protects against venous injury remodeling.

Objective To design and construct chimeric antigen receptor(CAR)T cells targeting Trop2,establish an in vitro cell exhaustion model through continuous antigen stimulation,and investigate their anti-tumor activity and exhaustion characteristics.Methods The second-generation CAR plasmid was constructed based on the single-chain variable fragment(scFv)sequence of Sacituzumab Govitecan targeting Trop2.The viral vector titer was determined by retroviral vector packaging and gradient dilution.Peripheral blood mononuclear cells(PBMCs)from healthy donors were isolated using Ficoll density gradient centrifugation,and CAR virus vectors were transduced into PBMCs activated with OKT-3/IL-2 to generate Trop2-targeted CAR T cells.CAR expression levels were assessed by flow cytometry using MYC tags.In vitro 3 tumor cell models were established,including human ovarian cancer cells(SKOV3),human breast cancer cells(MDA-MB-453),and human lung cancer cells(A549).The expression of the Trop2 antigen in these models was confirmed using flow cytometry.Additionally,luciferase assay was employed to evaluate the cytotoxic efficiency of Trop2-targeted therapy at various effector-to-target ratios.An in vitro CAR-T exhaustion model was developed,and the long-term killing ability of CAR-T cells was dynamically monitored using the Incucyte live-cell imaging system.The PD-1/TIM-3 phenotype of CAR-T cells was analyzed by flow cytometry,and cytokine secretion levels were quantified using the cytometric bead array(CBA).Transcriptomic sequencing and RT-qPCR were employed to validate the differentially expressed genes associated with exhaustion.Results The second-generation CAR T cells targeting Trop2 were successfully constructed.Compared to the P-T group,in vitro experiments demonstrated that these CAR T cells exhibited antigen-specific and dose-dependent cytotoxic effects against tumor cells with high Trop2 expression,such as MDA-MB-453 and SKOV3.A CAR-T cell exhaustion model established through repeated tumor antigen stimulation in vitro revealed that,compared to the initial state,the exhausted Trop2 CAR-T cells exhibited significantly reduced tumor-killing capacity while P-T cells showed almost no killing effect,the expression of inhibitory receptors(PD-1 and TIM-3)was up-regulated on the surface of exhausted CAR-T cells,and the secretion of effector cytokines was diminished.Transcriptomic analysis identified multiple differentially expressed genes in the exhausted CAR-T cells.Pathways related to immune response and T cell receptor signaling were down-regulated,while apoptosis-related pathways were activated.RT-qPCR further confirmed abnormal expression of immunoregulatory genes,including IL3,IL5,and IL13(P<0.05).Conclusion During continuous in vitro tumor antigen stimulation,the second-generation CAR-T cells targeting the Trop2 antigen demonstrate declined anti-tumor activity,weakened effector function and up-regulated expression of exhaustion-related molecules.

Objective To construct CD19-targeted chimeric antigen receptor natural killer(CAR-NK)cells armored with interleukin15(IL15)or IL15 receptor-linker(RLI)and preliminarily validate their proliferative capacity and anti-tumor activity in vitro.Methods Natural killer cells(NK92 and NK92MI)from patients with human malignant non-Hodgkin lymphoma were cultured,and CD19-targeted CAR-NK cells armored with IL15 or RLI were prepared using retroviral vector particles.IL15 secretion was measured by ELISA,and proliferative capacity was assessed via CFSE dilution assays.Human B-lymphocytic leukemia cells(Nalm6-GFP-Luc)and human colon cancer cells overexpressing CD19(hCD19-SW620-GFP-Luc)were cultured,with surface CD19 expression confirmed(>99%positivity for both).Anti-tumor activity was evaluated by measuring cytotoxicity at effector-to-target(E:T)ratios using luciferase-based assays(4/12 h),detecting surface CD107a expression via flow cytometry,and quantifying cytokine release using CBA assays.Results NK92/NK92MI-CD19 CAR cells armored with IL15 or RLI were successfully generated.IL15 secretion was significantly higher in armored groups versus non-armored controls(P<0.01).Without IL-2 stimulation,IL15/RLI enhanced CAR-NK proliferation(P<0.05).Both armored designs significantly increased tumor-killing efficiency(P<0.05)and CD107a degranulation.IL15/RLI-armored NK92-CD19 CAR cells exhibited elevated release of IL2,IL10,IL6,TNF-α,sFas,IFN-γ,and Granulysin(P<0.05),while armored NK92MI-CD19 CAR cells showed additional increases in Granzyme A,Granzyme B,and Perforin(P<0.05).Conclusion IL15/RLI-armored NK92/NK92MI-CD19 CAR cells demonstrate potent anti-tumor activity,supporting their combined clinical therapeutic potential for tumors.

Vein graft(VG)failure(VGF)is associated with VG intimal hyperplasia,which is characterized by abnormal accumulation of vascular smooth muscle cells(VSMCs),Most neointimal VSMCs are derived from pre-existing VSMCs via a process of VSMC phenotypic transition,also known as dedifferentiation.There is increasing evidence to suggest that ginger or its bioactive ingredients may block VSMC dedif-ferentiation,exerting vasoprotective functions;however,the precise mechanisms have not been fully characterized.Therefore,we investigated the effect of ginger on VSMC phenotypic transition in VG remodeling after transplantation.Ginger significantly inhibited neointimal hyperplasia and promoted lumen(L)opening in a 3-month VG,which was primarily achieved by reducing ferroptotic stress.Fer-roptotic stress is a pro-ferroptotic state.Contractile VSMCs did not die but instead gained a proliferative capacity and switched to the secretory type,forming neointima(NI)after vein transplantation.Ginger and its two main vasoprotective ingredients(6-gingerol and 6-shogaol)inhibit VSMC dedifferentiation by reducing ferroptotic stress.Network pharmacology analysis revealed that 6-gingerol inhibits fer-roptotic stress by targeting P53,while 6-shogaol inhibits ferroptotic stress by targeting 5-lipoxygenase(Alox5),both promoting ferroptosis.Furthermore,both ingredients co-target peroxisome proliferator-activated receptor gamma(PPARγ),decreasing PPARγ-mediated nicotinamide adenine dinucleotide phosphate(NADPH)oxidase 1(Nox1)expression.Nox1 promotes intracellular reactive oxygen species(ROS)production and directly induces VSMC dedifferentiation.In addition,Nox1 is a ferroptosis-promoting gene that encourages ferroptotic stress production,indirectly leading to VSMC dedifferenti-ation.Ginger,a natural multi-targeted ferroptotic stress inhibitor,finely and effectively prevents VSMC phenotypic transition and protects against venous injury remodeling.

Objective:To investigate the technical application pathways of artificial intelligence(AI)in laboratory information management systems(LIMS)and its role in promoting laboratory management efficiency and intelli-gence.Methods:Through the integration of traditional AI technologies(e.g.,machine learning,computer vision)with large language models,this study demonstrated the application of various AI technologies in scenarios such as intelligent Q&A for local knowledge bases,comprehensive review of inspection processes,intelligent data visualization,and image recognition.Results:Through the implementation of AI applications in laboratory settings,AI significantly enhanced management efficiency:the intelligent Q&A system achieved over 90％accuracy,auto-mated inspection processes reduced manual workload by 40％,and image recognition precision reached 89％-100％.Conclusion:AI provides efficient and precise solutions for laboratory management via multimodal integration and process optimization.Future efforts should focus on strengthening data security and model interpret-ability to promote comprehensive intelligent development.

Objective To explore the optimal conditions for stable transfer of reporter gene containing enhanced green fluorescent protein(EGFP)into human breast cancer cells MDA-MB-231 cells under different electrotransfection conditions.Methods A PiggyBac(PB)transposon system containing EGFP reporter gene was constructed,and transfection conditions such as waveform,voltage,shock time,shock times,plasmid concentration,cell density,transposon to transposase ratio were controlled during electrotransfection of MDA-MB-231 cells.The transfection efficiency was detected by flow cytometry fluorescein isothiocyanate(FITC)channel.The expression of fluorescent protein was observed under laser scanning confocal microscope,the electrotransfection efficiency and expression of EGFP reporter gene were verified,and the electrotransfer efficiency of MDA-MB-231 cells was analyzed under different electrotransfection conditions.Results The optimal transfection conditions of MDA-MB-231 cells were as follows:voltage 280V,exponential wave,electric shock once,concentration of transposon plasmid about 1 000 ng/μl,mass ratio of transposon to transpotase 1:1,number of cells 2×106,electrotransfection rate up to 60.23%±5.63%.The state of MDA-MB-231 cells was good.Conclusion The electrotransfection conditions of MDA-MB-231 cells were successfully optimized to achieve stable and efficient transfection,which provided efficient electrotransfer experimental parameters for the relevant basic research of MDA-MB-231 cell line.

Objective:To investigate the prognostic impact of perineural invasion in patients with stageⅢ colon cancer and to clarify its guidance value for the duration of postoperative adjuvant chemotherapy.Methods:This study employed a retrospective cohort study method. It analyzed 426 patients with stageⅢ colon cancer who underwent radical surgery at Sun Yat-sen University Cancer Center and Longyan First Affiliated Hospital of Fujian Medical University, between April 2008 and June 2020. Inclusion criteria: patients received at least 3 months of adjuvant CapeOX therapy post-surgery, had complete pathological data, and were followed up for at least 12 months after the last chemotherapy. Among these patients, 231 were male, the median age was 59 (50~67) years, and 263 tumors were located in the right-sided colon. Postoperative pathology indicated that 107 cases (25.12%) had neural invasion, and 131 patients (30.75%) had vascular tumor thrombus. All patients received at least 4 cycles of postoperative CapeOX adjuvant chemotherapy, with 193 patients receiving 8 cycles and 233 patients receiving 4 to 7 cycles of adjuvant chemotherapy. The study analyzed the impact of neural invasion status and the duration of adjuvant chemotherapy on disease-free survival (DFS). Furthermore, within subgroups stratified by different risk levels (referencing the criteria proposed by the IDEA study: high risk: T4, N2 or T4N2; low risk: T3N1) and different neural invasion statuses, the impact of the duration of adjuvant chemotherapy on prognosis was analyzed.Results:The median follow-up time for the entire cohort was 94.00 months (55.27-128.80 months). Multivariate Cox analysis indicated that pathological T stage T4 (HR = 2.457, 95%CI: 1.499-4.029, P<0.001) and postoperative pathological confirmation of perineural invasion (HR = 2.465, 95% CI: 1.519-4.000, P<0.001) were independent adverse prognostic factors for 5-year DFS. In the perineural invasion-positive group, the 5-year DFS for patients who received 8 cycles of postoperative adjuvant CapeOX chemotherapy was 86.90%, compared to 58.22% for those who received 4-7 cycles, with statistically significant differences (both P<0.05). In the perineural invasion-negative group, the 5-year DFS for patients who received 8 cycles was 88.66%, compared to 90.99% for those who received 4-7 cycles, with no statistically significant differences ( P=0.929). Among IDEA high-risk patients with perineural invasion, the 5-year DFS was 91.81% for those who received 8 cycles versus 50.66% for those who received 4-7 cycles, showing a statistically significant difference ( P=0.003). In IDEA high-risk patients without perineural invasion, the 5-year DFS for those who received 8 cycles was 82.28% compared to 87.32% for those who received 4-7 cycles, with no statistically significant difference ( P=0.806). In the IDEA low-risk patients, no differences were observed in the 5-year DFS between patients receiving 8 cycles and those receiving 4-7 cycles of adjuvant CapeOX chemotherapy in both perineural invasion-positive and negative subgroups (both P>0.05). Conclusion:Perineural invasion serves as a significant prognostic factor for 5-year DFS in stage Ⅲ colon cancer patients who have undergone radical surgery and postoperative adjuvant chemotherapy. It can also be considered an important reference factor in deciding the duration of postoperative adjuvant chemotherapy.

Objective:To investigate the technical application pathways of artificial intelligence(AI)in laboratory information management systems(LIMS)and its role in promoting laboratory management efficiency and intelli-gence.Methods:Through the integration of traditional AI technologies(e.g.,machine learning,computer vision)with large language models,this study demonstrated the application of various AI technologies in scenarios such as intelligent Q&A for local knowledge bases,comprehensive review of inspection processes,intelligent data visualization,and image recognition.Results:Through the implementation of AI applications in laboratory settings,AI significantly enhanced management efficiency:the intelligent Q&A system achieved over 90％accuracy,auto-mated inspection processes reduced manual workload by 40％,and image recognition precision reached 89％-100％.Conclusion:AI provides efficient and precise solutions for laboratory management via multimodal integration and process optimization.Future efforts should focus on strengthening data security and model interpret-ability to promote comprehensive intelligent development.