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 investigate the effects and mechanisms of Xuebijing injection (XBJ) on Chimeric antigen receptor-T (CAR-T) cell function and its therapeutic potential against CAR-T therapy-associated cytokine storms (CRS). Methods: Anti-CD19 CAR-T cells were established based on FMC63 antibodies. Different doses of XBJ (1 and 10 mg/mL) were added to the culture system. Untreated anti-CD19 CAR-T cells served as negative controls. After 48-h co-culture, the effects of XBJ on CAR-T cell function were assessed. Carboxyfluorescein diacetate succinimidyl ester staining was used to assess the effect of XBJ on CAR-T cell proliferation. Flow cytometry, luciferase reporter gene assays, and real time cellular analysis were employed to evaluate the effects of XBJ on CAR-T cell cytotoxicity in vitro. RNA-sequencing was performed to analyze the effects of XBJ on CAR-T cell gene expression. Network pharmacology predicted potential XBJ therapeutic targets for CRS, which were verified in a THP-1 macrophage inflammation model. Results: XBJ enhanced both the proliferation and tumor killing capacities of CAR-T cells. Transcriptome analysis showed that XBJ treatment affects multiple genes and pathways in CAR-T cells, with differential gene enrichment in multiple cell proliferation and growth factor pathways. Potential targets for CRS control by XBJ were predicted using network pharmacology, and the inhibitory effect of XBJ on the expression of relevant genes was verified using a macrophage model. Conclusion The results of this study indicate that XBJ can enhance the killing effect of CAR-T cells on tumor cells and that the mechanism is related to the regulation of T cell proliferation and activation. Moreover, XBJ inhibited excessive inflammation associated with CAR-T therapy. However, the current findings remain to be further validated through in vivo experiments.

[摘 要] 目的：设计和构建表达PD-1 shRNA的靶向CD19 CAR-T细胞并验证其体外肿瘤细胞杀伤能力。方法：设计并构建表达PD-1 shRNA的CD19 CAR分子基因，将其包装成逆转录病毒载体，通过qPCR法检测病毒载体拷贝数。将慢病毒转导人原代T细胞，获得三种CAR-T细胞，分别为RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞。采用qPCR法检测三种CAR-T细胞中PD-1 mRNA的表达水平，流式细胞术检测三种CAR-T细胞中PD-1表达水平，萤光素酶报告基因实验、流式细胞术检测在不同效靶比时CAR-T细胞对CD19阳性靶细胞（人淋巴瘤daudi细胞）的杀伤功能。结果：RNAU6-CD19 CAR、PD-1 shRNA1-CD19 CAR、PD-1 shRNA2-CD19 CAR三种CAR分子成功包装成逆转录病毒载体，病毒载体拷贝数均高于1×107拷贝/mL，转导人原代T细胞获得CAR-T细胞，RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞转导效率分别为43.1%、55.1%、41.7%。与RNAU6-CD19 CAR-T细胞相比，PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞中PD-1 mRNA表达水平均显著降低（均P<0.01）、细胞表面PD-1表达水平更低（均P<0.01）、体外对daudi细胞的杀伤率更高（P<0.05或P<0.01）。结论：成功构建表达PD-1 shRNA的靶向CD19 CAR-T细胞，其对CD19阳性靶细胞的杀伤率显著提高，PD-1 mRNA及其翻译产物PD-1的表达减少，CAR-T细胞的耗竭减缓。

ObjectiveTo explore whether the mechanism of Shuangshen Ningxin capsules （SSNX） in alleviating myocardial ischemia-reperfusion injury （MIRI） in rats is related to the regulation of mitochondrial fission and fusion. MethodThis study focused on Sprague Dawley （SD） rats and ligated the left anterior descending branch of the coronary artery to construct a rat model of MIRI. The rats were divided into the sham operation group， model group， SSNX group （90 mg·kg^-1） and trimetazidine group （5.4 mg·kg^-1）. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） were detected by micro method. Changes in mitochondrial membrane potential （△Ψm） and the degree of mitochondrial permeability transition pore （mPTP） opening were detected by the chemical fluorescence method. The intracellular adenosine triphosphate （ATP） level was detected by the luciferase assay. The messenger ribonucleic acid （mRNA） and protein expression levels of mitochondrial fission and fusion related factors dynamin-related protein 1 （DRP1）， mitochondrial fission 1 protein （FIS1）， optic atrophy protein 1 （OPA1）， mitochondrial outer membrane fusion protein 1 （MFN1）， and MFN2 were detected by real-time polymerase chain reaction （real-time PCR） and Western blot. ResultCompared with the sham operation group， the model group showed a decrease in serum SOD activity and an increase in MDA content. The opening level of mPTP， the level of △Ψm and ATP content decreased， the protein expressions of mitochondrial fission factors DRP1 and FIS1 increased， and the protein expressions and mRNA transcription levels of fusion related factors OPA1 and MFN1 decreased. Compared with the model group，SSNX significantly increased serum SOD activity， reduced MDA content， increased intracellular ATP level and △Ψm， reduced the opening level of mPTP， downregulated the protein expressions of mitochondrial fission factors DRP1 and FIS1， and increased the mRNA transcription levels and protein expressions of fusion related factors OPA1 and MFN1. ConclusionSSNX inhibits the expressions of mitochondrial fission factors DRP1 and FIS1， and increases the expressions of fusion related factors OPA1 and MFN1， inhibiting mitochondrial fission and increasing mitochondrial fusion， thereby alleviating MIRI.

Objective To construct Raji-Luc lymphoma cells with CD19 knockout using CRISPR/Cas9 technology and preliminarily validate their immune escape ability.Methods PB-CRISPR-CD19 small guide RNA(sgRNA)plasmids was constructed,the optimal sgRNA sequence was screened,and Raji-Luc cells with pCAG-PBase,PB-CD19 sgRNA,and PB-CRISPR-Cas9 were co-transfected.Stable knockout monoclonal cell lines were screened by flow sorting and limit dilution method and the knockout effect was verified through gene sequence testing.The expression of luciferase on the surface of the cell line was detected by microplate reader,CD19 CAR-T and CD38 CAR-T previously constructed in the laboratory were used as effector cells,and the immune escape ability of Raji-Luc CD19 KO cell line was verified by universal luciferase chemiluminescence method.Results The transfection efficiency of Raji-Luc CD19 KO cells prepared by electro transfection was high,and the knockout efficiency of the two monoclonal cells was more than 99%.There was no significant difference in luciferase expression compared to the original Raji-Luc cells,and CD19 CAR-T cells could not be activated to the kill them.Conclusion Successfully constructed Raji-Luc CD19 KO lymphoma cell line.

ObjectiveThis study observes the intervention effect of Longmu Piyan prescription on oxidative stress in BALB/c mice with atopic dermatitis （AD） induced by 2，4-dinitrochlorobenzene （DNCB） and explores its mechanism. MethodThe AD model was established using the method of DNCB sensitization on the back skin of BALB/c mice. Forty male BALB/c mice were randomly divided into a blank group， a model group， a vitamin C control group （0.5×10^-3 mg·kg^-1）， and a Longmu Piyan prescription group （26 g·kg^-1）. Except for the blank group， other groups were sensitized with different concentrations of DNCB on the back to induce AD， and the blank group was treated with matrix coating. The gastric administration was started on the seventh day after sensitization with 2% DNCB and on the 24th day after sensitization with 0.2% DNCB continuously for 21 days. The changes in skin lesions of each group were directly observed after the experiment. Enzyme-linked immunosorbent assay （ELISA） was used to detect the levels of interleukin （IL）-4， tumor necrosis factor （TNF）-α， immunoglobulin E （IgE）， and reactive oxygen species （ROS） in the serum of each group. The total antioxidant capacity determination kit-trace method (ABTS method) was used to measure the level of total antioxidant capacity （TAOC） in serum. The Hematoxylin eosin （HE） staining method was used to observe the pathological and morphological changes of the skin lesion site. The immunohistochemical method was used to detect the expression of thymic stromal lymphopoietin （TSLP） in the skin lesion site. Western blot was used to detect the expression of filaggrin （FLG） in the dorsal skin lesions. ResultThe results showed that compared with the blank group， the skin lesion score of the model group mice was significantly increased （P<0.01）， and HE staining showed characteristic pathological changes of AD in the skin lesion site. At the same time， the expression of TSLP in the skin lesion was significantly increased， and that of FLG was reduced （P<0.05）. The levels of TNF-α， IL-4， IgE， and ROS in serum increased， while the activity of TAOC decreased （P<0.01）. Compared with the model group， the Longmu Piyan prescription group showed a significant decrease in skin lesion scores and a significant improvement in skin lesion pathology. At the same time， the expression of TSLP decreased， and the expression of FLG increased in the skin lesions （P<0.05）. In addition， compared with the model group， the serum levels of TNF-α， IL-4， IgE， and ROS also decreased to varying degrees （P<0.05，P<0.01）， and TAOC activity increased in the Longmu Piyan prescription group （P<0.01）. ConclusionThere is a significant correlation among the degree of oxidative stress， the severity of skin lesions in AD， and the levels of inflammatory cytokines. Longmu Piyandu prescription can improve AD-like skin lesions in BALB/c mice by promoting ROS clearance， enhancing TAOC， and inhibiting oxidative stress， thus protecting the skin barrier and reducing inflammation.

OBJECTIVE To investigate the regulatory effects of icariin(ICA)on cardiac micro-vascular endothelial cells(CMEC)after oxygen-glucose deprivation reperfusion(OGD/R)injury.METHODS CMEC were subjected to OGD/R treatment to construct a myocardial ischemia-reperfusion model,and were divided into normal,model,low(10 μmol·L-1),medium(20 μmol·L-1)and high(40 μmol·L-1)ICA group,and high ICA+ inhibitor group(40 μmol·L-1+20 nmol·L-1).CCK-8 assay was used to assess the protective ability of ICA against CMEC,and cell migration assay and tube-formation assay were used to detect the migration and generation ability of CMEC.The TCMSP database,Swiss-Target database and literature mining methods were used to col-lect ICA-related targets,the GeneCards data-base was used to collect target genes related to myocardial ischemia/reperfusion,and Cytoscape 3.8.0 software was used to construct a"drug-tar-get-disease"network.The potential targets were imported into STRING 11.5 database to obtain the PPI network.GO and KEGG enrichment analyses were performed on the potential targets using the DAVID database.Molecular docking was performed using AutoDock-vina 1.1.2 soft-ware.Western blot detected the expression of related proteins.RESULTS After CMEC was subjected to OGD/R treatment,ICA had a protec-tive effect at 10-160 μmol·L-1;the results of the cell migration assay showed that each group of ICA could promote the migratory effect of CMEC(P＜0.01,P＜0.01);and the results of tube-for-mation assay showed that each group of ICA could significantly promote the generation of branches(P＜0.01)and the capillary length exten-sion(P＜0.05).Network pharmacology collected a total of 23 ICA action targets,1500 disease tar-gets and 12 key targets.GO function enrichment analysis found 85 results.KEGG pathway enrich-ment analysis found 53 results,involving AGE-RAGE signaling pathway,sphingolipid signaling pathway and VEGF signaling pathway.Molecu-lar docking results showed that ICA had better binding with core targets PRKCB,PRKCA and PTGS2.Western blot results showed that ICA could regulate the expression of PRKCB,PRKCA and PTGS2 proteins.The results of cell migra-tion assay,tube-formation assay and protein expression were reversed after addition of PKC inhibitor.CONCLUSION The potential mecha-nism of action of ICA against myocardial isch-emia-reperfusion injury may be related to the reg-ulation of processes such as CMEC migration and angiogenesis,and it functions through the key target gene PKC.

Objective:To evaluate the clinical value of different shimming methods at 7.0 T MR in two-dimensional (2D) and three-dimensional (3D) T 2WI. Methods:Totally 23 healthy volunteers were prospectively recruited from the First Medical Center of PLA General Hospital from November, 2022 to May, 2023, including 12 volunteers who underwent 2D shimming mode and 14 volunteers who underwent 3D shimming mode. 2D shimming mode included patient-specific (PS) mode, direct signal control (DSC) mode, the standard circularly polarized (CP) mode, and volume-specific (VS) mode. 3D shimming mode included universal pulses (UP) mode and CP mode. The image quality for the subtentorial and supratentorial region was assessed by the subjective image quality score and signal-to-noise ratio. Comparisons of quantitative indices between multiple groups were performed using repeated-measures ANOVA or Friedman′s test; comparisons of quantitative indices between 2 groups were performed using paired-samples t test or Wilcoxon signed-rank test. Results:The image quality of subtentorial region and SNR was significant differences in 2D T 2WI with PS mode, DSC mode, CP mode and VS mode ( F=26.74, P<0.001; F=28.24, P<0.001), and the image quality score and SNR of PS mode, DSC mode, VS mode were better than CP mode ( P<0.05). In 2D T 2WI, there was no significant difference in image quality score and SNR of supratentorial region in PS mode, DSC mode, CP mode ( P>0.05). Besides, in 3D T 2WI, the image quality score for subtentorial and supratentorial region of UP mode were better than those of CP mode ( Z=-2.74, P=0.006; Z=-3.24, P=0.001); SNR of subtentorial region was significantly better in UP mode than those in CP mode ( t=3.49, P=0.004). But there was no significant difference in SNR of supratentorial region between the UP mode and CP mode in 3D T 2WI ( P>0.05). Conclusion:T 2WI with different shimming methods at 7.0 T MR can provide data support for the clinical application, which is helpful for the accurate diagnosis of patients with subtentorial lesions.