ObjectiveThis paper aims to investigate the material basis of the anti-inflammatory efficacy and mechanism of action of Bushen Tongdu prescription （BSTDP）. MethodsThe chemical components of BSTDP and its blood-absorbed components in vivo were systematically identified by using ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry （UPLC-LIT-Orbitrap-MS）. Network pharmacology was employed to screen blood-absorbed bioactive components and potential targets of this formula. A protein-protein interaction （PPI） network of core targets was constructed to conduct enrichment analysis. Molecular docking was further utilized to verify the binding affinity between key components and targets. The inflammatory model was established and verified in vivo by using a transgenic zebrafish Tg （mpx： GFP）. At three days post-fertilization （3 dpf）， larvae of zebrafish were randomly assigned to blank group， model group， positive drug dexamethasone acetate group （75 μmol·L^-1）， and BSTDP groups with low， medium， and high doses （500， 1 000， and 2 000 mg·L^-1）. The distribution and quantity of neutrophils in the yolk sac region were observed under a fluorescence microscope. The mRNA expression levels of key genes in the toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor kappa-B （NF-κB） signaling pathway and inflammatory factors including interleukin （IL）-1β， IL-6， and tumor necrosis factor-α （TNF-α） were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsA total of 120 chemical components were identified in BSTDP， among which 26 original components were confirmed by using serum pharmacochemical methods. A total of 227 common targets linking rheumatoid arthritis （RA） and the blood-absorbed components were screened by network pharmacology. It is suggested that pseudobrucine， vomicine， sinapine， rehmannioside， cinnamyl alcohol glycoside， and methylephedrine exert anti-inflammatory effects by acting on core targets including protein kinase B1 （Akt1）， signal transducer and activator of transcription 3 （STAT3）， tumor necrosis factor （TNF）， TLR4， mitogen-activated protein kinase 14 （MAPK14）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit α （PIK3CA）， thereby modulating multiple signaling pathways such as TLR4 and NF-κB. In vivo verification in zebrafish demonstrates that the maximum tolerable concentration of Bushen Tongdu Formula is 2 000 mg·L^-1. Compared to those in the blank group， zebrafish in the model group showed a significantly higher number of neutrophils in the yolk sac region （P<0.01） and rising mRNA levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β （P<0.01）. Compared to that in the model group， the number of neutrophils was significantly reduced in BSTDP groups with medium and high doses， as well as the dexamethasone acetate group （P<0.05， P<0.01）. There was no statistically significant difference in the low dose group. The mRNA expression levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β were significantly down-regulated （P<0.05， P<0.01）. ConclusionThis paper identifies the material basis of the efficacy of BSTDP， demonstrating that the formula can exert an anti-inflammatory effect through the TLR4/MyD88/NF-κB signaling pathway. The results provide scientific experimental evidence for its further clinical application.

ObjectiveThis paper aims to investigate the material basis of the anti-inflammatory efficacy and mechanism of action of Bushen Tongdu prescription （BSTDP）. MethodsThe chemical components of BSTDP and its blood-absorbed components in vivo were systematically identified by using ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry （UPLC-LIT-Orbitrap-MS）. Network pharmacology was employed to screen blood-absorbed bioactive components and potential targets of this formula. A protein-protein interaction （PPI） network of core targets was constructed to conduct enrichment analysis. Molecular docking was further utilized to verify the binding affinity between key components and targets. The inflammatory model was established and verified in vivo by using a transgenic zebrafish Tg （mpx： GFP）. At three days post-fertilization （3 dpf）， larvae of zebrafish were randomly assigned to blank group， model group， positive drug dexamethasone acetate group （75 μmol·L^-1）， and BSTDP groups with low， medium， and high doses （500， 1 000， and 2 000 mg·L^-1）. The distribution and quantity of neutrophils in the yolk sac region were observed under a fluorescence microscope. The mRNA expression levels of key genes in the toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor kappa-B （NF-κB） signaling pathway and inflammatory factors including interleukin （IL）-1β， IL-6， and tumor necrosis factor-α （TNF-α） were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsA total of 120 chemical components were identified in BSTDP， among which 26 original components were confirmed by using serum pharmacochemical methods. A total of 227 common targets linking rheumatoid arthritis （RA） and the blood-absorbed components were screened by network pharmacology. It is suggested that pseudobrucine， vomicine， sinapine， rehmannioside， cinnamyl alcohol glycoside， and methylephedrine exert anti-inflammatory effects by acting on core targets including protein kinase B1 （Akt1）， signal transducer and activator of transcription 3 （STAT3）， tumor necrosis factor （TNF）， TLR4， mitogen-activated protein kinase 14 （MAPK14）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit α （PIK3CA）， thereby modulating multiple signaling pathways such as TLR4 and NF-κB. In vivo verification in zebrafish demonstrates that the maximum tolerable concentration of Bushen Tongdu Formula is 2 000 mg·L^-1. Compared to those in the blank group， zebrafish in the model group showed a significantly higher number of neutrophils in the yolk sac region （P<0.01） and rising mRNA levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β （P<0.01）. Compared to that in the model group， the number of neutrophils was significantly reduced in BSTDP groups with medium and high doses， as well as the dexamethasone acetate group （P<0.05， P<0.01）. There was no statistically significant difference in the low dose group. The mRNA expression levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β were significantly down-regulated （P<0.05， P<0.01）. ConclusionThis paper identifies the material basis of the efficacy of BSTDP， demonstrating that the formula can exert an anti-inflammatory effect through the TLR4/MyD88/NF-κB signaling pathway. The results provide scientific experimental evidence for its further clinical application.

ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

ObjectivePancreatic ductal adenocarcinoma (PDAC) exhibits a limited response to current treatments due to its dense fibrotic stroma and highly immunosuppressive tumor microenvironment. In recent years, advancements in cellular immunotherapy, particularly chimeric antigen receptor macrophage (CAR-M) therapy, have offered new hope for pancreatic cancer treatment. Although CAR-M therapy demonstrates dual potential in directly killing tumor cells and remodeling the immune microenvironment, it still faces challenges such as complex in vitro preparation processes and low in vivo targeting and delivery efficiency. Therefore, developing strategies for efficient and targeted in vivo delivery of CAR genes has become crucial for overcoming current therapeutic limitations. This study aims to develop an orally administrable nano-gene delivery system for the targeted delivery of CAR genes to pancreatic tumor sites. MethodsCore nano-gene particles (PNP/pCAR) were constructed by loading plasmid DNA encoding CAR (pCAR) with cationic polypeptides (PNP). Subsequently, PNP/pCAR was surface-modified with β-glucan to prepare the targeted nanoparticles (βGlus-PNP/pCAR). The loading efficiency of PNP for pCAR was quantitatively assessed by gel retardation assay. The particle size, Zeta potential, morphology, and storage stability of PNP/pCAR were characterized using a Malvern particle size analyzer and transmission electron microscopy. At the cellular level, RAW 264.7 macrophages were selected. The cytotoxicity of PNP/pCAR was evaluated using the CCK-8 assay. The cellular uptake efficiency and lysosomal escape ability of the nanoparticles were assessed via flow cytometry and confocal microscopy. Transfection efficiency was quantitatively evaluated by detecting the expression of the reporter gene GFP using flow cytometry. At the in vivo level, an orthotopic pancreatic cancer mouse model was established. Cy7-labeled βGlus-PNP/pCAR nanoparticles were administered orally, and the fluorescence distribution in mice was dynamically monitored at 1, 2, 4, 8, and 16 h post-administration using a small animal in vivo imaging system. Forty-eight hours after oral gavage, the mice were euthanized, and pancreatic tumor tissues were collected for further analysis of intratumoral fluorescence signals using the imaging system. Additionally, βGlus-PNP/pCAR-GFP nanoparticles loaded with the reporter gene (GFP) were administered orally. Forty-eight hours post-administration, pancreatic tumor tissues were harvested to prepare frozen sections, and GFP expression was observed and analyzed under a fluorescence microscope. ResultsThe PNP carrier exhibited a high loading capacity for pCAR. The successfully prepared PNP/pCAR nanoparticles were regular spheres with a hydrodynamic diameter of approximately (120±10) nm and a Zeta potential of about +(6±1) mV. They maintained good structural stability after incubation in PBS buffer for 7 d. Cell experiments demonstrated that PNP/pCAR exhibited no significant cytotoxicity in RAW 264.7 cells while being efficiently internalized and effectively escaping lysosomal degradation. The transfection positive rate of PNP/pCAR-GFP in RAW 264.7 cells reached (25±3)%, surpassing that of Lipofectamine 2000-loaded pCAR-GFP (Lipo/pCAR-GFP), which was (20±1)%.In vivo experiments revealed that, compared to unmodified PNP/pCAR, βGlus-PNP/pCAR exhibited strongerin situ pancreatic tumor targeting ability after oral administration. Furthermore, oral administration of βGlus-PNP/pCAR-GFP resulted in significant GFP protein expression detectable within pancreatic tumor tissues. ConclusionThis study successfully constructed and validated an orally administrable, pancreatic cancer-targeting polypeptide-based nano-gene delivery system. It provides an important technological foundation in delivery systems and experimental basis for the subsequent development of in situ CAR-M-based therapeutic strategies for pancreatic cancer.

BACKGROUND:Adipose-derived stem cells have anti-aging effects,but whether adipose-derived stem cells from donors of different ages are different needs further study. OBJECTIVE:To compare the biological properties of adipose-derived stem cells in old and young mice. METHODS:Adipose-derived stem cells were extracted from adipose tissue of C57BL mice aged 8 and 14 weeks,respectively.The differences of cell cycle,apoptosis,and proliferation of adipose-derived stem cells in old and young mice were compared.The expression levels of aging-related P21 and P27 genes and proteins of adipose-derived stem cells in old and young mice were detected by quantitative PCR and western blot assay. RESULTS AND CONCLUSION:Compared with old mouse adipose-derived stem cells,young mouse adipose-derived stem cells were more active,more regular in morphology,less apoptosis,faster proliferation,and lower in expression of age-related P21 and P27 genes and proteins.It has been proven that adipose-derived stem cells from young mice have better anti-aging effects.

AIM To establish a quantitative analysis of multi-components by single-marker(QAMS)method for the simultaneous content determination of baicalin,baicalin,hesperidin,naringin,neohesperidin,rosinic acid,glycyrrhizin,ammonium glycyrrhizate and prehumetin in Tongxuan Lifei Pills.METHODS The analysis was performed on a 30 ℃ thermostatic SVEA C18 column(4.6 mm × 250 mm,5 μm),with the mobile phase comprising of acetonitrile-water(containing 0.1％phosphoric acid)flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 250 nm.Baicalin was used as an internal standard to calculate the relative correction factors of the other eight constituents,after which the content determination was made.RESULTS Nine constituents showed good linear relationships within their own ranges(r ≥ 0.999 1),whose average recoveries were 96.15％-101.58％with the RSDs of 0.75％-1.74％.The result obtained by QAMS approximated those obtained by external standard method.CONCLUSION This accurate,stable and reproducible method can be used for the quality control of Tongxuan Lifei Pills.

Objective:To investigate the association between estimated cumulative low-density lipoprotein cholesterol (LDL-C) exposure and the severity of coronary artery disease and long-term adverse cardiovascular and cerebrovascular events (MACCE) in patients with acute coronary syndrome (ACS).Methods:The subjects were from the PROMISE study. This study was a prospective cohort study led by Fuwai Hospital, Chinese Academy of Medical Sciences, with participation from eight regional tertiary hospitals as sub-centers, and enrolled 18 701 patients with confirmed coronary heart disease between January 2015 and May 2019. Among them, 8 429 patients with ACS were included in this study. The estimated cumulative LDL-C exposure was calculated by multiplying LDL-C by age. Participants were then divided into four groups based on quartiles. Baseline data and coronary angiography data were collected, and participants were followed for 2 years. The primary endpoint was MACCE, which was composed of all-cause death, cardiac death, myocardial infarction, revascularization, and stroke. Spearman correlation analysis was used to estimate the correlation between cumulative LDL-C exposure and the severity of coronary artery disease. The differences in MACCE among the four groups were compared, and multivariate Cox regression was used to divide the estimated cumulative exposure LDL-C into two groups, three groups, and four groups to analyze its relationship with MACCE.Results:The 8 429 ACS patients included in the study had an age of (60.9±11.4) years, with 1 951(23.1%) females. Spearman correlation analysis revealed that estimated cumulative LDL-C exposure was positively associated with the preoperative SYNTAX score, three-vessel lesions disease, left main disease, and the number of target lesions (correlation coefficients r=0.14, 0.10, 0.04 and 0.03, respectively, with all P<0.05). The 2-year follow-up results indicated that the incidence rates of MACCE, all-cause death, cardiac death, myocardial infarction, and stroke in ACS patients grouped by different levels of estimated cumulative LDL-C exposure were statistically significant (all P<0.05). The results of the Cox multivariate regression analysis showed that when the estimated cumulative LDL-C exposure was treated as a continuous variable and analyzed in two, three, and four groups, with the lowest group as the reference, the risk of MACCE occurrence in the high-value group increased by 21% (95% CI 1.08-1.37, P=0.002), 24% (95% CI 1.07-1.43, P=0.004), and 21% (95% CI 1.02-1.43, P=0.025) respectively. Conclusions:A positive correlation was found between estimated cumulative LDL-C exposure and severity of coronary artery disease. High estimated cumulative LDL-C exposure level is a risk factor for MACCE in ACS patients within 2 years.

Aim To explore the effect of astragalosideⅣ(AS-Ⅳ)on lipopolysaccharide(LPS)-induced po-larization and migration of RAW 264.7 macrophages and the underlying mechanism.Methods 1 mg·L-1 LPS was used to construct cell migration model.Scratch assay was utilized to determine cell migration rate.Immunofluorescence staining was utilized to de-tect the expression and location of F4/80,iNOS and Arg-1.CCK-8 assay was used to determine the viabili-ty of RAW 264.7 cells.Griess assay was used to measure NO content.Molecular docking was used to analyze the interaction between AS-Ⅳ and the core tar-gets such as cGAS and STING protein.Western blot was employed to detect the expression of iNOS,Arg-1,cGAS,STING,NF-κB p65 and p-NF-κB p65 protein.Results AS-Ⅳ significantly inhibited the migration and M1 polarization of RAW 264.7 cells induced by LPS.Moreover,AS-Ⅳ could interact with cGAS and STING protein,especially cGAS.Further Western blot assay showed that AS-Ⅳ significantly downregulated the expression of iNOS,cGAS,STING and p-NF-κB p65 protein.Conclusions AS-Ⅳ could promote mac-rophage M1 to M2 polarization,thereby inhibited mac-rophage migration through restraining the cGAS/STING/NF-κB signaling pathway,which provides a new therapeutic target for AS-Ⅳ to improve the early inflammatory response of AS.

OBJECTIVE To investigate the result of human immunodeficiency virus(HIV)screening for the people visiting to a three-A hospital of Sichuan Province and analyze the prevalence of complications with hepatitis B virus(HBV)infection,hepatitis C virus(HCV)infection and Treponema pallidum(TP)infection in the emerging HIV infection patients.METHODS The result of HIV screening for the people who visited to Ziyang Central Hos-pital from Jan.1,2020 to Dec.31,2024 and the test results of HBV,HCV and TP for the emerging HIV infec-tion patients were collected and were summarized and statistically analyzed by SPSS.0 software.RESULTS Totally 289 891 case-times were tested for HIV,1529 cases were previously diagnosed with HIV,465 of whom were tested posi-tive for the first time,there was significant difference in the positive rate of test for the first time among the 5 years(x2=15.998,P=0.003).Totally 353 cases were confirmed positive among the 465 primary positive screening cases.Among the emerging HIV infection patients,the positive rate was higher in the male than in the female(x2=141.141,P＜0.001),and the positive rate was high among the population aged more than 40 year old(x2=11.448,P＜0.001),mi-grant workers(x2=270.110,P＜0.001)and low education level population(x2=25.911,P＜0.001).The detection rate of gp41 was up to 100.00％in strip type testing.The analysis of the ratio of relative light unit(RLU)to Cutoff val-ue(COI)in the initial screening experiment showed that when COI was greater than 50,all of the confirmed tests were positive,when COI ranged between 1 and 5,the false positive rate was 97.06％.The incidence of complica-tion with HBV infection in the emerging HIV infection patients was increased year by year(x2=20.355,P＜0.001),and the incidence of complication with HCV infection was increased in recent two years(x2=10.690,P=0.030).CONCLUSIONS There is no obvious rise of positive rate of HIV screening among the people visiting to the hospital in recent 5 years.The sensitivity of the primary screening of clinical laboratory is high without posi-tive missing test.The positive rates of HBV and HCV are increased among the emerging HIV infection patients.

Objective:To explore the prognostic value of assessing plasma thrombin-antithrombin complex (TAT) and D-dimer (D-D) in patients with acute ischemic stroke (AIS).Methods:This prospective cohort study enrolled 538 AIS patients admitted to the General Hospital of Tianjin Medical University from July 2021 to May 2024, including 306 males and 232 females, with an average age of (69.5±11.5) years. Among them, there were 147 cases of lacunar infarction, 166 cases of large artery atherosclerosis type, 122 cases of cardioembolic type, and 103 cases of cancer-related type. Follow-up observations were conducted on enrolled patients, data within 30 days after the initiation of acute phase treatment for one week were collected, thrombus-related cerebral ischemic events recurrence served as the endpoint event, starting from August 5, 2021, to June 19, 2024. During the follow-up period, 68 patients experienced endpoint events, including 14 cases of large artery atherosclerosis type, 23 cases of cardioembolic type, and 31 cases of cancer-related type. The plasma levels of TAT and D-D were determined by chemiluminescence method and enzyme-linked immunofluorescence assay respectively. The receiver operating characteristic (ROC) curve was used to analyze the prediction performance of TAT and D-dimer for recurrent ischemic events in AIS patients during the 30-day follow-up period, and the Kaplan-Meier curve was used for survival analysis.Results:Statistically differences were observed in the plasma TAT and D-D levels among patients with different types of AIS ( P<0.05), with cancer-related type had higher levels than cardioembolic type ( P<0.05), and cardioembolic type had higher levels than large artery atherosclerosis type ( P<0.05). In the non-recurrent ischemic event group, the plasma TAT and D-D levels of patients with large artery atherosclerosis type, cardioembolic type and cancer-related type were lower in post-treatment than pre-treatment ( P<0.05). In the recurrent ischemic event group, the plasma D-D levels were higher in post-treatment than pre-treatment in the patients with large artery atherosclerosis type ( P<0.05); there was no statistically difference between the post-treatment and pre-treatment in plasma TAT and D-D levels in patients with cardioembolic type ( P>0.05); in patients with cancer-related type, the TAT and D-D levels were lower in post-treatment than pre-treatment ( P<0.05), but higher than those in the non-recurrence group ( P<0.05). The ROC curve showed that the area under the curve for predicting the risk of ischemic event recurrence within 30 days in AIS patients by plasma TAT combined with D-D on day 7 after treatment was all >0.9 (0.950 for large artery atherosclerosis type, 0.965 for cardioembolic type, and 0.907 for cancer-related types). Survival analysis indicated that various patients with both indicators above the critical value had an increased cumulative risk probability of adverse events (log-rank χ 2=93.667, 109.266, and 58.433, respectively, with all P<0.001). Conclusion:The changes of plasma TAT and D-D levels in AIS patients are associated with stroke type and coagulation activation, and dynamic monitoring of these two indicators could help evaluate the treatment effect and predict the risk of recurrent ischemic events.