Objective: To develop a convenient, direct, and highly sensitive method for screening trace chemical additives in complex Chinese patent medicines, thereby addressing core technological bottlenecks in pharmaceutical analysis and quality control. Methods: A brush ionization probe device was independently designed and constructed, and an efficient detection method was established through systematic optimization of key parameters. Twenty-three Chinese patent medicine samples, representing 6 dosage forms (capsules, tablets, pills, granules, powders, and liquid preparations), were analyzed using 10 common chemical additives as target analytes. Results: All samples were successfully analyzed without complex pretreatment, and 5 chemical additives were detected in 7 Chinese patent medicines. The brush ionization probe device exhibited cost-effectiveness (~0.2 USD per probe), operational simplicity, rapid analysis (~10s per sample), high efficiency, and minimal reagent consumption (~10 μL per sample). Conclusion: This advancement is expected to provide an innovative scientific tool for improving the generality and convenience of on-site quality control, while promoting technological progress in disciplines such as pharmacology and traditional Chinese medicine.

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.

With the rising incidence of colon cancer, several studies have indicated that aquaporin 1 (AQP1) expression is associated with the development of colon cancer. This study aims to elucidate the potential molecular mechanisms between them. Methods: We screened data from The Cancer Genome Atlas (TCGA) database and retrospectively examined AQP1 protein expression in 127 colon cancer patients to analyze the relationship between AQP1 expression and pathological stages, prognosis. We created stable colon cancer cell lines with differential AQP1 expression, the effect of AQP1 expression on the proliferation and migration of colon cancer cells was assessed by in vitro and in vivo studies, and explored potential molecular mechanisms through Western blotting. Results: High AQP1 expression was associated with poorer survival (overall survival [OS], p = .028) in colon cancer patients from the TCGA database. Similarly, retrospective clinical data indicated that high AQP1 expression was associated with reduced disease-free survival and OS (p = .036 and p = .017, respectively). The low-expressing AQP1 colon cancer cells exhibited a decrease in proliferation and migration ability of colon cancer cells compared to the overexpressing AQP1 group (p < .05) in vitro and in vivo. Immunohistochemistry and western blotting experiments validated heightened expression of N-cadherin, vimentin, and claudin- 1 in the tumor tissues of the overexpressing AQP1 group. Conversely, reduced AQP1 expression resulted in decreased expression of claudin- 1. Conclusions: AQP1 correlates with unfavorable prognosis in colon cancer and potentially enhances the proliferation and migration of colon cancer by up-regulating claudin-1 expression.

Background: Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder. Cellular drug screening represents a key approach in the preclinical discovery of AD therapeutics. Moreover, selecting an appropriate cellular model is essential for exploring the mechanisms underlying AD pathogenesis. Objective: This study aimed to establish a progressive strategy integrating cell model construction, monomer evaluation, and effective component group-based drug discovery for the development of novel therapeutics targeting AD. Methods: A novel AD12 cell line stably expressing the double-transgenic genes amyloid precursor protein (APP)695/presenilin 1(PS1)-ΔE9 was first constructed and subjected to lipopolysaccharide (LPS) stimulation. The resulting LPS-induced neuroinflammatory response was characterized using metabolomic analysis. Subsequently, 16 alkaloids derived from traditional Chinese medicine(TCM) were screened with the LPS-AD12 cell model. Molecular docking was then performed to assess the binding affinity of benzoylaconitine (BAC) with APP and PS1 proteins. A targeted metabolomics approach focusing on amino acids and the tricarboxylic acid (TCA) cycle was applied to BAC-treated LPS-AD12 cells. Finally, aconite extract was administered to five-familial-AD-mutations(5xFAD) transgenic mice, and therapeutic efficacy was evaluated through phenotypic and metabolomic analyses. Results: LPS stimulation induced neuroinflammation in AD12 cells, characterized by elevated levels of interleukin-6, amyloid-β (Aβ ), and PS1. The primary metabolic pathways affected in AD12 cells included alanine, aspartate, and glutamate metabolism, as well as arginine biosynthesis. Notably, BAC significantly reduced the levels of Aβ and interleukin-6 in LPS-AD12 cells. Furthermore, molecular docking analyses revealed a strong binding affinity of BAC with APP and PS1 proteins. Targeted metabolomics analysis indicated that the pharmacological activity of BAC was primarily associated with its anti-inflammatory effects, modulation of the cholinergic signaling pathway, and regulation of energy metabolism. Behavioral experiments in 5xFAD mice, together with immunofluorescence assessments of brain tissues (amyloid-β, glial fibrillary acidic protein, and ionized calcium-binding adapter molecule 1), demonstrated that aconite extract treatment significantly alleviated AD-related symptoms. In addition, metabolomics-based mechanistic analyses revealed that serine, acetylcholine, ornithine, and gamma-aminobutyric acid exhibited consistent trends in both brain tissue and serum samples from treated mice. Conclusion: This study established a novel AD12 cell model and an effective, reliable LPS-induced cell-based screening system. Additionally, we proposed a new drug discovery strategy integrating cell model construction, monomer evaluation, and effective component group-based drug discovery. This screening system has the potential to accelerate the identification of anti-AD agents and provides a feasible framework for subsequent in-depth investigations into new drug development from TCM, thereby contributing to the modernization of TCM-derived therapeutics.

Objective:To analyze the gene mutation spectrum of children with T-acute lymphoblastic leukemia (T-ALL) using next generation sequencing technology and to evaluate the value of gene mutations in prognosis stratification.Methods:A case series analysis was made.The clinical data of newly diagnosed pediatric T-ALL patients in the First Affiliated Hospital of Zhengzhou University from January 1, 2019 to February 29, 2024 were analyzed retrospectively.T-ALL gene mutations were analyzed.The relationships of gene mutations with clinical features and induction of responses to therapy were studied.The effects of gene mutations on overall survival (OS) and event-free survival (EFS) were examined by the Kaplan-Meier method and COX regression model.Results:A total of 80 newly diagnosed pediatric T-ALL patients were enrolled in the study, with a male-to-female ratio of 3.4∶1.0 and a median age of 8 (range, 2-17) years.A total of 57 mutations were detected in 74 patients, 46.2% (37/74) of whom showed 3 or more gene mutations.The coexistence of mutated genes was obvious. PTEN mutations were more prevalent in male patients ( P=0.018).Initial leukocyte counts were higher in patients with PTEN mutations ( P=0.038) and lower in patients with JAK3 mutations ( P=0.002).Patients with NOTCH1 mutations had a higher positive rate of fusion genes ( P=0.043).Patients with PTEN mutations had a higher rate of minimal residual disease(MRD) remission after 15/19 d of treatment with induction therapy, respectively ( P=0.013).The rate of MRD remission after 33/46 d of treatment with induction therapy was higher in patients with the FBXW7 mutation ( P=0.004) and lower in patients with JAK3 mutations ( P=0.003).Multifactorial COX regression analysis showed that IL7R mutation and three or more gene mutations were independent risk factors for OS and EFS in T-ALL patients(OS: HR=3.252, 7.357, 95% CI: 1.020-10.372, 1.646-32.882; EFS: HR=3.372, 3.009, 95% CI: 1.234-9.214, 1.174-7.708; all P<0.05). Conclusions:Gene mutations are prevalent in T-ALL children and correlate with clinical manifestations and prognosis.The coexistence of mutated genes is obvious.Pediatric T-ALL patients with IL7R mutations and three or more gene mutations have a poorer prognosis.

Objective:To investigate the clinical experience and efficacy of unrelated umbilical cord blood hematopoietic stem cell transplantation (HSCT) for the treatment of congenital amegakaryocytic thrombocytopenia (CAMT).Methods:A case summary was conducted.The clinical data of 2 children with CAMT who were finally cured by unrelated umbilical cord blood HSCT in the Department of Pediatric Medicine, the First Affiliated Hospital of Zhengzhou University from March 2020 to August 2023 were retrospectively analyzed.Related studies were retrieved from databases CNKI, Wanfang and PubMed using search terms including " congenital amegakaryocytic thrombocytopenia" and " hematopoietic stem cell transplantation" from the inception to July 2024.The clinical characteristics, diagnosis and treatment processes, and prognosis of CAMT patients treated by HSCT were then summarized.Results:Both cases exhibited scattered skin haemorrhages throughout the body and carried 2 compound heterozygous mutations with pathogenicity in the MPL gene.Both patients were finally diagnosed with CAMT.Case 1 was a girl aged 3 at the time of transplantation, and case 2 was also a girl, who aged 5 at the time of transplantation.Both of them received unrelated umbilical cord blood HSCT and hematopoietic reconstruction was achieved.The time of neutrophil and platelet implantation was 21 and 40 days after transplantation in case 1, and 20 and 31 days in case 2, respectively.The chimerism rate of neutrophil implantation in both children was complete chimerism of donor cells.Implantation syndrome was detected in case 1 following transplantation.Case 2 suffered implantation syndrome, hypertensive encephalopathy, and cytomegalovirus infection following transplantation.Both children showed no graft-versus-host disease (GVHD).Both children had hematopoietic and immune reconstruction after transplantation and their primary diseases were cured.Cases 1 and 2 were followed up for more than 14 and 17 months, respectively.Both of them achieved disease-free survival during the follow up.Literature review of 26 cases with CAMT treated by HSCT, including the above-mentioned 2 cases was conducted, with an overall disease-free survival rate of 92.3%(24/26).Of 12 cases with CAMT typing, 10 were type Ⅰ and 2 were type Ⅱ.Of the 26 cases treated by HSCT, 17 had bone marrow HSCT, with an overall survival rate of 88.2%(15/17), and 2 had peripheral blood HSCT.Seven cases had umbilical cord blood HSCT (6 cases receiving unrelated umbilical cord blood HSCT and 1 case receiving related umbilical cord blood HSCT), with an overall survival rate of 100%.Unlike bone marrow and peripheral blood HSCT, unrelated umbilical cord blood HSCT did not result in 3-4 grade GVHD. Conclusions:Unrelated umbilical cord blood HSCT can achieve good therapeutic effects in CAMT patients when there is no suitable donor.Myeloablative pretreatment is conducive to CAMT patients.

This study explores whether the current external quality assessment (EQA) level and acceptable bias for basic semen analysis in China are clinically useful. We collected data of semen EQA from Andrology laboratories in the Hunan Province (China) in 2022 and searched for data in the published literature from January 2000 to December 2023 in China. On the basis of these data, we analyzed the coefficients of variation and acceptable biases of different quality control materials for basic semen analysis through robust statistics. We compared these findings with quality specifications based on biological variation from optimal, desirable, and minimum levels of bias to seek a unified and more suitable semen EQA bias evaluation standard for China's national conditions. Different sources of semen quality control material exhibited considerable variation in acceptable biases among laboratories, ranging from 8.2% to 56.9%. A total of 50.0% of the laboratories met the minimum quality specifications for progressive motility (PR), whereas 100.0% and 75.0% of laboratories met only the minimum quality specifications for sperm concentration and total motility (nonprogressive [NP] + PR), respectively. The Z value for sperm concentration and PR+NP was equivalent to the desirable performance specification, whereas the Z value for PR was equivalent only to the minimum performance specification. This study highlights the feasibility of operating external quality assessment schemes for basic semen analysis using quality specifications based on biological variation. These specifications should be unified among external quality control (EQC) centers based on biological variation.
Semen Analysis/standards* ; Humans ; China ; Male ; Quality Control ; Sperm Motility ; Sperm Count/standards*

Objective To explore the optimization strategy of Qishen Granules in treating coronary heart disease with heart failure(CHD-HF)based on data mining and the pathogenic"toxin"theory,and to predict its active components and mechanisms using network pharmacology.Methods Literature on traditional Chinese medicine(TCM)for treating CHD-HF was collected from relevant databases,and prescriptions were screened and established into a database according to inclusion and exclusion criteria.Frequency,association rules,and hierarchical clustering analyses were performed using the Ancient and Modern Medical Case Cloud Platform.Network pharmacology techniques were applied to screen potential targets of the optimized combination for treating CHD-HF,and carry out the targets and pathways enrichment analysis.Results A total of 336 articles and 339 prescriptions involving 191 herbs were included,with 12 herbs used more than 100 times.The core drug combinations for treating CHD-HF included Astragali Radix,Poria,Salviae Miltiorrhizae Radix et Rhizoma,Glycyrrhizae Radix et Rhizoma,Chuanxiong Rhizoma,etc,while commonly used detoxifying herbs included Leonuri Herb,Coptidis Rhizoma,etc.Association rule analysis yielded 10 two-item associations and 17 three-item associations;clustering analysis grouped the data into 5 categories.Based on data mining and the pathogenic"toxin"theory,the combination for treating CHD-HF was optimized to include Astragali Radix,Salviae Miltiorrhizae Radix et Rhizoma,Aconiti Lateralis Radix Praeparata,Glycyrrhizae Radix et Rhizoma,Coptidis Rhizoma,and Taraxaci Herba.Network pharmacology analysis identified 366 common targets between the optimized combination and CHD-HF,with 16 core targets screened out.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis revealed significant enrichment in pathways such as cancer pathways,lipid and atherosclerosis,Rap1 signaling pathway,hypoxia-inducible factor 1(HIF-1)signaling pathway,phosphatidylinositol-3-kinase/protein kinase B(PI3K/Akt)signaling pathway,Ras signaling pathway,and mitogen-activated protein kinase(MAPK)signaling pathway.Conclusion TCM treatment for CHD-HF primarily focuses on replenishing qi and warming yang,activating blood circulation and resolving fluid retention.Based on data mining results and the pathogenic"toxin"theory,the formulation strategy of Qishen Granules for treating CHD-HF was optimized,potentially exerting therapeutic effects through anti-inflammatory,anti-apoptotic,and anti-hypoxia physiological processes.

OBJECTIVE: To analyze the RHD genotyping and sequencing results of RhD serology negative samples in the clinic, and to further explore the laboratory methods for RhD detection, in order to provide a basis for clinical precision blood transfusion. METHODS: A total of 27 200 whole blood samples were screened for RhD blood group antigen using microcolumn gel card method.Serologic RhD-negative confirmation tests were performed on blood samples that were negative for RhD on initial screening using three different clonal strains of IgG anti-D reagents. The 10 exons of the RHD gene on chromosome 1 were also analyzed by PCR-SSP to determine RHD genotyping.When the PCR-SSP method did not yield definitive results, the RHD gene of the sample was analyzed by the third-generation sequencing. RESULTS: The results of the initial screening test by the microcolumn gel card method showed that 136 of the 27 200 samples were RhD-negative, of which 86 underwent RhD-negative confirmation testing and RHD genotyping, 88.37% (76/86 cases) of the RhD-negative confirmation test results were negative for the three anti-D reagents, and the results of RHD genotyping showed that 67.44% (58/86 cases) of the cases had a complete deletion of 10 exons, and the remaining 28 cases were RHD*711delC (1 case), RHD*D-CE(1-9)-D (1 case), RHD*D-CE(2-9-)D (2 cases), RHD*D-CE(3-9)-D (4 cases), RHD*DEL1 (c.1227G >A) mutation (16 cases), RHD*weak partial 15(845G >A) mutation (3 cases), and a mutation of c.165C >T base was found in 1 sample by three-generation sequencing. CONCLUSION RHD genotype testing of samples that are serologically negative for RhD antigen shows that some of the samples have RHD gene variants, not all of which are total deletions of RHD, suggesting that there are some limitations of the serologic method for RhD detection. Due to the polymorphism of the RHD gene structure, different RhD variants present different serologic features, which need to be further detected in combination with molecular biology testing, especially for the identification of Asian-type DELs, which is important for clinical precision blood transfusion.
Humans ; Rh-Hr Blood-Group System/genetics* ; Genotype ; Polymerase Chain Reaction ; Exons ; Blood Grouping and Crossmatching