Based on the pharmacokinetics theory, this study investigated the pharmacokinetic characteristics of albiflorin, paeoniflorin, wogonoside, and wogonin in normal and febrile rats and summarized absorption and elimination rules of Dayuanyin in them to provide reference for further development and clinical application of Dayuanyin. Blood samples were taken from the fundus venous plexus of normal and model rats after intragastric administration of Dayuanyin at different time points. The concentration of each substance in blood was determined by ultra performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-MS/MS) technique at different time points. DAS 2.0, a piece of pharmacokinetics software, was used to calculate the pharmacokinetic parameters of each component. The results show that the 4 components had good linear relationship in their respective ranges, and the results of methodological investigation met the requirements. The pharmacokinetic parameters of C_(max), T_(max), t_(1/2), AUC_(0-t), AUC_(0-∞), and MRT_(0-t) were calculated by the DAS 2.0 non-compartmental model. Compared with those in the normal group, C_(max) and AUC_(0-t) of the 4 components in the model group were significantly increased. There were significant differences in the pharmacokinetic characteristics between the normal and model groups, suggesting that the absorption and elimination of Dayuanyin may be affected by the changes of internal environment of the body in different physiological states.
Animals ; Rats ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Rats, Sprague-Dawley ; Fever/metabolism* ; Tandem Mass Spectrometry ; Chromatography, High Pressure Liquid ; Glucosides/pharmacokinetics* ; Monoterpenes

This study investigated the effects of Rehmanniae Radix Praeparata on striatal neuronal apoptosis in rats with attention deficit hyperactivity disorder(ADHD) based on the B-cell lymphoma-2(Bcl-2)/Bcl-2-associated X protein(Bax)/caspase-3 signaling pathway. Twenty-four 3-week-old male spontaneously hypertensive rats(SHR) were randomly divided into a model group, a methylphenidate group(2 mg·kg~(-1)·d~(-1)), and a Rehmanniae Radix Praeparata group(2.4 mg·kg~(-1)·d~(-1)). Age-matched male Wistar Kyoto(WKY) rats were used as the normal control group, with 8 rats in each group. The rats were administered by gavage for 28 days. Body weight and food intake were recorded for each group. The open field test and elevated plus maze test were used to assess hyperactivity and impulsive behaviors. Nissl staining was used to detect changes in striatal neurons and Nissl bodies. Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) fluorescence staining was used to detect striatal cell apoptosis. Western blot was employed to detect the expression levels of Bcl-2, Bax, and caspase-3 proteins in the striatum. The results showed that compared with the model group, Rehmanniae Radix Praeparata significantly reduced the total movement distance, average movement speed, and central area residence time in the open field test, and significantly reduced the ratio of open arm entries, open arm stay time, and head dipping in the elevated plus maze test. Furthermore, it increased the number of Nissl bodies in striatal neurons, significantly downregulated the apoptosis index, significantly increased Bcl-2 protein expression and the Bcl-2/Bax ratio, and reduced Bax and caspase-3 protein expression. In conclusion, Rehmanniae Radix Praeparata can reduce hyperactivity and impulsive behaviors in ADHD rats. Its mechanism may be related to the regulation of the Bcl-2/Bax/caspase-3 signaling pathway in the striatum, enhancing the anti-apoptotic capacity of striatal neurons.
Animals ; Male ; Apoptosis/drug effects* ; Rats ; Drugs, Chinese Herbal/administration & dosage* ; Caspase 3/genetics* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; bcl-2-Associated X Protein/genetics* ; Rehmannia/chemistry* ; Attention Deficit Disorder with Hyperactivity/physiopathology* ; Signal Transduction/drug effects* ; Neurons/cytology* ; Rats, Inbred SHR ; Rats, Inbred WKY ; Humans ; Corpus Striatum/cytology* ; Plant Extracts

Based on the ultra performance liquid chromatography-quadrupole Exactive Orbitrap mass spectrometry(UPLC-Q-Exactive Orbitrap-MS) technology, combined with related literature, databases, and reference material information, this study qualitatively analyzed the components of Dayuanyin in the tissue of rats after gavage and employed molecular docking technology to predict the rationality of the mechanism behind the antipyretic effect of the in vivo components in Dayuanyin. A total of 21, 26, 20, 21, 14, and 31 prototype components and 3, 16, 3, 7, 5, and 24 metabolites were identified from the heart, liver, spleen, lung, kidney, and hypothalamus of the rats, respectively, and the binding ability of key components and targets was further verified by molecular docking. The results showed that all components had good binding ability with targets. The established UPLC-Q-Exactive Orbitrap-MS could effectively and quickly identify the Dayuanyin components distributed in tissue and preliminarily identify their metabolites. Many components were identified in the hypothalamus, which suggested that the components delivered to the brain should be focused on in the study on Dayuanyin in the treatment of febrile diseases. The molecular docking technology was used to predict the rationality of the mechanism behind its antipyretic effect, which lays the foundation for the clarification of the material basis and action mechanism of Dayuanyin, the development of new preparations, and the prediction of quality markers.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Molecular Docking Simulation ; Male ; Antipyretics/metabolism* ; Rats, Sprague-Dawley ; Tissue Distribution ; Mass Spectrometry ; Chromatography, High Pressure Liquid ; Hypothalamus/metabolism*

The study investigated the intrinsic changes in material basis of Angelicae Sinensis Radix during wine processing by headspace-gas chromatography-ion mobility spectrometry(HS-GC-IMS), headspace-solid phase microextraction-gas chromatography-mass spectrometry(HS-SPME-GC-MS), and ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry(UPLC-Q-Orbitrap-MS) combined with chemometrics. HS-GC-IMS fingerprints of Angelicae Sinensis Radix before and after wine processing were established to analyze the variation trends of volatile components and characterize volatile small-molecule substances before and after processing. Principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA) were employed for differentiation and difference analysis. A total of 89 volatile components in Angelicae Sinensis Radix were identified by HS-GC-IMS, including 14 unsaturated hydrocarbons, 16 aldehydes, 13 ketones, 9 alcohols, 16 esters, 6 organic acids, and 15 other compounds. HS-SPME-GC-MS detected 118 volatile components, comprising 42 unsaturated hydrocarbons, 11 aromatic compounds, 30 alcohols, 8 alkanes, 6 organic acids, 4 ketones, 7 aldehydes, 5 esters, and 5 other volatile compounds. UPLC-Q-Orbitrap-MS identified 76 non-volatile compounds. PCA revealed distinct clusters of raw and wine-processed Angelicae Sinensis Radix samples across the three detection methods. Both PCA and OPLS-DA effectively discriminated between the two groups, and 145 compounds(VIP>1) were identified as critical markers for evaluating processing quality, including 4-methyl-3-penten-2-one, ethyl 2-methylpentanoate, and 2,4-dimethyl-1,3-dioxolane detected by HS-GC-IMS, angelic acid, β-pinene, and germacrene B detected by HS-SPME-GC-MS, and L-tryptophan, licoricone, and angenomalin detected by UPLC-Q-Orbitrap-MS. In conclusion, the integration of the three detection methods with chemometrics elucidates the differences in the chemical material basis between raw and wine-processed Angelicae Sinensis Radix, providing a scientific foundation for understanding the processing mechanisms and clinical applications of wine-processed Angelicae Sinensis Radix.
Wine/analysis* ; Gas Chromatography-Mass Spectrometry/methods* ; Chromatography, High Pressure Liquid/methods* ; Angelica sinensis/chemistry* ; Solid Phase Microextraction/methods* ; Drugs, Chinese Herbal/isolation & purification* ; Chemometrics ; Volatile Organic Compounds/chemistry* ; Principal Component Analysis ; Ion Mobility Spectrometry/methods*

OBJECTIVE: To investigate the protective effects of stir-fried Semen Armeniacae Amarum (SAA) against aristolochic acid I (AAI)-induced nephrotoxicity and DNA adducts and elucidate the underlying mechanism involved for ensuring the safe use of Asari Radix et Rhizoma. METHODS: In vitro, HEK293T cells overexpressing Flag-tagged multidrug resistance-associated protein 3 (MRP3) were constructed by Lentiviral transduction, and inhibitory effect of top 10 common pairs of medicinal herbs with Asari Radix et Rhizoma in clinic on MRP3 activity was verified using a self-constructed fluorescence screening system. The mRNA, protein expressions, and enzyme activity levels of NAD(P)H quinone dehydrogenase 1 (NQO1) and cytochrome P450 1A2 (CYP1A2) were measured in differentiated HepaRG cells. Hepatocyte toxicity after inhibition of AAI metabolite transport was detected using cell counting kit-8 assay. In vivo, C57BL/6 mice were randomly divided into 5 groups according to a random number table, including: control (1% sodium bicarbonate), AAI (10 mg/kg), stir-fried SAA (1.75 g/kg) and AAI + stir-fried SAA (1.75 and 8.75 g/kg) groups, 6 mice in each group. After 7 days of continuous gavage administration, liver and kidney damages were assessed, and the protein expressions and enzyme activity of liver metabolic enzymes NQO1 and CYP1A2 were determined simultaneously. RESULTS: In vivo, combination of 1.75 g/kg SAA and 10 mg/kg AAI suppressed AAI-induced nephrotoxicity and reduced dA-ALI formation by 26.7%, and these detoxification effects in a dose-dependent manner (P<0.01). Mechanistically, SAA inhibited MRP3 transport in vitro, downregulated NQO1 expression in vivo, increased CYP1A2 expression and enzymatic activity in vitro and in vivo, respectively (P<0.05 or P<0.01). Notably, SAA also reduced AAI-induced hepatotoxicity throughout the detoxification process, as indicated by a 41.3% reduction in the number of liver adducts (P<0.01). CONCLUSIONS Stir-fried SAA is a novel drug candidate for the suppression of AAI-induced liver and kidney damages. The protective mechanism may be closely related to the regulation of transporters and metabolic enzymes.
Aristolochic Acids/toxicity* ; Animals ; Humans ; NAD(P)H Dehydrogenase (Quinone)/genetics* ; HEK293 Cells ; Kidney/pathology* ; Cytochrome P-450 CYP1A2/genetics* ; Mice, Inbred C57BL ; DNA Adducts/drug effects* ; Male ; Kidney Diseases/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Mice ; Prunus armeniaca ; Plant Extracts

Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin regions, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at the single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences on TF binding. Notably, cFOOT-seq, combined with FootTrack analysis, enables de novo prediction of TF binding sites and tracking of TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.
Transcription Factors/genetics* ; Humans ; Chromatin/genetics* ; Animals ; Binding Sites ; Mice ; DNA Footprinting/methods*

OBJECTIVE: This study examines utilizes the advantages of machine learning algorithms to discern key determinants in prognosticate postoperative circulatory complications (PCCs) for older patients. METHODS: This secondary analysis of data from a randomized controlled trial involved 1,720 elderly participants in five tertiary hospitals in Beijing, China. Participants aged 60-90 years undergoing major non-cardiac surgery under general anesthesia. The primary outcome metric of the study was the occurrence of PCCs, according to the European Society of Cardiology and the European Society of Anaesthesiology diagnostic criteria. The analysis metrics contained 67 candidate variables, including baseline characteristics, laboratory tests, and scale assessments. RESULTS: Our feature selection process identified key variables that significantly impact patient outcomes, including the duration of ICU stay, surgery, and anesthesia; APACHE-II score; intraoperative average heart rate and blood loss; cumulative opioid use during surgery; patient age; VAS-Move-Median score on the 1st to 3rd day; Charlson comorbidity score; volumes of intraoperative plasma, crystalloid, and colloid fluids; cumulative red blood cell transfusion during surgery; and endotracheal intubation duration. Notably, our Random Forest model demonstrated exceptional performance with an accuracy of 0.9872. CONCLUSION We have developed and validated an algorithm for predicting PCCs in elderly patients by identifying key risk factors.
Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Cardiovascular Diseases/etiology* ; Machine Learning ; Postoperative Complications/etiology* ; Risk Factors ; Randomized Controlled Trials as Topic ; Secondary Data Analysis

As the simplest label-free single-molecule analytical technique in solution,nanopore technology has been widely used in rapid sequencing of protein and nucleic acid,and single-molecule sensing.However,the main challenge is to get this technology out of the lab and achieve automation,miniaturization as well as rapid analysis at high throughput.In this work,a new microfluidic tubing protein nanopore analysis system was designed and constructed by combining microfluidic and nanopore nanofluidic analysis techniques.An arcuate lipid bilayer was formed in the microfluidic device through lipid monolayer at the oil/water interface by developed aperture processing technology at 10 μm level.The arcuate lipid bilayer with the life span of more than 98 h had high biomimetic fluidity and biological activity by which protein channel could be produced and sealed at 18 GΩ level without leakage.When applied+40 mV potential,relative standard deviation of single proteinα-hemolysin mutant(α-HL(M113F/K147N))channel current was less than 0.16 pA(n=3)in 10 mmol/L Tris-HCl(pH 7.4)buffer containing 1 mol/L NaCl.Single-molecule detection for aptamer and its host-guest interactions with biotin in protein channel demonstrated that this microfluidic tube protein nanopore sensor could well work with high sensitivity without the need of anti-vibration table and shielding cage.

Objective To investigate the effect and mechnism of betaine(BET)in reversing chemotherapy resistance in prostate cancer(PCa)by inhibiting ATP-binding cassette subfamily B member 1(ABCB1).Methods The PCa chemotherapy-sensitive C4-2B cells were cultured,and the TaxR cells resistant to docetaxel(DTX)were established by gradient increase the concentration of DTX.The drug resistance of C4-2B and TaxR cells against DTX was assessed using CCK-8 and the colony formation experiment.Western blotting and qRT-PCR were used to detect ABCB1 expression.The TaxR cells were divided into:(1)Control group,negative control group(NC),siABCB1-1 group(transfected with siABCB1-1),and siABCB1-2 group(transfected with siABCB1-2).Western blotting was used to detect the effect of small interfering RNA on silencing ABCB1,and CCK-8 was used to detect the differences in DTX resistance between each group.(2)Different concentrations of BET(0,100,200,400,600,800 mmol/L)groups.These groups were subjected to CCK-8 to detect cell viability,and Western blotting was used to detect the protein expression of ABCB1.(3)Control group,DTX group(20 nmol/L DTX),BET group(200 mmol/L BET),and DTX+BET group(20 nmol/L DTX+200 mmol/L BET),flow cytometry was used to detect apoptosis rate and cell cycle,and Western blotting to detect the protein expression of apoptosis-related proteins(Bcl2,BAX,c-caspase-3).(4)Control group,BET group(200 mmol/L BET),wortmannin(WM)group(100 μmol/L WM),and BET+WM group(200 mmol/L BET+100 μmol/L WM).Western blotting was used to detect the protein expression of PI3K,Akt,and ABCB1.(5)Control group,BET group(200 mmol/L BET),and BAY group(10 μmol/L BAY),BAY+BET group(200 mmol/L BET+10 μmol/L BAY).Western blotting was used to detect the protein expression of NF-κB p65,p-ikBα and ABCB1.Network pharmacology combined with transcriptome sequencing was used to predict the possible pathways for BET to reverse chemotherapy resistance.Results Compared with C4-2B cells,TaxR cells showed significantly increased resistance to DTX(P<0.01),and high expression of ABCB1(P<0.01).After silencing ABCB1 with siRNA,TaxR cells'resistance to DTX was significantly inhibited(P<0.01).The inhibition rate of TaxR cells treated with 200 mmol/L BET was less than 20%,and it significantly decreased the expression of ABCB1 protein in TaxR cells(P<0.05).Compared with control group,the combination of 200 mmol/L BET and 20 nmol/L DTX resulted in higher apoptosis rate and higher S stage cell ratio,lower expression of Bcl-2 protein and higher expression of BAX and c-caspase-3 proteins than the two drugs used alone(P<0.05).Compared with control group,the combination of 200 mmol/L BET and 100 μmol/L WM significantly down-regulated the protein expression of PI3K,Akt and ABCB1(P<0.01).The combination of 200 mmol/L BET and 10 μmol/L BAY significantly down-regulated the protein expression of NF-κB p65,p-ikBα and ABCB1(P<0.01).Conclusion BET may reverse TaxR cells'chemotherapy resistance by down-regulating ABCB1 expression through the PI3K/Akt/NF-κB signaling pathway.

Objective To investigate the clinical characteristics of patients with clinical and subclinical Cushing's syndrome caused by primary bilateral macronodular adrenal hyperplasia(PBMAH).Methods A retrospective analysis was performed on the clinical data of 198 patients with Cushing's syndrome caused by PBMAH diagnosed in the First Medical Center of Chinese PLA General Hospital from January 2004 to October 2024.According to clinical manifestations,the patients were classified into clinical type Cushing's syndrome(n=61)and subclinical type Cushing's syndrome(n=137),and the clinical characteristics of the two types were compared.Results The mean age at diagnosis of patients with PBMAH-induced Cushing's syndrome was(53.5±10.4)years,including 118 males and 80 females,with a male-to-female ratio of 1.475:1.Compared with the subclinical type,the clinical type had a higher proportion of females,higher levels of serum cortisol,24-hour urine free cortisol(24 h UFC),and inhibited serum cortisol after low-dose dexamethasone suppression.Additionally,the clinical type had lower plasma ACTH,larger adrenal nodules and a higher risk of surgery(P<0.05)compared with those in subclinical type.The incidences of hypertension,dyslipidemia,obesity,diabetes mellitus,hypokalemia,vitamin D deficiency,osteoporosis,coronary heart disease,and cerebrovascular disease in patients with Cushing's syndrome caused by PBMAH were 87.9%,50.5%,37.1%,36.9%,27.8%,25.9%,18.7%,18.7%and 12.1%,respectively.Among them,compared with subclinical type patients,clinical type patients had higher incidence of hypokalaemia,vitamin D deficiency and osteoporosis(P<0.05),while there were no statistically significant differences in the incidences of other comorbidities between the two types(P>0.05).The results of postoperative follow-up for PBMAH patients showed that the short-term biochemical remission rate of unilateral total adrenalectomy was 41.5%(22/53)and the long-term biochemical remission rate was 32.0%(8/25).The short-term biochemical remission rate of unilateral partial(or nodular)adrenalectomy was 52.9%(9/17),and the long-term biochemical remission rate was 14.3%(1/7).All patients who underwent unilateral total adrenalectomy plus contralateral partial resection developed adrenal insufficiency(3/3),and 1 patient(1/3)relapsed 3.4 years after surgery.Conclusion Clinical and subclinical types of Cushing's syndrome caused by PBMAH have their distinct clinical characteristics.Surgery is an effective treatment for PBMAH,but a certain proportion of patients fail to achieve biochemical remission after non-bilateral total adrenalectomy.