Comparative Pharmacokinetics Research of 13 Bioactive Components of Jieyu Pills in Control and Attention Deficit Hyperactivity Disorder Model Rats Based on UPLC-Orbitrap Fusion MS.

Jieyu Pills (JYPs), a Chinese medicine consisting of 10 herbal elements, have displayed promising clinical effectiveness and low by-effects in the treatment of depression. Prior investigations mostly focused on elucidating the mechanism and therapeutic efficacy of JYPs. In our earlier study, we provided an analysis of the chemical composition, serum pharmacochemistry, and concentrations of the main bioactive chemicals found in JYPs. However, our precise understanding of the pharmacokinetics and metabolism remained vague. This study involved a comprehensive and meticulous examination of the pharmacokinetics of 13 bioactive compounds in JYPs. Using UPLC-Orbitrap Fusion MS, we analyzed the metabolic characteristics and established the pharmacokinetic parameters in both control rats and model rats with attention deficit hyperactivity disorder (ADHD) following oral administration of the drug. Before analysis, plasma samples that were collected at different time intervals after the administration underwent methanol pre-treatment with Puerarin used as the internal standard (IS) solution. Subsequently, the sample was chromatographed on a C18 column employing gradient elution. The mobile phase consisted of methanol solution containing 0.1% formic acid in water. The electrospray ionization source (ESI) was utilized for ionization, whereas the scanning mode employed was selected ion monitoring (SIM). The UPLC-Orbitrap Fusion MS method was subjected to a comprehensive validation process to assess its performance. The method demonstrated excellent linearity (r ≥ 0.9944), precise measurements (RSD < 8.78%), accurate results (RE: -7.88% to 8.98%), and appropriate extraction recoveries (87.83-102.23%). Additionally, the method exhibited minimal matrix effects (87.58-101.08%) and satisfactory stability (RSD: 1.52-12.42%). These results demonstrated adherence to the criteria for evaluating and determining biological material. The 13 bioactive compounds exhibited unique pharmacokinetic patterns in vivo. In control rats, all bioactive compounds except Ferulic acid exhibited linear pharmacokinetics within the dose ranges. In the ADHD model, the absorption rate and amount of most of the components were both observed to have increased. Essentially, this work is an important reference for examining the metabolism of JYPs and providing guidelines for clinical therapy.

Discovery of the Active Compounds of the Ethyl Acetate Extract Site of Ardisia japonica (Thunb.) Blume for the Treatment of Acute Lung Injury.

The objective of this study was to identify and evaluate the pharmacodynamic constituents of Ardisiae Japonicae Herba (AJH) for the treatment of acute lung injury (ALI). To fully analyze the chemical contents of various extraction solvents (petroleum ether site (PE), ethyl acetate site (EA), n-butanol site (NB), and water site (WS)) of AJH, the UPLC-Orbitrap Fusion-MS technique was employed. Subsequently, the anti-inflammatory properties of the four extracted components of AJH were assessed using the lipopolysaccharide (LPS)-induced MH-S cellular inflammation model. The parts that exhibited anti-inflammatory activity were identified. Additionally, a technique was developed to measure the levels of specific chemical constituents in the anti-inflammatory components of AJH. The correlation between the "anti-inflammatory activity" and the constituents was analyzed, enabling the identification of a group of pharmacodynamic components with anti-inflammatory properties. ALI model rats were created using the tracheal drip LPS technique. The pharmacodynamic indices were evaluated for the anti-inflammatory active portions of AJH. The research revealed that the PE, EA, NB, and WS extracts of AJH included 215, 289, 128, and 69 unique chemical components, respectively. Additionally, 528 chemical components were discovered after removing duplicate values from the data. The EA exhibited significant anti-inflammatory activity in the cellular assay. A further analysis was conducted to determine the correlation between anti-inflammatory activity and components. Seventeen components, such as caryophyllene oxide, bergenin, and gallic acid, were identified as potential pharmacodynamic components with anti-inflammatory activity. The pharmacodynamic findings demonstrated that the intermediate and high doses of the EA extract from AJH exhibited a more pronounced effect in enhancing lung function, blood counts, and lung histology in a way that depended on the dosage. To summarize, when considering the findings from the previous study on the chemical properties of AJH, it was determined that the EA contained a group of 13 constituents that primarily contributed to its pharmacodynamic effects against ALI. The constituents include bergenin, quercetin, epigallocatechingallate, and others.

A Pharmacokinetic Study of Sixteen Major Bioactive Components of Jinshui-Huanxian Granules in Pulmonary Fibrosis Model and Control Rats Using Orbitrap Fusion Mass Spectrometry.

Jinshui-Huanxian granules (JHGs), a Chinese herbal compound prescription, have shown a therapeutic effect in reducing lung tissue damage, improving the degree of pulmonary fibrosis, replenishing lungs and kidneys, relieving cough and asthma, reducing phlegm, and activating blood circulation. However, these active compounds' pharmacokinetics and metabolic processes were unclear. This study aimed to compare the pharmacokinetics, reveal the metabolic dynamic changes, and obtain the basic pharmacokinetic parameters of 16 main bioactive compounds after intragastric administration of JHGs in control and pulmonary fibrosis (PF) model rats by using Orbitrap Fusion MS. After administration of JHGs, the rat plasma was collected at different times. Pretreating the plasma sample with methanol and internal standard (IS) solution carbamazepine (CBZ), and it was then applied to a C18 column by setting gradient elution with a mobile phase consisting of methanol 0.1% formic acid aqueous solution. Detection was performed on an electrospray ionization source (ESI), and the scanning mode was SIM. Pharmacokinetic parameters were analyzed according to the different analytes' concentrations in plasma. The matrix effect was within the range of 79.01-110.90%, the extraction recovery rate was 80.37-102.72%, the intra-day and inter-day precision relative standard deviation (RSD) was less than 7.76%, and the stability was good, which met the requirements of biological sample testing. The method was validated (r ≥ 0.9955) and applied to compare the pharmacokinetic profiles of the control group and PF model group after intragastric administration of the JHGs. The 16 analytes exhibited different pharmacokinetic behaviors in vivo. In the pathological state of the PF model, most of the components were more favorable for metabolism and absorption, and it was more meaningful to study the pharmacokinetics. Above all, this study provided an essential reference for exploring the mechanism of action of JHGs and guided clinical medication as well.

Network pharmacology combined with pharmacodynamics revealed the anti-inflammatory mechanism of Tanreqing capsule against acute-exacerbation chronic obstructive pulmonary disease.

Acute-exacerbation chronic obstructive pulmonary disease (AECOPD) is mainly associated with acute respiratory tract infection. In recent years, a growing number of studies have found that Tanreqing capsule (TRQ) has a favorable anti-inflammatory effect. In this study, we used network pharmacology and pharmacodynamics to explore the molecular mechanism and effects of TRQ in AECOPD treatment. To further understand the molecular mechanism of TRQ in AECOPD treatment, we used the network pharmacology to predict components of TRQ, TRQ-related targets, AECOPD-related targets, and pathways. In addition, we used the cigarette-smoke/lipopolysaccharide -induced AECOPD experimental model in Sprague-Dawley rats (72 rats randomly divided into six groups [n = 12 each]: control, model, high-TRQ [TRQ-H], medium-TRQ [TRQ-M], low-TRQ, and dexamethasone [Dex]) to evaluate the therapeutic effects of TRQ and to verify the network pharmacology. We found that 59 overlapping targets based on component-and AECOPD-related targets were frequently involved in the advanced glycation end product-receptor for advanced glycation end product signaling pathway in diabetic complications, the phosphatidylinositol-3-kinase-protein kinase B signaling pathway, and the hypoxia-inducible factor 1 signaling pathway, which might play important roles in the anti-inflammatory mechanism of TRQ in AECOPD treatment. Moreover, TRQ groups exerted protective effects against AECOPD by reducing the infiltration of inflammatory cells. Meanwhile, TRQ-M and TRQ-H groups significantly downregulated or upregulated the expression of tumor necrosis factor, interleukin (IL) 6, C-reactive protein, IL10, and serum amyloid A, as key targets in network pharmacology, in the serum and bronchoalveolar lavage fluid to achieve anti-inflammatory efficacy. Our study showed that TRQ had better anti-inflammatory efficacy against AECOPD, and initially elucidated its molecular mechanism. Moreover, our study also provides a new strategy to explore effective mechanism of TRQ against AECOPD; and further studies are needed to validate the biological processes and pathways of TRQ against AECOPD.

Systematic characterization of the components and molecular mechanisms of Jinshui Huanxian granules using UPLC-Orbitrap Fusion MS integrated with network pharmacology.

Jinshui Huanxian granules (JSHX) is a clinical Chinese medicine formula used for treating pulmonary fibrosis (PF). However, the effective components and molecular mechanisms of JSHX are still unclear. In this study, a combination approach using ultra-high performance liquid chromatography-Orbitrap Fusion mass spectrometry (UPLC-Orbitrap Fusion MS) integrated with network pharmacology was followed to identify the components of JSHX and the underlying molecular mechanisms against PF. UPLC-Orbitrap Fusion MS was used to identify the components present in JSHX. On the basis of the identified components, we performed target prediction using the SwissTargetPrediction database, protein-protein interaction (PPI) analysis using STRING database, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using Metascape and constructed a component-target-pathway network using Cytoscape 3.7.2. Molecular docking technology was used to verify the affinity between the core components and targets. Finally, the pharmacological activities of three potentially bioactive components were validated in transforming growth factor ß1 (TGF-ß1)-induced A549 cell fibrosis model. As a result, we identified 266 components, including 56 flavonoids, 52 saponins, 31 alkaloids, 10 coumarins, 12 terpenoids and 105 other components. Of these, 90 validated components were predicted to act on 172 PF-related targets and they exhibited therapeutic effects against PF via regulation of cell migration, regulation of the mitogen-activated protein kinase (MAPK) cascade, reduction of oxidative stress, and anti-inflammatory activity. Molecular docking showed that the core components could spontaneously bind to receptor proteins with a strong binding force. In vitro, compared to model group, hesperetin, ruscogenin and liquiritin significantly inhibited the increase of α-smooth muscle actin (α-SMA) and fibronectin (FN) and the decrease of e-cadherin (E-cad) in TGF-ß1-induced A549 cells. This study is the first to show, using UPLC-Orbitrap Fusion MS combined with network pharmacology and experimental validation, that JSHX might exert therapeutic actions against PF by suppressing the expression of key factors in PF. The findings provide a deeper understanding of the chemical profiling and pharmacological activities of JSHX and a reference for further scientific research and clinical use of JSHX in PF treatment.

The Bufei Jianpi Formula Improves Mucosal Immune Function by Remodeling Gut Microbiota Through the SCFAs/GPR43/NLRP3 Pathway in Chronic Obstructive Pulmonary Disease Rats.

Purpose: Bufei Jianpi formula (BJF), a traditional Chinese medicine, is an effective and safe therapeutic formula for chronic obstructive pulmonary disease (COPD). BJF treatment is known to reduce the incidence of loose stools in rats with COPD. It is unclear whether BJF regulates gut microbiota. This study examined whether BJF improved mucosal immune function by remodeling the gut microbiota and modulating metabolites in COPD rats. Methods: Sixty Sprague Dawley (SD) rats were randomized into control, model, BJF, aminophylline (APL), and probiotics (PBT) groups. The stable COPD rat model was duplicated using repeated cigarette smoke inhalation and lipopolysaccharide (LPS) injection. Normal saline, BJF, APL, or PBT were intragastrically administered from weeks eight to twelve, and then the rats were sacrificed at week thirteen. Lung and colon tissues were removed; feces were collected. Pulmonary function, histopathology, levels of inflammatory factors, and activation of NF-κB in the lung tissues were evaluated. Gut microbiota were analyzed using 16S rRNA gene sequencing; fecal short-chain fatty acid (SCFA) concentrations were determined using gas chromatography/mass spectrometry. Mucosal immune response-related genes and proteins were determined using quantitative polymerase chain reaction and Western blotting. Results: BJF improved pulmonary function and reduced lung inflammation. Further, BJF treatment altered the gut microbiota composition and significantly increased the abundance of Firmicutes and the ratio of Firmicutes to Bacteroides, raising SCFA levels, including acetate, butyrate, and propionate levels. However, the abundance of Bacteroidetes, Proteobacteria, Spirochaetes, Clostridiaceae, and Treponema decreased after BJF administration. BJF decreased the gene and protein expression of NLRP3, Caspase-1, IL-8, and IL-1ß, and increased GPR43 expression. Conclusion: Overall, BJF administration improved mucosal immune function by remodeling the gut microbiota and suppressing the SCFAs/GPR43/NLRP3 pathway in COPD rats. This study provides evidence for the mechanisms underlying BJF-induced improvements in COPD and supports clinical application of BJF.

Systematic characterization of the effective constituents and molecular mechanisms of Ardisiae Japonicae Herba using UPLC-Orbitrap Fusion MS and network pharmacology.

OBJECTIVE: Ardisiae Japonicae Herba (AJH), the dried whole herb of Ardisia japonica (Thunb.) Blume [Primulaceae], has been used in treating chronic obstructive pulmonary disease (COPD) in China. However, the material basis and molecular mechanisms of AJH against COPD remain unclear. Therefore, in this study, we attempt to establish a systematic approach to elucidate the material basis and molecular mechanisms through compound identification, network analysis, molecular docking, and experimental validation. METHODS: Ultra-high performance liquid chromatography-Orbitrap Fusion mass spectrometry (UPLC-Orbitrap Fusion MS) was used to characterize the chemical compounds of AJH. The SwissTargetPrediction, String and Metascape databases were selected for network pharmacology analysis, including target prediction, protein-protein interaction (PPI) network analysis, GO and KEGG pathway enrichment analysis. Cytoscape 3.7.2 software was used to construct a component-target-pathway network to screen out the main active compounds. Autodock Vina software was used to verify the affinity between the key compounds and targets. TNF-α-stimulated A549 cell inflammation model was built to further verify the anti-inflammatory effects of active compounds. RESULTS: Altogether, 236 compounds were identified in AJH, including 33 flavonoids, 21 Phenylpropanoids, 46 terpenes, 7 quinones, 27 steroids, 71 carboxylic acids and 31 other compounds. Among them, 41 compounds were selected as the key active constituents, which might exhibit therapeutic effects against COPD by modulating 65 corresponding targets primarily involved in inflammation/metabolism/immune-related pathways. The results of molecular docking showed that the key compounds could spontaneously bind to the receptor proteins with a strong binding ability. Finally, the anti-inflammatory effects of the three active compounds were validated with the decreased levels of Interleukin-6 (IL-6) and Matrix Metalloproteinase 9 (MMP9) in TNF-α-induced A549 cells model. CONCLUSION: This study clarified that AJH may exert therapeutic actions for COPD via regulating inflammation/immune/metabolism-related pathways using UPLC-Orbitrap Fusion MS technology combined with network pharmacology for the first time. This study had a deeper exploration of the chemical components and pharmacological activities in AJH, which provided a reference for the further study and clinical application of AJH in the treatment of COPD.

Pharmacodynamics and Cellular Uptake of Peimine and Peiminine in Inflammatory Model Non-Small-Cell Lung Cancer Epithelial Cells (A549).

Peimine and peiminine are isosteroidal alkaloids with multiple biological activities, such as anticancer and anti-inflammatory activities, but their cellular uptake and pharmacodynamics are unclear. In this study, a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous quantification of peimine and peiminine concentrations in A549 cells. In the pharmacodynamic study, the selected inflammatory cytokines were IL-8, MMP-9, and TIMP-1. The results demonstrated that all calibration curves exhibited good linearity (r > 0.9970). The RSDs of intraday and interday precision and accuracy were less than 6.73% and 1.76% and 7.73% and 3.05% for peimine and peiminine, respectively. Moreover, the average analytic recoveries ranged from 83.85% to 113.67%, and the matrix effect was within 95.05%-111.29%. The uptake experiment showed a time-dependent characteristic in the A549 cells. The combination group had increased uptake and had a longer T max than the single group. In the experimental pharmacodynamics groups, the anti-inflammatory effects of the 100.0 µg/mL combination group were the most obvious. This investigation, for the first time, explores the cellular uptake profiles and pharmacodynamics of peimine and peiminine in A549 cell lines.

Network pharmacology analysis uncovers the effect on apoptotic pathway by Bu-Fei formula for COPD treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: The Bu-Fei formula (BFF) has a positive effect on chronic obstructive pulmonary disease (COPD). However, its therapeutic mechanisms against COPD remain unknown. AIM OF THE STUDY: To explore BFF's therapeutic effect on COPD and pharmacological mechanisms. MATERIALS AND METHODS: First, the effect of BFF on rats with COPD was studied. Rats were randomly assigned to the blank, COPD, BFF treatment, and aminophylline (APL) treatment groups. From weeks 1-8, the COPD model was established by Klebsiella pneumoniae (KP) and cigarette smoke. Then, rats were given corresponding treatment for 8 weeks. The lung function of the rats was analyzed by whole-body plethysmography and pulmonary function testing, lung histopathology by electron microscopy and hematoxylin and eosin staining, and protein levels by immunohistochemistry. Next, the key components and targets of BFF in COPD were screened by network pharmacology analysis. Finally, the possible mechanism was verified through molecular docking and in vivo experiments. RESULTS: BFF significantly improved lung function and lung histopathology in COPD rats and inhibit inflammation and collagen deposition in lung tissues. Also, 46 bioactive compounds and 136 BFF targets related to COPD were identified; among them, 3 compounds (quercetin, luteolin, and nobiletin) and 6 core targets (Akt1, BCL2, NF-κB p65, VEGFA, MMP9, and Caspase 8) were the key molecules associated with the mechanisms of BFF. The target enrichment analysis suggested that BFF's mechanisms might involve the apoptosis-related pathway; this possibility was supported by the molecular docking data. Lastly, BFF was indicated to increase the expression of core target genes and the production of apoptosis-related proteins. CONCLUSIONS: BFF affects COPD by regulating the apoptosis-related pathways and targets.

The pharmacokinetic study of Tanreqing and the interaction with cefixime in rat model of pneumonia by validated UPLC-MS/MS.

Combining traditional Chinese medicine and chemical drugs with antimicrobial activities has become more popular, but there is insufficient relevant research on such combinations. The Tanreqing injection (TRQI), a Chinese compound medicine, exhibits therapeutic effects in treating upper respiratory tract infections, severe influenza, and pneumonia. This research investigates the pharmacokinetics of TRQI in pneumonia model rats and explores the effect of the antibiotic cefixime on its metabolism. The pneumonia model rats were randomly divided into six groups: low, medium, and high (3, 6, and 12 mL kg-1) dose TRQI group, and a medium dose TRQI combined with cefixime (14.4 mg kg-1) group, with the remainder two groups were control group. Blood samples were collected from the tail vein at different time points between 0 and 24 h after injection. A sensitive and quick method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established for the simultaneous determination of the 13 TRQI components in the blood samples. The analytes were separated on an XBridge™C18 column (2.1 mm × 150 mm, 5 µm), with the flow phase consisting of methanol and 0.1% formic acid water at a flow rate of 0.3 mL/min. The assay method met the biological sample determination requirements, demonstrating good adaptability and practicability for application in the pharmacokinetic study of TRQI in pneumonia model rats. Moreover, the method was used successfully in the interaction study of TRQI with cefixime. The results indicated that co-administration results in a significant change in the pharmacokinetic parameters of the main TRQI components. However, the changes in the pharmacokinetic characteristics of multiple TRQI components were inconsistent. Thus, the results of this drug combination under different pathological conditions in clinical applications were unpredictable. Therefore, more attention should be paid to the combined use of cefixime and TRQI in clinical applications to avoid the risk of adverse drug reactions in future studies.

Tiaobu Feishen therapy inhibits inflammation induced by cigarette smoke extracts in a human monocyte/macrophage cell line.

OBJECTIVE: To study the mechanistic effects of Tiaobu Feishen therapy (TBFS) on inflammation induced by cigarette smoke extract (CSE) in a human monocyte/macrophage cell line. METHODS: The human monocyte/macrophage cell line THP-1 was stimulated with 10 % CSE in the presence or absence of Bufei Yishen formula (BYF), Bufei Jianpi formula (BJF) and Yiqi Zishen formula (YZF). All formulations contained serum. Pro-inflammatory cytokines were measured in the supernatants using enzyme-linked immunosorbent assay. The activity of STAT3 DNA binding was detected using electrophoretic mobility shift assay and janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation was assessed using Western blotting. RESULTS: The results showed that BYF, BJF and YZF treatment strongly decreased the CSE-induced secretion of interleukin (IL)-6, IL-8, tumor necrosis factor-α and matrix metalloproteinase-9 by THP-1 cells. Furthermore, BYF, BJF and YZF treatment attenuated STAT3 DNA binding capacity and JAK2 and STAT3 were shown to be phosphorylated. CONCLUSION: The data revealed that BYF, BJF and YZF effectively inhibited a CSE-induced inflammatory response in THP-1 cells by limiting activation of the JAK2/STAT3 pathway.

Pharmacokinetics, tissue distribution and excretion of five rhubarb anthraquinones in rats after oral administration of effective fraction of anthraquinones from rheum officinale.

Rhubarb, a famous traditional Chinese medicine, shows a wide range of physiological activities and pharmacological benefits. Rhubarb anthraquinones are perceived as the pharmacologically active compounds of Rhubarb, and understanding metabolism of them is crucial to assure safety and effectiveness of clinical application. In this study, the pharmacokinetics, tissue distribution and excretion of five rhubarb anthraquinones (aloe-emodin, rhein, emodin, chrysophanol, physcion) were systematically investigated after oral administration of rhubarb extract to rats.An HPLC method was developed and validated for quantitation of five rhubarb anthraquinones in rat plasma, tissues, urine and faeces to investigate the Pharmacokinetic characteristics. The results showed that the proposed method was suitable for the quantification of five anthraquinones in plasma, tissue and excreta samples with satisfactory linear (r > 0.99), precision (<10%) and recovery (85.12-104.20%). The plasma concentration profiles showed a quick absorption with the mean Tmax of 0.42-0.75 h and t1/2 of 6.60-15.11 h for five anthraquinones. The analytes were widely distributed in most of the tissues. Approximately 0.13-10.59% and 28.47-81.14% of five anthraquinones were recovered in urine and faeces within 132 h post-dosing, which indicated the major elimination route was faeces excretion.In summary, this study lays a foundation for elucidating the pharmacokinetic rule of rhubarb anthraquinone and the important data can provide reliable scientific resource for further research.

[Comparative study on HPLC fingerprints between crude and processed Ligustri Lucidi Fructus].

To establish high performance liquid chromatography(HPLC) fingerprints for crude and processed Ligustri Lucidi Fructus,and to evaluate their quality through the similarity calculation and chemical pattern recognition. The separation was performed with Syncronis C_(18) column(4.6 mm × 250 mm, 5 µm), with acetonitrile(A) and 0.1% phosphoric acid solution(B) as the mobile phase for gradient elution, and a detection wavelength of 280 nm. HPLC was used to detect 22 batches of crude and processed Ligustri Lucidi Fructus,and the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 Edition) was used to evaluate the similarity among 22 batches. The research on pattern recognition was conducted with cluster analysis(CA), principal component analysis(PCA), and partial least squares discriminate analysis(PLS-DA). HPLC fingerprints of crude and processed Ligustri Lucidi Fructus were established, with similarity ranging from 0.9 to 1.0. The crude and processed Ligustri Lucidi Fructus can be obviously distinguished by using CA, PCA and PLS-DA. According to the results of PLS-DA,11 constituents including hydroxytyrosol, tyrosol, specnuezhenide and oleuropein were the main marker components leading to the difference. The established fingerprint method is stable and reliable, and can provide method basis for quality control of crude and processed Ligustri Lucidi Fructus. Chemical pattern recognition is proved to be helpful in comprehensive quality control and evaluation of Ligustri Lucidi Fructus before and after the process.

Bufei Yishen Formula Restores Th17/Treg Balance and Attenuates Chronic Obstructive Pulmonary Disease via Activation of the Adenosine 2a Receptor.

Bufei Yishen formula (BYF) is a Traditional Chinese Medicine (TCM) reported to ameliorate chronic obstructive pulmonary disease (COPD) by regulating the balance between T helper (Th) 17 and regulatory T (Treg) cells. However, its mechanism remains unknown. Therefore, this study aimed to explore the underlying mechanisms of BYF. Naïve CD4+ T cells were exposed to anti-CD3, anti-CD28, transforming growth factor (TGF)-ß, and/or interleukin (IL)-6 to promote their differentiation into Th17 or Treg cells. A rat model of cigarette smoke- and bacterial infection-induced COPD was established and orally treated with BYF and/or an adenosine 2a receptor (A2aR) antagonist. Then, the rats were sacrificed, their lung tissues were removed for histological analysis, and their spleens were collected to evaluate Th17 and Treg cells. The results showed that BYF significantly suppressed Th17 cell differentiation and its related cytokines and enhanced Treg cell differentiation and its related cytokines. In addition, BYF activated the A2aR, increased the levels of p-signal transducer and activator of transcription (STAT)5, and decreased the level of p-STAT3 in Treg and Th17 cells. The A2aR antagonist suppressed the changes induced by BYF treatment in Th17 and Treg cells. Furthermore, the A2aR antagonist diminished the therapeutic effect of BYF on COPD, as indicated by the lung injury scores, bronchiole wall thickness, small pulmonary vessels wall thickness, bronchiole stenosis, alveolar diameters, decrease in inflammatory cytokines, increase in alveolar number, and lung functions. Similarly, the A2aR antagonist reversed the effects of BYF on the proportion of Th17 and Treg cells in the spleen. Additionally, BYF increased the protein and mRNA levels of A2aR and regulated the phosphorylation of STAT3 and STAT5 in spleen and lung tissues, which were inhibited by cotreatment with the A2aR antagonist. In conclusion, this study suggested that BYF exhibited its anti-COPD efficacy by restoring the Th17/Treg balance via activating A2aR, which may provide evidence for the clinical application of BYF in the treatment of COPD.

Effective-constituent compatibility-based analysis of Bufei Yishen formula, a traditional herbal compound as an effective treatment for chronic obstructive pulmonary disease.

OBJECTIVE: Critical effective constituents were identified from Bufei Yishen formula (BYF), a traditional herbal compound and combined as effective-constituent compatibility (ECC) of BYF I, which may have potential bioactive equivalence to BYF. METHODS: The active constituents of BYF were identified using four cellular models and categorised into Groups 1 (Bufeiqi), 2 (Bushen), 3 (Huatan) and 4 (Huoxue) according to Chinese medicinal theory. An orthogonal design and a combination method were used to determine the optimal ratios of effective constituents in each group and the ratios of "Groups 1 to 4" according to their pharmacological activity. We also comprehensively assessed bioactive equivalence between the BYF and the ECC of BYF I in a rat model of chronic obstructive pulmonary disease (COPD). RESULTS: We identified 12 active constituents in BYF. The numbers of constituents in Groups 1 to 4 were 3, 2, 5 and 2, respectively. We identified the optimal ratios of effective constituents within each group. In Group 1, total ginsenosides:Astragalus polysaccharide:astragaloside IV ratio was 9:5:2. In Group 2, icariin:schisandrin B ratio was 100:12.5. In Group 3, nobiletin:hesperidin:peimine:peiminine:kaempferol ratio was 4:30:6.25:0:0. In Group 4, paeoniflorin:paeonol ratio was 4:1. An orthogonal design was then used to establish the optimal ratios of Group 1, Group 2, Group 3 and Group 4 in ECC of BYF I. The ratio for total ginsenosides:Astragalus polysaccharide:astragaloside IV:icariin:schisandrin B:nobiletin:hesperidin:peimine:paeoniflorin:paeonol was determined to be 22.5:12.5:5:100:12.5:4:30:6.25:25:6.25. A comprehensive evaluation confirmed that ECC of BYF I presented with bioactive equivalence to the original BYF. CONCLUSION: Based on the ECC of traditional Chinese medicine formula method, the effective constituents of BYF were identified and combined in a fixed ratio as ECC of BYF I that was as effective as BYF itself in treating rats with COPD.

Three Tiaobu Feishen formulae reduces cigarette smoke-induced inflammation in human airway epithelial cells.

OBJECTIVE: To investigate the therapeutic efficacy of Tiaobu Feishen formulae (TBFS) on cigarette smoke-induced inflammation in vitro using lipopolysaccharide (LPS)-induced and cigarette smoke extract (CSE)-induced NCI-H292 cells. METHODS: We evaluated the inhibitory effects of Bufei Jianpi formula (BJF), Bufei Yishen formula (BYF), and Yiqi Zishen formula (YZF) on the expressions of inflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-8, matrix metalloproteinase (MMP)-9, tissue inhibitor of matrix metalloprotease (TIMP)-1, and superoxide dismutase (SOD) in H292 cells stimulated with LPS or CSE. Their related transcription factors and signaling pathways were also analyzed. RESULTS: BJF, BYF, and YZF significantly inhibited the LPS- or CSE-induced expressions of TNF-α, IL-8, MMP-9, TIMP-1, and SOD in H292 cells, and suppressed the activation of transcription factors including nuclear transcription factor (NF)-κB, activator protein (AP)-1, and signal transducers and activators of transcription (STAT) 3 and their corresponding pathways, including NF-κB, mitogen-activated protein kinase (MAPK), STAT3, and peroxisome proliferator-activated receptor (PPAR). CONCLUSION: BJF, BYF, and YZF effectively suppressed inflammatory responses, protease-antiprotease imbalance, and oxidative stress induced by LPS and CSE, an effect that was closely associated with the inhibition of the NF-κB, MAPK, STAT3, and PPAR pathways.

Effective-component compatibility of Bufei Yishen formula II inhibits mucus hypersecretion of chronic obstructive pulmonary disease rats by regulating EGFR/PI3K/mTOR signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: The effective-component compatibility of Bufei Yishen formula I (ECC-BYF I), a combination of 10 compounds, including total ginsenosides, astragaloside IV, icariin, and paeonol, etc., is derived from Bufei Yishen formula (BYF). The efficacy and safety of ECC-BYF I is equal to BYF. However, the composition of ECC-BYF I needs to be further optimized. Based on the beneficial effects of BYF and ECC-BYF I on chronic obstructive pulmonary disease (COPD), this study aimed to optimize the composition of ECC-BYF I and to explore the effects and mechanisms of optimized ECC-BYF I (ECC-BYF II) on mucus hypersecretion in COPD rats. MATERIALS AND METHODS: ECC-BYF I was initially optimized to six groups: optimized ECC-BYF I (OECC-BYF I)-A~F. Based on a COPD rat model, the effects of OECC-BYF I-A~F on COPD rats were evaluated. R-value comprehensive evaluation was used to evaluate the optimal formula, which was named ECC-BYF II. The changes in goblet cells and expression of mucins and the mRNA and proteins involved in the epidermal growth factor receptor/phosphoinositide-3-kinase/mammalian target of rapamycin (EGFR/PI3K/mTOR) pathway were evaluated to explore the effects and mechanisms of ECC-BYF II on mucus hypersecretion. RESULTS: ECC-BYF I and its six optimized groups, OECC-BYF I-A~F, had beneficial effects on COPD rats in improving pulmonary function and lung tissue pathology, reducing inflammation and oxidative stress, and improving the protease/anti-protease imbalance and collagen deposition. R-value comprehensive evaluation found that OECC-BYF I-E (paeonol, icariin, nobiletin, total ginsenoside, astragaloside IV) was the optimal formula for improving the comprehensive effects (lung function: VT, MV, PEF, EF50, FVC, FEV 0.1, FEV 0.1/FVC; histological changes: MLI, MAN; IL-1ß, IL-6, TNF-α, MMP-9, TIMP-1, T-AOC, LPO, MUC5AC, Collagen I and Collagen III). OECC-BYF I-E was named ECC-BYF II. Importantly, the effect of ECC-BYF II showed no significant difference from BYF and ECC-BYF I. ECC-BYF II inhibited mucus hypersecretion in COPD rats, which manifested as reducing the expression of MUC5AC and MUC5B and the hyperplasia rate of goblet cells. The mRNA and protein expression levels of EGFR, PI3K, Akt, and mTOR were increased in COPD rats and were obviously downregulated after ECC-BYF II administration. CONCLUSION: ECC-BYF II, which consists of paeonol, icariin, nobiletin, total ginsenoside and astragaloside IV, has beneficial effects equivalent to BYF and ECC-BYF I on COPD rats. ECC-BYF II significantly inhibited mucus hypersecretion, which may be related to the regulation of the EGFR/PI3K/mTOR pathway.

Ethanol extracts from Ilex pubescens promotes cerebral ischemic tolerance via modulation of TLR4-MyD88/TRIF signaling pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pubescent Holly Root is the dry root of Ilex pubescens Hook. et Arn. It is clinically using in the treatment for stroke and coronary artery disease. It remains unclear whether the ethanol extracts of Ilex pubescens(IPEE) treatment can promote cerebral ischemic tolerance (CIT) and exert endogenous neuroprotective effects and thus to alleviate the nerve injury caused by the subsequent persistent cerebral ischemic attacks. AIM OF THE STUDY: To investigate the effects of IPEE on CIT and its underlying molecular mechanisms. MATERIALS AND METHODS: Adult male Wistar rats were used in the present study. The bilateral common carotid arteries were blocked for 10 min followed a subsequent reperfusion to create the cerebral ischemic preconditioning (CIP); After 3 days post CIP, rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R)-injury. Rats were continuously fed with IPEE for 5 days throughout the experiment period at the dose of 100 mg/kg and 200 mg/kg, respectively. Then, the brain infarct volume, histopathology, neurological deficits, and the gene/protein expression related with the TLR4-MyD88/TRIF signaling pathway were evaluated after 24 h of MCAO/R experiment. RESULTS: IPEE pretreatment significantly reduced the cerebral infarct volume, the neurological deficit scores, and the plasma level of neuron specific enolase (NSE) at the dose of 100 mg/kg. Meanwhile, IPEE pretreatment significantly decreased the levels of inflammatory cytokines including TNF-α, IL-6, MCP-1, MIP-1α and RANTES, while it increased the levels of anti-inflammatory cytokines, such as IL-10 and TGF-ß, when compared with the group with CIP treatment alone. Moreover, the effect of IPEE treatment on CIT was in a dose-dependent manner, showing as a better effect in the group pretreated with IPEE with the dose of 100 mg/kg than that in group pretreated with IPEE with the dose of 200 mg/kg. In addition, IPEE pretreatment significantly inhibited the expressions of MyD88 mRNA and the protein expression of COX-2 and NF-κBp65, while it strengthened the expressions of TRIF mRNA and protein. The effects of IPEE pretreatment on the expression of these genes were better than that in the group treated with CIP alone. CONCLUSIONS: The present study demonstrates that IPEE pretreatment can enhance cerebral ischemic tolerance with a underlying mechanism involved in the toll-like receptor 4 (TLR4) signaling pathway through inhibiting the production of proteins or cytokines in the downstream of MyD88 and activating TRIF dependent anti-inflammatory pathways.

Bufei Jianpi Formula Improves Mitochondrial Function and Suppresses Mitophagy in Skeletal Muscle via the Adenosine Monophosphate-Activated Protein Kinase Pathway in Chronic Obstructive Pulmonary Disease.

Skeletal muscle dysfunction, a striking systemic comorbidity of chronic obstructive pulmonary disease (COPD), is associated with declines in activities of daily living, reductions in health status and prognosis, and increases in mortality. Bufei Jianpi formula (BJF), a traditional Chinese herbal formulation, has been shown to improve skeletal muscle tension and tolerance via inhibition of cellular apoptosis in COPD rat models. This study aimed to investigate the mechanisms by which BJF regulates the adenosine monophosphate-activated protein kinase (AMPK) pathway to improve mitochondrial function and to suppress mitophagy in skeletal muscle cells. Our study showed that BJF repaired lung function and ameliorated pathological impairment in rat lung and skeletal muscle tissues. BJF also improved mitochondrial function and reduced mitophagy via the AMPK signaling pathway in rat skeletal muscle tissue. In vitro, BJF significantly improved cigarette smoke extract-induced mitochondrial functional impairment in L6 skeletal muscle cells through effects on mitochondrial membrane potential, mitochondrial permeability transition pores, adenosine triphosphate production, and mitochondrial respiration. In addition, BJF led to upregulated expression of mitochondrial biogenesis markers, including AMPK-α, PGC-1α, and TFAM and downregulation of mitophagy markers, including LC3B, ULK1, PINK1, and Parkin, with increased expression of downstream markers of the AMPK pathway, including mTOR, PPARγ, and SIRT1. In conclusion, BJF significantly improved skeletal muscle and mitochondrial function in COPD rats and L6 cells by promoting mitochondrial biogenesis and suppressing mitophagy via the AMPK pathway. This study suggests that BJF may have therapeutic potential for prophylaxis and treatment of skeletal muscle dysfunction in patients with COPD.

Pharmacodynamics of Five Anthraquinones (Aloe-emodin, Emodin, Rhein, Chysophanol, and Physcion) and Reciprocal Pharmacokinetic Interaction in Rats with Cerebral Ischemia.

(1) Background: Rhubarb anthraquinones-a class of components with neuroprotective function-can be used to alleviate cerebral ischemia reperfusion injury. (2) Methods: The three pharmacodynamic indicators are neurological function score, brain water content, and cerebral infarction area; UPLC-MS/MS was used in pharmacokinetic studies to detect plasma concentrations at different time points, and DAS software was used to calculate pharmacokinetic parameters in a noncompartmental model. (3) Results: The results showed that the pharmacodynamics and pharmacokinetics of one of the five anthraquinone aglycones could be modified by the other four anthraquinones, and the degree of interaction between different anthraquinones was different. The chrysophanol group showed the greatest reduction in pharmacodynamic indicators comparing with other four groups where the rats were administered one of the five anthraquinones, and there was no significant difference between the nimodipine group. While the Aloe-emodin + Physcion group showed the most obvious anti-ischemic effect among the groups where the subjects were administered two of the five anthraquinones simultaneously. Emodin, rhein, chrysophanol, and physcion all increase plasma exposure levels of aloe-emodin, while aloe-emodin lower their plasma exposure levels. (4) Conclusions: This experiment provides a certain preclinical basis for the study of anthraquinone aglycones against cerebral ischemia and a theoretical basis for the study of the mechanism of interaction between anthraquinones.