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.

Bufei Yishen formula protects the airway epithelial barrier and ameliorates COPD by enhancing autophagy through the Sirt1/AMPK/Foxo3 signaling pathway.

OBJECT: Bufei Yishen formula (BYF), a traditional Chinese medicine alleviates COPD symptoms and suppresses airway epithelial inflammation. In this study, we determined whether BYF protects the airway epithelial barrier from destruction in COPD rats. METHODS: The protective effects of BYF on the airway epithelial barrier were examined in a rat COPD model. BEAS-2B epithelial cells were exposed to cigarette smoke extract (CSE) to determine the effect of BYF on epithelial barrier function. Transcriptomic and network analyses were conducted to identify the protective mechanisms. RESULTS: Oral BYF reduced the severity of COPD in rats by suppressing the decline in lung function, pathological changes, inflammation, and protected airway epithelial barrier function by upregulating apical junction proteins, including occludin (OCLN), zonula occludens (ZO)-1, and E-cadherin (E-cad). BYF treatment reduced epithelial permeability, and increased TEER as well as the apical junction proteins, OCLN, ZO-1, and E-cad in BEAS-2B cells exposed to CSE. Furthermore, 58 compounds identified in BYF were used to predict 421 potential targets. In addition, the expression of 572 differentially expressed genes (DEGs) was identified in CSE-exposed BEAS-2B cells. A network analysis of the 421 targets and 572 DEGs revealed that BYF regulates multiple pathways, of which the Sirt1, AMPK, Foxo3, and autophagy pathways may be the most important with respect to protective mechanisms. Moreover, in vitro experiments confirmed that nobiletin, one of the active compounds in BYF, increased apical junction protein levels, including OCLN, ZO-1, and E-cad. It also increased LC3B and phosphorylated AMPK levels and decreased the phosphorylation of FoxO3a. CONCLUSIONS: BYF protects the airway epithelial barrier in COPD by enhancing autophagy through regulation of the SIRT1/AMPK/FOXO3 signaling pathway.

Effective-component compatibility of Bufei Yishen formula alleviates chronic obstructive pulmonary disease inflammation by regulating GSK3ß-mediated NLRP3 inflammasome activation.

Glycogen synthase kinase 3ß (GSK3ß) has been associated with sensing many different stimuli to trigger the NLRP3 inflammasome, which plays a crucial role in promoting the inflammatory response in diseases, including chronic obstructive pulmonary disease (COPD). Bufei Yishen formula (BYF), a traditional Chinese herbal medicine, has beneficial effects on COPD. Effective-component compatibility of BYF (ECC-BYF), optimized from BYF, is equally effective as BYF in inhibiting COPD inflammation. However, the exact mechanism by which ECC-BYF regulates the activation of NLRP3 inflammasome to inhibit COPD inflammation remains unclear. Hence, we investigated the mechanisms underlying the alleviation of COPD inflammation by ECC-BYF through the inhibition of GSK3ß-mediated NLRP3 inflammasome activation by experimental rat model of COPD and lipopolysaccharide/adenosine triphosphate (LPS/ATP) induced macrophages. The data showed that ECC-BYF significantly improved the lung function, attenuated histopathological damage, and alleviated inflammatory cell infiltration and alveolar destruction. Further, it significantly inhibited inflammatory cytokine production and downregulated the phosphorylation of GSK3ß by inhibiting the activation of NLRP3 inflammasome in the rat model of COPD. Moreover, ECC-BYF suppressed the activation of the NLRP3 inflammasome by increasing the phosphorylation at serine 9 and decreasing the phosphorylation at tyrosine 216 of GSK3ß, followed by the inhibition of IL-1ß secretion in macrophages. Together, ECC-BYF effectively ameliorates COPD by suppressing inflammation, which is dependent on the regulation of GSK3ß-mediated NLRP3 inflammasome activation.

Integrated bioinformatics analysis for the identification of idiopathic pulmonary fibrosis-related genes and potential therapeutic drugs.

OBJECTIVE: The pathogenesis of idiopathic pulmonary fibrosis (IPF) remains unclear. We sought to identify IPF-related genes that may participate in the pathogenesis and predict potential targeted traditional Chinese medicines (TCMs). METHODS: Using IPF gene-expression data, Wilcoxon rank-sum tests were performed to identify differentially expressed genes (DEGs). Protein-protein interaction (PPI) networks, hub genes, and competitive endogenous RNA (ceRNA) networks were constructed or identified by Cytoscape. Quantitative polymerase chain reaction (qPCR) experiments in TGF-ß1-induced human fetal lung (HFL) fibroblast cells and a pulmonary fibrosis mouse model verified gene reliability. The SymMap database predicted potential TCMs targeting IPF. The reliability of TCMs was verified in TGF-ß1-induced MRC-5 cells. MATERIALS: Multiple gene-expression profile data of normal lung and IPF tissues were downloaded from the Gene Expression Omnibus database. HFL fibroblast cells and MRC-5 cells were purchased from Wuhan Procell Life Science and Technology Co., Ltd. (Wuhan, China). C57BL/12 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). RESULTS: In datasets GSE134692 and GSE15197, DEGs were identified using Wilcoxon rank-sum tests (both p < 0.05). Among them, 1885 DEGs were commonly identified, and 87% (1640 genes) had identical dysregulation directions (binomial test, p < 1.00E-16). A PPI network with 1623 nodes and 8159 edges was constructed, and 18 hub genes were identified using the Analyze Network plugin in Cytoscape. Of 18 genes, CAV1, PECAM1, BMP4, VEGFA, FYN, SPP1, and COL1A1 were further validated in the GeneCards database and independent dataset GSE24206. ceRNA networks of VEGFA, SPP1, and COL1A1 were constructed. The genes were verified by qPCR in samples of TGF-ß1-induced HFL fibroblast cells and pulmonary fibrosis mice. Finally, Sea Buckthorn and Gnaphalium Affine were predicted as potential TCMs for IPF. The TCMs were verified by qPCR in TGF-ß1-induced MRC-5 cells. CONCLUSION: This analysis strategy may be useful for elucidating novel mechanisms underlying IPF at the transcriptome level. The identified hub genes may play key roles in IPF pathogenesis and therapy.

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.

Maintenance of airway epithelial barrier integrity via the inhibition of AHR/EGFR activation ameliorates chronic obstructive pulmonary disease using effective-component combination.

BACKGROUND: Airway epithelial barrier dysfunction is highly related to the pathogenesis of chronic obstructive pulmonary disease (COPD). Effective-component combination (ECC) derived from Bufei Yishen formula (BYF) is an effective treatment regimen for patients with COPD and has previously been found to attenuate COPD and airway epithelial inflammation in rats. PURPOSE: To determine the mechanism underlying the protective effects of ECC-BYF against the disruption of the airway epithelial barrier in COPD. METHODS: The protective effects of ECC-BYF on the airway epithelial barrier were investigated in a rat COPD model. BEAS-2B epithelial cells were stimulated with cigarette smoke extract (CSE) to determine the direct effects of ECC-BYF on epithelial barrier function and aryl hydrocarbon receptor (AHR)/ epidermal growth factor receptor (EGFR) signaling. RESULTS: The results revealed that ECC-BYF attenuated COPD in rats and maintained the airway epithelial barrier by upregulating the expression of apical junction proteins, including occludin (OCC), zonula occludens (ZO)-1, and E-cadherin (E-cad). In BEAS-2B cells, ECC-BYF decreased permeability, increased transepithelial electrical resistance, and prevented the decrease in OCC, ZO-1, and E-cad expression induced by CSE exposure. In addition, transcriptomics and network analysis revealed that the protective effects of ECC-BYF may be related to multiple signaling pathways, including ErbB, AHR, and PI3K-Akt-mTOR pathways. ECC-BYF treatment suppressed the protein levels of p-EGFR and p-ERK1/2 and mRNA levels of CYP1A1 in CSE-exposed BEAS-2B cells as well as the protein levels of p-EGFR, p-ERK1/2, and CYP1A1 in the lungs of rats with COPD. In BEAS-2B cells, the AHR agonist FICZ weakened the protective effect of ECC-BYF on the epithelial barrier by suppressing the increase in ZO-1 and OCC expression induced by ECC-BYF and preventing the inhibitory effects of ECC-BYF on EGFR phosphorylation. CONCLUSIONS: This is the first study to demonstrate the protective effect of ECC-BYF on airway epithelial barrier function. The underlying mechanism may be associated with the suppression of the AHR/EGFR pathway to promote apical junction protein adhesion.

Yangqing Chenfei formula alleviates silica-induced pulmonary inflammation in rats by inhibiting macrophage M1 polarization.

BACKGROUND: Yangqing Chenfei formula (YCF) is a traditional Chinese medicine formula for early-stage silicosis. However, the therapeutic mechanism is unclear. The purpose of this study was to determine the mechanism for the effects of YCF on early-stage experimental silicosis. METHODS: The anti-inflammatory and anti-fibrotic effects of YCF were determined in a silicosis rat model, which was established by intratracheal instillation of silica. The anti-inflammatory efficacy and molecular mechanisms of YCF were examined in a lipopolysaccharide (LPS)/interferon (IFN)-γ-induced macrophage inflammation model. Network pharmacology and transcriptomics were integrated to analyze the active components, corresponding targets, and anti-inflammatory mechanisms of YCF, and these mechanisms were validated in vitro. RESULTS: Oral administration of YCF attenuated the pathological changes, reduced inflammatory cell infiltration, inhibited collagen deposition, decreased the levels of inflammatory factors, and reduced the number of M1 macrophages in the lung tissue of rats with silicosis. YCF5, the effective fraction of YCF, significantly attenuated the inflammatory factors induced by LPS and IFN-γ in M1 macrophages. Network pharmacology analysis showed that YCF contained 185 active components and 988 protein targets, which were mainly associated with inflammation-related signaling pathways. Transcriptomic analysis showed that YCF regulated 117 reversal genes mainly associated with the inflammatory response. Integrative analysis of network pharmacology and transcriptomics indicated that YCF suppressed M1 macrophage-mediated inflammation by regulating signaling networks, including the mTOR, mitogen-activated protein kinases (MAPK), PI3K-Akt, NF-κB, and JAK-STAT signaling pathways. In vitro studies confirmed that the active components of YCF significantly decreased the levels of p-mTORC1, p-P38, and p-P65 by suppressing the activation of related-pathways. CONCLUSION: YCF significantly attenuated the inflammatory response in rats with silicosis via the suppression of macrophage M1 polarization by inhibiting a "multicomponent-multitarget-multipathway" network.

Uncovering mechanisms of Baojin Chenfei formula treatment for silicosis by inhibiting inflammation and fibrosis based on serum pharmacochemistry and network analysis.

BACKGROUND: Baojin Chenfei formula (BCF), a Chinese herbal formula, has significant effects on improving the clinical symptoms of patients with silicosis. However, its active compounds and the underlying mechanisms have not yet fully been elucidated. PURPOSE: This study aimed to explore the underlying mechanisms of BCF in treating silicosis. METHODS: The rat model of silicosis was developed via a single intratracheal instillation of SiO2 suspension to examine the therapeutic impacts of BCF on silicosis. Subsequently, the active compounds, targets, and mechanisms of BCF were analyzed based on serum pharmacochemistry and network analysis. Finally, the underlying mechanisms of representative compounds of BCF were validated in vitro experiments. RESULTS: BCF significantly alleviated SiO2-induced silicosis in rats, evidenced by improved lung function, decreased pathological injury, and reduced inflammatory response and fibrosis. 19 active compounds were identified from the rat serum samples after BCF gavage. Subsequently, 299 targets for these 19 compounds in BCF and 257 genes related to silicosis were collected. 26 overlapping targets, including AKT1, TNF, IL6, MAPK3, EGFR, and others, were obtained from the intersection of the 299 BCF-related targets and 257 silicosis-associated genes. These overlapping targets mainly corresponded to glycyrrhetic acid and paeoniflorin and were mainly associated with positive regulation of smooth muscle cell proliferation, positive regulation of MAP kinase activity, and inflammatory response. In vitro experiments also demonstrated that the representative compounds of BCF (glycyrrhetic acid and paeoniflorin) could suppress inflammatory response by the MAPK pathway, and also inhibited fibroblast activation by the EGFR-PI3K-AKT pathway. CONCLUSION: Active compounds of BCF, such as glycyrrhetic acid and paeoniflorin, could suppress inflammatory response by the MAPK pathway and suppress fibroblast activation by the EGFR-PI3K-AKT pathway. These might be the mechanisms of BCF in treating silicosis.

Tong Sai granule improves AECOPD via regulation of MAPK-SIRT1-NF-κB pathway and cellular senescence alleviation.

ETHNOPHARMACOLOGICAL RELEVANCE: Tong Sai granule (TSG) a traditional Chinese medicine, are used to treat acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Cellular senescence is considered the mechanism underlying AECOPD progression. AIM OF THE STUDY: This study aimed to investigate the therapeutic mechanisms of TSG in an AECOPD rat model (established using cigarette smoke exposure and bacterial infection) and focused on the inhibition of cellular senescence in vivo and in vitro. MATERIALS AND METHODS: Histological changes and levels of inflammatory cytokines, matrix metalloproteinases (MMPs), p53, and p21 were determined. A cellular senescence model was established by challenging airway epithelial cells with cigarette smoke extract (CSE) and lipopolysaccharide (LPS). Quantitative PCR, western blotting, and immunofluorescence were used to measure mRNA and protein levels. Additionally, UPLC-Q-Extractive-Orbitrap MS analysis, network analysis, and transcriptomics were used to analyze the potential compounds and molecular mechanisms of TSG. RESULTS: The results showed that oral administration of TSG significantly reduced the severity of AECOPD in rats by ameliorating lung function decline and pathological injuries and increasing the levels of C-reactive protein and serum amyloid A, two well-known proinflammatory mediators of the acute phase response. Oral TSG administration also decreased the expression levels of proinflammatory cytokines (e.g., IL-6, IL-1ß, and TNF-α), MMPs (e.g., MMP-2 and MMP-9), critical regulators of senescence such as p21 and p53, and the apoptotic marker γH2AX, all of which are factors in cellular senescence in lung tissue. TSG4 was isolated from TSGs using macroporous resin and found to significantly suppress cellular senescence in CSE/LPS-induced bronchial epithelial cells. Furthermore, 26 of 56 compounds identified in TSG4 were used to predict 882 potential targets. Additionally, 317 differentially expressed genes (DEGs) were detected in CSE/LPS-treated bronchial epithelial cells. Network analysis of the 882 targets and 317 DEGs revealed that TSG4 regulated multiple pathways, among which the mitogen-activated protein kinase-sirtuin 1-nuclear factor kappa B (MAPK-SIRT1-NF-κB) pathway is important in terms of antisenescent mechanisms. Moreover, in CSE/LPS-induced bronchial epithelial cells, p-p38, p-ERK1/2, p-JNK, and p-p65 levels were increased and SIRT1 levels were decreased after TSG4 treatment. Additionally, oral TSG administration decreased p-p38 and p-p65 levels and increased SIRT1 levels in the lung tissues of AECOPD model rats. CONCLUSION: Collectively, these results indicate that TSGs ameliorate AECOPD by regulating the MAPK-SIRT1-NF-κB signaling pathway and subsequently suppressing cellular senescence.

Effective-compounds of Jinshui Huanxian formula ameliorates fibroblast activation in pulmonary fibrosis by inhibiting the activation of mTOR signaling.

BACKGROUND: Jinshui Huanxian formula (JHF) ameliorates idiopathic pulmonary fibrosis patients. Active compounds, including icariin, isoliquiritigenin, nobiletin, peimine, and paeoniflorin, deriving from JHF were combined as effective-component compatibility ECC of JHF II (ECC-JHF II), which is an effective therapeutic strategy for pulmonary fibrosis (PF) induced by bleomycin (BLM) in rats. PURPOSE: This study aimed to explore the underlying mechanism of ECC-JHF II on pulmonary fibrosis. METHODS: A model of PF in rats was established through intratracheal instillation of BLM. Pulmonary function, pathological changes, and collagen deposition were examined. The gene and protein expressions in fibroblast activation were detected by quantitative real-time PCR and western blotting respectively. RESULTS: ECC-JHF II significantly improved BLM-induced PF in rats, manifested as decreased collagen deposition, reduced pathological damage and improved pulmonary function. Furthermore, ECC-JHF II inhibited fibroblast activation by reducing the expression of α-smooth muscle actin (α-SMA) and fibronectin. We analyzed the targets of ECC-JHF II and differentially expressed genes (DEGs) of fibroblast activation induced by transforming growth factor-ß1 (TGF-ß1) and found that ECC-JHF II might regulate fibroblast activation by EGFR, PI3K-Akt or mTOR signaling pathway. In vitro experiments, we also found that ECC-JHF II suppressed the mTOR pathway, such as downregulating the phosphorylation levels of p70S6K in fibroblast activation induced by TGF-ß1. After activating mTOR signaling, the inhibition of ECC-JHF II on fibroblast activation was blocked. These results suggested that ECC-JHF II potently ameliorated pulmonary fibrosis in rats and effectively suppressed fibroblast activation by interfering with mTOR signaling. CONCLUSION: We combined transcriptomics with the network analysis to predict the mechanism underlying ECC-JHF II suppression of fibroblast activation. In summary, ECC-JHF II improved BLM-induced pulmonary fibrosis, which might be associated with the suppression of fibroblast activation by inhibiting the mTOR signaling.

Yangqing Chenfei formula attenuates silica-induced pulmonary fibrosis by suppressing activation of fibroblast via regulating PI3K/AKT, JAK/STAT, and Wnt signaling pathway.

BACKGROUND: Yangqing Chenfei formula (YCF) has been demonstrated its clinical efficiency on silicosis patients. However, the effect of YCF against silicotic fibrosis and its mechanism remain unclear. PURPOSE: This study is aimed to investigate active compounds and molecular mechanism of YCF in treating silicosis. METHOD: YCF was orally administrated to silicosis rats induced by crystalline silica. The effective fraction of YCF and the compounds was isolated and identified by using macroporous resin and HPLC-MS, respectively. The targets and potential molecular mechanism of YCF against silicotic fibrosis were investigated through pharmacological network and RNA-sequencing analysis and in vitro-experimental validation. RESULTS: YCF could remarkably improve the lung function and pathological changes of silicotic rats, reduce the aggregation of fibrocytes and deposition of ECM, such as collagen I, III, FN, and α-SMA, and suppress the TGF-ß/Smad3 signaling. Furthermore, YCF6, the effective fraction derived from YCF, could significantly inhibit fibroblast activation induced by TGF-ß. Then, 135 compounds were identified from YCF6 by using HPLC-MS, and Network pharmacology analysis predicted total 941 targets for these compounds. Moreover, 409 differentially expressed genes of fibroblast activation induced by TGF-ß were identified. Then, integrated analysis of the 941 targets with 409 differentially expressed genes showed that YCF6 contains multiple compounds, such as tangeretin, L-Malic acid, 2-Monolinolein etc., which inhibits fibroblast activation probably by targeting different proteins, such as PIK3CA, AKT1, JAK2, STAT3, GSK3ß, leading to regulate the signal network, such as PI3K/AKT signaling pathway, JAK/STAT signaling pathway, and Wnt signaling pathway. Finally, in vitro experiment indicated that tangeretin, the active compound contained in YCF6, could significantly inhibit TGF-ß induced fibroblast activation. Moreover, YCF6 and tangeretin could markedly inhibit the activation of PI3K/AKT, JAK/STAT, and Wnt pathway. CONCLUSION: YCF contained multiple compounds and targeted various proteins that regulated the fibroblast activation, which might be the molecular mechanisms of it in treating silicosis.

Network Pharmacology and Experimental Validation to Reveal Effects and Mechanisms of Icariin Combined with Nobiletin against Chronic Obstructive Pulmonary Diseases.

Background: Chronic obstructive pulmonary disease (COPD) is a long-term respiratory disorder marked by restricted airflow and persistent respiratory symptoms. According to previous studies, icariin combined with nobiletin (I&N) significantly ameliorates COPD, but the therapeutic mechanisms remain unclear. Purpose: The aim of the study is to investigate the therapeutic mechanisms of I&N against COPD using network pharmacology and experimental validation. Methods: The targets of I&N and related genes of COPD were screened and their intersection was selected. Next, the protein-protein interaction (PPI) networks, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Further, a COPD rat model was established to validate the effect and mechanisms of I&N. Results: 445 potential targets I&N were obtained from SwissTargetPrediction, STITCH 5.0, and PharmMapper databases. 1831 related genes of COPD were obtained from GeneCards, DrugBank, and DisGeNet databases. 189 related genes were screened via matching COPD targets with I&N. 16 highest score targets among 189 targets were obtained according to PPI networks. GO and KEGG pathway enrichment analyses of 16 highest score targets suggested that these key genes of I&N were mostly enriched in the tumor necrosis factor (TNF) pathway, mitogen-activated protein kinase (MAPK) pathway, and phosphatidyl inositol 3-kinase (PI3K)-protein kinase B (AKT) pathway. Therefore, the treatments of I&N for COPD were connected with inflammation-related pathways. In in vivo experiments, the studies indicated that I&N improved the lung function and alleviated the damage of pulmonary histopathology. Moreover, I&N reduced levels of interleukin (IL)-6, IL-1ß, and TNF-α in lung tissues of COPD rats and inhibited the activation of the MAPK pathway and PI3K-Akt pathway. Conclusions: Icariin combined with nobiletin has therapeutic effects on COPD by inhibiting inflammation. The potential mechanisms of I&N may relate to the MAPK pathway and PI3K-Akt pathway.

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.

Identification of cancer risk assessment signature in patients with chronic obstructive pulmonary disease and exploration of the potential key genes.

It is essential to assess the cancer risk for patients with chronic obstructive pulmonary disease (COPD). Comparing gene expression data from patients with lung cancer (a total of 506 samples) and those with cancer-adjacent normal lung tissues (a total of 370 samples), we generated a qualitative transcriptional signature consisting of 2046 gene pairs. The signature was verified in an evaluation dataset comprising 18 subjects with severe disease and 52 subjects with moderate disease (Wilcoxon rank-sum test; p = 7.33 × 10-5). Similar results were obtained in other independent datasets. Among the gene pairs in the signature, 326 COPD stage-related gene pairs were identified based on Spearman's rank correlation tests and those gene pairs comprised 368 unique genes. Of these 368 genes, 16 genes were significantly dysregulated in COPD rat model data compared with control data. Some of these genes (Dhx16, Upf2, Notch3, Sec61a1, Dyrk2, and Hmmr) were altered when the COPD rat model was treated with traditional Chinese medicines (TCM), including Bufei Yishen formula, Bufei Jianpi formula, and Yiqi Zishen formula. Overall, the signature could predict the cancer incidence-risk of COPD and the identified key genes might provide guidance regarding both the treatment of COPD using TCM and the prevention of cancer in patients with COPD. KEY MESSAGESA cancer risk assessment signature was identified in patients with COPD.The signature is insensitive to batch effects and is well verified.COPD key genes identified in this study might play a crucial role in TCM treatment and cancer prevention.

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.

Effective-Component Compatibility of Bufei Yishen Formula III Combined with Electroacupuncture Suppresses Inflammatory Response in Rats with Chronic Obstructive Pulmonary Disease via Regulating SIRT1/NF-κB Signaling.

Objective: To explore more efficient treatments for chronic obstructive pulmonary disease (COPD), effective-component compatibility of Bufei Yishen formula III (ECC-BYF III) and electroacupuncture were tested on rats with COPD, and silent information regulator transcript-1 (SIRT1)/nuclear factor-kappaB (NF-κB) signaling was further investigated to interpret the therapy. Methods: In total, 70 rats were randomly divided into control (Control), model (Model), aminophylline (APL), ECC-BYF III, electroacupuncture (EA), ECC-BYF III+EA, and sham electroacupuncture (SA) groups. Cigarette smoke exposure combined with repeated bacterial infections was used to establish COPD models in 1-12 weeks. From 13 to 20 weeks, the ECC-BYF III and APL groups received corresponding drugs; the EA group received electroacupuncture therapy, wherein Dazhui (GV 14), Feishu (BL 13), and Shenshu (BL 23) points were selected; the ECC-BYF III+EA group received ECC-BYF III intragastrically combined with electroacupuncture; and the SA group received simulated electroacupuncture (nonacupoint). Pulmonary function, pulmonary histopathology, the expressions of SIRT1/NF-κB signaling, and inflammation-related mRNA and protein were detected. Results: Significant deterioration was observed in pulmonary function and pulmonary histopathology in rats with COPD (P < 0.01), and inflammatory state was illustrated by increased levels of interleukin- (IL-) 6 and tumor necrosis factor alpha (TNF-α) and decreased levels of IL-10 (P < 0.01). After the intervention of APL, ECC-BYF III, EA, and ECC-BYF III+EA, both pulmonary function and pulmonary histopathology were improved (P < 0.05 and P < 0.01), whereas the levels of IL-6 and TNF-α were decreased and IL-10 was increased (P < 0.05 and P < 0.01). Additionally, the mRNA expressions of IL-6, TNF-α, NF-κB, and acetylated NF-κBp65 (Ac-NF-κB) were noted to decrease, and SIRT1 and IL-10 were increased (P < 0.05 and P < 0.01); the protein expression of SIRT1 was upregulated, and NF-κBp65 and Ac-NF-κB were downregulated (P < 0.05 and P < 0.01). The effect of ECC-BYF III+EA was better in terms of improving pulmonary function and alleviating inflammation than that of the other treatment groups (P < 0.01 and P < 0.05). Conclusions: ECC-BYF III, electroacupuncture, and their combination can suppress inflammation, among which the combination therapy has been proven to be the most effective treatment, and the mechanism may be involved in activating SIRT1/NF-κB signaling.

Electroacupuncture attenuates pulmonary vascular remodeling in a rat model of chronic obstructive pulmonary disease via the VEGF/PI3K/Akt pathway.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation that is not fully reversible. Pulmonary vascular remodeling is the main pathological feature of COPD. Vascular endothelial growth factor (VEGF), the key regulator of angiogenesis, mediates activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, which regulates the proliferation and migration of vascular endothelial cells and plays important roles in pulmonary angiogenesis and remodeling in COPD. Here, the efficacy of electroacupuncture (EA) with respect to regulation of microvascular remodeling induced by VEGF/PI3K/Akt was evaluated in a rat model of COPD. METHODS: Rats were randomly assigned to blank, COPD model, EA and sham acupuncture (SA) groups. Rats in the EA group received EA at GV14, BL13 and BL23 three times per week, while those in the SA group, as a control, received shallow and minimal electrostimulation at sites 5-10 mm away from the traditional acupuncture point locations. After 2, 4 and 8 weeks of treatment, the optimal treatment duration was determined according to the results of lung function, lung pathology and inflammatory factor levels. Then, microvessel density, protein levels and mRNA expression of selected VEGF/PI3K/Akt pathway intermediates were determined by immunofluorescence, immunohistochemistry and Western blot analysis, and mRNA qRT-PCR, respectively. RESULTS: EA improved lung function and lung tissue histopathology, with the best effect after 8 weeks of treatment, as noted by reduced density of lung microvessels and expression of angiogenesis-related factors (VEGF and endothelin (ET)-1). EA-treated COPD rats exhibited reduced VEGF, VEGF receptor 2 (VEGFR2), ET-1 mRNA and VEGF, VEGFR2, phosphorylated (p)-VEGFR2, PI3K, Akt, p-Akt, mammalian target of rapamycin (mTOR), and p-mTOR at the protein level in comparison with untreated and SA-treated COPD model rats. CONCLUSION: EA had beneficial effects on COPD in this animal model including reduced pulmonary vascular remodeling via mechanisms possibly related to the VEGF/PI3K/Akt pathway.