Integrating serum pharmacochemistry, network pharmacology and untargeted metabolomics strategies to reveal the material basis and mechanism of action of Feining keli in the treatment of chronic bronchitis.

Feining keli (FNKL) is herbal preparation mainly made from Senecio cannabifolius Less., In recent years, more and more studies have found that FNKL has excellent therapeutic effects on chronic bronchitis (CB). Nevertheless, its pharmacodynamic material basis and mechanism of action are still unknown. This study aimed to explore the pharmacodynamic material basis and mechanism of action of FNKL in treating CB. The CB rat model was induced using nasal drops of lipopolysaccharide (LPS) in combination with smoking. Various assessments including behavioral and body mass examination, lung index measurement, enzyme linked immunosorbent assay (ELISA), as well as histological analyses using hematoxylin and eosin (H&E) and Masson staining were conducted to validate the reliability of the CB model. The serum components of FNKL in CB rats were identified using ultra-high-performance liquid chromatography Orbitrap Exploris mass spectrometer (UHPLC-OE-MS). Network pharmacology was used to predict the network of action of the active ingredients in FNKL based on these serum components. Signaling pathways were enriched and analyzed, and molecular docking was conducted for key targets. Molecular dynamics simulations were performed using GROMACS software. The mechanism was confirmed through a series of experiments including Western blot (WB), immunofluorescence (IF), and reverse transcription (RT)-PCR. Additionally, untargeted metabolomics was employed to identify biomarkers and relevant metabolic pathways associated with the treatment of CB with FNKL. In CB rats, FNKL improved body mass, lung index, and pathological damage of lung tissues. It also decreased interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), malonaldehyde (MDA) levels, and percentage of lung collagen fiber area. Furthermore, FNKL increased IL-10 and superoxide dismutase (SOD) levels, which helped alleviate bronchial inflammation in the lungs. A total of 70 FNKL chemical components were identified in CB rat serum. Through network pharmacology analysis, 5 targets, such as PI3K, AKT, NF-κB, HIF-1α, and MYD88, were identified as key targets of FNKL in the treatment of CB. Additionally, the key signaling pathways identified were PI3K/AKT pathway、NF-κB/MyD88 pathway、HIF-1α pathway. WB, IF, and RT-PCR experiments were conducted to confirm the findings. Molecular docking studies demonstrated successful docking of 16 potential active components with 5 key targets. Additionally, molecular dynamics simulations indicated the stability of quercetin-3-galactoside and HIF-1α. Metabolomics analysis revealed that FNKL primarily regulated pathways related to alpha-linolenic acid metabolism, primary bile acid biosynthesis, bile secretion, arachidonic acid metabolism, neuroactive ligand-receptor interaction, and folate biosynthesis. Furthermore, the expression levels of traumatic acid, traumatin, alpha linolenic acid, cholic acid, 2-arachidonoylglycerol, deoxycholic acid, 7,8-dihydroneopterin, and other metabolites were found to be regulated. FNKL exhibits positive therapeutic effects on CB, with quercetin-3-galactoside identified as a key active component. The mechanism of FNKL's therapeutic action on CB involves reducing inflammatory response, oxidative stress, and regulating metabolism, and its molecular mechanism was better elucidated in a holistic manner. This study serves as a reference for understanding the pharmacodynamic material basis and mechanism of action of FNKL in treating CB, and provides avenues for exploring the effects of compounded herbal medicines on CB.

Zhu Z ，Feng YD ，Zou YL ，Xiao YH ，Wu JJ ，Yang YR ，Jiang XX ，Wang L ，Xu W ... -  《-》

Zhisou powder displays therapeutic effect on chronic bronchitis through inhibiting PI3K/Akt/HIF-1α/VEGFA signaling pathway and reprograming metabolic pathway of arachidonic acid.

Zhisou Powder (ZP), one of the most common prescriptions in traditional Chinese medicine, has been widely used in the treatment of acute or chronic bronchitis and chronic cough. The ZP was composed of Ziwan (Aster tataricus L. f.), Jiegeng (Platycodon grandiflorus (Jacq.) A. DC.), Jingjie (Nepeta cataria L.), Baibu (Stemona sessilifolia (Miq.) Miq.), Baiqian (Vincetoxicum glaucescens (Decne.) C. Y. Wu & D. Z. Li), Chenpi (Citrus × aurantium f. deliciosa (Ten.) M. Hiroe) and Gancao (Glycyrrhiza uralensis Fisch. ex DC.), with plant names among it checked with MPNS (http://mpns.kew.org). But until now, the key active components and targets of ZP, and related mechanism of ZP in the treatment of chronic bronchitis (CB) remain unclear. This study combined UPLC-Q-Exactive-Orbitrap-MS, network pharmacology, metabonomics with experiment verification to explore potential mechanism of ZP in the treatment of CB. UPLC-Q-Exactive-Orbitrap-MS was performed to analyze the chemical components of ZP. The potentially effective components, attractive targets and critical signaling pathways of Zhisou Powder in the treatment of CB were screened by UPLC-Q-Exactive-Orbitrap-MS combined with network pharmacology. Additionally, the CB model rats induced by SO2 were used to evaluate the anti-chronic bronchitis activity of ZP in vivo. The pulmonary pathology was determined by hematoxylin-eosin staining. Meanwhile, PI3K/Akt/HIF-1α/VEGFA signaling pathway predicted from network pharmacology was verified by Western blot and RT-PCR. Lastly, the metabolic changes of arachidonic acid (AA) in ZP-treated rats were quantitatively analyzed by LC-MS targeted metabonomics, and the proteins expression involved in AA metabolic pathway were detected by immunohistochemistry, immunofluorescence and Western blot. The main active components of ZP in the treatment of CB selected by network pharmacology and UPLC-Q-Exactive-Orbitrap-MS technology were quercetin, kaempferol, luteolin, galangin, isorhamnetin, naringenin, nobiletin, formononetin and so on. The core targets of these components were predicted to be TP53, TNF, IL-6, VEGFA, CASP3, IL-1β, JUN, PTGS2. Enrichment of KEGG pathway analysis found that PI3K/Akt/HIF-1α/VEGFA signaling pathway might play a key role in the treatment of CB with ZP. The in vivo study showed that ZP significantly improved the pathological changes of SO2-treated lung tissue and inhibited the activation of PI3K/Akt/HIF-1α/VEGFA signaling pathway. The changes of AA and its metabolites in vivo were studied by targeted metabonomics, and it showed that ZP could reprogram the disorder of AA metabolism which contributed to the treatment of CB with ZP. ZP displayed good therapeutic effect on CB model rats through inhibiting PI3K/Akt/HIF-1α/VEGFA signaling pathway to exhibit anti-inflammatory effect and reprogramming disordered metabolic pathway of arachidonic acid.

Dong Y ，Liu Y ，Tang J ，Du J ，Zhuang X ，Tan S ，Yang Y ，Yin D ... -  《-》

Gleditsiae Sinensis Fructus ingredients and mechanism in anti-asthmatic bronchitis research.

Gleditsiae Sinensis Fructus (GSF) is commonly used in traditional medicine to treat respiratory diseases such as bronchial asthma. However, there is a lack of research on the chemical composition of GSF and the pharmacological substance and mechanism of action for GSF in treating bronchial asthma. The chemical constituents of GSF were analyzed using ultrahigh-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). In this study, we combined network pharmacology, molecular docking techniques, and experimental validation to explore the therapeutic efficacy and underlying mechanism of GSF in the treatment of bronchial asthma. Characterization of the chemical constituents of GSF was conducted using UHPLC-Q-Orbitrap HRMS. The identified chemical components were subjected to screening for active ingredients in the Swiss Absorption, Distribution, Metabolism, and Excretion (ADME) database. Relevant databases were utilized to retrieve target proteins for the active ingredients and targets associated with bronchial asthma disease, and the common targets between the two were selected. Subsequently, the protein-protein interaction (PPI) network was constructed using the String database and Cytoscape software to identify key targets. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the Metascape database. The "component-common target" network was constructed using Cytoscape to identify the primary active ingredients. Molecular docking validation was conducted using AutoDock software. The bronchial asthma mouse model was established using ovalbumin (OVA), and the lung organ index of the mice was measured. Lung tissue pathological changes were observed using hematoxylin and eosin (HE), Periodic Acid-Schiff (PAS), and Masson staining. The respiratory resistance (Penh) of the mice was assessed using a pulmonary function test instrument. An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of IgE, IL-4, IL-5, and IL-13 in the mouse serum. Immunofluorescence staining was performed to detect the protein expression levels of AKT and PI3K in the lung tissues. An in vitro experiment was performed to observe the effects of echinocystic acid (EA) on IL-4 stimulated Human ASMCs (hASMCs). Cell viability was measured using a CCK-8 assay to calculate the IC50 value of the EA. A wound healing test was conducted to observe the effect of EA on degree of healing. RT-qPCR was performed to detect the influence of EA on the mRNA expression levels of ALB, SRC, TNF-α, AKT1, and IL6 in the cells. A total of 95 chemical constituents were identified from the GSF. Of these, 37 were identified as active ingredients. There were 169 overlapping targets between the active ingredients and the disease targets. A topological analysis of the protein-protein interaction (PPI) network identified the core targets as IL6, TNF, ALB, AKT1, and SRC. An enrichment analysis revealed that the treatment of bronchial asthma with GSF primarily involved the AGE-RAGE signaling pathway and the PI3K-Akt signaling pathway, among others. The primary active ingredients included 13(s)-HOTRE, linolenic acid, and acacetin. The molecular docking results demonstrated a favorable binding activity between the critical components of GSF and the core targets. Animal experimental studies indicated that GSF effectively improved symptoms, lung function, and lung tissue pathological changes in the OVA-induced asthmatic mice, while alleviating inflammatory responses. GSF decreased the fluorescent intensity of the AKT and PI3K proteins. The IC50 value of EA was 30.02μg/ml. EA (30) significantly promoted the proliferation of IL4-stimulated hASMCs cells. EA (30) significantly increased the expression of ALB and SRC mRNA and decreased the expressions of TNF-α, AKT, and IL6 mRNA. The multiple active ingredients found in GSF exerted their anti-inflammatory effects through multiple targets and pathways. This preliminary study revealed the core target and the mechanism of action underlying its treatment of bronchial asthma. These findings provided valuable insights for further research on the pharmacological substances and quality control of GSF.

Li H ，Kang L ，Dou S ，Zhang Y ，Zhang Y ，Li N ，Cao Y ，Liu M ，Han D ，Li K ，Feng W ... -  《-》

Metabolomics and serum pharmacochemistry combined with network pharmacology uncover the potential effective ingredients and mechanisms of Yin-Chen-Si-Ni Decoction treating ANIT-induced cholestatic liver injury.

Yin-Chen-Si-Ni Decoction is a classical traditional Chinese medicine (TCM) prescription that is used clinically for treating cholestatic liver injury (CLI) and other hepatic diseases. However, the material basis and underlying mechanisms of YCSND are not clear. To investigate effective components and mechanisms of YCSND in the treatment of CLI using serum pharmacochemistry, metabolomics, and network pharmacology. Biochemical indicators, liver index, and histopathology analysis were adopted to evaluate the protective effect of YCSND on ANIT-induced CLI rats. Then, a UPLC-Q-Exactive Orbitrap MS/MS analysis of the migrant components in serum and liver including prototype and metabolic components was performed in YCSND. In addition, a study of the endogenous metabolites using serum and liver metabolomics was performed to discover potential biomarkers, metabolic pathways, and associated mechanisms. Further, the network pharmacology oriented by in vivo migrant components was also used to pinpoint the active ingredients, core targets, and signaling pathways of YCSND. Finally, molecular docking and molecular dynamics simulation (MDS) were used to predict the binding ability between components and core targets, and a real-time qPCR (RT-qPCR) experiment was used to measure the mRNA expression of the core target genes. Pharmacodynamic studies suggest that YCSND could exert obvious hepatoprotective effects on CLI rats. Furthermore, 68 compounds, comprising 32 prototype components and 36 metabolic components from YCSND, were found by serum pharmacochemistry analysis. Network pharmacology combining molecular docking and MDS showed that apigenin, naringenin, 18β-glycyrrhetinic acid, and isoformononetin have better binding ability to 6 core targets (EGFR, AKT1, IL6, MMP9, CASP3, PPARG). Additionally, PI3K, TNF-α, MAPK3, and six core target genes in liver tissues were validated with RT-qPCR. Metabolomics revealed the anti-CLI effects of YCSND by regulating four metabolic pathways of primary bile acid and biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis, taurine and hypotaurine metabolism, and arachidonic acid metabolism. Integrating metabolomics and network pharmacology identified four pathways related to CLI, including the PI3K-Akt, HIF-1, MAPK, and TNF signaling pathway, which revealed multiple mechanisms of YCSND against CLI that might involve anti-inflammatory and apoptosis. The research based on serum pharmacochemistry, network pharmacology, and metabolomics demonstrates the beneficial hepatoprotective effects of YCSND on CLI rats by regulating multiple components, multiple targets, and multiple pathways, and provides a potent means of illuminating the material basis and mechanisms of TCM prescriptions.

Liu Y ，Chen H ，Yang G ，Feng F ... -  《-》

Explore bioactive ingredients and potential mechanism of Houpo Mahuang decoction for chronic bronchitis based on UHPLC-Q exactive orbitrap HRMS, network pharmacology, and experiment verification.

Chronic bronchitis (CB) affects a growing number of people and may be linked to lung function impairment. The traditional Chinese medicine formula Houpo Mahuang Decoction (HPMHD) has been used for clinical treatment of respiratory diseases for thousands of years. Until now, its bioactive ingredients, potential targets and molecular mechanism remain unclear. To investigate the effect of HPMHD on the treatment of CB and explore the bioactive ingredients and possible mechanisms of HPMHD against CB. UHPLC-Q Exactive Orbitrap HRMS was performed to analyze the chemical components of HPMHD. The mechanism of multiple components, targets and pathways of HPMHD in the treatment of chronic bronchitis were explored by network pharmacology. Additionally, CB mice model induced by lipopolysaccharide (LPS) and smoking was used to evaluate the anti-chronic bronchitis activity of HPMHD in vivo. Pulmonary pathology was determined by hematoxylin and eosin (H&E) measurement. The levels of TNF-α and IL-6 in lung were measured by ELISA. The immunofluorescence experiments were carried out for the expression of IL-1β, TNF-α, IL-6 and NF-κB p-P65/P65 in lung. Western blot assays were performed to quantify and visualize the protein expression of NF-κB p-P65/P65 in mice lung. Data showed that 79 compounds were identified in HPMHD. The network pharmacology results showed 53 compounds were hinted their effectivity for the treatment of chronic bronchitis with HPMHD, such as ephedrine, schisantherin A, and honokiol. The main targets were predicted as 37 genes, including TNF, TP53, IL6 and so on. HPMHD ameliorated lung damages in mice and inhibited the NF-κB signaling pathway, one of the pathways plotted by KEGG pathway enrichment analysis, by reducing IL-1β, TNF-α and IL-6 expression and significantly downregulating the NF-κB p-P65/P65. In summary, the complex chemical components of HPHMD was successfully elucidate by UHPLC-Q Exactive Orbitrap HRMS. The study based on network pharmacology and experiment verification indicated that HPMHD can decreased inflammatory response in lung to treat CB. The underlying mechanism may be related to the reduction of inflammation by down-regulated the NF-κB pathways.

Gao F ，Zhang T ，Zhang H ，Dai Z ，Gu Y ，Lu M ，Zhang Z ，Zeng Q ，Shang B ，Gao S ，Wang N ，Xu B ，Lei H ... -  《-》