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 ... -  《-》

Network pharmacology-based mechanism prediction and pharmacological validation of Xiaoyan Lidan formula on attenuating alpha-naphthylisothiocyanate induced cholestatic hepatic injury in rats.

The well-known Chinese prescription, Xiaoyan Lidan Formula (XYLDF), possesses efficiency of heat-clearing, dampness-eliminating and jaundice-removing. It has long been used clinically for the treatment of hepatobiliary diseases due to intrahepatic cholestasis (IHC). However, the mechanism of XYLDF for its therapeutic effects remains elusive. The study aimed to explore the potential targets for liver protective mechanism of XYLDF based on network pharmacology and experimental assays in ANIT-induced cholestatic hepatic injury (CHI) in rats. On the basis of the 29 serum migrant compounds of XYLDF elucidated by UPLC-TOF-MS/MS, a network pharmacology approach was applied for the mechanism prediction. Systematic networks were constructed to identify potential molecular targets, biological processes, and signaling pathways. And the interactions between significantly potential targets and active compounds were simulated by molecular docking. For the mechanism validation, an ANIT-induced rat model was used to evaluate the effects of XYLDF on CHI according to serum biochemistry, bile flow rates, histopathological examination, and the gene and protein expression including enzymes related to synthesis, export, and import of bile acid in liver and ileum, and those of inflammatory cytokines, analyzed by RT-qPCR and WB. The results of network pharmacology research indicated TNF (TNF-α), RELA (NF-κB), NR1H4 (FXR), and ICAM1 (ICAM-1) to be the important potential targets of XYLDF for cholestatic liver injury, which are related to bile metabolism and NF-κB-mediated inflammatory signaling. And the molecular docking had pre-validated the prediction of network pharmacology, as the core active compounds of XYLDF had shown strong simulation binding affinity with FXR, followed by NF-κB, TNF-α, and ICAM-1. Meanwhile, the effects of XYLDF after oral administration on ANIT-induced CHI in rats exhibited the decreased levels of transaminases (ALT and AST), TBA, and TBIL in serum, raised bile flow rates, and markedly improved hepatic histopathology. Furthermore, consistent to the above targets prediction and molecular docking, XYLDF significantly up-regulated the expression of FXR, SHP, BSEP, and MRP2, and down-regulated CYP7A1 and NTCP in liver, and promoted expression of IBABP and OSTα/β in ileum, suggesting the activation of FXR-mediated pathway referring to bile acid synthesis, transportation, and reabsorption. Moreover, the lower levels of TNF-α in plasma and liver, as well as the reduced hepatic gene and protein expression of NF-κB, TNF-α, and ICAM-1 after XYLDF treatment revealed the suppression of NF-κB-mediated inflammatory signaling pathway, as evidenced by the inhibition of nuclear translocation of NF-κB. XYLDF exhibited an ameliorative liver protective effect on ANIT-induced cholestatic hepatic injury. The present study has confirmed its mechanism as activating the FXR-regulated bile acid pathway and inhibiting inflammation via the NF-κB signaling pathway.

Wang M ，Liu F ，Yao Y ，Zhang Q ，Lu Z ，Zhang R ，Liu C ，Lin C ，Zhu C ... -  《-》

Investigation into the anti-inflammatory mechanism of Pothos chinensis (Raf.) Merr. By regulating TLR4/MyD88/NF-κB pathway: Integrated network pharmacology, serum pharmacochemistry, and metabolomics.

Inflammation is directly related to disease progression and contributes significantly to the global burden of disease. Pothos chinensis (Raf.) Merr. (PCM) is commonly used in Yao medicine in China to treat tumors, and orthopedic illnesses such as knee osteoarthritis, and rheumatic bone discomfort. PCM was found to have significant anti-inflammatory properties in previous studies. To explore the active compounds of PCM and their anti-inflammatory pharmacological mechanisms through an integrated strategy of serum pharmacochemistry, network pharmacology, and serum metabolomics. The qualitative and quantitative analyses of the chemical components of PCM were performed using UPLC-QTOF-MS/MS and UPLC, respectively, and the prototype components of PCM absorbed into the blood were analyzed. Based on the characterized absorbed into blood components, potential targets and signaling pathways of PCM anti-inflammatory were found using network pharmacology. Furthermore, metabolomics studies using UPLC-QTOF-MS/MS identified biomarkers and metabolic pathways related to the anti-inflammatory effects of PCM. Finally, the hypothesized mechanisms were verified by in vivo and in vitro experiments. Forty chemical components from PCM were identified for the first time, and seven of them were quantitatively analyzed, while five serum migratory prototype components were found. Network pharmacology KEGG enrichment analysis revealed that arachidonic acid metabolism, Tyrosine metabolism, TNF signaling pathway, NF-κB signaling pathway, and phenylalanine metabolism were the main signaling pathways of PCM anti-inflammatory. Pharmacodynamic results showed that PCM ameliorated liver injury and inflammatory cell infiltration and downregulated protein expression of IL-1β, NF-κB p65, and MyD88 in the liver. Metabolomics studies identified 53 different serum metabolites, mainly related to purine and pyrimidine metabolism, phenylalanine metabolism, primary bile acid biosynthesis, and glycerophospholipid metabolism. The comprehensive results demonstrated that the anti-inflammatory modulatory network of PCM was related to 5 metabolites, 3 metabolic pathways, 7 targets, and 4 active components of PCM. In addition, molecular docking identified the binding ability between the active ingredients and the core targets, and the anti-inflammatory efficacy of the active ingredients was verified by in vitro experiments. Our study demonstrated the anti-inflammatory effect of PCM, and these findings provide new insights into the active ingredients and metabolic mechanisms of PCM in anti-inflammation.

Xiao G ，Yang M ，Zeng Z ，Tang R ，Jiang J ，Wu G ，Xie C ，Jia D ，Bi X ... -  《-》

Discovery of the toxicity-related quality markers and mechanisms of Zhi-Zi-Hou-Po decoction based on Chinmedomics combined with differentially absorbed components and network pharmacology.

Zhi-Zi-Hou-Po decoction (ZZHPD), as a representative traditional Chinese medicine (TCM) formula for the treatment of depression, has frequently triggered hepatorenal toxicity in recent years. However, its toxic effect, material basis, and underlying mechanisms have not been fully elucidated. To explore the hepatorenal toxicity-material basis-quality markers (Q-markers) and multiple mechanisms of ZZHPD. ZZHPD-induced rat model of toxicity was evaluated by behavioral indicators, biochemical parameters, and histopathological sections. Then, UHPLC-Q-Exactive Orbitrap-MS combined with multivariate data analysis was utilized to identify the endogenous differential metabolites and the prototype components of ZZHPD in the plasma. A comprehensive strategy integrating in-house library, diagnostic ions, Compound Discover software, and network databases was constructed to identify the chemical constituents of ZZHPD. Additionally, the differentially absorbed components of ZZHPD were screened out based on the spectrum-effect relationship (toxic state and normal state), feature extraction of exogenous components, and variable influence on projection (VIP). Further, Chinmedomics and network pharmacology oriented by differentially absorbed components were performed to predict toxicity-related Q-markers and core targets, as well as relevant pathways. Finally, the binding ability between components and targets was predicted using molecular docking, and the mRNA expression of core target genes was determined by real-time qPCR experiment. ZZHPD exerted significant hepatotoxicity and nephrotoxicity in rats accompanied by body weight loss, abnormal biochemical indicators, and pathologic characteristics with mild inflammation and cell damage. The results of plasma metabolomics indicated that 22 differential metabolites interfered by ZZHPD mainly involved in primary bile acid biosynthesis, arginine and proline metabolism, phenylalanine metabolism and biosynthesis, sphingolipid metabolism, pyrimidine and purine metabolism. Firstly, 106 chemical substances of ZZHPD were identified, 44 of them were absorbed into the blood, mainly including 7 iridoid glycosides, 15 flavonoids, 5 lignans, and others. Then, the correlation analysis results suggested that 12 of 19 differentially absorbed constituents were highly correlated with 22 differential metabolites and recognized as potential Q-markers. Finally, 9 toxicity-related Q-markers were predicted and confirmed with better binding ability to 5 core targets (PTGS2, CASP3, TNF, PPARG, HMOX1), including 3 flavonoids (naringin, hesperidin, and neohesperidin), 2 iridoid glycosides (geniposide and genipin-1-β-D-gentiobioside), 2 lignans (honokiol and magnolol), organic acid (chlorogenic acid), and crocin (crocetin). The real-time qPCR results showed that the mRNA levels of CASP3, TNF-α, and PPARG significantly increased in the damaged liver. Combining metabolomics and network pharmacology results, the multiple mechanisms of toxicity might involve in oxidative damage, inflammation, and apoptosis pathways. Taken together, the toxicity-related Q-markers of ZZHPD screened for the first time in this work were reliable, and the holistic intervention for hepatorenal toxicity further revealed the multi-component, multi-target, and multi-pathway features in TCM. The integrated approach provides a novel perspective for the discovery of toxicity/efficacy-related substances and mechanistic studies in TCM.

Wan S ，Xie X ，Yang G ，Feng F ... -  《-》

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 ... -  《-》