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.

ETHNOPHARMACOLOGICAL RELEVANCE: 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. AIM OF THE STUDY: 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. MATERIALS AND METHODS: 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. RESULTS: 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-1ß, 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. CONCLUSION: 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.

Integrated Serum Pharmacochemistry, Network Pharmacology, and Molecular Docking to Study the Mechanism of Rhubarb against Atherosclerosis.

BACKGROUND: Atherosclerosis (AS) is a chronic inflammatory disease characterized by the accumulation of lipids, the formation of lesion plaques, and the narrowing of arterial lumens. Rhubarb has significant effects against AS, but there is a lack of analysis and exploration of the mechanism of action of the transitional components in serum containing rhubarb. OBJECTIVE: This work aims to combine serum pharmacochemistry, network pharmacology, and molecular docking to explore active ingredients and mechanism of rhubarb against AS. METHOD: Firstly, the components of rhubarb in blood samples were identified using HPLC-QTOF/MS. The ingredients-targets-disease interaction network of rhubarb was constructed through network pharmacology. Then, molecular docking between the ingredients and the core targets was carried out using the Autodock Vina software. RESULTS: Eleven active ingredients and five metabolites were preliminarily identified. The network pharmacology results showed that chrysophanol, resveratrol, and emodin might have potential pharmacological effects on AS. The PPI network showed that the key proteins were PTGS2, ESR1, PTGS1, and ELANE. GO analysis revealed that genes were mainly enriched in the inflammatory response and response to exogenous stimuli. Moreover, these genes were related to IL-17 signaling pathways, lipid and atherosclerosis, and other pathways. Molecular docking analyses showed that chrysophanol and emodin have strong binding affinities with the target proteins PTGS2 and PTGS1. CONCLUSION: A comprehensive strategy combining serum pharmacochemistry with network pharmacology and molecular docking was employed to investigate the active ingredients and the mechanism of rhubarb in treating AS, which provided a basis for studying the pharmacological effects and action mechanisms of rhubarb.

Material basis and pharmacodynamic mechanism of YangshenDingzhi granules in the intervention of viral pneumonia: Based on serum pharmacochemistry and network pharmacology.

BACKGROUND: YangshenDingzhi granules (YSDZ) are clinically effective in preventing and treating COVID-19. The present study elucidates the underlying mechanism of YSDZ intervention in viral pneumonia by employing serum pharmacochemistry and network pharmacology. METHODS: The chemical constituents of YSDZ in the blood were examined using ultra-performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry (UPLC-Q-Exactive Orbitrap MS). Potential protein targets were obtained from the SwissTargetPrediction database, and the target genes associated with viral pneumonia were identified using GeneCards, DisGeNET, and Online Mendelian Inheritance in Man (OMIM) databases. The intersection of blood component-related targets and disease-related targets was determined using Venny 2.1. Protein-protein interaction networks were constructed using the STRING database. The Metascape database was employed to perform enrichment analyses of Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways for the targets, while the Cytoscape 3.9.1 software was utilized to construct drug-component-disease-target-pathway networks. Further, in vitro and in vivo experiments were performed to establish the therapeutic effectiveness of YSDZ against viral pneumonia. RESULTS: Fifteen compounds and 124 targets linked to viral pneumonia were detected in serum. Among these, MAPK1, MAPK3, AKT1, EGFR, and TNF play significant roles. In vitro tests revealed that the medicated serum suppressed the replication of H1N1, RSV, and SARS-CoV-2 replicon. Further, in vivo testing analysis shows that YSDZ decreases the viral load in the lungs of mice infected with RSV and H1N1. CONCLUSION: The chemical constituents of YSDZ in the blood may elicit therapeutic effects against viral pneumonia by targeting multiple proteins and pathways.

Lianweng Granules Alleviate Intestinal Barrier Damage via the IL-6/STAT3/PI3K/AKT Signaling Pathway with Dampness-Heat Syndrome Diarrhea.

Dampness-heat syndrome diarrhea (DHSD) is a common clinical disease with a high prevalence but still has no satisfactory therapeutic medicine, so the search for a safe and effective drug candidate is ongoing. This study aims to explore the efficacy and mechanisms of Lianweng granules (LWG) in the treatment of DHSD and to identify the blood transport components of LWG. We assessed the efficacy of LWG in DHSD by various in vivo metrics such as body weight, disease activity index (DAI), histopathologic examination, intestinal barrier function, levels of inflammatory, apoptotic biomarkers, and oxidative stress. We identified the blood components of LWG using ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS), and the resolved key components were used to explore the relevant targets. We next predicted the potential mechanisms of LWG in treating DHSD using network pharmacology and molecular docking based on the relevant targets. Finally, the mechanisms were validated in vivo using RT-qPCR, Western blotting, ELISA, and immunofluorescence and evaluated in vitro using Cell Counting Kit-8 (CCK-8), small interfering RNA, cellular enthusiasm transfer assay (CETSA), and drug affinity response target stability (DARTS). Ninety-one pharmacodynamic components of LWG enter the bloodstream and exert possible therapeutic effects. In vivo, LWG treatment improved body weight, reduced colonic injury and DAI scores, lowered inflammation, oxidative stress, and apoptosis markers, and partially restored intestinal barrier function in DHSD mice. Guided by network pharmacology and molecular docking, it is suggested that LWG may exert therapeutic effects by inhibiting IL-6/STAT3/PI3K/AKT signaling. LWG significantly decreased the expression of IL-6, p-STAT3, p-PI3K, p-AKT, and other proteins. These findings were supported by in vitro experiments, where CETSA, DARTS, and siRNA evidenced LWG's targeting of STAT3. LWG targeted STAT3 to inhibit inflammation, oxidative stress, and apoptosis in the colon, thereby restoring the intestinal barrier function to some extent and exerting a therapeutic effect on DHSD.

To elucidate the mechanism of "Scrophulariae Radix-Fritillaria" in goiter by integrated metabolomics and serum pharmaco-chemistry.

The pharmacodynamic substances in "Scrophulariae Radix-Fritillaria" and the molecular mechanisms underlying its therapeutic effects against goiter were analyzed through metabolomics and serum pharmaco-chemistry. A rat model of goiter was established using propylthiouracil (PTU), and the animals were treated using "Scrophulariae Radix-Fritillaria." The efficacy of the drug pair was evaluated in terms of thyroid gland histopathology and blood biochemical indices. Serum and urine samples of the rats were analyzed by UPLC-Q-TOF/MS. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed to screen potential biomarkers in urine and the corresponding metabolic pathways. The blood components of "Scrophulariae Radix-Fritillaria" were also identified, and their correlation with urine biomarkers was analyzed in order to screen for potential bioactive compounds. "Scrophulariae Radix-Fritillaria" mitigated injury to thyroid tissues and normalized the levels of the thyroid hormones FT3, FT4, and TSH. We also identified 22 urine biomarkers related to goiter, of which 19 were regulated by "Scrophulariae Radix-Fritillaria." Moreover, urine biomarkers are involved in tryptophan metabolism, steroid hormone biosynthesis, and beta-alanine metabolism, and these pathways may be targeted by the drug pair. In addition, 47 compounds of "Scrophulariae Radix-Fritillaria" were detected by serum pharmacochemistry, of which nine components, namely, syringic acid, paeonol, cedrol, and cis-ferulic acid, fetisinine, aucubigenin, linolenic acid, ussuriedine, and 5-(methylsulfanyl)pentanenitrile, were identified as potential effective substances against goiter. To summarize, we characterized the chemical components and mechanisms of "Scrophulariae Radix-Fritillaria" involved in the treatment of goiter, and our findings provide an experimental basis for its clinical application.

Xintongtai Granule: Investigating the serum pharmacology and mechanisms of action against atherosclerosis.

PURPOSE: A serum medicinal chemistry analysis was performed to investigate the pharmacological basis of Xintongtai granule and to predict the potential mechanism of anti-atherosclerotic action based on the blood components. METHODS: UPLC-Q-TOF-MS/MS was used to analyze the in vitro chemical composition and in vivo blood components of Xintongtai granule, and to detect the blood drug concentration. The PPI network was constructed by collecting blood components and disease targets through the network pharmacology method, and the key targets were subjected to GO and KEGG functional enrichment analyses, so as to construct the topology network of drug-component-target-disease, and to validate the network by molecular docking. RESULTS: The UPLC-Q-TOF-MS/MS analysis identified 69 chemical components in Xintongtai granule, including 19 prototype circulating components and 9 metabolites in the bloodstream. Network pharmacology analysis revealed 115 intersecting targets for the circulating components, from which 10 core targets were selected. GO and KEGG analyses unveiled associated signaling pathways and biological processes. The construction of a topology network and preliminary molecular docking provided insights into its mechanism of action. CONCLUSION: The mechanism underlying the anti- atherosclerosis effect of Xintongtai granule may be associated with the intervention of active components such as Cryptotanshinone, Kaempferitrin, and Puerarin in pathways targeting CXCL8, STAT3, TNF, and other related targets.

Integrated serum pharmacochemistry with network pharmacology and pharmacological validation to elucidate the mechanism of yiqitongmai decoction (YQTMD) against myocardial infarction.

ETHNOPHARMACOLOGICAL RELEVANCE: Yiqitongmai decoction (YQTMD), a classic TCM, has been widely used in clinical treatment for MI. However, it is still difficult to clarify the potential active compounds and pharmacological mechanisms of it in treating MI. AIM OF THE STUDY: To explore the active ingredients, pharmacological effects, potential targets and mechanisms of YQTMD against MI. MATERIALS AND METHODS: Serum pharmacochemistry by UPLC-MS/MS was applied to analyze the phytochemical components in serum from YQTMD. These components were then used to predict the potential targets using network pharmacology approach and molecular dynamics simulations, and then the protective effect of them on H9c2 cells following hypoxic conditions was assessed. Afterwards, the pharmacological effects of YQTMD on MI in mice were tested by determining electrocardiogram (ECG), echocardiography, cardiac biomarkers, oxidative stress, inflammation and pathophysiological changes. The protein levels involving STAT3 signal were detected using Western blot and immunofluorescence assays. Furthermore, STAT3 inhibitor Sttatic was employed to further elucidate the underlying mechanisms. RESULTS: Totally, 26 compounds derived from YQTMD were identified in mice serum, and 201 genes associated with the compounds were collected. The compounds including safflomin A, ferulic acid, gypenoside XVII, ginsenoside Rg1 and glycyrrhizic acid were identified as the critical compounds of YQTMD to regulate STAT3 pathway. In vitro, compounds combination significantly enhanced the viability of H9c2 cells and reduced ROS level compared to model cells. The in vivo results showed that YQTMD effectively reduced myocardial injury, as evidenced by the decreased serum cardiac injury markers, reduction in the size of myocardial infarct, restoration of abnormal alterations in ECG and decrease in cardiomyocyte apoptosis. Additionally, YQTMD attenuated MI-induced cardiac dysfunction, alleviated pathological changes, reduced MDA levels, and enhanced SOD and GSH levels compared with model mice. Significantly, the levels of IL-6, IL-1ß, and TNF-α were observed to decrease in the YQTMD group. The expression levels of key proteins (p-STAT3, HIF-1α, NOX2, TLR5 and Caspase3) in STAT3 pathway were also regulated by YQTMD. However, the cardioprotective effects of YQTMD on MI were attenuated by STAT3 inhibitor Sttatic. CONCLUSIONS: This study investigated the active ingredients and potential mechanisms of YQTMD for MI treatment based on serum pharmacochemistry and network pharmacology approaches, revealing that YQTMD exerts its therapeutic effects on MI by alleviating oxidative stress, inflammation and apoptosis through adjusting STAT3 signaling pathway.

Multicomponent qualitative and quantitative analysis of Farfarae Flos in serum using UHPLC-Q TOF-MS.

Farfarae Flos is a traditional herb widely employed for treating coughs, bronchitis, and asthmatic disorders. In the current study, we utilized SWATH and IDA data acquisition modes in combination with multiple data processing techniques to identify Farfarae Flos metabolites in mice serum. A total of 56 compounds were characterized, including 31 phenolic acids, 13 flavonoids, 11 sesquiterpenoids and 1 alkaloid. Further quantitative analysis was conducted on 12 absorbed metabolites, utilizing a newly developed and rigorously validated analytical method. Our approach demonstrated an acceptable level of specificity, accuracy, precision, and stability. When applied to compare the serum of mice treated with FF, all 12 metabolites showed the highest concentration at 0.5 h. Overall, this study presented a novel strategy for unraveling the active compounds of FF via serum pharmacochemistry analysis, which made a foundation for exploring the pharmacodynamic material basis of FF.

Integrated serum pharmacochemistry, network pharmacology, and pharmacokinetics to clarify the effective components and pharmacological mechanisms of the proprietary Chinese medicine Jinkui Shenqi Pill in treating kidney yang deficiency syndrome.

The proprietary Chinese medicine Jinkui Shenqi Pill (PCM-JKSQP) is a classic compound used for the effective clinical treatment of kidney yang deficiency syndrome (KYDS), a metabolic disease accompanied by kidney injury. However, its active ingredients and therapeutic mechanisms are not clear. This study employed serum pharmacochemistry, network pharmacology, and pharmacokinetics (PK) to identify the bioactive components of PCM-JKSQP and preliminarily clarify its mechanism in treating KYDS. One hundred and forty chemical components of PCM-JKSQP, 47 (20 parent compouds and 27 metabolites) of which were absorbed into the blood, were identified by ultra-high-performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). The topological parameters of network pharmacology and high concentrations in blood found six parent components as PK markers (cinnamic acid, paeonol, loganin, morroniside, apigenin, and poricoic acid A). PK analysis further identified these six compounds as active ingredients. Protein-protein interaction (PPI) analysis and molecular docking simulation predicted and verified eight core targets (TP53, ESR1, CTNNB1, EP300, EGFR, AKT1, ERBB2, and TNF). Most were concentrated in the MAPK, HIF-1, and PI3K-AKT signaling pathways, indicating that these six active ingredients may mainly exert therapeutic effects through these three pathways via their core targets. The PK results also showed these six components were absorbed quickly, although cinnamic acid and paeonol were rapidly metabolized, with a short half-life and retention time. Loganin and morroniside did not have high peak concentrations, and apigenin and poricoic acid A had long retention times. This study provides a new overall perspective for exploring the bioactive components and mechanisms underlying the effects of PCM-JKSQP in treating KYDS.

Sangbaipi decoction exerted in vitro and in vivo anti-influenza effect through inhibiting viral proteins.

HEADINGS ETHNOPHARMACOLOGICAL RELEVANCE: Sangbaipi Decoction (SBPD) is an effective treatment for lung diseases caused by phlegm-heat obstruction according to Jingyue Quanshu, and soothes panting by purging the lung meridian. It is composed of anti-pyretic herbs (e.g., Scutellaria baicalensis Georgi and Coptis chinensis Franch.) and antitussive herbs (e.g., Cortex Mori and Armeniacae Semen Amarum). Therefore, we hypothesized that SBPD has therapeutic effects on lung injury caused by influenza virus. AIM OF THE STUDY: This study aimed to explore anti-influenza activity, active components, and mechanisms of SBPD. MATERIALS AND METHODS: The anti-influenza activities of SBPD were determined in 48 h drug-treated MDCK cell model using CPE and plaque reduction assays, and 24 h drug-treated A549 cells using qRT-PCR. The in vivo efficacy of SBPD (1.0 g/kg/day and 0.5 g/kg/day) was evaluated in PR8 infected BALB/c mice. The chemical component was assessed through HPLC-Q-TOF MS/MS analysis. Network pharmacology was built via TCMSP, GeneCards, DisgeNet, OMIM, DrugBank databases, and Cytoscape software. Additionally, TOA, HI and NAI assays were employed to investigate impact on the virus replication cycle with different concentrations of SBPD (2.5 mg/mL, 1.25 mg/mL, or 0.625 mg/mL). RESULTS: In MDCK infected with viruses A/PR/8/34, A/Hong Kong/1/68, or A/California/4/2009, the IC50 values of SBPD were 0.80 mg/mL, 1.20 mg/mL, and 1.25 mg/mL. In A549 cells, SBPD treatment reduced cytokine expression (e.g., TNF-α, IL-6, IL-1ß) (p < 0.05). In PR8 infected BALB/c mice, SBPD improved the survival rate of infected mice, reduced lung index (p < 0.05), protected lung tissue from pathological damage, and regulated cytokine overexpression (p < 0.05). 29 components of SBPD were identified in SBPD treated mouse serum including some phytochemicals targeting influenza proteins. HI and NAI assays suggested the potential antiviral mechanism of SBPD through inhibition of HA and NA. CONCLUSION: This study is the first to demonstrate the anti-influenza and the anti-inflammatory effects of SBPD in vitro and in vivo. Its major anti-influenza phytochemicals were explored and its inhibitory effects on HA and NA protein were proved. It provides more options for anti-influenza drug discovery.

Exploring the inhibitory impact of Mongolian medicinal He-Zi-3 soup on mammary gland hyperplasia in rats induced by estrogen and progestogen.

ETHNOPHARMACOLOGICAL RELEVANCE: Mammary gland hyperplasia, a prevalent benign breast condition, often serves as a precursor to various other breast diseases. He-Zi-3 soup (HZ-3), a traditional Mongolian remedy, is utilized for treating this condition. AIM OF THE STUDY: To explore the effect and underlying mechanism of HZ-3, a Mongolian medicinal preparation, on mammary gland hyperplasia. MATERIALS AND METHODS: This study aimed to assess the impact of different doses of HZ-3 in a rat model of mammary hyperplasia. The active components within HZ-3 drug serum were identified and analyzed through network pharmacology and target prediction. To elucidate the underlying mechanism of HZ-3 in addressing mammary hyperplasia, we conducted a series of investigations on estradiol-induced mammary hyperplasia in model rates. Assessments included measurements of papilla width and height, hematoxylin and eosin staining, Masson staining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunohistochemistry. RESULTS: Our investigation revealed the identification of 21 compounds, primarily terpenoids, through serum medicinal chemistry screening. Utilizing network pharmacological analysis, we observed predominant regulation through the estrogen pathway, closely associated with key genes including esr1,esr2, ncoa1, krt 19, ctsd, ebag 9, and bcl-2. Assessments encompassing nipple height and width, histological examination, immunohistochemical analysis, and serum hormone levels via enzyme-linked immunosorbent assay demonstrated the inhibitory effect of HZ-3 on mammary hyperplasia in rat models. RT-qPCR and Western blot analyses corroborated these findings, affirming the suppression of mammary hyperplasia by HZ-3 through the activation of estrogen pathway signaling.

Integrated Serum Pharmacochemistry and Network Pharmacological Analysis Used To Explore Possible Anti-Aging Mechanisms of the Ginsenosides

BACKGROUND: Although constitutive ginsenosides are credited with ginseng's remarkable anti-aging efficacy, the mechanism of action and bioactive components of ginsenosides are unclear. OBJECTIVE: The goal of the study was to examine the effect of ginsenosides on D-galactose (D-gal)-induced aging in rats and to figure out the underlying molecular mechanism using serum pharmacochemistry and network pharmacology. METHODS: Using behavioral, biochemical indexes, and histological analysis, ginsenosides were evaluated for their anti-aging effects in rats induced by D-gal, and effective ingredients absorbed in the blood were examined by ultra-performance liquid chromatography quadrupole time of flight coupled with mass spectrometry (UPLC-Q/TOF-MS) before being subjected to network pharmacology analysis. RESULTS: As well as improving spatial learning and memory skills, Ginsenosides are known to regulate malondialdehyde (MDA), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity. In addition, it improved the ultrastructure of neurons in D-gal-induced rats' hippocampus. Seventy-four absorption components and metabolites of ginsenosides were identified in aging rat serum. According to a network pharmacology study, ginsenosides have anti-aging properties by modulating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinases (MAPK) signaling pathways. CONCLUSION: The potential mechanisms of the anti-aging effect of ginsenosides involve multiple components, targets, and pathways. These findings serve as a foundation for further research into the processes behind ginsenoside's anti-aging impact.

A LC-MS-based serum pharmacochemistry approach to reveal the compatibility features of mutual promotion/assistance herb pairs in Xijiao Dihuang decoction.

Xijiao Dihuang decoction (XDT), a famous formula, was usually used to improve the prognosis of patients with blood-heat and blood-stasis syndrome-related diseases. There were some mutual promotion and mutual assistance herb pairs in XDT. However, the exact functions of these herb pairs in the compatibility of XDT were not elucidated due to the lack of appropriate methodologies. Based on the theory of serum pharmacochemistry, a systematic method was established for the qualitative and quantitative analysis of characteristic components in the extracts and drug-containing plasma samples of XDT and its relational mutual promotion/assistance herb pairs. For qualitative analysis, 85 characteristic components were identified using the liquid chromatography with triple time-of-flight mass/mass spectrometry (LC-Triple QTOF-MS/MS) based on the mass defect filtering, product ion filtering, neutral loss filtering and isotope pattern filtering techniques. For quantitative detection, a relative quantitation assay using an extract ion chromatogram (EIC) of the full scan MS experiment was validated and employed to assess the quantity of the 85 identified compounds in the test samples of single herb, herb pairs and XDT. The results of multivariate statistical analyses indicated that both the assistant and guide herbs could improve the solubilization of active compounds from the sovereign and minister herbs in XDT in vitro, might change the trans-membrane transportation, and regulate metabolism in vivo. The methods used in present study might be also valuable for the investigation of multiple components from other classic TCM formulas for the purpose of compatibility feature study.

A Serum Pharmacochemistry and Network Pharmacology-based Approach to Study the Anti-depressant Effect of Chaihu-Shugan San.

AIMS: The aim of this study is to explore the anti-depressant mechanism of Chaihu- Shugan San based on serum medicinal chemistry and network pharmacology methods. BACKGROUND: Depression lacks effective treatments, with current anti-depressants ineffective in 40% of patients. Chaihu-Shugan San (CHSGS) is a well-known traditional Chinese medicine compound to treat depression. However, the chemical components and the underlying mechanisms targeting the liver and brain in the anti-depressant effects of CHSGS need to be elucidated. METHODS: The chemical components of CHSGS in most current network pharmacology studies are screened from TCMSP and TCMID databases. In this study, we investigated the mechanism and material basis of soothing the liver and relieving depression in the treatment of depression by CHSGS based on serum pharmacochemistry. The anti-depressant mechanism of CHSGS was further verified by proteomics and high-throughput data. RESULTS: Through serum medicinal chemistry, we obtained 9 bioactive substances of CHSGS. These ingredients have good human oral bioavailability and are non-toxic. Based on liver ChIPseq data, CHSGS acts on 8 targets specifically localized in the liver, such as FGA, FGB, and FGG. The main contributors to CHSGS soothing the liver qi targets are hesperetin, nobiletin, ferulic acid, naringin and albiflorin. In addition, network pharmacology analysis identified 9 blood components of CHSGS that corresponded to 63 anti-depressant targets in the brain. Among them, nobiletin has the largest number of anti-depressant targets, followed by glycyrrhizic acid, ferulic acid, albiflorin and hesperetin. We also validated the anti-depressant mechanism of CHSGS based on hippocampal proteomics. CHSGS exerts anti-depressant effects on synaptic structure and neuronal function by targeting multiple synapse related proteins. CONCLUSION: This study not only provides a theoretical basis for further expanding the clinical application of CHSGS, but also provides a series of potential lead compounds for the development of depression drugs.

Multiple-matrices metabolomics combined with serum pharmacochemistry for discovering the potential targets and active constituents of Qifu decoction against heart failure.

Qifu decoction (QFD) is an ancient traditional Chinese medicine (TCM) prescription for the treatment of heart failure. However, the mechanisms and active constituents of QFD are poorly understood. In this study, multi-matrices metabolomics (serum, urine, and myocardial mitochondria) based on ultra-high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOFMS), were employed for exploring the mechanisms of QFD against heart failure in rat model. Twenty-one, seventeen, and fifteen endogenous metabolite biomarkers associated with heart failure were identified from serum, urine, and myocardial mitochondria datasets, respectively. Fourteen, twelve, and ten of the identified serum, urine, and mitochondria biomarkers were significantly reversed by QFD, respectively. QFD-targeted pathways were involved in TCA cycle, branched chain amino acids metabolism, fatty acid ß-oxidation, sphingolipid metabolism, glycerophospholipid metabolism, arachidonic acid metabolism, tryptophan metabolism, purine metabolism. In addition, QFD-derived constituents in serum were fully analyzed by UHPLC-Q-TOFMS and SUS-plot, and 24 QFD-derived components were identified in serum. Then, the correlation analysis between the QFD-reversed serum biomarkers and QFD-derived constituents in serum was employed to dissect the active constituents of QFD. It was found that eight prototypical components and three metabolites were highly correlated with efficacy and could serve as the active constituents of QFD against heart failure. Finally, neoline and calycosin, which highly correlated with branched-chain amino acid metabolism and fatty acid ß-oxidation, were selected to validate in Na2S2O4-induced cell model. It was found that neoline and calycosin provided a significant protective effect against Na2S2O4-induced cell death in a low dose-dependent manner and increased the expressions of the pathway-related protein CPT1B and BCAT2 in the cell model. In conclusions, these findings provided light on the mechanisms and active constituents of QFD against heart failure. Neoline and calycosin could be selected as potential quality-markers of QFD against heart failure.

Explore the active ingredients and potential mechanism of action on Actinidia arguta leaves against T2DM by integration of serum pharmacochemistry and network pharmacology.

BACKGROUND: Actinidia arguta leaves (AAL) are traditionally consumed as a vegetable and as tea in folk China and Korea. Previous studies have reported the anti-diabetic effect of AAL, but its bioactive components and mechanism of action are still unclear. AIM OF THE STUDY: This study aims to identify the hypoglycemic active components of AAL by combining serum pharmacochemistry and network pharmacology and to elucidate its possible mechanism of action. METHODS: Firstly, the effective components in mice serum samples were characterized by UPLC-Q/TOF-MSE. Furthermore, based on these active ingredients, network pharmacology analysis was performed to establish an "H-C-T-P-D" interaction network and reveal possible biological mechanisms. Finally, the affinity between serum AAL components and the main proteins in the important pathways above was investigated through molecular docking analysis. RESULTS: Serum pharmacochemistry analysis showed that 69 compounds in the serum samples were identified, including 23 prototypes and 46 metabolites. The metabolic reactions mainly included deglycosylation, dehydration, hydrogenation, methylation, acetylation, glucuronidation, and sulfation. Network pharmacology analysis showed that the key components quercetin, pinoresinol diglucoside, and 5-O-trans-p-coumaroyl quinic acid butyl ester mainly acted on the core targets PTGS2, HRAS, RELA, PRKCA, and BCL2 targets and through the PI3K-Akt signaling pathway, endocrine resistance, and MAPK signaling pathway to exert a hypoglycemic effect. Likewise, molecular docking results showed that the three potential active ingredients had good binding effects on the five key targets. CONCLUSION: This study provides a basis for elucidating the pharmacodynamic substance basis of AA against T2DM and further exploring the mechanism of action.

Integrated serum pharmacochemistry and network pharmacology to explore the mechanism of Yi-Shan-Hong formula in alleviating chronic liver injury.

BACKGROUND: Chronic liver injury (CLI) is a complex condition that requires effective therapeutic interventions. The Yi-Shan-Hong (YSH) formula is an empirically derived remedy that has shown effectiveness and safety in the management of chronic liver damage. However, the bioactive components and multifaceted mechanisms of YSH remain inadequately understood. PURPOSE: To examine the bioactive compounds and functional processes that contribute to the therapeutic benefits of YSH against CLI. METHODS: Serum pharmacochemistry and network pharmacology were employed to identify active compounds and possible targets of YSH in CLI. In addition, YSH was also given in three doses to d-(+)-galactosamine hydrochloride (D-GalN) -induced CLI rats to test its therapeutic efficacy. RESULTS: The analysis of serum samples successfully detected 25 compounds from YSH. Searches on the databases resulted in 277 genes as being correlated with chemicals in YSH, and 397 genes associated with CLI. In vivo experiments revealed that YSH displayed a notable therapeutic impact on liver injury caused by d-GalN. This was evidenced by enhanced liver function and histopathological improvements, reduced oxidative stress response, proinflammatory factors, and fibrosis levels. Importantly, no discernible adverse effects were observed. Furthermore, the administration of YSH treatment reversed the activation of AKT phosphorylation caused by d-GalN, aligning with the findings of the network pharmacology study. CONCLUSION: These findings provide preclinical evidence of YSH's therapeutic value in CLI and highlight its hepatoprotective action via the PI3K/AKT signaling pathway.

Integrating serum pharmacochemistry and network pharmacology to explore the molecular mechanisms of Acanthopanax senticosus (Rupr. & Maxim.) Harms on attenuating doxorubicin-induced myocardial injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Acanthopanax senticosus (Rupr. & Maxim.) Harms (AS), also known as Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. or Siberian ginseng, has a rich history of use as an adaptogen, a substance believed to increase the body's resistance to stress, fatigue, and infectious diseases. As a traditional Chinese medicine, AS is popular for its cardioprotective effects which can protect the cardiovascular system from hazardous conditions. Doxorubicin (DOX), on the other hand, is a first-line chemotherapeutic agent against a variety of cancers, including breast cancer, lung cancer, gastric cancer, and leukemia, etc. Despite its effectiveness, the clinical use of DOX is limited by its side effects, the most serious of which is cardiotoxicity. Considering AS could be applied as an adjuvant to anticancer agents, the combination of AS and DOX might exert synergistic effects on certain malignancies with mitigated cardiotoxicity. Given this, it is necessary and meaningful to confirm whether AS would neutralize the DOX-induced cardiotoxicity and its underlying molecular mechanisms. AIM OF THE STUDY: This paper aims to validate the cardioprotective effects of AS against DOX-induced myocardial injury (MI) while deciphering the molecular mechanisms underlying such effects. MATERIALS AND METHODS: Firstly, the cardioprotective effects of AS against DOX-induced MI were confirmed both in vitro and in vivo. Secondly, serum pharmacochemistry and network pharmacology were orchestrated to explore the in vivo active compounds of AS and predict their ways of functioning in the treatment of DOX-induced MI. Finally, the predicted mechanisms were validated by Western blot analysis during in vivo experiments. RESULTS: The results demonstrated that AS possessed excellent antioxidative ability, and could alleviate the apoptosis of H9C2 cells and the damage to mitochondria induced by DOX. In vivo experiments indicated that AS could restore the conduction abnormalities and ameliorate histopathological changes according to the electrocardiogram and cardiac morphology. Meanwhile, it markedly downregulated the inflammatory factors (TNF-α, IL-6, and IL-1ß), decreased plasma ALT, AST, LDH, CK, CK-MB, and MDA levels, as well as increased SOD and GSH levels compared to the model group, which collectively substantiate the effectiveness of AS. Afterward, 14 compounds were identified from different batches of AS-dosed serum and selected for mechanism prediction through HPLC-HRMS analysis and network pharmacology. Consequently, the MAPKs and caspase cascade were confirmed as primary targets among which the interplay between the JNK/Caspase 3 feedback loop and the phosphorylation of ERK1/2 were highlighted. CONCLUSIONS: In conclusion, the integrated approach employed in this paper illuminated the molecular mechanism of AS against DOX-induced MI, whilst providing a valuable strategy to elucidate the therapeutic effects of complicated TCM systems more reliably and efficiently.

Based on Serum Pharmacochemistry and Virtual Screening Technique Analysis Used to Explore the Potential Active Ingredients and Possible Anti- Coronary Heart Disease Mechanisms of Salvia miltiorrhiza Bunge.

AIMS AND OBJECTIVE: This study aimed to identify the bioactive compounds and explore the multi-target mechanisms of Salvia miltiorrhiza Bunge (SMB) against coronary heart disease (CHD) using an integrated serum pharmacochemistry and network pharmacology approach. MATERIALS AND METHODS: The chemical constituents of SMB were characterized by UPLC-MS. The absorbed ingredients and metabolites after oral SMB administration were identified in rat serum. Therapeutic targets of SMB against CHD were predicted by intersecting the targets of absorbed compounds from databases and CHD-associated genes. Protein-protein interaction network, pathway analysis, molecular docking, and molecular dynamic simulation were performed. RESULTS: A total of 61 SMB-derived compounds were identified in rat serum. Network analysis revealed 111 candidate targets highly related to CHD pathways. Further topological analysis identified 10 hub targets and 20 key active compounds, constructing an informative compoundtarget- pathway network. PTGS2 and TNF were predicted as primary targets of SMB against CHD based on molecular dynamic simulation. CONCLUSION: This integrated approach identified bioactive compounds and multi-target mechanisms of SMB against CHD. The results provide scientific evidence supporting SMB's clinical efficacy and reveal potential anti-CHD targets.

Rationality of the ethanol precipitation process in modern preparation production of Zishui-Qinggan decoction evaluated by integrating UPLC-QTOF-MS/MS-based chemical profiling/serum pharmacochemistry and network pharmacology.

INTRODUCTION: Zishui-Qinggan decoction (ZQD) is a classical traditional Chinese medicine formula (TCMF) for alleviating menopausal symptoms (MPS) induced by endocrine therapy in breast cancer patients. In the production of TCMF modern preparations, ethanol precipitation (EP) is a commonly but not fully verified refining process. OBJECTIVES: Chemical profiling/serum pharmacochemistry and network pharmacology approaches were integrated for exploring the rationality of the EP process in the production of ZQD modern preparations. MATERIAL AND METHODS: Ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) was applied to identify the chemical profiles and absorbed components of ZQD. Network pharmacology was used to identify targets and pathways related to MPS-relieving efficacy. RESULTS: The chemicals of ZQDs without/with EP process (referred to as ZQD-W and ZQD-W-P, respectively) were qualitatively similar with 89 and 87 components identified, respectively, but their relative contents were different; 51 components were detectable in the serum of rats orally administered with ZQD-W, whereas only 19 were detected in that administered with ZQD-W-P. Key targets, such as AKT1, and pathways, such as the PI3K-Akt signalling pathway, affected by ZQD-W and ZQD-W-P were similar, while the neuroactive ligand-receptor interaction pathway among others and the MAPK signalling pathway among others were specific pathways affected by ZQD-W and ZQD-W-P, respectively. The specifically absorbed components of ZQD-W could combine its specific key targets. CONCLUSION: The EP process quantitatively altered the chemical profiles of ZQD, subsequently affected the absorbed components of ZQD, and then affected the key targets and pathways of ZQD for relieving MPS. The EP process might result in variation of the MPS-relieving efficacy of ZQD, which deserves further in vivo verification.