Naringenin attenuated airway cilia structural and functional injury induced by cigarette smoke extract via IL-17 and cAMP pathways.

BACKGROUND: Cigarette smoke impairs mucociliary clearance via mechanisms such as inflammatory response and oxidative injury, which in turn induces various respiratory diseases. Naringenin, a naturally occurring flavonoid in grapes and grapefruit, has exhibited pharmacological properties such as anti-inflammatory, expectorant, and antioxidant properties. However, it is still unclear whether naringenin protects airway cilia from injury caused by cigarette smoke. PURPOSE: This study aimed to investigate the effect of naringenin on cigarette smoke extract (CSE)-induced structural and functional abnormalities in airway cilia and highlight the potential regulatory mechanism. METHODS: Initially, network pharmacology was used to predict the mechanism of action of naringenin in ciliary disease. Next, HE staining, immunofluorescence, TEM, qRT-PCR, western blot, and ELISA were performed to assess the effects of naringenin on airway cilia in tracheal rings and air-liquid interface (ALI) cultures of Sprague Dawley rats after co-exposure to CSE (10% or 20%) and naringenin (0, 25, 50, 100 µM) for 24 h. Finally, transcriptomics and molecular biotechnology methods were conducted to elucidate the mechanism by which naringenin protected cilia from CSE-induced damage in ALI cultures. RESULTS: The targets of ciliary diseases regulated by naringenin were significantly enriched in inflammation and oxidative stress pathways. Also, the CSE decreased the number of cilia in the tracheal rings and ALI cultures and reduced the ciliary beat frequency (CBF). However, naringenin prevented CSE-induced cilia damage via mechanisms such as the downregulation of cilia-related genes (e.g., RFX3, DNAI1, DNAH5, IFT88) and ciliary marker proteins such as DNAI2, FOXJ1, and ß-tubulin IV, the upregulation of inflammatory factors (e.g., IL-6, IL-8, IL-13), ROS and MDA. IL-17 signaling pathway might be involved in the protective effect of naringenin on airway cilia. Additionally, the cAMP signaling pathway might also be related to the enhancement of CBF by naringenin. CONCLUSION: In this study, we first found that naringenin reduces CSE-induced structural disruption of airway cilia in part via modulation of the IL-17 signaling pathway. Furthermore, we also found that naringenin enhances CBF by activating the cAMP signaling pathway. This is the first report to reveal the beneficial effects of naringenin on airway cilia and the potential underlying mechanisms.

Naringenin regulates cigarette smoke extract-induced extracellular vesicles from alveolar macrophage to attenuate the mouse lung epithelial ferroptosis through activating EV miR-23a-3p/ACSL4 axis.

BACKGROUND: Alveolar macrophages are one of the momentous regulators in pulmonary inflammatory responses, which can secrete extracellular vesicles (EVs) packing miRNAs. Ferroptosis, an iron-dependent cell death, is associated with cigarette smoke-induced lung injury, and EVs have been reported to regulate ferroptosis by transporting intracellular iron. However, the regulatory mechanism of alveolar macrophage-derived EVs has not been clearly illuminated in smoking-related pulmonary ferroptosis. Despite the known anti-ferroptosis effects of naringenin in lung injury, whether naringenin controls EVs-mediated ferroptosis has not yet been explored. PURPOSE: We explore the effects of EVs from cigarette smoke-stimulated alveolar macrophages in lung epithelial ferroptosis, and elucidate the EV miRNA-mediated pharmacological mechanism of naringenin. STUDY DESIGN AND METHODS: Differential and ultracentrifugation were conducted to extract EVs from different alveolar macrophages treatment groups in vitro. Both intratracheal instilled mice and treated epithelial cells were used to investigate the roles of EVs from alveolar macrophages involved in ferroptosis. Small RNA sequencing analysis was performed to distinguish altered miRNAs in EVs. The ferroptotic effects of EV miRNAs were examined by applying dual-Luciferase reporter assay and miRNA inhibitor transfection experiment. RESULTS: Here, we firstly reported that EVs from cigarette smoke extract-induced alveolar macrophages (CSE-EVs) provoked pulmonary epithelial ferroptosis. The ferroptosis inhibitor ferrostatin-1 treatment reversed these changes in vitro. Moreover, EVs from naringenin and CSE co-treated alveolar macrophages (CSE+Naringenin-EVs) markedly attenuated the lung epithelial ferroptosis compared with CSE-EVs. Notably, we identified miR-23a-3p as the most dramatically changed miRNA among Normal-EVs, CSE-EVs, and CSE+Naringenin-EVs. Further experimental investigation showed that ACSL4, a pro-ferroptotic gene leading to lipid peroxidation, was negatively regulated by miR-23a-3p. The inhibition of miR-23a-3p diminished the efficacy of CSE+Naringenin-EVs. CONCLUSION: Our findings firstly provided evidence that naringenin elevated the EV miR-23a-3p level from CSE-induced alveolar macrophages, thereby inhibiting the mouse lung epithelial ferroptosis via targeting ACSL4, and further complemented the mechanism of cigarette-induced lung injury and the protection of naringenin in a paracrine manner. The administration of miR-23a-3p-enriched EVs has the potential to ameliorate pulmonary ferroptosis.

Identification of the active compounds in the Yi-Fei-San-Jie formula using a comprehensive strategy based on cell extraction/UPLC-MS/MS, network pharmacology, and molecular biology techniques.

BACKGROUND: Chinese herbal formulae has multiple active constituents and targets, and the good clinical response is encouraging more scientists to explore the bio-active ingredients in such complex systems. Yi-Fei-San-Jie formula (YFSJF) is commonly used to treat patients with lung cancer in South China; however, its bio-active ingredients remain unknown. PURPOSE: We investigated the bio-active ingredients of the YFSJF using a novel comprehensive strategy. METHODS: A549 cell extraction coupled with ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was used for the screening of potential bio-active ingredients. Network pharmacology approach and molecular dynamics simulation were performed for the screening of targets. Surface plasmon resonance (SPR) assay and molecular biology techniques were used to verify the targets. RESULTS: Nine A549 cell membrane-binding compounds were identified through cell extraction/UPLC-MS/MS. Five compounds, namely ginsenoside Ro, ginsenoside Rb1, ginsenoside Rc, peimisine, and peimine were cytotoxic to A549 cells, and they were considered the bio-active ingredients of the YFSJF in vitro. Network pharmacology analysis revealed that TGFBR2 is the key target and the TGFß pathway is the key pathway targeted by YFSJF in non-small cell lung cancer. Peimisine showed an affinity to TGFBR2 using molecular docking and dynamic stimulation, which was confirmed using surface plasmon resonance spectroscopy. The molecular biology-based analysis further confirmed that peimisine targets TGFBR2 and can reverse A549 epithelial-mesenchymal transition by inhibiting the TGFß pathway. CONCLUSION: Taken together, cell extraction/UPLC-MS/MS, network pharmacology, and molecular biology-based analysis comprise a feasible strategy to explore active ingredients in YFSJF.

Integrative network fusion-based multi-omics study for biomarker identification and patient classification of rheumatoid arthritis.

BACKGROUND: Cold-dampness Syndrome (RA-Cold) and Hot-dampness Syndrome (RA-Hot) are two distinct groups of rheumatoid arthritis (RA) patients with different clinical symptoms based on traditional Chinese medicine (TCM) theories and clinical empirical knowledge. However, the biological basis of the two syndromes has not been fully elucidated, which may restrict the development of personalized medicine and drug discovery for RA diagnosis and therapy. METHODS: An integrative strategy combining clinical transcriptomics, phenomics, and metabolomics data based on clinical cohorts and adjuvant-induced arthritis rat models was performed to identify novel candidate biomarkers and to investigate the biological basis of RA-Cold and RA-Hot. RESULTS: The main clinical symptoms of RA-Cold patients are joint swelling, pain, and contracture, which may be associated with the dysregulation of T cell-mediated immunity, osteoblast differentiation, and subsequent disorders of steroid biosynthesis and phenylalanine metabolism. In contrast, the main clinical symptoms of RA-Hot patients are fever, irritability, and vertigo, which may be associated with various signals regulating angiogenesis, adrenocorticotropic hormone release, and NLRP3 inflammasome activation, leading to disorders of steroid biosynthesis, nicotinamide, and sphingolipid metabolism. IL17F, 5-HT, and IL4I1 were identified as candidate biomarkers of RA-Cold, while S1P and GLNS were identified as candidate biomarkers of RA-Hot. CONCLUSIONS: The current study presents the most comprehensive metabonomic and transcriptomic profiling of serum, urine, synovial fluid, and synovial tissue samples obtained from RA-Cold and RA-Hot patients and experimental animal models to date. Through the integration of multi-omics data and clinical independent validation, a list of novel candidate biomarkers of RA-Cold and RA-Hot syndromes were identified, that may be useful in improving RA diagnosis and therapy.

The activation of histone deacetylases 4 prevented endothelial dysfunction: A crucial mechanism of HuangqiGuizhiWuwu Decoction in improving microcirculation dysfunction in diabetes.

ETHNOPHARMACOLOGICAL RELEVANCE: The regulation of epigenetic factors is considered a crucial target for solving complex chronic diseases such as cardio-cerebrovascular diseases. HuangqiGuizhiWuwu Decoction (HGWWD), a classic Chinese prescription, is mainly used to treat various vascular diseases. Although our previous studies reported that HGWWD could effectively prevent vascular dysfunction in diabetic rodent models, the precise mechanism is still elusive. AIM OF THE STUDY: In this study, we investigated the epigenetic mechanisms of modulating the damage of vascular endothelial cells in diabetes by HGWWD. METHODS: We first analyzed common active components of HGWWD by using HPLC-Q-TOF-MS/MS analysis, and predicted the isoforms of histone deacetylase (HDAC) that can potentially combine the above active components by systems pharmacology. Next, we screened the involvement of specific HDAC isoforms in the protective effect of HGWWD on vascular injury by using pharmacological blockade combined with the evaluation of vascular function in vivo and in vitro. RESULTS: Firstly, HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, HDAC7, SIRT2, and SIRT3 have been implicated with the possibility of binding to the thirty-one common active components in HGWWD. Furthermore, the protective effect of HGWWD is reversed by both TSA (HDAC inhibitor) and MC1568 (class II HDAC inhibitor) on vascular impairment accompanied by reduced aortic HDAC activity in STZ mice. Finally, inhibition of HDAC4 blocked the protective effect of HGWWD on microvascular and endothelial dysfunction in diabetic mice. CONCLUSIONS: These results prove the key role of HDAC4 in diabetes-induced microvascular dysfunction and underlying epigenetic mechanisms for the protective effect of HGWWD in diabetes.

A Mechanism Exploration for the Yi-Fei-San-Jie Formula against Non-Small-Cell Lung Cancer Based on UPLC-MS/MS, Network Pharmacology, and In Silico Verification.

Non-small-cell lung cancer (NSCLC) is one of the most prevalent cancers worldwide. A Yi-Fei-San-Jie formula (YFSJF), widely used in NSCLC treatment in south China, has been validated in clinical studies. However, the pharmacological mechanism behind it remains unclear. In this study, 73 compounds were identified using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), with 58 enrolled in network pharmacology. The protein-protein interaction network, functional enrichment analysis, and compound-target-pathway network were constructed using 74 overlapping targets from 58 drugs and NSCLC. YFSJF has many targets and pathways in the fight against NSCLC. PIK3R1, PIK3CA, and AKT1 were identified as key targets, and the PI3K/AKT pathway was identified as the key pathway. According to the Human Protein Atlas (THPA) database and the Kaplan-Meier Online website, the three key targets had varying expression levels in normal and abnormal tissues and were linked to prognosis. Molecular docking and dynamics simulations verified that hub compounds have a strong affinity with three critical targets. This study revealed multiple compounds, targets, and pathways for YFSJF against NSCLC and suggested that YFSJF might inhibit PIK3R1, PIK3CA, and AKT1 to suppress the PI3K/AKT pathway and play its pharmacological role.

Value of dynamic contrast-enhanced magnetic resonance imaging in combination with mammography for screening early-stage breast cancer.

Objective: To study the value of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in combination with mammography for screening early-stage breast cancer. Methods: Ninety-three female patients visiting Zhejiang Zhuji Hospital of Traditional Chinese Medicine from January 2020 to March 2022 were enrolled to receive DCE-MRI and mammography. The diagnostic efficiencies of different methods were assessed with pathological diagnosis as the golden standard. The factors affecting diagnostic sensitivity were investigated based on clinicopathological characteristics. Results: Forty-one patients were diagnosed as malignant pathological changes by DCE-MRI, and the signs were unclear boundary with surrounding tissues and irregular or unsmooth edges. The maximum linear slope and ratio of the maximum linear SlopeR of malignant pathological changes were significantly larger than those of benign pathological changes (P<0.05). Forty-five patients were diagnosed as malignant pathological changes by mammography combined with DCE-MRI. Compared to single diagnosis method, the combined diagnosis had significantly increased sensitivity, specificity, accuracy, positive predictive value and negative predictive value, and decreased rates of missed diagnosis and misdiagnosis (P<0.05). Lesion diameter was an independent risk factor affecting the diagnostic sensitivity (P<0.05). Conclusion: Mammography and DCE-MRI play key roles in the early diagnosis of breast cancer, and their combination can increase the diagnostic efficiency.

A Novel Drug Combination of Mangiferin and Cinnamic Acid Alleviates Rheumatoid Arthritis by Inhibiting TLR4/NFκB/NLRP3 Activation-Induced Pyroptosis.

Growing evidence shows that Baihu-Guizhi decoction (BHGZD), a traditional Chinese medicine (TCM)-originated disease-modifying anti-rheumatic prescription, may exert a satisfying clinical efficacy for rheumatoid arthritis (RA) therapy. In our previous studies, we verified its immunomodulatory and anti-inflammatory activities. However, bioactive compounds (BACs) of BHGZD and the underlying mechanisms remain unclear. Herein, an integrative research strategy combining UFLC-Q-TOF-MS/MS, gene expression profiling, network calculation, pharmacokinetic profiling, surface plasmon resonance, microscale thermophoresis, and pharmacological experiments was carried out to identify the putative targets of BHGZD and underlying BACs. After that, both in vitro and in vivo experiments were performed to determine the drug effects and pharmacological mechanisms. As a result, the calculation and functional modularization based on the interaction network of the "RA-related gene-BHGZD effective gene" screened the TLR4/PI3K/AKT/NFκB/NLRP3 signaling-mediated pyroptosis to be one of the candidate effective targets of BHGZD for reversing the imbalance network of "immune-inflammation" during RA progression. In addition, both mangiferin (MG) and cinnamic acid (CA) were identified as representative BACs acting on that target, for the strong binding affinities between compounds and target proteins, good pharmacokinetic features, and similar pharmacological effects to BHGZD. Notably, both BHGZD and the two-BAC combination of MG and CA effectively alleviated the disease severity of the adjuvant-induced arthritis-modified rat model, including elevating pain thresholds, relieving joint inflammation and bone erosion via inhibiting NF-κB via TLR4/PI3K/AKT signaling to suppress the activation of the NLRP3 inflammasome, leading to the downregulation of downstream caspase-1, the reduced release of IL-1ß and IL-18, and the modulation of GSDMD-mediated pyroptosis. Consistent data were obtained based on the in vitro pyroptosis cellular models of RAW264.7 and MH7A cells induced by LPS/ATP. In conclusion, these findings offer an evidence that the MG and CA combination identified from BHGZD may interact with TLR4/PI3K/AKT/NFκB signaling to inhibit NLRP3 inflammasome activation and modulate pyroptosis, which provides the novel representative BACs and pharmacological mechanisms of BHGZD against active RA. Our data may shed new light on the mechanisms of the TCM formulas and promote the modernization development of TCM and drug discovery.

Platycladus orientalis seed extract as a potential triple reuptake MAO inhibitor rescue depression phenotype through restoring monoamine neurotransmitters.

ETHNOPHARMACOLOGY RELEVANCE: Platycladus orientalis seeds are recorded in traditional Chinese medicine (TCM) formulations for modulation of mood and physical activity in "Shen Nong Ben Cao Jing" and "Compendium of Materia Medica" and so on. Recently, we identified its extracting components and looked for the potentials in treatment for depression by improving the function of monoamine neurotransmitters. AIM OF THE STUDY: We investigated the mechanism of action of the seed extracts of P. orientalis (S4) to rescue depressive behavior in a chronic, unpredicted, mild stress (CUMS)-induced model in rats. MATERIALS AND METHODS: We used ultra-fast liquid chromatography coupled with triple quadrupole-time of flight tandem mass spectrometry to analyze the chemical constituents in S4. An assay platform in zebrafish and molecular docking were used to analyze if S4 regulated rest/wake behavior and predict the biological targets which correlated with monoamine neurotransmitters. Depressive-behavior tests (body weight, sucrose preference test, tail-suspension test, forced-swimming test) were carried in the CUMS model. After behavior tests and killing, rat brains were separated into the hippocampus, frontier cortex and dorsal raphe nucleus. The main monoamine neurotransmitters and their metabolite concentrations in these three brain regions were measured by rapid resolution liquid chromatography coupled with triple quadrupole tandem mass spectrometry. RESULTS: Forty-one compounds were identified in S4, including fatty acids, terpenoids, amino acids, plant sterols and flavonoids. S4 could increase the total rest time and decrease the waking activity of zebrafish. S4 showed high correlation with adrenaline agonists, 5-hydroxytryptamine (5-HT) reuptake inhibitors and dopamine agonists. CUMS-group rats, compared with controls, had significantly decreased body weight and preference for sucrose water, whereas the immobility time in the tail-suspension test and forced-swimming test was increased. S4 could significantly rescue the increased levels of 5-HT, noradrenaline and dopamine in the prefrontal cortex and dorsal raphe nucleus. CONCLUSIONS: We demonstrated that S4 was a potential inhibitor of MAO reuptake that could rescue depression in a CUMS-model rats by restoring monoamine neurotransmitters in different encephalic regions.

Chemical components analysis and in vivo metabolite profiling of Jian'er Xiaoshi oral liquid by UHPLC-Q-TOF-MS/MS.

Jian'er Xiaoshi oral liquid (JEXS), a traditional Chinese medicine (TCM) prescription, has been principally applied to treat spleen deficiency with gastrointestinal dysfunction in children caused by improper diet. However, due to a lack of research on the holistic component and metabolism of JEXS, the bioactive components of it remain unclear, hindering further study on its quality control and in vivo activity mechanism. In present study, an integrated analysis strategy based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) was established to systematically screen the components and the in vivo xenobiotics of JEXS. Totally 142 compounds in JEXS were characterized, 54 of which were identified. Besides, 178 xenobiotics were detected, including 52 prototypes and 126 metabolites, while the in vivo metabolic modes of chrysin-C-glycosyls and sinapinic acid derivatives were elucidated for the first time. Our investigation gave a comprehensive analysis of the compounds and metabolic characteristics of JEXS which indicated the direction of finding the bioactive ingredients and will provide an important basis for quality control and further study on the pharmacodynamic mechanism of JEXS.

Chemical composition, quality control, pharmacokinetics, pharmacological properties and clinical applications of Fufang Danshen Tablet: A systematic review.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang Danshen Tablet (FDT) is a traditional Chinese medicine (TCM) formula composed of three Chinese medicinal materials comprising Salviae Miltiorrhizar Radix et Rhizoma (Dan-Shen in Chinese), Notoginseng Radix et Rhizoma (San-Qi), and Borneolum Syntheticum (Bing-Pian). It has been documented to exert significant effects in promoting blood circulation and removing blood stasis, and become a frequently used formula in the treatment of cardiovascular and cerebrovascular diseases. AIM OF THE REVIEW: To systematically analyze and summarize the research findings concerning the chemical composition, quality control, pharmacokinetics, pharmacological properties, clinical applications, and toxicity of FDT, so as to point out some typical problems and provides opinions for future study. MATERIALS AND METHODS: Literatures involving FDT were collected from online scientific databases including China National Knowledge Infrastructure, WanFang Data, PubMed, Science Direct, Scopus, Web of Science, Springer Link, SciFinder, and Google Scholar up to March 2021. All eligible studies are analyzed and summarized in this review. RESULTS: This review summarizes reported results concerning the post-marketing quality and efficacy of FDT. Some problems are pointed out for FDT. Hereon we propose several directions for future study: (a) improvement of quality control based on exact overall chemical profiles, entire production process monitoring, and biopotency-associated multi-index content determination method; (b) clarification of functional mechanisms focused on pharmacokinetic profiles in human, interplay with gut microbiota, and integration of multi-omics technologies; (c) reconfirmation of clinical effectiveness and safety from large-scale clinical studies based on evidence-based medicine. CONCLUSIONS: FDT is a typical TCM formula in treating cardiovascular and cerebrovascular diseases, but there are also some troubles. Future studies should focus on the improvement of quality control, the clarification of functional mechanisms, as well as the reconfirmation of clinical effectiveness and safety.

Common mechanism of Citrus Grandis Exocarpium in treatment of chronic obstructive pulmonary disease and lung cancer.

Objective: "Same treatment for different diseases" is a unique treatment strategy in traditional Chinese medicine. Two kinds of malignant respiratory diseases endanger human health-chronic obstructive pulmonary disease (COPD) and lung cancer. Citrus Grandis Exocarpium (Huajuhong in Chinese, HJH), a famous herbal, is always applied by Chinese medicine practitioners to dispersion the lung to resolve phlegm based on "syndrome differentiation and treatment" theory. However, the common mechanism for HJH's treatment of COPD and lung cancer is not clear. Methods: In this study, based on network pharmacology and molecular docking technology, the common mechanism of HJH in the treatment of COPD and lung cancer was studied. The active ingredients and related targets of HJH were integrated from TCMSP, BATMAN-TAM, STP, and Pubchem databases. The standard names of these targets were united by UniProt database. Targets of COPD and lung cancer were enriched through GeneCards, NCBI (Gene), Therapeutic Target Database, and DisGeNET (v7.0) databases. Then the intersection targets of HJH and diseases were obtained. The STRING network and the Cytoscape 3.7.2 were used to construct PPI network, the DAVID database was used to perform GO and KEGG analysis. Then Cytoscape 3.6.1 was used to build "ingredient-target-signal pathway" network. Finally, AutoDock 1.5.6 software was used to perform molecular docking of key proteins and molecules. Results: Eleven active ingredients in HJH were obtained by searching the database, corresponding to 184 HJH-COPD-lung cancer targets intersection. The results of biological network analysis showed that naringenin, the active component in HJH, could mainly act on target proteins such as AKT1, EGFR. Then through positive regulation of vasoconstriction and other biological processes, naringenin could regulate estrogen signaling pathway, VEGF signaling pathway, HIF-1 signaling pathway, ErbB signaling pathway, PI3K-Akt signaling pathway to play an important role in the treatment of both COPD and lung cancer. Conclusion: Network pharmacology was employed to systematically investigate the active ingredients and targets of HJH in treatment of COPD and lung cancer. And then, the common pharmacodynamic network of HJH for the two malignant respiratory diseases was firstly described. Furthermore, naringenin was proved to strongly bind with AKT1 and EGFR. It may provide the scientific basis for understanding the "Same treatment for different diseases" strategy in traditional Chinese medicine and inspirit subsequent drug discovery for COPD, lung cancer and other malignant lung diseases.

A review on the chemical profiles, quality control, pharmacokinetic and pharmacological properties of Fufang Xueshuantong Capsule.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang Xueshuantong Capsule (FXC) is a traditional Chinese medicine (TCM) formula composed of four herbs including Panax notoginseng, Astragalus membranaceus, Salvia miltiorrhiza, and Scrophularia ningpoensis. Long-term and extensive clinical applications have confirmed that FXC could exert significant effects on fundus, cardiovascular and cerebrovascular occlusive diseases. AIM OF THE REVIEW: To systematically analyze and summarize the existing researches involving quality and efficacy re-evaluation of FXC, point out the typical problems, and further propose some opinions to contribute to future study. MATERIALS AND METHODS: Literatures concerning FXC were collected from online scientific databases including China National Knowledge Infrastructure, WanFang Data, PubMed, Science Direct, Scopus, Web of Science, Springer Link up to June 2020. All eligible studies are analyzed and summarized in this review. RESULTS: This review outlines the chemical profiles, quality control, pharmacokinetic and pharmacological properties of FXC based on reported results. Some problems are pointed out for FXC: the quality control needs further improvement, the pharmacokinetic properties have not been comprehensively investigated, and in-depth and systematic mechanism researches are scarce. Hereon we propose several directions for future study: (a) establishment of feasible HPLC or LC-MS based quantitative methods for simultaneous determination of multiple components to monitor the overall quality; (b) pharmacokinetic studies concerning humans, drug-drug interactions, and correlation with pharmacodynamics; (c) pharmacological mechanism researches integrating multi-omics technologies (gut microbiome, metabolomics, etc.). CONCLUSIONS: This review highlights the researches on quality and efficacy re-evaluation of FXC, and points out some typical problems. Further in-depth studies should focus on the promotion of quality control, pharmacokinetic properties, and pharmacological mechanism.

Pharmacokinetics and biotransformation investigation in beagle dog of active compounds from naoxintong capsule.

Naoxintong Capsule (NXTC), a standardized herbal medicine, has been widely applied in treating cardiovascular and cerebrovascular diseases with remarkable efficacy. However, the efficacy contributing components of NXTC are unclear, and the in vivo absorption and metabolism processes of NXTC remain largely obscured. In this study, using beagle dog as model species, we have identified and tentatively characterized 25 prototype and 15 catabolites of NXTC in beagle dog plasma by ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS). We have proposed the in vivo bio-transformation pathways of these absorbed constituents. In addition, for six crucial components, we have developed a quantitative method and conducted plasma pharmacokinetic study of these six components by rapid resolution liquid chromatography tandem triple quadrupole mass spectrometry (RRLC-QQQ-MS/MS). In conclude, our study provided comprehensive insights into the understanding of the plasma absorbed components profiling of NXTC as well as their in vivo transformation behaviors, which would be of great value for identifying efficacy contributing critical components as well as mechanism related investigations of NXTC in the future.

Integrating Pharmacology and Gut Microbiota Analysis to Explore the Mechanism of Citri Reticulatae Pericarpium Against Reserpine-Induced Spleen Deficiency in Rats.

Citri Reticulatae Pericarpium (CRP), dried peels of Citrus reticulata Blanco and its cultivars, is an important traditional Chinese medicine for the treatment of spleen deficiency-related diseases. To date, the mechanism of CRP alleviating spleen deficiency has not been well investigated. This study aimed to explore corresponding mechanisms with integrating pharmacology and gut microbiota analysis. Firstly, the therapeutic effects of CRP against spleen deficiency were evaluated in reserpine-treated rats. CRP was found to effectively relieve the typical symptoms of spleen deficiency, including poor digestion and absorption capacity, and disorder in gastrointestinal hormones, immune cytokines and oxidative stress. Secondly, high throughput 16S rRNA gene sequencing revealed that CRP could not only up-regulate some short-chain fatty acids producing and anti-inflammatory bacteria but also down-regulate certain spleen deficiency aggravated related bacteria, eventually led to the rebalance of gut microbiota in spleen deficiency rats. In addition, a total of 49 compounds derived from CRP were identified in rat urine using ultra-high performance liquid chromatography-quadrupole- time of flight tandem mass spectrometry. Network pharmacology analysis showed that apigenin, luteolin, naringenin, hesperidin, hesperetin, homoeriodictyol, dihydroxy-tetramethoxyflavone, and monohydroxy-tetramethoxyflavone were the core bioactive components for CRP against spleen deficiency. Further Gene Ontology analysis and pathway enrichment suggested that therapeutic effects of CRP against spleen deficiency involved multiple pathways such as tumor necrosis factor signaling, hypoxia-inducible factor-1 signaling and Toll-like receptor signaling pathway. These results would help to understand the mechanism of CRP alleviating spleen deficiency and provide a reference for further studies.

Modulation of the Aß-Peptide-Aggregation Pathway by Active Compounds From Platycladus orientalis Seed Extract in Alzheimer's Disease Models.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neuronal loss, cognitive impairment, and aphasia. Aggregation of ß-amyloid (Aß) peptide in the brain is considered a key mechanism in the development of AD. In the past 20 years, many compounds have been developed to inhibit Aß aggregation and accelerate its degradation. Platycladus orientalis seed is a traditional Chinese medicine used to enhance intelligence and slow aging. We previously found that Platycladus orientalis seed extract (EPOS) inhibited Aß-peptide aggregation in the hippocampus and reduced cognitive deficits in 5×FAD mice. However, the mechanisms of these effects have not been characterized. To characterize the protective mechanisms of EPOS, we used a transgenic Caenorhabditis elegans CL4176 model to perform Bioactivity-guided identification of active compounds. Four active compounds, comprising communic acid, isocupressic acid, imbricatolic acid, and pinusolide, were identified using 13C-and 1H-NMR spectroscopy. Furthermore, we showed that isocupressic acid inhibited Aß generation by modulating BACE1 activity via the GSK3ß/NF-κB pathway in HEK293-APPsw cells. These findings showed that EPOS reduced cognitive deficits in an AD model via modulation of the Aß peptide aggregation pathway.

Protective effects of Naoxintong capsule alone and in combination with ticagrelor and atorvastatin in rats with Qi deficiency and blood stasis syndrome.

CONTEXT: Naoxintong Capsule (NXT), a Chinese medicine, has been widely used for the treatment of coronary heart disease (CHD) in clinics. OBJECTIVE: This study evaluated the cardioprotective effects of NXT alone and in combination with ticagrelor (TIC) and atorvastatin (ATO). MATERIALS AND METHODS: Qi deficiency and blood stasis rats were established by 8 weeks high fat diet feeding and 16 days exhaustive swimming and randomly divided into seven groups, that is, NXT (250, 500 and 1000 mg/kg/d), TIC (20 mg/kg/d), ATO (8 mg/kg/d), NXT (500 mg/kg/d)+TIC (20 mg/kg/d) and NXT (500 mg/kg/d)+ATO (8 mg/kg/d) group, with oral administration for 12 weeks. The contents of TC, TG, LDL-C, HDL-C, IL-1ß, IL-6, IL-8, TNF-α, AST, ALT, SOD, MDA, CK-MB, LDH, TXA2, PGI2, IgA, IgG, IgM and C3 in serum were measured. RESULTS: NXT + TIC group was significantly superior to the TIC group in decreasing the levels of TC (4.34 vs. 5.54), TG (3.37 vs. 4.66), LDL-C (1.21 vs. 1.35), LDH (4919.71vs. 5367.19) and elevating SOD level (248.54 vs. 192.04). NXT + ATO group was significantly superior to the ATO group in decreasing the levels of AST (195.931 vs. 241.63), ALT (71.26 vs. 83.16), LDH (4690.05 vs. 5285.82), TXA2 (133.73 vs. 158.67), IgG (8.08 vs. 9.80), C3 (2.03 vs. 2.35) and elevating the levels of HDL-C (1.19 vs. 0.91), SOD (241.91vs. 209.49). CONCLUSIONS: The present findings demonstrate that the combined use of NXT with TIC and ATO had better integrated regulating effects than TIC and ATO, respectively. The mechanism of action requires further research.

Discovery of the possible mechanisms in kouyanqing granule for treatment of oral ulcers based on network pharmacology.

BACKGROUND: Oral ulcer diseases are complex inflammatory diseases caused by multi-factors, which severely impact patient quality of life. Kouyanqing Granule (KYQG) has been used to treat inflammatory diseases of the mouth and throat, including recurrent aphthous stomatitis (RAS), traumatic ulcers, oral leukoplakia and so on. However, the underlying molecular mechanisms of KYQG in treating these diseases are still unclear. We aimed to explore the possible mechanisms in KYQG for the treatment of oral ulcers. METHODS: An innovative network pharmacology method was established by incorporating targets searching and fishing, network analysis, and silico validation to discover the pharmacological mechanisms of action of KYQG for the treatment of oral ulcers. Then, we verified the reliability of this method by an animal experiment. RESULTS: Our data indicated that a total of 47 key targets were screened, which mainly involved in three function modules including the inhibition of inflammation, the regulation of immunological response, and the suppression of oxidative stress. The implementation of these functions relies on the complex multi-pathways network, especially TNF signaling pathway and HIF-1 signaling pathway. The results of the experimental verification indicated that KYQG significantly inhibited the serum levels of cyclooxygenase-2 (COX2), matrix metalloproteinase 9 (MMP9) and tumor necrosis factor-alpha (TNF-α) in rats with oral ulcer. CONCLUSION: KYQG exhibited the therapeutic effects on oral ulcers probably by inhibiting inflammation, regulating immunological response, and suppressing oxidative stress through a complex multi-pathways network. Particularly, TNF signaling pathway and HIF-1 signaling pathway may play crucial roles in the protection of KYQG against oral ulcers. This work not only offers a method for understanding the functional mechanisms of KYQG for treating oral ulcer diseases from a multi-scale perspective but also may provide an efficient way for research and development of complex composition formula.

Integration of molecular networking and fingerprint analysis for studying constituents in Microctis Folium.

Microctis Folium is the dried leaves of a plant (Microcos paniculata L.) used to improve the digestive system, alleviate diarrhoea, and relieve fever, but information regarding its chemical composition has rarely been reported. The traditional research approach of determining chemical composition has included isolating, purifying, and identifying compounds with high-cost and time-consuming processes. In this study, molecular networking (MN) and fingerprint analysis were integrated as a comprehensive approach to study the chemical composition of Microctis Folium by an ultra fast liquid chromatography-photo diode array detector-triple-time of flight-tandem mass spectrometry (UFLC-DAD-Triple TOF-MS/MS). Large numbers of mass spectrometric data were processed to identify constituents, and the identified compounds and their unknown analogues were comprehensively depicted as visualized figures comprising distinct families by MN. A validated fingerprint methodology was established to quantitatively determine compounds in Microctis Folium. Ultimately, 165 constituents were identified in Microctis Folium for the first time and the identified compounds and approximately five hundred unknown analogues were applied to create visualized figures by MN, indicating compound groups and their chemical structure analogues in Microctis Folium. The validated fingerprint methodology was indicated to be specific, repeatable, precise, and stable and was used to determine 15 batches of samples during three seasons in three districts. Furthermore, seasonal or geographic environmental influences on the chemical profile were estimated by principal coordinate analysis. The results can be used to control the quality of Microctis Folium, observe seasonal or geographic environmental influences on the chemical profiles, and provide a reference for further exploitation of potential active unknown analogues in the future.

Pharmacodynamic and Metabolomics Studies on the Effect of Kouyanqing Granule in the Treatment of Phenol-Induced Oral Ulcer Worsened by Sleep Deprivation.

Oral ulcers are the most prevalent oral mucosal diseases globally, and no specific treatment schemes are currently available due to the complexity of oral ulcer diseases. Sleep deprivation increases the risk of a deterioration in oral health. Kouyanqing Granule (KYQG) has been used for decades in China to treat inflammatory diseases of the mouth and throat associated with the hyperactivity of fire due to yin deficiency syndrome. However, the mechanisms underlying the effects of KYQG in the treatment of oral ulcers are still unclear. The aims of this study were to investigate whether KYQG treatment could attenuate the symptoms of oral ulcers worsened by sleep deprivation and identify the involved metabolic pathways. First, we conducted chemical profiling of KYQG via UPLC-MS analysis. We then combined pharmacological and metabolomics approaches in a phenol-induced rat model of oral ulcers worsened by sleep deprivation. A total of 79 compounds were initially identified. Our observations showed that KYQG treatment induced a significantly higher healing rate in oral ulcers worsened by sleep deprivation. KYQG significantly reduced the levels of 5-HT and GABA in serum, and only decreased the 5-HT level in brain tissue after phenol injury followed by sleep deprivation. Moreover, KYQG administration significantly suppressed systemic inflammation by inhibiting TNF-α, IL-1ß, IL-6, IL-18, and MCP-1. Immunohistochemical analysis further revealed that KYQG inhibited IL-6 expression in buccal mucosa tissues. KYQG treatment also significantly decreased the serum levels of ACTH, CORT, IgM, and 8-OHdG. Serum metabolomics analysis showed that a total of 30 metabolites showed significant differential abundances under KYQG intervention, while metabolic pathway analysis suggested that the altered metabolites were associated with the dysregulation of eight metabolic pathways. Taken together, our results indicated that KYQG attenuates the symptoms of oral ulcers worsened by sleep deprivation probably through the regulation of the neuroimmunoendocrine system, oxidative stress levels, and tryptophan metabolism. This study also provides a novel approach for addressing the increased health risks resulting from sleep deficiency using an herbal medicine formula.