Identification and functional analysis of the hub Ferroptosis-Related gene EZH2 in diabetic kidney disease.

BACKGROUND: Diabetic kidney disease (DKD) is a common microvascular complication and one of the main causes of death in diabetes. Ferroptosis, an iron-dependent mode of cell death characterized by lipid ROS accumulation, was found to be associated with a number of diseases and has great potential for kidney diseases. It has great value to identify potential ferroptosis-related genes and their biological mechanisms in DKD. METHODS: We obtained the GSE30122 dataset from Gene Expression Omnibus (GEO) database and ferroptosis-related genes from the Ferrdb database. After differential expression analysis, and three machine learning algorithms, the hub ferroptosis-related gene EZH2 was identified. In order to investigate the function of EZH2, Gene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA) and single cell analysis were conducted. The expression of EZH2 was validated in DKD patients, HK-2 cell models and DKD mouse models. EZH2 knockdown HK-2 cells and HK-2 cells treated with GSK126 were performed to verify whether EZH2 affected ferroptosis in DKD. CHIP assay was used to detect whether EZH2 regulated ferroptosis by suppressing SLC7A11. Molecular docking was performed to explore EZH2 and four traditional Chinese medicine (Sennoside A, Berberine, Umbelliferone, Platycodin D) related to ferroptosis in DKD treatment. RESULTS: According to the GSE30122 dataset in GEO and ferroptosis-related genes from the Ferrb database, we obtained the hub ferroptosis-related gene EZH2 in DKD via diversified machine learning methods. The increasing of EZH2 expression was shown in single cell analysis, DKD patients, DKD mouse models and high glucose induced DKD cell models. Further study showed that EZH2 knockdown and inhibition can alleviate HG-induced ferroptosis in vitro. CHIP assay showed EZH2-mediated epigenetic silencing regulated the expression of SLC7A11. Molecular docking results showed that EZH2 had strong binding stability with Sennoside A, Berberine, Umbelliferone, and Platycodin D. CONCLUSION: Overall, our data shouwed that histone H3K27 methyltransferase EZH2 could regulate the renal tubular epithelial cell ferroptosis by suppressing SLC7A11 in DKD, which may serve as a credible reliable indicator for diagnosing DKD and a potential target for treatment.

Integrated strategy of LC-MS and network pharmacology for predicting active constituents and pharmacological mechanisms of Ranunculus japonicus Thunb. for treating rheumatoid arthritis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ranunculus japonicus Thunb. (short for R. japonicus) is a topically applied herb with the activities of removing jaundice, nebula and edema, preventing malaria, stopping asthma, promoting diuresis and relieving pain. It was firstly recorded in Zhouhou Beiji Fang and has been used for the treatment of malaria, ulcers, carbuncle, jaundice, migraine, stomachache, toothache and arthritis for over 1800 years. AIM OF THE STUDY: This study aimed to uncover the potentially effective components of R. japonicus and the pharmacological mechanisms against rheumatoid arthritis (RA) by combing LC-MS and network pharmacology. MATERIALS AND METHODS: Firstly, the chemical constituents of R. japonicus were qualitatively identified by UPLC-ESI-LTQ-Orbitrap MS. Then we performed target prediction by PharmMapper, protein-protein interaction (PPI) analysis via String, GO and KEGG pathway enrichment analysis by DAVID and constructed the compound-target-pathway network using Cytoscape. Thirdly, crucial compounds in the network were quantitatively analyzed to achieve quality control of R. japonicus. Finally, the pharmacological activities of R. japonicus and two potentially bioactive ingredients were validated in RA-FLSs (Rheumatoid Arthritis Fibroblast-like Synoviocytes) in vitro. RESULTS: Overall fifty-four ingredients of R. japonicus were identified and forty-five components were firstly discovered in R. japonicus. Among them, twenty-seven validated compounds were predicted to act on twenty-five RA-related targets and they might exhibit therapeutic effects against RA via positive regulation of cell migration, etc. Nine potentially bioactive components of R. japonicus which played important roles in the compound-target-pathway network were simultaneously quantified by an optimized UPLC-ESI-Triple Quad method. In vitro, compared to control group, R. japonicus extract, berberine and yangonin significantly inhibited the migration capacity of RA-FLSs after 24 h treatment. CONCLUSION: This study clarified that R. japonicus and the bioactive ingredients berberine and yangonin might exert therapeutic actions for RA via suppressing the aggressive phenotypes of RA-FLSs through combined LC-MS technology and network pharmacology tools for the first time. The present research provided deeper understanding into the chemical profiling, pharmacological activities and quality control of R. japonicus and offered reference for further scientific research and clinical use of R. japonicus in treating RA.

Effect of Huangqin Tang on Urine Metabolic Profile in Rats with Ulcerative Colitis Based on UPLC-Q-Exactive Orbitrap MS.

As a classic prescription, Huangqin Tang (HQT) has been widely applied to treat ulcerative colitis (UC), although its pharmacological mechanisms are not clear. In this study, urine metabolomics was first analysed to explore the therapeutic mechanisms of HQT in UC rats induced by TNBS. We identified 28 potential biomarkers affected by HQT that might cause changes in urine metabolism in UC rats, mapped the network of metabolic pathways, and revealed how HQT affects metabolism of UC rats. The results showed that UC affects amino acid metabolism and biosynthesis of unsaturated fatty acids and impairs the tricarboxylic acid cycle (TCA cycle). UC induced inflammatory and gastrointestinal reactions by inhibiting the transport of fatty acids and disrupting amino acid metabolism. HQT plays key roles via regulating the level of biomarkers in the metabolism of amino acids, lipids, and so on, normalizing metabolic disorders. In addition, histopathology and other bioinformatics analysis further confirm that HQT altered UC rat physiology and pathology, ultimately affecting metabolic function of UC rats.

Effect of Huangqin Tang on Colonic Gene Expression in Rats with Ulcerative Colitis.

In this study, we explored the pharmacological mechanisms of Huangqin Tang (HQT; a traditional Chinese medicine formula) in ulcerative colitis (UC) and provided evidence for potential roles HQT plays by gene expression profiling. The UC rat model was made via a compound method (trinitrobenzene sulfonic acid plus ethanol). After a ten-day treatment, microarray analysis was performed from the colon segment of the rats. Biological functions and specific signaling pathways were enriched based on differentially expressed genes (DEG), and corresponding gene networks were constructed via Ingenuity Pathway Analysis (IPA). Through the network, we screened the potential "candidate targets," such as ITGB1, FN1, CASP3, and ITGA5 and FABP1, ABCB1, FABP2, and SLC51B. These potential candidate targets were functionally related to immune responses, inflammation, and metabolism. Moreover, HQT significantly decreased serum levels of proinflammatory factors nitrogen monoxide (NO), proinflammatory cytokines interleukin- (IL-) 17, and prostaglandin E2 (PGE2). The degree of HE staining of colonic tissue was severe in the model group but reduced significantly in the HQT group. HQT exhibited protective effects against colon damage by inhibiting the inflammatory response.

Hepatotoxicity evaluation of Euphorbia kansui on zebrafish larvae in vivo.

BACKGROUND: Euphorbia kansui is effective in treating various diseases, such as ascites and edema, but its liver toxicity is a major obstacle in its wide use in the clinic. However, further investigations have suggested that Euphorbia kansui can cause liver injury. HYPOTHESIS: The study aims to investigate the effect of Euphorbia kansui exposure on zebrafish, and explain the underlying toxicity mechanisms from a comprehensive perspective. STUDY DESIGN: The 4dpf zebrafish larvae were exposed to Euphorbia kansui at a sub-lethal concentration. METHODS: We evaluated the effect of Euphorbia kansui on the ultrastructure and function of the liver, apoptosis of liver cells by PCR and western blot, and metabolic profile by GC-MS based on sub-lethal concentrations. RESULTS: Our results suggested Euphorbia kansui could lead to liver injury and significant alteration of the metabolomics of the zebrafish larvae in sub-lethal concentration conditions. It could also induce alterations in liver microstructure, hepatic function, gene expression and protein associated with the apoptosis process, as well as endogenous metabolism. KEGG pathway analysis identified some biological processes on the basis of different metabolisms and their associated processes especially for amino acid metabolism. CONCLUSION: The results bring us closer to an in-depth understanding of the toxic effects of Euphorbia kansui on zebrafish liver, which will be significantly helpful in effectively guiding safer clinical application of this herb in the clinic. Furthermore, our results also showed the zebrafish model is reliable for evaluation of Euphorbia kansui extract hepatotoxicity and as a methodological reference for the evaluation of Traditional Chinese Medicine with underlying liver toxicity.

Analysis of pharmacological mechanisms and targets mining of Wuzi-Yanzong-Wan for treating non-obstructive oligoasthenospermia.

As a classical traditional Chinese medicine, Wuzi-Yanzong-Wan (WZYZW) has been widely applied for several centuries to treat non-obstructive oligoasthenozoospermia (NOA), although its pharmacological mechanisms remain largely unknown. In this study, both plasma and urine metabolomics profiling was first analysed to explore the therapeutic mechanisms of WZYZW in NOA rats induced by removal of the unilateral testicle. Then, 106 identified compounds comprising WZYZW (our previous work), for which putative targets were discovered using systems pharmacology, were systematically analysed via mRNA microarrays to validate their putative targets. Finally, metabolomics-tested WZYZW-regulated metabolites were connected with validated targets using Spearman correlation analysis to further confirm the targets from a biological perspective. The results suggested that WZYZW plays key roles in modulating the concentrations of 18 metabolites in the metabolism of amino acids, lipids and so on, normalizing the metabolic phenotype and regulating metabolic disorders. Moreover, 27 targets of WZYZW (23 compounds) against NOA were validated, and metabolomics-tested metabolites were also found to be significantly related to these identified targets, suggesting that these targets and compounds are worthy of further research. This work offers the first systematic investigation of the efficacy of WZYZW against NOA and illustrates a practicable approach for explaining the molecular mechanisms of multicomponent drugs.

Phytochemistry, pharmacology, quality control and future research of Forsythia suspensa (Thunb.) Vahl: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Forsythiae Fructus (called Lianqiao in Chinese), the fruit of Forsythia suspensa (Thunb.) Vahl, is utilized as a common traditional medicine in China, Japan and Korea. It is traditionally used to treat pyrexia, inflammation, gonorrhea, carbuncle and erysipelas. Depending on the different harvest time, Forsythiae Fructus can be classified into two forms, namely Qingqiao and Laoqiao. The greenish fruits that start to ripen are collected as Qingqiao, while the yellow fruits that are fully ripe are collected as Laoqiao. Both are applied to medical use. This review aims to provide a systematic summary of F. suspensa (Forsythia suspensa (Thunb.) Vahl) and to reveal the correlation between the traditional uses and pharmacological activities so as to offer inspiration for future research. MATERIALS AND METHODS: All corresponding information about F. suspensa was searched by Scifinder and obtained from scientific databases including Springer, Science Direct, Wiley, Pubmed and China Knowledge Resource Integrated (CNKI). Local dissertations and books were searched as well. RESULTS: According to classical Chinese herbal texts and Chinese Pharmacopoeia, Forsythiae Fructus dominantly displays heat-clearing and detoxifying effects in TCM prescriptions. In modern research, more than 230 compounds were separated and identified from F. suspensa. 211 Of them were isolated from fruits. Lignans and phenylethanoid glycosides are considered as the characteristic and active constituents of this herb, such as forsythiaside, phillyrin, rutin and phillygenin. They exhibited anti-inflammatory, antioxidant, antibacterial, anti-virus, anti-cancer and anti-allergy effects, etc. Currently, there is no report on the toxicity of Forsythiae Fructus, despite slight toxicity of forsythiaside reported in local publications. Compared to Laoqiao, Qingqiao contains higher levels of forsythiaside, forsythoside C, cornoside, rutin, phillyrin, gallic acid and chlorogenic acid and lower levels of rengyol, ß-glucose and S-suspensaside methyl ether. CONCLUSION: Heat-clearing actions of Forsythiae Fructus are based on the anti-inflammatory and antioxidant properties of lignans and phenylethanoid glycosides. Detoxifying effects attribute to the antibacterial, antiviral and anti-cancer activities of Forsythiae Fructus. And traditional Chinese medicine (TCM) characteristics of Forsythiae Fructus (bitter flavor, slightly cold nature and lung meridian) supported its strong anti-inflammatory effects. In addition, the remarkable anti-inflammatory and antioxidant capacities of Forsythiae Fructus contribute to its anti-cancer and neuroprotective activities. The higher proportion of lignans and phenylethanoid glycosides in Qingqiao than Laoqiao might explain the better antioxidant ability of Qingqiao and more frequent uses of Qingqiao in TCM prescriptions. For future research, more in vivo experiments and clinical studies are encouraged to further clarify the relation between traditional uses and modern applications. Regarding to Qingqiao and Laoqiao, they remain to be differentiated by all-round quality control methods, and the chemical compositions and clinical effects between them should be compared.

Analysis of Chemical Constituents in Wuzi-Yanzong-Wan by UPLC-ESI-LTQ-Orbitrap-MS.

Wuzi-Yanzong-Wan (WZYZW), a classical traditional Chinese medicine (TCM) prescription containing Fructus Lych, Semen Cuscutae (fried), Fructus Rubi, Fructus Schisandrae chinensis (steamed) and Semen Plantaginis (fried with salt), is widely used to treat impotence, sterility, spermatorrhea, premature ejaculation, lumbago and post-micturation dribble. However, the chemical profile of WZYZW has not been established yet. In this work, a rapid and sensitive method for systematically screening and identifying the chemical constituents of WZYZW in both positive and negative ion modes using Ultra-Performance LC coupled with ESI-linear ion trap-Orbitrap tandem mass spectrometry (UPLC-ESI-LTQ-Orbitrap-MS) has been developed. Based on the chromatographic and spectrometric data, and referring to the literature, we could tentatively identify 106 compounds, including organic acids, flavonoids, phenylpropanoids, alkaloids and terpenoids. Fourteen ingredients from Fructus Lych were identified, while 10 ingredients were from Semen Cuscutae (fried), 33 ingredients were from Fructus Rubi, 37 ingredients were from Fructus Schisandrae chinensis (steamed), and 20 ingredients were from Semen Plantaginis (fried with salt). The results may provide essential data for further quality control, pharmacological research and clinical evaluation of WZYZW. Furthermore, this study indicates the developed approach based on UPLC-ESI-LTQ-Orbitrap-MS is suitable for characterizing the chemical profiles of TCM prescriptions. This is the first report to provide a comprehensive analysis of the chemical constituents of WZYZW.

Metabolite profiling and pathway analysis of acute hepatitis rats by UPLC-ESI MS combined with pattern recognition methods.

BACKGROUND & AIMS: Metabolomics is comprehensive analysis of low-molecular-weight endogenous metabolites in a biological sample. It could enable mapping of perturbations of early biochemical changes in diseases and hence provide an opportunity to develop predictive biomarkers that could provide valuable insights into the mechanisms of diseases. The aim of this study was to elucidate the changes in endogenous metabolites and to phenotype the metabolic profiling of d-galactosamine (GalN)-inducing acute hepatitis in rats by UPLC-ESI MS. METHODS: The systemic biochemical actions of GalN administration (ip, 400 mg/kg) have been investigated in male wistar rats using conventional clinical chemistry, liver histopathology and metabolomic analysis of UPLC- ESI MS of urine. The urine was collected predose (-24 to 0 h) and 0-24, 24-48, 48-72, 72-96 h post-dose. Mass spectrometry of the urine was analysed visually and via conjunction with multivariate data analysis. RESULTS: Results demonstrated that there was a time-dependent biochemical effect of GalN dosed on the levels of a range of low-molecular-weight metabolites in urine, which was correlated with developing phase of the GalN-inducing acute hepatitis. Urinary excretion of beta-hydroxybutanoic acid and citric acid was decreased following GalN dosing, whereas that of glycocholic acid, indole-3-acetic acid, sphinganine, n-acetyl-l-phenylalanine, cholic acid and creatinine excretion was increased, which suggests that several key metabolic pathways such as energy metabolism, lipid metabolism and amino acid metabolism were perturbed by GalN. CONCLUSION: This metabolomic investigation demonstrates that this robust non-invasive tool offers insight into the metabolic states of diseases.

Metabolomics coupled with proteomics advancing drug discovery toward more agile development of targeted combination therapies.

To enhance the therapeutic efficacy and reduce the adverse effects of traditional Chinese medicine, practitioners often prescribe combinations of plant species and/or minerals, called formulae. Unfortunately, the working mechanisms of most of these compounds are difficult to determine and thus remain unknown. In an attempt to address the benefits of formulae based on current biomedical approaches, we analyzed the components of Yinchenhao Tang, a classical formula that has been shown to be clinically effective for treating hepatic injury syndrome. The three principal components of Yinchenhao Tang are Artemisia annua L., Gardenia jasminoids Ellis, and Rheum Palmatum L., whose major active ingredients are 6,7-dimethylesculetin (D), geniposide (G), and rhein (R), respectively. To determine the mechanisms underlying the efficacy of this formula, we conducted a systematic analysis of the therapeutic effects of the DGR compound using immunohistochemistry, biochemistry, metabolomics, and proteomics. Here, we report that the DGR combination exerts a more robust therapeutic effect than any one or two of the three individual compounds by hitting multiple targets in a rat model of hepatic injury. Thus, DGR synergistically causes intensified dynamic changes in metabolic biomarkers, regulates molecular networks through target proteins, has a synergistic/additive effect, and activates both intrinsic and extrinsic pathways.