Integration of metabolomics and network pharmacology to reveal the protective mechanism underlying Wogonoside in acute myocardial ischemia rats.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional medicine, both Scutellaria baicalensis Georgi (SBG) and the traditional formulas composed of it have been used to treat a wide range of diseases, including cancer and cardiovascular. Wogonoside (Wog) is the biologically active flavonoid compound extracted from the root of SBG, with potential cardiovascular protective effects. However, the mechanisms underlying the protective effect of Wog on acute myocardial ischemia (AMI) have not yet been clearly elucidated. AIM OF THE STUDY: To explore the protective mechanism of Wog on AMI rats by comprehensively integrating traditional pharmacodynamics, metabolomics, and network pharmacology. METHODS: The rat was pretreatment with Wog at a dose of 20 mg/kg/d and 40 mg/kg/d once daily for 10 days and then ligated the left anterior descending coronary artery of rats to establish the AMI rat model. Electrocardiogram (ECG), cardiac enzyme levels, heart weight index (HWI), Triphenyltetrazolium chloride (TTC) staining, and histopathological analyses were adopted to evaluate the protective effect of Wog on AMI rats. Moreover, a serum metabolomic-based UHPLC-Q-Orbitrap MS approach was performed to find metabolic biomarkers and metabolic pathways, and network pharmacology analysis was applied to predict targets and pathways of Wog in treating AMI. Then, the network pharmacology and metabolomic results were integrated to elucidate the mechanism of Wog in treating AMI. Finally, RT- PCR was used to detect the mRNA expression levels of PTGS1, PTGS2, ALOX5, and ALOX15 to validate the result of integrated metabolomics and network analysis. RESULTS: Pharmacodynamic studies suggest that Wog could effectively prevent the ST-segment of electrocardiogram elevation, reduce the myocardial infarct size, heart weight index, and cardiac enzyme levels, and alleviate cardiac histological damage in AMI rats. Metabolomics analysis showed that the disturbances of metabolic profile in AMI rats were partly corrected by Wog and the cardio-protection effects on AMI rats involved 32 differential metabolic biomarkers and 4 metabolic pathways. In addition, the integrated analysis of network pharmacology and metabolomics showed that 7 metabolic biomarkers, 6 targets, and 6 crucial pathways were the main mechanism for the therapeutic application of Wog for AMI. Moreover, the results of RT-PCR showed that PTGS1, PTGS2, ALOX5, and ALOX15 mRNA expression levels were reduced after treatment with Wog. CONCLUSION: Wog exerts cardio-protection effects on AMI rats via the regulation of multiple metabolic biomarkers, multiple targets, and multiple pathways, our current study will provide strong scientific evidence supporting the therapeutic application of Wog for AMI.

Metabolomics Study of the Hepatoprotective Effects and Mechanism of Aqueous Extract of Dendrobium nobile Lindl. on Alcoholic Liver Injury in Rats.

BACKGROUND: Dendrobium nobile Lindl. (DNL) is effective for the treatment of alcoholic liver disease (ALD), but the underly mechanism is still unclear. OBJECTIVES: This research aimed to investigate the effects and mechanism of the aqueous extract of Dendrobium nobile Lindl (AEDNL) in ALD rats based on a metabolomics approach. MATERIALS AND METHODS: In this study, 18 Sprague-Dawley male rats were randomly divided into control, model, and AEDNL groups (n=six). Rats in the AEDNL group were given AEDNL (152 mg/kg) intragastric administration from the first day for 30 consecutive days. From day 15 to day 30, model and AEDNL groups were given 30% ethanol (10 ml/kg) after 4 h of daily administration. Then, serum and liver samples were collected for biochemical analysis, histopathological examination, and Ultra Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-TOF/MS) determination for metabolomic analysis. RESULTS: Compared with the model group, the liver/body weight index and serum levels of TC, LDL-C, and TBIL in the AEDNL group were significantly decreased. Hepatocyte cord arrangement, hepatocyte balloon, and fat vacuolization were significantly improved in the AEDNL group. Metabolism profiles were changed in the model and AEDNL groups. Seven and two common differential metabolites (Guanosine3',5'-cyclic monophosphate, and Glutaric acid) were found in serum and liver, respectively. In addition, the hepatoprotective effect of AEDNL on ALD was related to steroid hormone biosynthesis, riboflavin metabolism, and glycerophospholipid metabolism. CONCLUSION: The research could provide novel evidence of the protective effects of AEDNL on ALD.

Mice kidney biometabolic process analysis after cantharidin exposure using widely-targeted metabolomics combined with network pharmacology.

Cantharidin (CTD) is a principal bioactive component of traditional Chinese medicine Mylabris used in cancer treatment. However, CTD clinical application is limited due to nephrotoxicity, and the mechanism is unknown. The present study used widely-targeted metabolomics, network pharmacology, and cell experiments to investigate the nephrotoxicity mechanism after CTD exposure. In mice exposed to CTD, serum creatinine and urea nitrogen levels increased with renal injury. Then, 74 differential metabolites were detected, including 51 up-regulated and 23 down-regulated metabolites classified as amino acids, small peptides, fatty acyl, arachidonic acid metabolite, organic acid, and nucleotides. Sixteen metabolic pathways including tyrosine, sulfur, and pyrimidine metabolism were all disrupted in the kidney. Furthermore, network pharmacology revealed that 258 metabolic targets, and pathway enrichment indicated that CTD could activate oxidative phosphorylation and oxidative stress (OS). Subsequently, HK-2 cell experiments demonstrated that CTD could reduce superoxide dismutase while increasing malondialdehyde levels. In conclusion, after CTD exposure, biometabolic processes may be disrupted with renal injury in mice, resulting in oxidative phosphorylation and OS.

Exploring the protective mechanisms of total tannins from Geum japonicum var. chinense F.Bolle in mice with hematopoietic dysfunction via the JAK2/STAT3/5 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Geum japonicum var. chinense F.Bolle (Rnglish name Gei herba, GH), a traditional Miao medicine, promotes hematopoiesis. Emerging evidence shows that total tannins of GH (TGH) can treat ischemic diseases. AIM OF THE STUDY: To explore the protective mechanism of TGH in hematopoietic dysfunction (HD) mice. MATERIALS AND METHOD: Forty-eight female mice were randomly assigned to 6 groups: control, model, Zhenqi Fuzheng positive, and three doses TGH. Cyclophosphamide was injected in mice to establish an HD model. Spleen tissue was examined histomorphologically, peripheral hemograms and organ index were calculated, and serum hematopoietic factor levels were determined. The expression of proteins in the Janus kinase 2 (JAK2)/transcription 3/5 (STAT3/5) pathway, as well as upstream and downstream proteins, was examined using western blot to elucidate the underlying protective mechanisms of TGH. RESULTS: TGH could effectively alleviate spleen tissue damage in HD mice, improve peripheral hemogram and antagonize organ atrophy, and increase levels of Granulocyte-macrophage Colony Stimulating Factor (GM-CSF) and Erythropoietin (EPO) in HD mouse serum. Furthermore, after TGH treatment, the protein expression levels of P-JAK2, P-STAT3, P-STAT5, M-CSF, G-CSF, Bcl-2, and Bcl-xL were significantly higher than in the model group. At the same time, following TGH treatment, the protein expression levels of LC3 A/B, Beclin1, ATG5, and ATG7 were significantly lower than in the model group. CONCLUSIONS: TGH has been shown to protect HD mice through a mechanism linked to the activation of the JAK2/STAT3/5 pathway, as well as autophagy inhibition and apoptosis activation.

Endoplasmic reticulum stress contributes to autophagy and apoptosis in cantharidin-induced nephrotoxicity.

Mylabris, as a natural product of traditional Chinese medicine (TCM), exhibiting typical antitumor activity, and cantharidin (CTD) is the major bioactive component. However, drug-induced nephrotoxicity (DIN) extremely limited its clinical application. In this study, we proved that activation of the endoplasmic reticulum (ER) stress-dependent PERK/CHOP pathway exerts a toxic role in rats and HK-2 cells through inducing autophagy and apoptosis. Results showed that CTD could cause renal function damage, cytotoxicity, and apoptosis. The ER dilatation and autolysosomes were observed after CTD treatment. Furthermore, the distribution of LC3, ATF4, and CHOP proteins was observed in the nucleus and cytoplasm. In addition, the mRNA levels of ER stress-regulated genes (PERK, eIF2α, CHOP, and ATF4) were increased, and the expression levels of GRP78, ATF4, CHOP, LC3, Beclin-1, Atg3, Atg7, Caspase 3, and Bax/Bcl-2 proteins were increased both in vitro and in vivo. Consistently, this upregulation could be inhibited by an ER stress inhibitor 4-Phenylbutyric acid (4-PBA), indicating that ER stress is partly responsible for activation of autophagy and apoptosis in CTD-induced DIN. In conclusion, CTD could induce DIN by triggering ER stress, further activating autophagy and apoptosis both in vivo and in vitro.

Effective Material Basis and Mechanism Analysis of Compound Banmao Capsule against Tumors Using Integrative Network Pharmacology and Molecular Docking.

PURPOSE: Compound banmao capsule (CBC), a well-known traditional Chinese medical material, is known to inhibit various tumors. However, its material basis and pharmacological mechanisms remain to be elucidated. This study aimed to investigate the effective material basis and mechanisms of action of CBC against tumors. METHODS: Active compounds of CBC were identified using public database and reports to build a network. The corresponding targets of active compounds were retrieved from online databases, and the antitumor targets were identified by GeneCards database. The antitumor hub targets were generated via protein-protein interaction analysis using String, and key compounds and targets from the integrative network were detected by molecular docking and ADMET. Top targets in hepatocellular carcinoma were confirmed by quantitative real-time PCR (qPCR). Finally, the multivariate biological network was built to identify the integrating mechanisms of action of CBC against tumor cells. RESULTS: A total of 128 compounds and 436 targets of CBC were identified successfully. Based on the generated multivariate biological network analysis, 25 key compounds, nine hub targets, and two pathways were further explored. Effective material bases of cantharidin, baicalein, scutellarin, sesamin, and quercetin were verified by integrative network analysis. PTGS2, ESR1, and TP53 were identified as hub targets via multivariate biological network analysis and confirmed using qPCR. Furthermore, VEGF and estrogen signaling pathways seem to play a role in the antitumor activity of CBC. Thus, breast cancer may be a potential clinical indication of CBC. CONCLUSION: This study successfully identified the material basis of CBC and its synergistic mechanisms of action against tumor cells.

Pharmacokinetics and tissue distribution of cantharidin after oral administration of aqueous extracts from Mylabris in rats.

A sensitive gas chromatography-mass spectroscopy method was established for the determination of cantharidin (CTD) in rat plasma and liver homogenates. During the experiment, rats were randomly divided into two groups (low, high) and were administered aqueous extract of Mylabris compound for 7 days. Then, plasma and tissue samples were taken at different time points to study the pharmacokinetics and tissue distribution of CTD in rats. The selected reaction monitoring transitions for CTD and clofibrate (internal standard) were m/z 128 → 85 and m/z 169 → 141, respectively. The calibration curve ranged from 10.26 to 3,078 ng/ml for plasma and from 10.26 to 246.24 ng/ml for liver homogenates. The lower limits of quantification were 10.26 ng/ml for both plasma and liver. The intra- and inter-day precision and accuracy were <20% for both plasma and liver homogenates. Extraction recovery ranged from 89.21 to 103.61% for CTD in rat plasma and liver and from 83.79 to 102.74% for IS in rat plasma and liver. Matrix effects ranged from 93.06 to 110.44% for CTD and from 91.65 to 110.80% for IS.

A novel approach combining metabolomics and molecular pharmacology to study the effect of Gei Herba on mouse hematopoietic function.

Gei Herba, Chinese named Lanbuzheng (LBZ), is a traditional Chinese medicine promotes hematopoiesis, yet the underlying mechanism for this effect remains largely unknown. In the present study, a novel approach combining LC-MS metabolomics and molecular pharmacology was developed to investigate the hematopoietic effect and mechanism of LBZ on hematopoietic dysfunction (HD) caused by cyclophosphamide (CTX) in treated mice. The results show that LBZ can reduce damage in the spleen, a result consistent with the peripheral hemogram. Fourteen potential biomarkers were identified in the spleen by metabolic profiles analysis, including 5-hydroxymethyluracil, ascorbalamic acid, adenosine 5'-monophosphate, menadiol disulfate, l-homocysteine sulfonic acid and l-carnitine. Change in biomarker levels suggest that LBZ mainly affects ß-oxidation of very-long-chain fatty acids, oxidation of branched chain fatty acids and carnitine synthesis, and those metabolites produced along with related metabolic pathways are closely associated with anti-apoptosis. A molecular pharmacology approach was simultaneously developed to examine accompanying cellular signaling mechanisms. LBZ activates PI3K/Akt signaling pathways and granulocyte-colony-stimulating-factor (G-CSF)-mediated Janus kinase 2 (JAK2)/transcription 3 (STAT3), resulting in inhibiting the release of cytochrome c. Further, LBZ inhibits caspase-mediated mitochondrial-dependent apoptosis mediated by caspase-9 and caspase-3. LBZ can thus reduce CTX-induced HD via G-CSF-mediated JAK2/STAT3 signaling and PI3K/Akt mitochondrial-dependent apoptotic pathways. The present study combines metabolomic and molecular pharmacological methods to elucidate mechanisms for the protective effect of LBZ on mouse HD following CTX-induced damage. This approach may be useful for exploring mechanisms of action of other drugs.

Cantharidin-induced LO2 cell autophagy and apoptosis via endoplasmic reticulum stress pathway in vitro.

Cantharidin (CTD), an important active compound derived from the traditional Chinese medicine Mylabris (also called Banmao), has been used in the treatment of diseases such as tumors and dermatosis. However, Mylabris has been shown to induce hepatotoxicity in clinical practice and animal experiments, limiting its use. Further, a detailed mechanism underlying CTD-induced hepatotoxicity has not been determined. In the present study, we aimed to explore the effect of endoplasmic reticulum stress (ERS), autophagy, and apoptosis on CTD-induced hepatotoxicity. We found that CTD could inhibit the proliferation of LO2 cells; increase alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and malondialdehyde levels; and reduce glutathione peroxidase and superoxide dismutase activities. Western blotting showed that low concentrations of CTD induced the expressions of ERS-related proteins [GRP78, ATF4, PERK, p-PERK, XBP1-1 s, and CHOP], but high concentrations of CTD inhibited their expressions. Furthermore, high concentrations of CTD activated autophagy (LC3, Beclin-1, Atg3, Atg4A, Atg4B, and Atg7), induced the expressions of apoptotic proteins (Bax/Bcl-2 and caspase-3), and increased LO2 toxicity. Taken together, these results indicated that CTD can induce LO2 cytotoxicity by inhibiting ERS and inducing autophagy and apoptosis, which provides a scientific basis for CTD-induced hepatotoxicity.

Protective effects of aqueous extract from Gei Herba on blood-deficiency mice: insights gained by a metabolomic approach.

With increasing tumor incidence, anemia (categorized as a blood deficiency in traditional Chinese medicine) caused by chemotherapy has become a major side effect worldwide. Gei Herba, a traditional Miao nation herb, has a prominent effect on the treatment of blood deficiency (BD). However, its application is limited owing to little fundamental research. Therefore, a GC-MS metabolomic approach was used to study the protective effect of aqueous extract from Gei Herba (AEG) on BD mice and its putative mechanism. In this study, 32 male mice were divided into four groups: a control group, a BD model group, and two groups subjected to AEG treatment at a daily dose of 0.15 or 0.30 g kg-1 for 8 d. After AEG treatment, the HGB and HCT levels in the blood of BD mice were significantly increased, the activity of superoxide dismutase was increased, and the histomorphology of the liver was improved. Furthermore, compared with those in the model group, the levels of eight significant metabolites [phosphoric acid, glycine, l-proline, ribitol, (Z,Z)-9,12-octadecadienoic acid, oleic acid, uridine and 4B2H-carbamic acid] in the liver were significantly changed by AEG. The findings of this study provide sound evidence regarding the protective effects of AEG in BD mice from both classical and metabolomic perspectives. The mechanisms of action of AEG could be related to regulation of linoleic acid metabolism and that of glycine, serine, and threonine metabolism.