Metabonomic profiles delineate the effect of traditional Chinese medicine sini decoction on myocardial infarction in rats

Background

In spite of great advances in target-oriented Western medicine for treating myocardial infarction (MI), it is still a leading cause of death in a worldwide epidemic. In contrast to Western medicine, Traditional Chinese medicine (TCM) uses a holistic and synergistic approach to restore the balance of Yin-Yang of body energy so the body''s normal function can be restored. Sini decoction (SND) is a well-known formula of TCM which has been used to treat MI for many years. However, its holistic activity evaluation and mechanistic understanding are still lacking due to its complex components.

Methodology/Principal Findings

A urinary metabonomic method based on nuclear magnetic resonance and ultra high-performance liquid chromatography coupled to mass spectrometry was developed to characterize MI-related metabolic profiles and delineate the effect of SND on MI. With Elastic Net for classification and selection of biomarkers, nineteen potential biomarkers in rat urine were screened out, primarily related to myocardial energy metabolism, including the glycolysis, citrate cycle, amino acid metabolism, purine metabolism and pyrimidine metabolism. With the altered metabolism pathways as possible drug targets, we systematically analyze the therapeutic effect of SND, which demonstrated that SND administration could provide satisfactory effect on MI through partially regulating the perturbed myocardial energy metabolism.

Conclusions/Significance

Our results showed that metabonomic approach offers a useful tool to identify MI-related biomarkers and provides a new methodological cue for systematically dissecting the underlying efficacies and mechanisms of TCM in treating MI.     

Metabolic Profiling Provides a System Understanding of Hypothyroidism in Rats and Its Application

Background

Hypothyroidism is a chronic condition of endocrine disorder and its precise molecular mechanism remains obscure. In spite of certain efficacy of thyroid hormone replacement therapy in treating hypothyroidism, it often results in other side effects because of its over-replacement, so it is still urgent to discover new modes of treatment for hypothyroidism. Sini decoction (SND) is a well-known formula of Traditional Chinese Medicine (TCM) and is considered as efficient agents against hypothyroidism. However, its holistic effect assessment and mechanistic understanding are still lacking due to its complex components.

Methodology/Principal Findings

A urinary metabonomic method based on ultra performance liquid chromatography coupled to mass spectrometry was employed to explore global metabolic characters of hypothyroidism. Three typical hypothyroidism models (methimazole-, propylthiouracil- and thyroidectomy-induced hypothyroidism) were applied to elucidate the molecular mechanism of hypothyroidism. 17, 21, 19 potential biomarkers were identified with these three hypothyroidism models respectively, primarily involved in energy metabolism, amino acid metabolism, sphingolipid metabolism and purine metabolism. In order to avert the interference of drug interaction between the antithyroid drugs and SND, the thyroidectomy-induced hypothyroidism model was further used to systematically assess the therapeutic efficacy of SND on hypothyroidism. A time-dependent recovery tendency was observed in SND-treated group from the beginning of model to the end of treatment, suggesting that SND exerted a recovery effect on hypothyroidism in a time-dependent manner through partially regulating the perturbed metabolic pathways.

Conclusions/Significance

Our results showed that the metabonomic approach is instrumental to understand the pathophysiology of hypothyroidism and offers a valuable tool for systematically studying the therapeutic effects of SND on hypothyroidism.     

N-3 polyunsaturated fatty acids decrease levels of doxorubicin-induced reactive oxygen species in cardiomyocytes -- involvement of uncoupling protein UCP2

Background

Use of the chemotherapeutic drug doxorubicin (DOX) is associated with serious cardiotoxicity, as it increases levels of reactive oxygen species (ROS). N-3 polyunsaturated fatty acid dietary supplements can be of benefit to patients undergoing cancer therapy. The aims of this study were to determine whether DOX-induced cardiotoxicity is related to mitochondrial uncoupling proteins and whether eicosapentaenoic acid (EPA, C20:5 n-3) or docosahexaenoic acid (DHA, C22:6 n-3) affects DOX-induced cardiomyocyte toxicity.

Results

Treatment of H9C2 cells with DOX resulted in decreased cell viability and UCP2 expression. Treatment with 100 μM EPA or 50 μM DHA for 24 h resulted in a maximal mitochondria concentration of these fatty acids and increased UCP2 expression. Pretreatment with 100 μM EPA or 50 μM DHA prevented the DOX-induced decrease in UCP2 mRNA and protein levels, but these effects were not seen with EPA or DHA and DOX cotreatment. In addition, the DOX-induced increase in ROS production and subsequent mitochondrial membrane potential change (∆ψ) were significantly attenuated by pretreatment with EPA or DHA.

Conclusion

EPA or DHA pre-treatment inhibits the DOX-induced decrease in UCP2 expression, increase in ROS production, and subsequent mitochondrial membrane potential change that contribute to the cardiotoxicity of DOX.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-014-0101-3) contains supplementary material, which is available to authorized users.     

Senescence Marker Protein 30 Has a Cardio-Protective Role in Doxorubicin-Induced Cardiac Dysfunction

Background

Senescence marker protein 30 (SMP30), which was originally identified as an aging marker protein, is assumed to act as a novel anti-aging factor in the liver, lungs and brain. We hypothesized that SMP30 has cardio-protective function due to its anti-aging and anti-oxidant effects on doxorubicin (DOX)-induced cardiac dysfunction.

Methods and Results

SMP30 knockout (SMP30 KO) mice, SMP30 transgenic (SMP30 TG) mice with cardiac-specific overexpression of SMP30 gene and wild-type (WT) littermate mice at 12–14 weeks of age were given intra-peritoneal injection of DOX (20 mg/kg) or saline. Five days after DOX injection, echocardiography revealed that left ventricular ejection fraction was more severely reduced in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but was preserved in the DOX-treated SMP30 TG mice. Generation of reactive oxygen species and oxidative DNA damage in the myocardium were greater in the DOX-treated SMP30 KO mice than in the DOX-treated WT mice, but much less in the SMP30 TG mice. The numbers of deoxynucleotidyltransferase-mediated dUTP nick end-labeling positive nuclei in the myocardium, apoptotic signaling pathways such as caspase-3 activity, Bax/Bcl-2 ratio and phosphorylation activity of c-Jun N-terminal kinase were increased in SMP30 KO mice and decreased in SMP30 TG mice compared with WT mice after DOX injection.

Conclusions

SMP30 has a cardio-protective role by anti-oxidative and anti-apoptotic effects in DOX-induced cardiotoxicity, and can be a new therapeutic target to prevent DOX-induced heart failure.     

Quercetin Potentiates Doxorubicin Mediated Antitumor Effects against Liver Cancer through p53/Bcl-xl

Background

The dose-dependent toxicities of doxorubicin (DOX) limit its clinical applications, particularly in drug-resistant cancers, such as liver cancer. In this study, we investigated the role of quercetin on the antitumor effects of DOX on liver cancer cells and its ability to provide protection against DOX-mediated liver damage in mice.

Methodology and Results

The MTT and Annexin V/PI staining assay demonstrated that quercetin selectively sensitized DOX-induced cytotoxicity against liver cancer cells while protecting normal liver cells. The increase in DOX-mediated apoptosis in hepatoma cells by quercetin was p53-dependent and occurred by downregulating Bcl-xl expression. Z-VAD-fmk (caspase inhibitor), pifithrin-α (p53 inhibitor), or overexpressed Bcl-xl decreased the effects of quercetin on DOX-mediated apoptosis. The combined treatment of quercetin and DOX significantly reduced the growth of liver cancer xenografts in mice. Moreover, quercetin decreased the serum levels of alanine aminotransferase and aspartate aminotransferase that were increased in DOX-treated mice. Quercetin also reversed the DOX-induced pathological changes in mice livers.

Conclusion and Significance

These results indicate that quercetin potentiated the antitumor effects of DOX on liver cancer cells while protecting normal liver cells. Therefore, the development of quercetin may be beneficial in a combined treatment with DOX for increased therapeutic efficacy against liver cancer.     

Assay of the multiple energy-producing pathways of mammalian cells

Background

To elucidate metabolic changes that occur in diabetes, obesity, and cancer, it is important to understand cellular energy metabolism pathways and their alterations in various cells.

Methodology and Principal Findings

Here we describe a technology for simultaneous assessment of cellular energy metabolism pathways. The technology employs a redox dye chemistry specifically coupled to catabolic energy-producing pathways. Using this colorimetric assay, we show that human cancer cell lines from different organ tissues produce distinct profiles of metabolic activity. Further, we show that murine white and brown adipocyte cell lines produce profiles that are distinct from each other as well as from precursor cells undergoing differentiation.

Conclusions

This technology can be employed as a fundamental tool in genotype-phenotype studies to determine changes in cells from shared lineages due to differentiation or mutation.     

Arginase Inhibition Ameliorates Hepatic Metabolic Abnormalities in Obese Mice

Objectives

We examined whether arginase inhibition influences hepatic metabolic pathways and whole body adiposity in diet-induced obesity.

Methods and Results

After obesity induction by a high fat diet (HFD), mice were fed either the HFD or the HFD with an arginase inhibitor, N^ω-hydroxy-nor-L-arginine (nor-NOHA). Nor-NOHA significantly prevented HFD-induced increases in body, liver, and visceral fat tissue weight, and ameliorated abnormal lipid profiles. Furthermore, nor-NOHA treatment reduced lipid accumulation in oleic acid-induced hepatic steatosis in vitro. Arginase inhibition increased hepatic nitric oxide (NO) in HFD-fed mice and HepG2 cells, and reversed the elevated mRNA expression of hepatic genes in lipid metabolism. Expression of phosphorylated 5′ AMPK-activated protein kinase α was increased by arginase inhibition in the mouse livers and HepG2 cells.

Conclusions

Arginase inhibition ameliorated obesity-induced hepatic lipid abnormalities and whole body adiposity, possibly as a result of increased hepatic NO production and subsequent activation of metabolic pathways involved in hepatic triglyceride metabolism and mitochondrial function.     

Mitochondrial reactive oxygen species modulate mosquito susceptibility to Plasmodium infection

Background

Mitochondria perform multiple roles in cell biology, acting as the site of aerobic energy-transducing pathways and as an important source of reactive oxygen species (ROS) that modulate redox metabolism.

Methodology/Principal Findings

We demonstrate that a novel member of the mitochondrial transporter protein family, Anopheles gambiae mitochondrial carrier 1 (AgMC1), is required to maintain mitochondrial membrane potential in mosquito midgut cells and modulates epithelial responses to Plasmodium infection. AgMC1 silencing reduces mitochondrial membrane potential, resulting in increased proton-leak and uncoupling of oxidative phosphorylation. These metabolic changes reduce midgut ROS generation and increase A. gambiae susceptibility to Plasmodium infection.

Conclusion

We provide direct experimental evidence indicating that ROS derived from mitochondria can modulate mosquito epithelial responses to Plasmodium infection.     

Metabolome-Wide Association Study of Neovascular Age-Related Macular Degeneration

Purpose

To determine if plasma metabolic profiles can detect differences between patients with neovascular age-related macular degeneration (NVAMD) and similarly-aged controls.

Methods

Metabolomic analysis using liquid chromatography with Fourier-transform mass spectrometry (LC-FTMS) was performed on plasma samples from 26 NVAMD patients and 19 controls. Data were collected from mass/charge ratio (m/z) 85 to 850 on a Thermo LTQ-FT mass spectrometer, and metabolic features were extracted using an adaptive processing software package. Both non-transformed and log2 transformed data were corrected using Benjamini and Hochberg False Discovery Rate (FDR) to account for multiple testing. Orthogonal Partial Least Squares-Discriminant Analysis was performed to determine metabolic features that distinguished NVAMD patients from controls. Individual m/z features were matched to the Kyoto Encyclopedia of Genes and Genomes database and the Metlin metabolomics database, and metabolic pathways associated with NVAMD were identified using MetScape.

Results

Of the 1680 total m/z features detected by LC-FTMS, 94 unique m/z features were significantly different between NVAMD patients and controls using FDR (q = 0.05). A comparison of these features to those found with log2 transformed data (n = 132, q = 0.2) revealed 40 features in common, reaffirming the involvement of certain metabolites. Such metabolites included di- and tripeptides, covalently modified amino acids, bile acids, and vitamin D-related metabolites. Correlation analysis revealed associations among certain significant features, and pathway analysis demonstrated broader changes in tyrosine metabolism, sulfur amino acid metabolism, and amino acids related to urea metabolism.

Conclusions

These data suggest that metabolomic analysis can identify a panel of individual metabolites that differ between NVAMD cases and controls. Pathway analysis can assess the involvement of certain metabolic pathways, such as tyrosine and urea metabolism, and can provide further insight into the pathophysiology of AMD.     

In Vitro and In Vivo Antitumor Activity of a Novel pH-Activated Polymeric Drug Delivery System for Doxorubicin

Background

Conventional chemotherapy agent such as doxorubicin (DOX) is of limited clinical use because of its inherently low selectivity, which can lead to systemic toxicity in normal healthy tissue.

Methods

A pH stimuli-sensitive conjugate based on polyethylene glycol (PEG) with covalently attachment doxorubicin via hydrazone bond (PEG-hyd-DOX) was prepared for tumor targeting delivery system. While PEG-DOX conjugates via amid bond (PEG-ami-DOX) was synthesized as control.

Results

The synthetic conjugates were confirmed by proton nuclear magnetic resonance (NMR) spectroscopy, the release profile of DOX from PEG-hyd-DOX was acid-liable for the hydrazone linkage between DOX and PEG, led to different intracellular uptake route; intracellular accumulation of PEG-hyd-DOX was higher than PEG-ami-DOX due to its pH-triggered profile, and thereby more cytotoxicity against MCF-7, MDA-MB-231 (breast cancer models) and HepG2 (hepatocellular carcinoma model) cell lines. Following the in vitro results, we xenografted MDA-MB-231 cell onto SCID mice, PEG-hyd-DOX showed stronger antitumor efficacy than free DOX and was tumor-targeting.

Conclusions

Results from these in vivo experiments were consistent with our in vitro results; suggested this pH-triggered PEG-hyd-DOX conjugate could target DOX to tumor tissues and release free drugs by acidic tumor environment, which would be potent in antitumor drug delivery.     

Cytoplasmic p21 induced by p65 prevents doxorubicin-induced cell death in pancreatic carcinoma cell line

Background

Studies have shown the existence of p21 induction in a p53-dependent and -independent pathway. Our previous study indicates that DOX-induced p65 is able to bind the p21 promoter to activate its transactivation in the cells.

Methods

Over-expression and knock-down experiments were performed in Human Pancreatic Carcinoma (PANC1) cells. Cell cycle and cell death related proteins were assessed by Western Blotting. Cytotoxicity assay was checked by CCK-8 kit. Cell growth was analyzed by flow cytometers.

Results

Here we showed that over-expression of p65 decreased the cytotoxic effect of DOX on PANC1 cells, correlating with increased induction of cytoplasmic p21. We observed that pro-caspase-3 physically associated with cytoplasmic p21, which may be contribution to prevent p21 translocation into the nucleus. Our data also suggested that no clear elevation of nuclear p21 by p65 provides a survival advantage by progression cell cycle after treatment of DOX. Likewise, down-regulation of p65 expression enhanced the cytotoxic effect of DOX, due to a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1), and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2), leading to efficient induction of caspase-3 cleavage in the cells. More, we present evidence that over-expression of p53 or p53/p65 in the PANC1 cells were more sensitive to DOX treatment, correlated with activation of caspase-3 and clear elevation of nuclear p21 level. Our previous data suggested that expression of p21 increases Gefitinib-induced cell death by blocking the cell cycle at the G1 and G2 phases. The present findings here reinforced this idea by showing p21''s ability of potentiality of DOX-induced cell death correlated with its inhibition of cell cycle progression after over-expression of p53 or p53/p65.

Conclusion

Our data suggested p65 could increase p53-mediated cell death in response to DOX in PANC1 cells. Thus, it is worth noting that in p53 null or defective tumors, targeting in down-regulation of p65 may well be useful, leading to the potentiality of chemotherapeutic drugs.     

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern

Background

Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome.

Results

PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y’s phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism.

Conclusions

In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-384) contains supplementary material, which is available to authorized users.     

Chronic Endocannabinoid System Stimulation Induces Muscle Macrophage and Lipid Accumulation in Type 2 Diabetic Mice Independently of Metabolic Endotoxaemia

Aims

Obesity and type 2 diabetes are characterised by low-grade inflammation, metabolic endotoxaemia (i.e., increased plasma lipopolysaccharides [LPS] levels) and altered endocannabinoid (eCB)-system tone. The aim of this study was to decipher the specific role of eCB-system stimulation or metabolic endotoxaemia in the onset of glucose intolerance, metabolic inflammation and altered lipid metabolism.

Methods

Mice were treated with either a cannabinoid (CB) receptor agonist (HU210) or low-dose LPS using subcutaneous mini-pumps for 6 weeks. After 3 weeks of the treatment under control (CT) diet, one-half of each group of mice were challenged with a high fat (HF) diet for the following 3-week period.

Results

Under basal conditions (control diet), chronic CB receptor agonist treatment (i.e., 6 weeks) induced glucose intolerance, stimulated metabolic endotoxaemia, and increased macrophage infiltration (CD11c and F4/80 expression) in the muscles; this phenomenon was associated with an altered lipid metabolism (increased PGC-1α expression and decreased CPT-1b expression) in this tissue. Chronic LPS treatment tended to increase the body weight and fat mass, with minor effects on the other metabolic parameters. Challenging mice with an HF diet following pre-treatment with the CB agonist exacerbated the HF diet-induced glucose intolerance, the muscle macrophage infiltration and the muscle''s lipid content without affecting the body weight or the fat mass.

Conclusion

Chronic CB receptor stimulation under basal conditions induces glucose intolerance, stimulates metabolic inflammation and alters lipid metabolism in the muscles. These effects worsen following the concomitant ingestion of an HF diet. Here, we highlight the central roles played by the eCB system and LPS in the pathophysiology of several hallmarks of obesity and type 2 diabetes.     

Time course of gene expression profiling in the liver of experimental mice infected with Echinococcus multilocularis

Background

Alveolar echinococcosis (AE) is a severe chronic parasitic disease which behaves like a slow-growing liver cancer. Clinical observations suggest that the parasite, Echinococcus multilocularis (E. multilocularis) influences liver homeostasis and hepatic cell metabolism. However, this has never been analyzed during the time course of infection in the common model of secondary echinococcosis in experimental mice.

Methodology/Principal Findings

Gene expression profiles were assessed using DNA microarray analysis, 1, 2, 3 and 6 months after injection of E. multilocularis metacestode in the liver of susceptible mice. Data were collected at different time points to monitor the dynamic behavior of gene expression. 557 differentially expressed genes were identified at one or more time points, including 351 up-regulated and 228 down-regulated genes. Time-course analysis indicated, at the initial stage of E. multilocularis infection (month 1–2), that most of up-regulated pathways were related to immune processes and cell trafficking such as chemokine-, mitogen-activated protein kinase (MAPK) signaling, and down-regulated pathways were related to xenobiotic metabolism; at the middle stage (month 3), MAPK signaling pathway was maintained and peroxisome proliferator-activated receptor (PPAR) signaling pathway emerged; at the late stage (month 6), most of up-regulated pathways were related to PPAR signaling pathway, complement and coagulation cascades, while down-regulated pathways were related to metabolism of xenobiotics by cytochrome P450. Quantitative RT-PCR analysis of a random selection of 19 genes confirmed the reliability of the microarray data. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was increased in the liver of E. multilocularis infected mice from 2 months to 6 months.

Conclusions

E. multilocularis metacestode definitely exerts a deep influence on liver homeostasis, by modifying a number of gene expression and metabolic pathways. It especially promotes hepatic cell proliferation, as evidenced by the increased PCNA constantly found in all the experimental time-points we studied and by an increased gene expression of key metabolic pathways.