Characterization of the metabolism of eupalinolide A and B by carboxylesterase and cytochrome P450 in human liver microsomes

Eupalinolide A (EA; Z-configuration) and eupalinolide B (EB; E-configuration) are bioactive cis-trans isomers isolated from Eupatorii Lindleyani Herba that exert anti-inflammatory and antitumor effects. Although one pharmacokinetic study found that the metabolic parameters of the isomers were different in rats, metabolic processes relevant to EA and EB remain largely unknown. Our preliminary findings revealed that EA and EB are rapidly hydrolyzed by carboxylesterase. Here, we investigated the metabolic stability and enzyme kinetics of carboxylesterase-mediated hydrolysis and cytochrome P450 (CYP)-mediated oxidation of EA and EB in human liver microsomes (HLMs). We also explored differences in the hydrolytic stability of EA and EB in human liver microsomes and rat liver microsomes (RLMs). Moreover, cytochrome P450 reaction phenotyping of the isomers was performed via in silico methods (i.e., using a quantitative structure-activity relationship model and molecular docking) and confirmed using human recombinant enzymes. The total normalized rate approach was considered to assess the relative contributions of five major cytochrome P450s to EA and EB metabolism. We found that EA and EB were eliminated rapidly, mainly by carboxylesterase-mediated hydrolysis, as compared with cytochrome P450-mediated oxidation. An inter-species difference was observed as well, with faster rates of EA and EB hydrolysis in rat liver microsomes. Furthermore, our findings confirmed EA and EB were metabolized by multiple cytochrome P450s, among which CYP3A4 played a particularly important role

Schizandrin A Inhibits Microglia-Mediated Neuroninflammation through Inhibiting TRAF6-NF-κB and Jak2-Stat3 Signaling Pathways.

Microglial-mediated neuroinflammation has been established as playing a vital role in pathogenesis of neurodegenerative disorders. Thus, rational regulation of microglia functions to inhibit inflammation injury may be a logical and promising approach to neurodegenerative disease therapy. The purposes of the present study were to explore the neuroprotective effects and potential molecular mechanism of Schizandrin A (Sch A), a lignin compound isolated from Schisandra chinesnesis. Our observations showed that Sch A could significantly down-regulate the increased production of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-6 induced by lipopolysaccharide (LPS) both in BV-2 cells and primary microglia cells. Moreover, Sch A exerted obvious neuroprotective effects against inflammatory injury in neurons when exposed to microglia-conditioned medium. Investigations of the mechanism showed the anti-inflammatory effect of Sch A involved the inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression levels and inhibition of the LPS-induced TRAF6-IKKβ-NF-κB pathway. Furthermore, inhibition of Jak2-Stat3 pathway activation and Stat3 nuclear translocation also was observed. In conclusion, SchA can exert anti-inflammatory and neuroprotective effects by alleviating microglia-mediated neuroinflammation injury through inhibiting the TRAF6-IKKβ-NF-κB and Jak2-Stat3 signaling pathways

Cucurbitacin E Inhibits Huh7 Hepatoma Carcinoma Cell Proliferation and Metastasis via Suppressing MAPKs and JAK/STAT3 Pathways

Cucurbitacin E (CuE), a highly oxygenated tetracyclic triterpene from Cucurbitaceae, has shown to exhibit potent cytotoxic and anti-proliferative properties against several human cancer cells. However, the underlying effects and mechanisms of CuE regarding hepatocellular carcinoma (HCC) have not been well understood. In the current study, unbiased RNA-sequencing (RNA-seq) and bioinformatics analysis was applied to elucidate the underlying molecular mechanism. CuE could significantly inhibit cell proliferation and migration of Huh7 cells, meanwhile CuE exhibited potent anti-angiogenic activity. RNA-seq analysis revealed that CuE negatively regulated 241 differentially expressed genes (DEGs) involved in multiple processes including cytoskeleton formation, angiogenesis and focal adhesion. Further analysis revealed that CuE effectually regulated diversified pharmacological signaling pathways such as MAPKs and JAK-STAT3. Our findings demonstrated the role of CuE in inhibiting proliferation and migration, providing an insight into the regulation of multiple signaling pathways as a new paradigm for anti-cancer treatment strategy

Sch A inhibition of inflammation response by down-regulating expression of inflammatory mediators.

<p>(A) BV-2 cells were treated with LPS (1 μg/ml) with or without Sch A (10, 20 and 50 μM) for 24 h, and then cell viability was determined by the MTT assay. (B) BV-2 cells were treated with LPS (1 μg/ml) with or without Sch A (10, 20 and 50 μM) for 24 h, and then NO production was quantified by an assay kit. (C) BV-2 cells were treated with LPS (1 μg/ml) with or without Sch A (10, 20 and 50 μM) for 8 h, followed analysis of IL-6 expression. (D) BV-2 cells were treated with LPS (1 μg/ml) with or without Sch A (10, 20 and 50 μM) for 4 h, followed analysis of TNF-α expression. (E) BV-2 cells were treated with LPS (1 μg/ml) for 2 h, and then incubated with Sch A (10, 20 and 50 μM) for 24 h. NO production was tested by an assay kit. (F) BV-2 cells were treated with LTA (20 μg/ml) with or without Sch A (10, 20 and 50 μM) for 24 h, followed by analysis of NO production. (G) BV-2 cells were treated with LPS (1 μg/ml) with or without Sch A (10μM) for 24 h, followed by analysis of IL-10 production. (H) After BV-2 cells were treated with LPS (1 μg/ml) with or without Sch A (10, 20 and 50 μM) for 24 h, western blot analysis of IL-1β expression was done. (I) BV-2 cells were treated with LPS (1 μg/ml) with or without Sch A (10, 20 and 50 μM) for 24 h, and then iNOS and COX-2 protein levels were analyzed by Western blot. All data are shown as the mean ± S.D. from independent experiments performed in triplicate. ^##<i>P</i> < 0.01 relative to control group; *<i>P</i> < 0.05, **<i>P</i> < 0.01, relative to LPS group.</p

Integration of Metabonomics and Transcriptomics Reveals the Therapeutic Effects and Mechanisms of Baoyuan Decoction for Myocardial Ischemia

Myocardial ischemia (MI) is an escalating public health care burden worldwide. Baoyuan decoction (BYD) is a traditional Chinese medicine formula with cardioprotective activity; however, its pharmacological characteristics and mechanisms are obscured. Herein, a multi-omics strategy via incorporating the metabonomics, transcriptomics, and pharmacodynamics was adopted to investigate the effects and molecular mechanisms of BYD for treating MI in a rat model of left anterior descending coronary artery (LADCA) ligation. The results indicated that BYD has a significantly cardioprotective role against MI by decreasing the infarct size, converting the echocardiographic abnormalities and myocardial enzyme markers, and reversing the serum metabolic disorders and myocardial transcriptional perturbations resulting from MI. Integrated bioinformatics analysis and literature reports constructed the interaction network based on the changes of the key MI targeted-metabolites and transcripts after BYD treatment and disclosed that the cardioprotection of BYD is mainly involved in the regulation of energy homeostasis, oxidative stress, apoptosis, inflammation, cardiac contractile dysfunction, and extracellular matrix remodeling. The results of histopathological examination, quantitative RT-PCR assay, cardiac energy synthesis, and serum antioxidant assessment complemented the multi-omics findings, and indicated the multi-pathway modulation mechanisms of BYD. Our investigation demonstrated that the multi-omics approach could achieve a complementary and verified view for the comprehensive evaluation of therapeutic effects and complex mechanisms of TCMF like BYD

Azukisapogenol Triterpene Glycosides from Oxytropis chiliophylla Royle

Eight azukisapogenol triterpene glycosides, including five new compounds, oxychiliotriterpenosides A&ndash;E (1&ndash;5), two new methyl glucuronide derivatives that proved to be artifacts, oxychiliotriterpenoside E-glucuronic acid methyl ester (6) and myrioside B-glucuronic acid methyl ester (7), and a known one, myrioside B (8), was isolated from the aerial part of Oxytropis chiliophylla Royle. Their structures were elucidated based on extensive spectroscopic analyses and chemical methods. Triterpene glycosides were first obtained from O. chiliophylla, and those containing a galactose unit (1, 2, 5 and 6) and diglucosidic or triglucosidic linkage at C-29 (1&ndash;4), were reported from Oxytropis species for the first time, which might be recognized as a chemotaxonomic feature of O. chiliophylla. All isolated compounds were evaluated for their anti-inflammatory activities against NO production using lipopolysaccharide (LPS)-induced RAW 264.7 cells, but no compounds showed potent inhibition on NO production

Microfluidic Coculture Device for Monitoring of Inflammation-Induced Myocardial Injury Dynamics

Emerging awareness of cardiac macrophages’ role in inflammation after myocardial infarction indicates that overabundant proinflammatory macrophages induce accentuated myocardial injury. The investigation of the macrophages–cardiomyocytes interaction and inflammation-induced dynamic damage in myocardial infarction, especially in a spatiotemporally controlled manner, remains a huge challenge. Here, we developed an in vitro model using a microfluidic coculture system to mimic inflammatory cardiac injury. To our knowledge, on-chip pathological models focused on inflammation-induced myocardial injury have not been reported. The device consists of two sets of thin interconnecting grooves that isolate heterogeneous cells spatially but maintain their soluble factors communication. The mass transportation is visually characterized, and the complete diffusion reaches equilibrium within 100 s. We investigate the dynamic interaction between the macrophages and the cardiomyocytes in the spatiotemporal controlled microenvironment, mimicking a key aspect of the in vivo pathophysiological process. The results show that the activated macrophages induce time-lapsed apoptotic responses of the cardiac cells and damage mitochondria membrane integrity. The anti-inflammatory and cardio-protective effects of quercetin were explored on the chip. The extent of caspase-3 activation is asynchronous in the individual cardiac cells, suggesting the different apoptosis dynamics. We further demonstrate that the mechanism of activated inflammation is associated with the upregulation of several inflammatory cytokines and NF-κB pathway. Thus, the developed microfluidic coculture device provides a useful tool for real-time monitoring of inflammatory response for myocardial disease and holds potential for anti-inflammatory drug screening

Proposed anti-neuroinflammatory mechanism of Sch A via TRAF6-NF-κB and Jak2-Stat3 pathways.

<p>Proposed anti-neuroinflammatory mechanism of Sch A via TRAF6-NF-κB and Jak2-Stat3 pathways.</p

Chemical structure of Sch A.

<p>Chemical structure of Sch A.</p

Sch A inhibition of Jak2-Stat3 pathway activation in LPS-induced BV-2 cells.

<p>(A) BV-2 cells were treated with LPS (1 μg/ml) with or without Sch A (10, 20 and 50 μM) for 0.5 h, Western blot analysis was performed to detect phosphorylated and total protein levels of Jak2, Stat3 Tyr705 and Stat3 Ser727. (B) BV-2 cells were treated with LPS (1 μg/ml) with or without Sch A (50 μM) for 2 h, and Stat3 nuclear translocation was detected by immunocytochemistry. Red fluorescence represents Stat3, and blue fluorescence represents nuclear DAPI staining (bar = 100 μm). All data are shown as the mean ± S.D. from independent experiments performed in triplicate. ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01 relative to control group; *<i>P</i> < 0.05, **<i>P</i> < 0.01 relative to LPS group.</p