Hong Xu

Study on interaction between curcumin and pepsin by spectroscopic and docking methods

The interaction between curcumin and pepsin was investigated by fluorescence, synchronous fluorescence, UV-vis absorption, circular dichroism (CD), and molecular docking. Under physiological pH value in stomach, the fluorescence of pepsin can be quenched effectively by curcumin via a combined quenching process. Binding constant (Ka) and binding site number (n) of curcumin to pepsin were obtained. According to the theory of Försters non-radiation energy transfer, the distance r between pepsin and curcumin was found to be 2.45 nm within the curcumin-pepsin complex, which implies that the energy transfer occurs between curcumin and pepsin, leading to the quenching of pepsin fluorescence. Fluorescence experiments also suggest that curcumin is located more closely to tryptophan residues than tyrosine residues. CD spectra together with UV-vis absorbance studies show that binding of curcumin to pepsin results in the extension of peptide strands of pepsin with loss of some β-sheet structures. Thermodynamic parameters calculated from the binding constants at different temperatures reveal that hydrophobic force plays a major role in stabilizing the curcumin-pepsin complex. In addition, docking results support the above experimental findings and suggest the possible hydrogen bonds of curcumin with Thr-77, Thr-218, and Glu-287 of pepsin, which help further stabilize the curcumin-pepsin complex.

Investigation on interaction between Ligupurpuroside A and pepsin by spectroscopic and docking methods

Ligupurpuroside A is one of the major glycoside in Ku-Din-Cha, a type of Chinese functional tea. In order to better understand its digestion and metabolism in humans, the interaction between Ligupurpuroside A and pepsin has been investigated by fluorescence spectra, UV-vis absorption spectra and synchronous fluorescence spectra along with molecular docking method. The fluorescence experiments indicate that Ligupurpuroside A can effectively quench the intrinsic fluorescence of pepsin through a combined quenching way at the low concentration of Ligupurpuroside A, and a static quenching procedure at the high concentration. The binding constant, binding sites of Ligupurpuroside A with pepsin have been calculated. The thermodynamic analysis suggests that non-covalent reactions, including electrostatic force, hydrophobic interaction and hydrogen bond are the main forces stabilizing the complex. According to the Försters non-radiation energy transfer theory, the binding distance between pepsin and Ligupurpuroside A was calculated to be 3.15 nm, which implies that energy transfer occurs between pepsin and Ligupurpuroside A. Conformation change of pepsin was observed from UV-vis absorption spectra and synchronous fluorescence spectra under experimental conditions. In addition, all these experimental results have been validated by the protein-ligand docking studies which show that Ligupurpuroside A is located in the cleft between the domains of pepsin.

Copper(II)-selective chelation improves function and antioxidant defences in cardiovascular tissues of rats as a model of diabetes: comparisons between triethylenetetramine and three less copper-selective transition-metal-targeted treatments

Aims/hypothesisTreatment with the Cu(II)-selective chelator triethylenetetramine (TETA) improves cardiovascular disease in human patients, and cardiac and vascular/renal disease in rats used as a model of diabetes. Here we tested two hypotheses: first, that TETA elicits greater improvement in organ function than less Cu-selective transition-metal-targeted treatments; second, that the therapeutic actions of TETA are consistent with mediation through suppression of oxidative stress.MethodsRats were made diabetic with streptozotocin (55xa0mg/kg, i. v.) and treated from 8xa0weeks after disease induction for the following 8xa0weeks with effective dosages of oral TETA, or one of three less Cu-selective transition-metal-targeted treatments: d-penicillamine, deferiprone or Zn acetate. Treatment effects were measured in ex vivo cardiac and aortic tissues, plasma and urine.ResultsDiabetes damaged both cardiac and renal/vascular function by impairing the ability of cardiac output to respond physiologically to rising afterload, and by significantly elevating the urinary albumin/creatinine ratio. Diabetes also lowered total antioxidant potential and heparan sulphate levels in cardiac and arterial tissues, and serum ferroxidase activity, whereas it elevated urinary heparan sulphate excretion. TETA treatment rectified or partially rectified all these defects, whereas the other three experimental treatments were ineffectual. By contrast, none of the four drug treatments lowered diabetes-mediated elevations of plasma glucose or lipid concentrations.Conclusions/interpretationTETA may limit the cardiac and renal/vascular damage inflicted by diabetes through its actions to reinforce antioxidant defence mechanisms, probably acting through selective chelation of ‘loosely-bound’/chelatable Cu(II). It may also improve heparan sulphate homeostasis and bolster antioxidant defence by increasing vascular extracellular superoxide dismutase activity. Urinary albumin/creatinine ratio might prove useful for monitoring TETA treatment.

Treatment with a copper-selective chelator causes substantive improvement in cardiac function of diabetic rats with left-ventricular impairment

BackgroundDefective copper regulation is implicated as a causative mechanism of organ damage in diabetes. Treatment with trientine, a divalent-copper-selective chelator, improves arterial and renal structure/function in diabetes, wherein it also ameliorates left-ventricular (LV) hypertrophy. However, direct in vivo evidence that trientine can improve cardiac function in heart failure has hitherto been lacking.MethodsTo determine whether trientine treatment could improve in vivo outcome, we measured cardiac function in groups of trientine-treated diabetic (TETA-DIA), non-drug-treated diabetic (DIA) and sham-treated control (SHAM) rats, by using in vivo high-field cardiac magnetic-resonance imaging (cMRI) and an ex vivo isolated-perfused working heart method. Forty age-matched animals underwent a cMRI scan after which 12 were randomized to the SHAM group and 28 underwent streptozotocin-injection; of these, 25 developed stable diabetes, and 12 were then randomized to receive no treatment for 16xa0weeks (DIA) and the other 13 to undergo 8-weeks’ untreated diabetes followed by 8-weeks’ drug treatment (TETA-DIA). Animals were studied again by cMRI at 8 and 16xa0weeks following disease induction, and finally by measurement of ex vivo cardiac function.ResultsAfter eight weeks diabetes, rats (DIA/TETA-DIA) had developed significant impairment of LV function, as judged by impairment of ejection fraction (LVEF), cardiac output (CO), and LV mass (LVM)/body-mass (all Pu2009<u20090.001), as well as other functional indexes. LVEF, CO (both Pu2009<u20090.001) and the other indexes deteriorated further at 16xa0weeks in DIA, whereas trientine (TETA-DIA) improved cardiac function by elevating LVEF and CO (both Pu2009<u20090.001), and also partially reversed the increase in LVM/body-mass (Pu2009<u20090.05). In ex vivo hearts from DIA, the CO response to increasing preload pressure was deficient compared with SHAM (Pu2009<u20090.001) whereas the preload-CO relationship was significantly improved in TETA-DIA animals (Pu2009<u20090.001).ConclusionsTrientine treatment significantly improved cardiac function in diabetic rats with substantive LV impairment. These results implicate impaired copper regulation in the pathogenesis of impaired cardiac function caused by diabetic cardiomyopathy, and support ongoing studies of trientine treatment in patients with heart failure.

Identification and activation of TLR4-mediated signalling pathways by alginate-derived guluronate oligosaccharide in RAW264.7 macrophages

Alginate, a natural acidic polysaccharide extracted from marine brown seaweeds, is composed of different blocks of β-(1, 4)-D-mannuronate (M) and its C-5 epimer α-(1, 4)-L-guluronate (G). Alginate-derived guluronate oligosaccharide (GOS) readily activates macrophages. However, to understand its role in immune responses, further studies are needed to characterize GOS transport and signalling. Our results show that GOS is recognized by and upregulates Toll-like receptor 4 (TLR4) on RAW264.7 macrophages, followed by its endocytosis via TLR4. Increased expression of TLR4 and myeloid differentiation protein 2 (MD2) results in Akt phosphorylation and subsequent activation of both nuclear factor-κB (NF-κB) and mechanistic target of rapamycin (mTOR). Moreover, GOS stimulates mitogen-activated protein kinases (MAPKs); notably, c-Jun N-terminal kinase (JNK) phosphorylation depends on TLR4 initiation. All these events contribute to the production of inflammatory mediators, either together or separately. Our findings also reveal that GOS induces cytoskeleton remodelling in RAW264.7 cells and promotes macrophage proliferation in mice ascites, both of which improve innate immunity. Conclusively, our investigation demonstrates that GOS, which is dependent on TLR4, is taken up by macrophages and stimulates TLR4/Akt/NF-κB, TLR4/Akt/mTOR and MAPK signalling pathways and exerts impressive immuno-stimulatory activity.

Interaction mechanism of pepsin with a natural inhibitor gastrodin studied by spectroscopic methods and molecular docking

The interaction of gastrodin with pepsin has been investigated by enzyme activity assay, fluorescence, UV–Visible, circular dichroism spectra, and molecular docking. The pepsin activity results suggest that gastrodin is an inhibitor of pepsin. The fluorescence experiments show that gastrodin can quench the fluorescence of pepsin via a static quenching process. The thermodynamic analysis suggests that hydrophobic interaction is the main force between pepsin and gastrodin. UV–Visible and circular dichroism spectra studies suggest that the binding of gastrodin leads to a loosening and unfolding of pepsin backbone with partial α-helix being transformed into β-sheet. All these experimental results have been validated by docking studies, which further show that besides hydrophobic interaction, hydrogen bond also help stabilize the gastrodin–pepsin complex. The results reveal the potential to develop the natural compound gastrodin for the treatment of diseases related to the excessive activity of pepsin.

Mechanism and Nature of Inhibition of Trypsin by Ligupurpuroside A, a Ku-Ding Tea Extract, Studied by Spectroscopic and Docking Methods

Ligupurpuroside A is a glycoside extracted from Ku-Ding tea. As extracts from Ku-Ding tea exhibit anti-inflammatory property, we hypothesize that Ligupurpuroside A may be an active compound which inhibits trypsin activity during the anti-inflammatory process. The mechanism and nature of inhibition of trypsin by Ligupurpuroside A have been studied by multi-spectroscopic method, enzyme-activity assay and molecular docking. Enzyme activity assay reveals that Ligupurpuroside A significantly inhibits the activity of trypsin through a competitive manner with an IC50 value of 3.08xa0×xa010−3xa0molxa0L−1. Fluorescence titration together with thermodynamic analysis indicate that a Ligupurpuroside A-trypsin complex is formed, and that hydrophobic force and hydrogen bonding are the main forces stabilizing the complex. UV-vis absorption, synchronous fluorescence and circular dichroism spectra show that the interaction between Ligupurpuroside A and trypsin induces conformational changes of trypsin with a decrease in the contents of α-helix and β-sheet. Finally, molecular docking further suggests that Ligupurpuroside A molecule binds within the active pocket of trypsin via hydrophobic force and hydrogen bond. Results from this study of the interaction of trypsin with its natural inhibitor should be useful to minimize the antinutritional effects and make full use of tea extracts in the food industry, and be also helpful to the design of the drugs for the diseases related to overexpression of trypsin.

Study on the mechanism of the interaction between acteoside and pepsin using spectroscopic techniques

The interaction of acteoside with pepsin has been investigated using fluorescence spectra, UV/vis absorption spectra, three-dimensional (3D) fluorescence spectra and synchronous fluorescence spectra, along with a molecular docking method. The fluorescence experiments indicate that acteoside can quench the intrinsic fluorescence of pepsin through combined quenching at a low concentration of acteoside, and static quenching at high concentrations. Thermodynamic analysis suggests that hydrogen bonds and van der Waals forces are the main forces between pepsin and acteoside. According to the theory of Försters non-radiation energy transfer, the binding distance between pepsin and acteoside was calculated to be 2.018 nm, which implies that energy transfer occurs between acteoside and pepsin. In addition, experimental results from UV/vis absorption spectra, 3D fluorescence spectra and synchronous fluorescence spectra imply that pepsin undergoes a conformation change when it interacts with acteoside.

Gypenoside L inhibits autophagic flux and induces cell death in human esophageal cancer cells through endoplasm reticulum stress-mediated Ca 2+ release

Esophageal cancer is one of the leading cause of cancer mortality in the world. Due to the increased drug and radiation tolerance, it is urgent to develop novel anticancer agent that triggers nonapoptotic cell death to compensate for apoptosis resistance. In this study, we show that treatment with gypenoside L (Gyp-L), a saponin isolated from Gynostemma pentaphyllum, induced nonapoptotic, lysosome-associated cell death in human esophageal cancer cells. Gyp-L-induced cell death was associated with lysosomal swelling and autophagic flux inhibition. Mechanistic investigations revealed that through increasing the levels of intracellular reactive oxygen species (ROS), Gyp-L triggered protein ubiquitination and endoplasm reticulum (ER) stress response, leading to Ca2+ release from ER inositol trisphosphate receptor (IP3R)-operated stores and finally cell death. Interestingly, there existed a reciprocal positive-regulatory loop between Ca2+ release and ER stress in response to Gyp-L. In addition, protein synthesis was critical for Gyp-L-mediated ER stress and cell death. Taken together, this work suggested a novel therapeutic option by Gyp-L through the induction of an unconventional ROS-ER-Ca2+-mediated cell death in human esophageal cancer.

Gypenoside L, Isolated from Gynostemma pentaphyllum, Induces Cytoplasmic Vacuolation Death in Hepatocellular Carcinoma Cells through Reactive-Oxygen-Species-Mediated Unfolded Protein Response.

Exploring novel anticancer agents that can trigger non-apoptotic or non-autophagic cell death is urgent for cancer treatment. In this study, we screened and identified an unexplored anticancer activity of gypenoside L (Gyp-L) isolated from Gynostemma pentaphyllum. We showed that treatment with Gyp-L induces non-apoptotic and non-autophagic cytoplasmic vacuolation death in human hepatocellular carcinoma (HCC) cells. Mechanically, Gyp-L initially increased the intracellular reactive oxygen species (ROS) levels, which, in turn, triggered protein ubiquitination and unfolded protein response (UPR), resulting in Ca(2+) release from endoplasm reticulum (ER) inositol trisphosphate receptor (IP3R)-operated stores and finally cytoplasmic vacuolation and cell death. Interruption of the ROS-ER-Ca(2+) signaling pathway by chemical inhibitors significantly prevented Gyp-L-induced vacuole formation and cell death. In addition, Gyp-L-induced ER stress and vacuolation death required new protein synthesis. Overall, our works provide strong evidence for the anti-HCC activity of Gyp-L and suggest a novel therapeutic option by Gyp-L through the induction of a unconventional ROS-ER-Ca(2+)-mediated cytoplasmic vacuolation death in human HCC.