A novel fluorescent sensor was developed in this study based on glutathione-functionalized graphene quantum dots (GQDs@GSH) to detect organophosphorus pesticide residues in Radix Angelica Sinensis. GQDs@GSH was synthesized by a one-step pyrolysis method with a fluorescence quantum yield as high as 33.9% and its structure was characterized by transmission electron microscopy and X-ray photoelectron spectroscopy. GQDs@GSH exhibited excellent fluorescence property showing strong blue fluorescence under UV irradiation. The fluorescence of GQDs@GSH could be quenched by Fe(3+) by electron transfer and the quenched fluorescence could be recovered due to the strong chelating and reducing ability of phytic acid (PA). Under the catalyzation of acetylcholinesterase (AChE) and choline oxidase (ChOx), acetylcholine (ACh) could be decomposed to H(2)O(2), which could further oxidize Fe(2+) to Fe(3+) thus quenching the fluorescence of GQDs@GSH once again. Coumaphos, a kind of organophosphorus pesticide, could inhibit AChE activity, thus making the quenched fluorescence turn on again. Several parameters influencing the fluorescence response such as Fe(3+), PA, ACh and coumaphos concentration, pH value and reaction time were optimized. Based on such a fluorescence "off-on-off-on" ngkmechanism, GQDs@GSH was successfully applied to the detection of coumaphos in Radix Angelica Sinensis. A good linear relationship between the fluorescence intensity and coumaphos concentration was obtained in the range of 0.1-10.0 micromol.L(-1). By a standard addition method, the recoveries were measured to be 101.44-117.90% with RSDs lower than 1.98%. The biosensor system is simple, sensitive and accurate. It has a good application prospect in the detection of organophosphorus pesticide residues in traditional Chinese medicine and agricultural products, and also expanded the application scope for glutathione as a highly selective biological molecule.
BACKGROUND: It has been established that the dipeptidyl peptidase-4 (DPP-4) inhibitor Diprotin A TFA can reduce vascular endothelial (VE)-cadherin disruption by inhibiting the increase in cleaved beta-catenin in response to hypoxia, thereby protecting the vascular barrier of human umbilical vein endothelial cells. In this study, we sought to investigate the possible effect of Diprotin A TFA on the VE barrier after cerebral ischemic stroke in mice. METHODS: C57BL/6J mice were divided into five groups, namely, (1) sham, (2) stroke, (3) stroke + dimethyl sulfoxide (DMSO), (4) stroke + Diprotin A TFA, and (5) stroke + Diprotin A TFA + XAV-939. First, the cerebral ischemia model was established by photothrombotic ischemia, followed by intraperitoneal injection with Diprotin A TFA and XAV-939 at doses of 70 microg/kg and 40 mg/kg 30 min once in the morning and once in the evening for 3 days. Immunofluorescence staining and Western blot methods were used to analyze the expression of vascular and blood-brain barrier (BBB)-associated molecular markers in the peri-infarct area. RESULTS: Compared with the vehicle control group, we found that mice injected with Diprotin A TFA exhibited reduced cerebral infarction volume, increased vascular area and length around the brain injury, increased pericyte and basement membrane coverage, upregulated expression of BBB tight junction proteins, and improved their BBB permeability, whereas the group injected with both drug and inhibitor exhibited significantly aggravated vascular injury and BBB permeability. CONCLUSION: Diprotin A TFA can reduce VE-cadherin disruption by inhibiting ischemia-hypoxia-induced beta-catenin cleavage to protect blood vessels.
Magnolol, the most abundant bioactive constituent of the Chinese herb Magnolia officinalis, has been found with multiple biological activities, including anti-oxidative, anti-inflammatory and enzyme-regulatory activities. In this study, the inhibitory effects and inhibition mechanism of magnolol on human carboxylesterases (hCEs), the key enzymes responsible for the hydrolytic metabolism of a variety of endogenous esters as well as ester-bearing drugs, have been well-investigated. The results demonstrate that magnolol strongly inhibits hCE1-mediated hydrolysis of various substrates, whereas the inhibition of hCE2 by magnolol is substrate-dependent, ranging from strong to moderate. Inhibition of intracellular hCE1 and hCE2 by magnolol was also investigated in living HepG2 cells, and the results showed that magnolol could strongly inhibit intracellular hCE1, while the inhibition of intracellular hCE2 was weak. Inhibition kinetic analyses and docking simulations revealed that magnolol inhibited both hCE1 and hCE2 in a mixed manner, which could be partially attributed to its binding at two distinct ligand-binding sites in each carboxylesterase, including the catalytic cavity and the regulatory domain. In addition, the potential risk of the metabolic interactions of magnolol via hCE1 inhibition was predicted on the basis of a series of available pharmacokinetic data and the inhibition constants. All these findings are very helpful in deciphering the metabolic interactions between magnolol and hCEs, and also very useful for avoiding deleterious interactions via inhibition of hCEs.
Human carboxylesterase 2 (CES2A), one of the most abundant hydrolases distributed in human small intestine and colon, play key roles in the hydrolysis of a wide range of prodrugs and other esters. Recent studies have demonstrated that CES2A inhibitors may ameliorate irinotecan-induced severe diarrhea, but the specific and efficacious inhibitors targeting intracellular CES2A are rarely reported. Herein, a large-scale screening campaign was conducted for discovery of potent and specific CES2A inhibitor(s). Following screening of more than one hundred of natural products, glabridin (a bioactive compound of Glycyrrhiza glabra L.) was found displaying potent inhibition on CES2A and high specificity over CES1A (>500-fold) and other serine hydrolases. Further investigation showed that glabridin was cell permeable and low cytotoxic, as well as capable of inhibiting intracellular CES2A in living cells, with the IC50 value of 0.52muM. Molecular dynamics simulations showed that glabridin formed strong and stable interactions with both the catalytic cavity and Z site of CES2A via hydrophobic interactions. In summary, glabridin was a potent and specific inhibitor targeting intracellular CES2A, which could be used as an ideal lead compound to develop more efficacious CES2A inhibitors for modulating the pharmacokinetic behaviors of CES2A-substrate drugs and alleviating irinotecan-induced diarrhea.
Clopidogrel, a clinically used antiplatelet agent, can be readily hydrolyzed by human carboxylesterase 1A (CES1A) to release an inactive metabolite clopidogrel carboxylic acid (CCA). In this study, clopidogrel was used as a tool substrate to investigate the interspecies variation of clopidogrel hydrolysis in hepatic microsomes from various mammals including human and six laboratory animals (such as mouse, rat, rabbit, beagle dog, minipig and cynomolgus monkey). The results demonstrated that clopidogrel could be hydrolyzed into CCA by all tested hepatic microsomes from human or other mammals, but the hydrolytic rates greatly varied among species. Inhibition assays demonstrated that BNPP (an inactivator of mammalian CES) strongly inactivated clopidogrel hydrolytic activity in all tested hepatic microsomes, suggested that mammalian CES were major contributor(s) responsible for clopidogrel hydrolysis in hepatic preparations from all above-mentioned species. By contrast, the response of a reversible inhibitor of human CES1A on clopidogrel hydrolysis in these liver preparations varied significantly among different species. Moreover, the enzymatic kinetics and the apparent kinetic parameters of clopidogrel hydrolysis in hepatic microsomes from various animal species were evaluated and compared to each other. These findings provide crucial information for deeply understanding the differences in catalytic behaviors of mammalian CES, which will be very helpful for choosing suitable laboratory animal(s) for whole tests of CES1A substrate-drugs.
Human carboxylesterase 1 (hCE1) is a key enzyme responsible for the hydrolysis of a wide range of endogenous and xenobiotic esters, but the highly selective inhibitors against hCE1 are rarely reported. This study aimed to assess the inhibitory effects of natural flavonoids against hCE1 and to find potential specific hCE1 inhibitors. To this end, fifty-eight natural flavonoids were collected and their inhibitory effects against both hCE1 and hCE2 were assayed. Among all tested compounds, nevadensin, an abundant natural constitute from Lysionotus pauciflorus Maxim., displayed the best combination of inhibition potency and selectivity towards hCE1. The inhibition mechanism of nevadensin on hCE1 was further investigated using two site-specific hCE1 substrates including D-luciferin methyl ester (DME) and 2(2benzoyloxy3methoxyphenyl)benzothiazole (BMBT). Furthermore, docking simulations demonstrated that the binding area of nevadensin on hCE1 was highly overlapped with that of DME but was far away from that of BMBT, which was highly consistent with the inhibition modes of nevadensin. These findings found a natural occurring specific inhibitor of hCE1, which could be served as a lead compound for the development of novel hCE1 inhibitor with improved properties, and also hold great promise for investigating hCE1-ligand interactions.
Title: Cannabinoid receptor agonist WIN55,212-2 and fatty acid amide hydrolase inhibitor URB597 may protect against cognitive impairment in rats of chronic cerebral hypoperfusion via PI3K/AKT signaling Su SH, Wang YQ, Wu YF, Wang DP, Lin Q, Hai J Ref: Behavioural Brain Research, 313:334, 2016 : PubMed
The present study further investigated the protective effects of cannabinoid receptor agonist WIN55,212-2 (WIN) and fatty acid amide hydrolase (FAAH) inhibitor URB597 (URB) on chronic cerebral hypoperfusion (CCH)-induced cognitive impairment in rats. Spatial learning and memory were assessed with the Morris water maze and by measuring Long-term potentiation. The expression of microtubule-associated protein-2 (MAP)-2, growth-associated protein-43 (GAP)-43, synaptophysin, cannabinoid receptor 1 (CB1), brain-derived neurotrophic factor (BDNF), FAAH, N-acylphosphatidylethanolamine phospholipase D(NAPE-PLD) and monoacyl glycerol lipase (MGL) as well as phosphoinositide 3-kinase (PI3K)/AKT signaling pathway molecules and downstream targets including AKT, phosphorylated (p-)AKT, cyclic AMP response element- binding protein (CREB), p-CREB, Bcl-2-associated death protein (BAD), p-BAD, glycogen synthase kinase (GSK)-3beta, p-GSK-3beta, forkhead box protein (FOXO) 3A and p-FOXO3A was determined by western blotting. WIN and URB treatment improved learning and memory performance, effects that were abolished by co-administration of the PI3K/AKT inhibitor LY294002. Moreover, WIN and URB reversed the decreases in MAP-2 and synaptophysin expression resulting from CCH, and stimulated BDNF and CB1 expression as well as CREB, FOXO3A, GSK-3beta, and BAD phosphorylation, confirming that WIN and URB mediate neuroprotection by preventing neuronal apoptosis and improving cognition via PI3K/AKT signaling. These findings suggest that WIN and URB are promising agents for therapeutic management of CCH.
Mitochondrial ferritin (FtMt) has a significant effect on the regulation of cytosolic and mitochondrial iron levels. However, because of the deficiency of iron regulatory elements (IRE) in FtMt's gene sequence, the exact function of FtMt remains unclear. In the present study, we found that FtMt dramatically inhibited SH-SY5Y cell proliferation and tumor growth in nude mice. Interestingly, excess FtMt did not adversely affect the development of drosophila. Additionally, we found that the expression of FtMt in human normal brain tissue was significantly higher than that of neuroblastoma, but not higher than that of neurospongioma. However, the expression of transferrin receptor 1 is completely opposite. We therefore hypothesized that increased expression of FtMt may negatively affect the vitality of neuronal tumor cells. Therefore, we further investigated the underlying mechanisms of FtMt's inhibitory effects on neuronal tumor cell proliferation. As expected, FtMt overexpression disturbed the iron homeostasis of tumor cells and significantly downregulated the expression of proliferating cell nuclear antigen. Moreover, FtMt affected cell cycle, causing G1/S arrest by modifying the expression of cyclinD1, cyclinE, Cdk2, Cdk4 and p21. Remarkably, FtMt strongly upregulated the expression of the tumor suppressors, p53 and N-myc downstream-regulated gene-1 (NDRG1), but dramatically decreased C-myc, N-myc and p-Rb levels. This study demonstrates for the first time a new role and mechanism for FtMt in the regulation of cell cycle. We thus propose FtMt as a new candidate target for inhibiting neuronal tumor cell proliferation. Appropriate regulation of FtMt expression may prevent tumor cell growth. Our study may provide a new strategy for neuronal cancer therapy.
Excessive release of high mobility group box-1 (HMGB1) protein from ischemic cardiomyocytes activates inflammatory cascades and enhances myocardial injury after reperfusion. Here we report evidence that electroacupuncture of mice at Neiguan acupoints can inhibit the up-regulation of cardiac HMGB1 following myocardial ischemia and attenuate the associated inflammatory responses and myocardial injury during reperfusion. These benefits of electroacupuncture were partially reversed by administering recombinant HMGB1 to the mice, and further potentiated by administering anti-HMGB1 antibody. Electroacupuncture-induced inhibition of HMGB1 release was markedly reduced by unilateral vagotomy or administration of nicotinic receptor antagonist, but not by chemical sympathectomy. The cholinesterase inhibitor neostigmine mimicked the effects of electroacupuncture on HMGB1 release and myocardial ischemia reperfusion injury. Culture experiments with isolated neonatal cardiomyocytes showed that acetylcholine, but not noradrenaline, inhibited hypoxia-induced release of HMGB1 via a alpha7nAchR-dependent pathway. These results suggest that electroacupuncture acts via the vagal nerve and its nicotinic receptor-mediated signaling to inhibit HMGB1 release from ischemic cardiomyocytes. This helps attenuate pro-inflammatory responses and myocardial injury during reperfusion.
Title: Design, synthesis and evaluation of novel 4-dimethylamine flavonoid derivatives as potential multi-functional anti-Alzheimer agents Luo W, Su YB, Hong C, Tian RG, Su LP, Wang YQ, Li Y, Yue JJ, Wang CJ Ref: Bioorganic & Medicinal Chemistry, 21:7275, 2013 : PubMed
A series of 4-dimethylamine flavonoid derivatives 5a-5r were designed, synthesized and evaluated as potential multi-functional anti-Alzheimer agents. The results showed that most of the synthesized compounds exhibited high acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity at the micromolar range (IC50, 1.83-33.20muM for AChE and 0.82-11.45muM for BChE). A Lineweaver-Burk plot indicated a mixed-type inhibition for compound 5j with AChE, and molecular modeling study showed that 5j targeted both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. Besides, the derivatives showed potent self-induced Abeta aggregation inhibitory activity at 20muM with percentage from 25% to 48%. In addition, some compounds (5j-5q) showed potent oxygen radical absorbance capacity (ORAC) ranging from 1.5- to 2.6-fold of the Trolox value. These compounds should be further investigated as multi-potent agents for the treatment of Alzheimer's disease.
Rg1 and Rb1 are two major active compounds of ginseng that facilitate learning and memory. The present study aimed to compare the nootropic effects of Rg1 and Rb1 in a scopolamine induced dementia mice model. After 6 and 12 mg/kg of Rg1 and Rb1 intraperitoneal administration to mice for 7 days, their effects were assessed using the step-down passive avoidance (SD) and the Morris water maze (MWM) tests, the acetylcholinesterase (AChE) activity, acetylcholine (ACh) content and serotonin (5-HT) level in the hippocampus were analysed after SD and MWM tests. The results showed that Rg1 and Rb1 ameliorated cognition-deficiency in mice with dementia. Rg1 showed stronger effects than Rb1 on escape acquisition in MWM. Both Rg1 and Rb1 increased ACh levels in the hippocampus, but Rg1 inhibited AChE activity while Rb1 had no effect on AChE activity. Both Rg1 and Rb1 inhibited the decrease of 5-HT induced by scopolamine, but Rb1 was more active than the same dose of Rg1. These results demonstrate that multiple administrations of Rg1 and Rb1 are effective in improving memory deficiency induced by scopolamine. Rg1 appears to be more potent than Rb1 in improving acquisition impairment, and the two ginsenosides may act through different mechanisms.
