[Huangpu Tongqiao Capsule improves cognitive impairment in rats with Wilson disease by inhibiting endoplasmic reticulum stress-mediated apoptosis pathway].

OBJECTIVE: To investigate the neuroprotective effect of Huangpu Tongqiao Capsule (HPTQ) in a rat model of Wilson disease (WD) and explore the underlying mechanisms. METHODS: SD rat models of WD were established by feeding of coppersupplemented chow diet and drinking water for 12 weeks, and starting from the 9th week, the rats were treated with low-, moderate- and high-dose HPTQ, penicillamine, or normal saline by gavage on a daily basis for 3 weeks. Copper levels in the liver and 24-h urine of the rats were detected, and their learning and memory abilities were evaluated using Morris water maze test. HE staining was used to observe morphological changes of CA1 region neurons in the hippocampus, and neuronal apoptosis was detected with TUNEL staining. Hippocampal expressions of endoplasmic reticulum stress (ERS)-mediated apoptosis pathway-related proteins GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 at both the mRNA and protein levels were detected using RT-qPCR, immunofluorescence assay or Western blotting. RESULTS: Compared with normal control rats, the rat models with copper overload-induced WD exhibited significantly increased copper levels in both the liver and 24-h urine, impaired learning and memory abilities, obvious hippocampal neuronal damage in the CA1 region and increased TUNEL-positive neurons (P<0.01), with also lowered mRNA and protein expressions of GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 in the hippocampus (all P<0.01). Treatments with HPTQ and penicillamine significantly lowered copper level in the liver but increased urinary copper level, improved learning and memory ability, alleviated neuronal damage and apoptosis in the hippocampus, and decreased hippocampal expressions of GRP78, CHOP, caspase-12, cleaved caspase-9, and cleaved caspase-3 in the rat models (P<0.01 or 0.05). CONCLUSION: HPTQ Capsule has neuroprotective effects in rat models of WD possibly by inhibiting ERS-mediated apoptosis pathway.

Antioxidant effects of penicillamine against in vitro-induced oxidative stress in human spermatozoa.

Oxidative stress contributes importantly to the aetiology of male infertility, impairing sperm function. The protective effect of antioxidants on seminal parameters has been established, and the antioxidant penicillamine has shown beneficial effects; however, its protective effect on human spermatozoa exposed to oxidative stress has not been reported. The objective of this work was to evaluate the effect of penicillamine on human spermatozoa exposed in vitro to oxidative stress. First, the effect of penicillamine on spermatozoa from normozoospermic donors was evaluated. Then, the effect of penicillamine on spermatozoa exposed to oxidative stress induced separately by ionomycin and hydrogen peroxide (H2 O2 ) was analysed. An untreated control and a control treated only with the oxidative stress inducer were included. Reactive oxygen species (ROS) levels, viability, mitochondrial membrane potential (MMP) and motility were analysed. The results showed that penicillamine, added to the incubation medium, decreased the ROS levels induced by ionomycin and H2 O2 , and this effect was associated with better preservation of MMP, motility, and ATP levels. These results highlight the potential advantages of penicillamine supplementation of sperm culture medium, especially for semen samples with high ROS levels and also in circumstances where laboratory handling can cause an increase in ROS production.

Nitric oxide-releasing vascular grafts: A therapeutic strategy to promote angiogenic activity and endothelium regeneration.

Small-diameter vascular grafts (SDVGs) are associated with a high incidence of failure due to infection and obstruction. Although several vascular grafts are commercially available, specific anatomical differences of defect sites require patient-based design and fabrication. Design and fabrication of such custom-tailored grafts are possible with 3d-printing technology. The aim of this study is to develop 3d-printed SDVGs with a nitric oxide (NO)-releasing coating to improve the success rate of implantation. The SDVGs were printed from polylactic acid and coated with blending of 10 wt% S-nitroso-N-acetyl-D-penicillamine into the polymeric substrate consisting of poly (ethylene glycol) and polycaprolactone. Our results show that NO is released in the physiological range (0.5-4 × 10-10 mol·cm-2·min-1) for 14 days and NO-releasing coating showed significant antibacterial potential against Gram-positive and Gram-negative strains. It was shown that both NO-releasing and control grafts are biocompatible in-vitro and in-vivo. Interestingly, the NO-releasing SDVGs dramatically enhanced ECs proliferation and significantly enhanced ECs migration in-vitro compared to control grafts. In addition, the NO-releasing SDVGs showed angiogenic potential in-vivo which can further prove the results of our in-vitro study. These findings are expected to facilitate tissue regeneration and integration of custom-made vascular implants with enhanced clinical success. STATEMENT OF SIGNIFICANCE: A series of 3d-printed small-diameter vascular grafts (SDVGs, <6 mm) with controlled release of nitric oxide (NO) were prepared to combine the advantages of 3D printing technology and NO-releasing systems. The resulting NO-releasing grafts were promisingly showing sustained NO release in the physiological range over a two weeks period. In addition to the evaluation of endothelial cell migration in-vitro, we implanted for the first time the NO-releasing vascular grafts in a chick chorioallantoic membrane (CAM) to investigate the effect of the prepared grafts on the angiogenesis in-vivo. The fabricated grafts also exhibited bactericidal properties which prevent the formation of a biofilm layer and can thereby enhance the chance of endothelialization on the surface. Taken together, the innovative combination of rapid and highly accurate 3d-printing technology as a patient-specific fabrication method with NO-releasing coating represents a promising approach to develop bactericidal SDVGs with improved endothelialization.

Copper(I)-binding properties of de-coppering drugs for the treatment of Wilson disease. α-Lipoic acid as a potential anti-copper agent.

Wilson disease is an autosomal recessive genetic disorder caused by loss-of-function mutations in the P-type copper ATPase, ATP7B, which leads to toxic accumulation of copper mainly in the liver and brain. Wilson disease is treatable, primarily by copper-chelation therapy, which promotes copper excretion. Although several de-coppering drugs are currently available, their Cu(I)-binding affinities have not been quantitatively characterized. Here we determined the Cu(I)-binding affinities of five major de-coppering drugs - D-penicillamine, trientine, 2,3-dimercapto-1-propanol, meso-2,3-dimercaptosuccinate and tetrathiomolybdate - by exploring their ability to extract Cu(I) ions from two Cu(I)-binding proteins, the copper chaperone for cytochrome c oxidase, Cox17, and metallothionein. We report that the Cu(I)-binding affinity of these drugs varies by four orders of magnitude and correlates positively with the number of sulfur atoms in the drug molecule and negatively with the number of atoms separating two SH groups. Based on the analysis of structure-activity relationship and determined Cu(I)-binding affinity, we hypothesize that the endogenous biologically active substance, α-lipoic acid, may be suitable for the treatment of Wilson disease. Our hypothesis is supported by cell culture experiments where α-lipoic acid protected hepatic cells from copper toxicity. These results provide a basis for elaboration of new generation drugs that may provide better therapeutic outcomes.

Spatial investigation of the elemental distribution in Wilson's disease liver after d-penicillamine treatment by LA-ICP-MS.

At present, the copper chelator d-penicillamine (DPA) is the first-line therapy of Wilson's disease (WD), which is characterized by an excessive copper overload. Lifelong DPA treatments aim to reduce the amount of detrimental excess copper retention in the liver and other organs. Although DPA shows beneficial effect in many patients, it may cause severe adverse effects. Despite several years of copper chelation therapy, discontinuation of DPA therapy can be linked to a rapidly progressing liver failure, indicating a high residual liver copper load. In order to investigate the spatial distribution of remaining copper and additional elements, such as zinc and iron, in rat and human liver samples after DPA treatment, a high resolution (spotsize of 10µm) laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) imaging method was applied. Untreated LPP-/- rats, an established animal model for WD, appeared with a high overall copper concentration and a copper distribution of hotspots distributed over the liver tissue. In contrast, a low (>2-fold decreased) overall copper concentration was detected in liver of DPA treated animals. Importantly, however, copper distribution was highly inhomogeneous with lowest concentrations in direct proximity to blood vessels, as observed using novel zonal analysis. A human liver needle biopsy of a DPA treated WD patient substantiated the finding of an inhomogeneous copper deposition upon chelation therapy. In contrast, comparatively homogenous distributions of zinc and iron were observed. Our study indicates that a high resolution LA-ICP-MS analysis of liver samples is excellently suited to follow efficacy of chelator therapy in WD patients.

D-penicillamine combined with inhibitors of hydroperoxide metabolism enhances lung and breast cancer cell responses to radiation and carboplatin via H2O2-mediated oxidative stress.

D-penicillamine (DPEN), a copper chelator, has been used in the treatment of Wilson's disease, cystinuria, and rheumatoid arthritis. Recent evidence suggests that DPEN in combination with biologically relevant copper (Cu) concentrations generates H2O2 in cancer cell cultures, but the effects of this on cancer cell responses to ionizing radiation and chemotherapy are unknown. Increased steady-state levels of H2O2 were detected in MB231 breast and H1299 lung cancer cells following treatment with DPEN (100µM) and copper sulfate (15µM). Clonogenic survival demonstrated that DPEN-induced cancer cell toxicity was dependent on Cu and was significantly enhanced by depletion of glutathione [using buthionine sulfoximine (BSO)] as well as inhibition of thioredoxin reductase [using Auranofin (Au)] prior to exposure. Treatment with catalase inhibited DPEN toxicity confirming H2O2 as the toxic species. Furthermore, pretreating cancer cells with iron sucrose enhanced DPEN toxicity while treating with deferoxamine, an Fe chelator that inhibits redox cycling, inhibited DPEN toxicity. Importantly, DPEN also demonstrated selective toxicity in human breast and lung cancer cells, relative to normal untransformed human lung or mammary epithelial cells and enhanced cancer cell killing when combined with ionizing radiation or carboplatin. Consistent with the selective cancer cell toxicity, normal untransformed human lung epithelial cells had significantly lower labile iron pools than lung cancer cells. These results support the hypothesis that DPEN mediates selective cancer cell killing as well as radio-chemo-sensitization by a mechanism involving metal ion catalyzed H2O2-mediated oxidative stress and suggest that DPEN could be repurposed as an adjuvant in conventional cancer therapy.

Protective Effects of D-Penicillamine on Catecholamine-Induced Myocardial Injury.

Iron and copper release participates in the myocardial injury under ischemic conditions and hence protection might be achieved by iron chelators. Data on copper chelation are, however, sparse. The effect of the clinically used copper chelator D-penicillamine in the catecholamine model of acute myocardial injury was tested in cardiomyoblast cell line H9c2 and in Wistar Han rats. D-Penicillamine had a protective effect against catecholamine-induced injury both in vitro and in vivo. It protected H9c2 cells against the catecholamine-induced viability loss in a dose-dependent manner. In animals, both intravenous D-penicillamine doses of 11 (low) and 44 mg/kg (high) decreased the mortality caused by s.c. isoprenaline (100 mg/kg) from 36% to 14% and 22%, respectively. However, whereas the low D-penicillamine dose decreased the release of cardiac troponin T (specific marker of myocardial injury), the high dose resulted in an increase. Interestingly, the high dose led to a marked elevation in plasma vitamin C. This might be related to potentiation of oxidative stress, as suggested by additional in vitro experiments with D-penicillamine (iron reduction and the Fenton reaction). In conclusion, D-penicillamine has protective potential against catecholamine-induced cardiotoxicity; however the optimal dose selection seems to be crucial for further application.

Liquid storage of equine semen: Assessing the effect of d-penicillamine on longevity of ejaculated and epididymal stallion sperm.

Short-term storage of equine sperm at 5°C in an extender containing milk and/or egg yolk components is common practice in the equine breeding industry. Sperm motility, viability, DNA integrity and, consequently, fertilizing ability decline over time, partly due to reactive oxygen species (ROS) generation. We investigated whether adding the anti-oxidant d-penicillamine to a commercial milk/egg yolk extender delayed the decrease in semen quality. Semen was recovered on four consecutive days from eight 3-year old Warmblood stallions. On day 5, seven of the stallions were castrated and sperm recovered from the caudae epididymides. Ejaculated samples were split, and one portion was centrifuged and re-suspended to reduce seminal plasma content. All samples were diluted to 50millionsperm/ml and divided into two portions, one of which was supplemented with 0.5mM d-penicillamine. After 48h, 96h, 144h and 192h storage, sperm motility was assessed by computer-assisted semen analysis (CASA), viability by SYBR14/PI staining, and DNA integrity using the sperm chromatin structure assay (SCSA). d-Penicillamine had no effect on motility of ejaculated sperm (P>0.05) but reduced total and progressive motility of epididymal sperm. Sperm chromatin integrity was not influenced by storage time, seminal plasma or d-penicillamine. In short, adding d-penicillamine to a commercial semen extender was neither beneficial nor detrimental to the maintenance of quality in ejaculated semen stored at 5°C. The negative effect on motility of epididymal sperm may reflect differences in (membrane) physiology of spermatozoa that have not been exposed to seminal plasma.

Effects of tetrathiomolybdate and penicillamine on brain hydroxyl radical and free copper levels: a microdialysis study in vivo.

Wilson disease is an inherited disorder of excessive copper accumulation. The commonly used drug d-penicillamine (PA) or trientine both cause a high incidence (10-50%) of neurological worsening, which rarely occurs with tetrathiomolybdate (TM) treatment. To investigate the mechanisms of neurologic deterioration after the initiation of chelation therapy, brain hydroxyl radical and free copper were assessed in vivo in this study. On days 3, 7, 14, and 21 after PA or TM administration, striatal hydroxyl radical levels of both TX mice and controls were assessed by terephthalic acid (TA) combined with microdialysis and high-performance liquid chromatography (HPLC). Within the same microdialysis samples, free copper was measured by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that both hydroxyl radical and free copper markedly increased in the striatum of TX mice during PA administration but were not elevated when administering TM. These results suggested that the further increased free copper in the brain and oxidative stress caused by some chelators might contribute to the neurological deterioration.

Effects of D-penicillamine on pentylenetetrazole-induced seizures in mice: involvement of nitric oxide/NMDA pathways.

Besides the clinical applications of penicillamine, some reports show that use of D-penicillamine (D-pen) has been associated with adverse effects such as seizures. So, the purpose of this study was to evaluate the effects of D-pen on pentylenetetrazole (PTZ)-induced seizures in male NMRI mice. It also examined whether N-methyl-D-aspartate (NMDA) receptor/nitrergic system blockage was able to alter the probable effects of D-pen. Different doses of D-pen (0.1, 0.5, 1, 10, 100, 150, and 250 mg/kg) were administered intraperitoneally (i.p.) 90 min prior to induction of seizures. D-Penicillamine at a low dose (0.5 mg/kg, i.p.) had anticonvulsant effects, whereas at a high dose (250 mg/kg, i.p.), it was proconvulsant. Both anti- and proconvulsant effects of D-pen were blocked by a single dose of a nonspecific inhibitor of nitric oxide synthase (NOS), L-NAME (10 mg/kg, i.p.), and a single dose of a specific inhibitor of neuronal nitric oxide synthase (nNOS), 7-nitroindazole (30 mg/kg, i.p.). A selective inhibitor of iNOS, aminoguanidine (100 mg/kg, i.p.), had no effect on these activities. An NMDA receptor antagonist, MK-801 (0.05 mg/kg, i.p.), alters the anti- and proconvulsant effects of D-pen. The results of the present study showed that the nitric oxide system and NMDA receptors may contribute to the biphasic effects of D-pen, which remain to be clarified further.

Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development.

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development.

Treatment with D-penicillamine or zinc sulphate affects copper metabolism and improves but not normalizes antioxidant capacity parameters in Wilson disease.

Copper accumulation in tissues due to a biallelic pathogenic mutation of the gene: ATP7B results in a clinical phenotype known as Wilson disease (WD). Aberrations in copper homeostasis can create favourable conditions for superoxide-yielding redox cycling and oxidative tissue damage. Drugs used in WD treatment aim to remove accumulated copper and normalise the free copper concentration in the blood. In the current study the effect of decoppering treatment on copper metabolism and systemic antioxidant capacity parameters was analyzed. Treatment naïve WD patients (TNWD) (n = 33), those treated with anti-copper drugs (TWD) (n = 99), and healthy controls (n = 99) were studied. Both TNWD and TWD patients characterised with decreased copper metabolism parameters, as well as decreased total antioxidant potential (AOP), glutathione (GSH) level, activity of catalase, glutathione peroxidase (GPx), and S-transferase glutathione, compared to controls. TWD patients had significantly lower copper metabolism parameters, higher total AOP and higher levels of GSH than TWD individuals; however, no difference was observed between these two patient groups with respect to the rest of the antioxidant capacity parameters. Patients who had undergone treatment with D-penicillamine or zinc sulphate did not differ with respect to copper metabolism or antioxidant capacity parameters, with the exception of GPx that was lower in D-penicillamine treated individuals. These data suggest that anti-copper treatment affects copper metabolism as well as improves, but does not normalize, natural antioxidant capacity in patients with WD. We propose to undertake studies aimed to evaluate the usefulness of antioxidants as well as selenium as a supplemental therapy in WD.

Wilson's disease: changes in methionine metabolism and inflammation affect global DNA methylation in early liver disease.

UNLABELLED: Hepatic methionine metabolism may play an essential role in regulating methylation status and liver injury in Wilson's disease (WD) through the inhibition of S-adenosylhomocysteine hydrolase (SAHH) by copper (Cu) and the consequent accumulation of S-adenosylhomocysteine (SAH). We studied the transcript levels of selected genes related to liver injury, levels of SAHH, SAH, DNA methyltransferases genes (Dnmt1, Dnmt3a, Dnmt3b), and global DNA methylation in the tx-j mouse (tx-j), an animal model of WD. Findings were compared to those in control C3H mice, and in response to Cu chelation by penicillamine (PCA) and dietary supplementation of the methyl donor betaine to modulate inflammatory and methylation status. Transcript levels of selected genes related to endoplasmic reticulum stress, lipid synthesis, and fatty acid oxidation were down-regulated at baseline in tx-j mice, further down-regulated in response to PCA, and showed little to no response to betaine. Hepatic Sahh transcript and protein levels were reduced in tx-j mice with consequent increase of SAH levels. Hepatic Cu accumulation was associated with inflammation, as indicated by histopathology and elevated serum alanine aminotransferase (ALT) and liver tumor necrosis factor alpha (Tnf-α) levels. Dnmt3b was down-regulated in tx-j mice together with global DNA hypomethylation. PCA treatment of tx-j mice reduced Tnf-α and ALT levels, betaine treatment increased S-adenosylmethionine and up-regulated Dnmt3b levels, and both treatments restored global DNA methylation levels. CONCLUSION: Reduced hepatic Sahh expression was associated with increased liver SAH levels in the tx-j model of WD, with consequent global DNA hypomethylation. Increased global DNA methylation was achieved by reducing inflammation by Cu chelation or by providing methyl groups. We propose that increased SAH levels and inflammation affect widespread epigenetic regulation of gene expression in WD.

Sperm motility is lost in vitro as a consequence of mitochondrial free radical production and the generation of electrophilic aldehydes but can be significantly rescued by the presence of nucleophilic thiols.

The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic aldehydes, as a means of ensuring the long-term survival of spermatozoa in vitro. Four nucleophilic compounds were consequently assessed (penicillamine, homocysteine, N-acetylcysteine, and mercaptosuccinate) in three species (human, rat, and horse). The results of this analysis revealed drug and species specificity in the manner in which these compounds affected sperm function, with penicillamine conferring the most consistent, effective support. This prosurvival effect was achieved downstream of mitochondrial reactive oxygen species generation and was associated with the stabilization of 4-hydroxynonenal generation, the preservation of sperm thiols, and a reduction in 8-hydroxy-2'-deoxyguanosine formation. Theoretical calculations of Fe-S and Cu-S bond distances and corresponding binding energies suggested that the particular effectiveness of penicillamine may, in part, reflect the ability of this nucleophile to form stable complexes with transition metals that catalyze lipid peroxidation. The practical implications of these findings were indicated by the effective preservation of equine spermatozoa for 8 days at ambient temperature when the culture medium was supplemented with penicillamine.

Chelation therapy in Wilson's disease: from D-penicillamine to the design of selective bioinspired intracellular Cu(I) chelators.

Wilson's disease is an orphan disease due to copper homeostasis dysfunction. Mutations of the ATP7B gene induces an impaired functioning of a Cu-ATPase, impaired Cu detoxification in the liver and copper overload in the body. Indeed, even though copper is an essential element, which is used as cofactor by many enzymes playing vital roles, it becomes toxic when in excess as it promotes cytotoxic reactions leading to oxidative stress. In this perspective, human copper homeostasis is first described in order to explain the mechanisms promoting copper overload in Wilson's disease. We will see that the liver is the main organ for copper distribution and detoxification in the body. Nowadays this disease is treated life-long by systemic chelation therapy, which is not satisfactory in many cases. Therefore the design of more selective and efficient drugs is of great interest. A strategy to design more specific chelators to treat localized copper accumulation in the liver will then be presented. In particular we will show how bioinorganic chemistry may help in the design of such novel chelators by taking inspiration from the biological copper cell transporters.

Comparison of therapeutic effects of garlic and d-Penicillamine in patients with chronic occupational lead poisoning.

Previous studies on animals have revealed that garlic (Allium sativum) is effective in reducing blood and tissue lead concentrations. The aim of this study was to investigate therapeutic effects of garlic and compare it with d-penicillamine in patients with chronic lead poisoning. After coordination and obtaining informed consent, clinical examinations and blood lead concentration (BLC) of 117 workers at a car battery industry were investigated. BLC was determined by heated graphite atomization technique of an atomic absorption spectrometer. The workers were randomly assigned into two groups of garlic (1200 µg allicin, three times daily) and d-penicillamine (250 mg, three times daily) and treated for 4 weeks. BLC was determined again 10days post-treatment. Clinical signs and symptoms of lead poisoning were also investigated and compared with the initial findings. Clinical improvement was significant in a number of clinical manifestations including irritability (p = 0.031), headache (p = 0.028), decreased deep tendon reflex (p=0.019) and mean systolic blood pressure (0.021) after treatment with garlic, but not d-penicillamine. BLCs were reduced significantly (p=0.002 and p=0.025) from 426.32±185.128 to 347.34±121.056 µg/L and from 417.47±192.54 to 315.76±140.00µg/L in the garlic and d-penicillamine groups, respectively, with no significant difference (p=0.892) between the two groups. The frequency of side effects was significantly (p=0.023) higher in d-penicillamine than in the garlic group. Thus, garlic seems safer clinically and as effective as d-penicillamine. Therefore, garlic can be recommended for the treatment of mild-to-moderate lead poisoning.

Isolation and quantitative analysis of peroxynitrite scavengers from Artemisia princeps var. orientalis.

Young and mature Artemisia princeps var. orientalis (APO, Compositae) are used as a health food and a medicinal plant, respectively, in Korea. Here, we identified the in vitro potent peroxynitrite (ONOO(-))-scavenging effect (IC(50), 0.26 microg/mL) of the components from the EtOAc fraction. Octadecylsilane column chromatography on the EtOAc fraction yielded two caffeoylquinic acid compounds, 3,5-di-O-caffeoyl-muco-quinic acid (1) and methyl 4,5-di-O-caffeoylquinate (2) by NMR spectroscopic data, which have not been reported before from APO. The IC(50) values of compounds 1 and 2 were 0.18 +/- 0.01 microg/mL and 0.12 +/- 0.00 microg/mL, respectively, lower than that of the positive control (L-penicillamine). HPLC data indicated that young APO (1: 30.3 mg/g dried weight, 2: 27.7 mg/g) contained considerably higher quantities of the two caffeoylquinic acids than mature APO (1: 1.77 mg/g dried weight, 2: 4.10 mg/g).

[Effect of Wenyang Huazhuo Tongluo recipe contained serum on proliferation and cell cycle of systemic sclerosis skin fibroblasts].

OBJECTIVE: To investigate the effect of "Wenyang Huazhuo Tongluo recipe" (WYHZTLR) on proliferation and cell cycle of systemic sclerosis (SSc) skin fibroblasts in vitro by serum pharmacologic technique. METHODS: Prednisone, D-penicillamine and WYHZTLR contained serum from patients with SSc were respectively added into cultured normal dermal fibroblast cell-line and SSc lesional dermal fibroblast cell-line. Then the cells were incubated for 48 hours. Cell proliferation and cell cycle were examined by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. RESULTS: Western medicine Chinese medicine and integrated Chinese and western medicine could markedly inhibit the proliferation of normal dermal fibroblasts and SSc lesional dermal fibroblasts in vitro compared with control group (P < 0.05 or P < 0.01). But 20% serum in integrated Chinese and western medicine group could markedly inhibit the proliferation of fibroblasts compared with western medicine group (P < 0.01). Different drug-contained serum of WYHZTLR could markedly increase the percentage of G0-G1 stage cells (P < 0.01) and depress the percentage of sphase cells and G2-M stage cells compared with control group (P < 0.05 or P < 0.01). CONCLUSION: WYHZTLR can block the progression of SSc skin fibroblasts into sphase and G2-M stage and inhibit the proliferation of SSc skin fibroblasts, and WYHZTLR was unselectively interfered with normal and SSc fibroblasts in vitro.

Glucocorticoids regulate glutamate and GABA synapse-specific retrograde transmission via divergent nongenomic signaling pathways.

Glucocorticoids exert an opposing rapid regulation of glutamate and GABA synaptic inputs to hypothalamic magnocellular neurons via the activation of postsynaptic membrane-associated receptors and the release of retrograde messengers. Glucocorticoids suppress synaptic glutamate release via the retrograde release of endocannabinoids and facilitate synaptic GABA release via an unknown retrograde messenger. Here, we show that the glucocorticoid facilitation of GABA inputs is due to the retrograde release of neuronal nitric oxide and that glucocorticoid-induced endocannabinoid synthesis and nitric oxide synthesis are mediated by divergent G-protein signaling mechanisms. While the glucocorticoid-induced, endocannabinoid-mediated suppression of glutamate release is dependent on activation of the G(alpha)s G-protein subunit and cAMP-cAMP-dependent protein kinase activation, the nitric oxide facilitation of GABA release is mediated by G(beta)gamma signaling that leads to activation of neuronal nitric oxide synthase. Our findings indicate, therefore, that glucocorticoids exert opposing rapid actions on glutamate and GABA release by activating divergent G-protein signaling pathways that trigger the synthesis of, and glutamate and GABA synapse-specific retrograde actions of, endocannabinoids and nitric oxide, respectively. The simultaneous rapid stimulation of nitric oxide and endocannabinoid synthesis by glucocorticoids has important implications for the impact of stress on the brain as well as on neural-immune interactions in the hypothalamus.

Effects of chelators on mercury, iron, and lead neurotoxicity in cortical culture.

Chelation therapy for the treatment of acute, high dose exposure to heavy metals is accepted medical practice. However, a much wider use of metal chelators is by alternative health practitioners for so called "chelation therapy". Given this widespread and largely unregulated use of metal chelators it is important to understand the actions of these compounds. We tested the effects of four commonly used metal chelators, calcium disodium ethylenediaminetetraacetate (CaNa2EDTA), D-penicillamine (DPA), 2,3 dimercaptopropane-1-sulfonate (DMPS), and dimercaptosuccinic acid (DMSA) for their effects on heavy metal neurotoxicity in primary cortical cultures. We studied the toxicity of three forms of mercury, inorganic mercury (HgCl2), methyl mercury (MeHg), and ethyl mercury (thimerosal), as well as lead (PbCl2) and iron (Fe-citrate). DPA had the worst profile of effects, providing no protection while potentiating HgCl2, thimerosal, and Fe-citrate toxicity. DMPS and DMSA both attenuated HgCl2 toxicity and potentiated thimerosal and Fe toxicity, while DMPS also potentiated PbCl2 toxicity. CaNa2EDTA attenuated HgCl2 toxicity, but caused a severe potentiation of Fe-citrate toxicity. The ability of these chelators to attenuate the toxicity of various metals is quite restricted, and potentiation of toxicity is a serious concern. Specifically, protection is provided only against inorganic mercury, while it is lacking against the common form of mercury found in food, MeHg, and the form found in vaccines, thimerosal. The potentiation of Fe-citrate toxicity is of concern because of iron's role in oxidative stress in the body. Potentiation of iron toxicity could have serious health consequences when using chelation therapy.