Tanshinone ⅡA inhibits endothelin-1 production in TNF-α-induced brain microvascular endothelial cells through suppression of endothelin-converting enzyme-1 synthesis

Aim： To investigate the effects of tanshinone ⅡA （Tan ⅡA） on the regulation of the production of endothelin （ET）- 1 （including large ET- 1）, mRNA levels of ET- 1, endothelin-converting enzyme- 1 （ECE- 1）, endothelin-A receptor （ETA） and endothelin-B receptor （ETB） induced by TNF-α in rat brain microvascular endothelial cells （BMVEC）. Methods： The ET-1 release （including large ET-1） into the culture medium was determined by enzyme immunoassay. The levels of ET-1, ECE- 1, ETA, and ETB mRNA were measured by RT-PCR. Endothelin receptor binding was also tested. Results： The induction of ET- 1 release by TNF-α from cultured BMVEC was dose-dependently reduced by Tan ⅡA, but large ET-1 levels progressively increased in response to Tan ⅡA; the mRNA expression of ET- 1 was unaffected. Tan ⅡA also caused a decrease in ETA receptor mRNA and ECE- 1 expression in a dose-dependent manner. Endothelin receptor binding was unaltered in BMVEC stimulated with TNF-α alone or a combination of TNF-α and Tan ⅡA. Conclusion： These findings suggest that Tan ⅡA may inhibit ET-1 production in TNF-α-induced BMVEC through the suppression of ECE-1 synthesis.     

MgCl2和Gpp（NH）p对腺苷A1受体激动剂和拮抗剂结合的作用比较

AIM: To investigate modulation of antagonist and agonist binding to adenosine A1 receptors by MgCI2 and 5'-guanylimidodiphosphate (Gpp(NH)p) using rat brain membranes and the A1 antagonist [^3H]-8-cyclopentyl-1,3-dipropylxanthine ([^3H]DPCPX) and the A1 agonist [^3H]-2-chloro-N^6-cyclopentyladenosine ([^3H]CCPA). METHODS:Parallel saturation and inhibition studies were performed using well-characterised radioligand binding assays and aBrandel Cell Harvester. RESULTS: MgCI2 produced a concentration-dependent decrease (44%), whereasGpp(NH)p increased [^3H]DPCPX binding (19%). In [^3H]DPCPX competition studies, agonist affinity was 1.5-14.6-fold higher and 4.6-10-fold lower in the presence of l0 mmol/L MgCl2 and l0μmol/L Gpp(NH)p respectively;antagonist affinity was unaffected. The decrease in agonist affinity with increasing Gpp(NH)p concentrations was due to a reduction in the proportion of binding to the high affinity receptor state. In contrast to [^3H]DPCPX, MgCl2produced a concentration-dependent increase (72%) and Gpp(NH)p a decrease (85%) in [^3H]CCPA binding.Using [^3H]CCPA, agonist affinities were 5-17-fold higher than those for [^3H]DPCPX, consistent with binding onlyto the high affinity receptor state. Agonist affinity was 1.3-10.5-fold higher and 2.4-4.7-fold lower on addingMgCl2 or Gpp(NH)p respectively; antagonist affinities were as for [^3H]DPCPX. CONCLUSION: The inconsistencies surrounding the effects of MgCl2 and guanine nucleotides on radioligand binding to adenosine A1 receptorswere systematically examined. The effects of MgCl2 and Gpp(NH)p on agonist binding to A1 receptors are consistent with their roles in stimulating GTP-hydrolysis at the G-protein α-subunit and in blocking formation of the highaffinity agonist-receptor-G protein complex.     

Induction of CRMP-4 in striatum of adult rat after transient brain ischemia

AIM: To study the expression of collapsing response mediated protein-4 (CRMP-4) and nestin in the ischemic adult rat brain following transient brain ischemia. METHODS: Brain ischemia was induced by transient left middle cerebral artery occlusion (MCAO) for 60 min in adult rats. The expression of CRMP-4, nestin and bromodeoxyuridine (BrdU) was analyzed by immunohistochemical method. The co-localization of CRMP-4 and nestin or BrdU was analyzed by double staining combined with confocal laser scanning microscopy. RESULTS: CRMP-4, a marker of immature neuron, could be expressed in the ipsilateral striatum and cerebral cortex at 1st and 2nd week after the ischemia-reperfusion; nestin, a marker of neural stem cell, occurred in above regions from several hours to 2 weeks. CRMP-4 costained with nestin and with BrdU incorporation. CONCLUSION: Neural stem cells may present in the striatum and cerebral cortex of adult rat and can be triggered to differentiate into newborn neuron there by ischemic brain trauma.     

Modulation of 5 -HT2C receptor on transcription of neprilysin gene and secretion of neprilysin in human glioma cell line

Neprilysin （NEP） , a member of zinc metallopeptidase family, has recently been focused in pharmacological research. Besides cleaving peptide signaling molecules in cardiovascular, inflammatory and immune system such as atrial natriuretic peptide, bradykinin, tachykinin, substance P and enkephalin, it has been proved to be a principal enzyme capable of degrading extracellular Aβin the brain. Since the results of cell and animal experiments suggested that stimulation of serotonin receptor subtype 5 - HT2C can increase secretion of soluble amyloid precursor protein （sAPP） and reduce accumulation of Aβpeptide both in vitro and in vivo, it is not known whether activation of 5 - HT2C receptor can also -affect NEP expression. We investigated NEP expression in human glioma cell Line U251. U251 was incubated for 24h with different concentrations of m - CPP （5 - HT2C receptor agonist） or RS 102221 （5 - HT2C receptor antagonist）. Real time quantitative PCR （RT - QPCR） was used to measure the mRNA level of neprilysin and western blot was used to analyse the protein level of neprilysin. Compared with the control group, a significant dose - dependent increase in NEP mRNA level was observed ＇after treated with m - CPP., On the contrary, RS 102221 caused NEP mRNA dose - dependent reduction. Relative levels of neprilysin protein generally paralleled those of the mRNA. These results suggest that activation of 5 - HT2C receptor can increase NEP expression in human glioma cell line, which might be of value in the exploration of etiology of and therapy of AD.     

Protective effect of carnosine against NMDA -induced neurotoxicity in differentiated rat PC12 cells

Aim： To investigat the neuroprotective effects of carnosine and its mechanisms of action in an in vitro model of neurotoxicity. Methods： Cultured differentiated PC12 cells were exposed to N -methyl -D -aspartate （NMDA） in the presence or absence of carnosine or other histaminergic drugs. Cell viability was assayed by the MTT assay. Changes in the levels of carnosine, histidine, histamine, glutamate and GABAin PC12 cells were measured by high - performance liquid chromatography combined with electrochemical detection. Results： Pretreatment with carnosine effectively reduced neuronal cell death and decreased the number of apoptotic and necrotic cells induced by NMDA insult. The neuroprotection by carnosine was reversed by pyrilamine and thioperamide, selective central histamine H1 and H3 antagonists, α - fluoromethylhistidine, a selective and irreversible inhibitor of histidine decarboxylase （HDC） also significantly reversed the protection of carnosine. Pretreatment with carnosine significantly increased HDC activity and the intracellular and extracellular content of carnosine, histidine and histamine. Further, it inhibited glutamate release in the presence or absence of excitotoxic concentrations of NMDA, an effect that was reversed by thioperamide. Conclusions： Carnosine protects against NMDA - induced neurotoxicity in differentiated PC12 cells and its mechanism of action may not only involve the camosine- histidine -histamine pathway, but also H1/H3 receptors and the effective inhibition of glutamate release. This study indicates that carnosine may be an endogenous neuroprotective factor in the brain and may be useful as a new antiexcitotoxic drug.     

Protective effects of compound FLZ,a novel synthetic analogue of squamosamide,on β-amyloid-induced rat brain mitochondrial dysfunction in vitro

Aim： The aim of the present study was to assess the effects of N-[2-（4-hydroxyphenyl）ethyl]-2-（2,5-dimethoxyphenyl）-3-（3- methoxy-4-hydroxyphenyl） acrylamide （compound FLZ）, a novel synthetic analogue of squamosamide, on the dysfunction of rat brain mitochondria induced by Aβ25-35 in vitro. Methods： Isolated rat brain mitochondria were incubated with aged Aβ25-35 for 30 rain in the presence and absence of FLZ （1-100 μmol/L）. The activities of.key mitochondrial enzymes, the production of hydrogen peroxide （H2O2） and superoxide anion （O2^-）, and the levels of glutathione （GSH） in mitochondria were examined. Mitochondrial swelling and the release of cytochrome c from mitochondria were assessed by biochemical and Western blot methods, respectively. Results： Incubation of mitochondria with aged Aβ25-35 inhibited the activities of α-ketoglutarate dehydrogenase （α-KGDH）, pyruvate dehydrogenase （PDH） and respiratory chain complex IV. It also resulted in increased H2O2 and O2^- production, and decreased the GSH level in mitochondria. Furthermore, it induced mitochondrial swelling and cytochrome c release from the mitochondria. The addition of FLZ （100 μmol/L） prior to treatment with Aβ25-35 significantly prevented these toxic effects of Aβ25-35 on the mitochondria. Conclusion： FLZ has a protective effect against Aβ25-35-induced mitochondrial dysfunction in vitro.     

rho家族的小分子量G蛋白介导缓激肽引起的猪冠状动脉松弛

AIM：To determine whether or not low molecular G-proteins are involved in the endothelium-dependent relaxations to bradykinin. METHODS: The effects of botulinum ADP-ribosyltranferase C3 were studied in porcine coronary arteries and endothelial cells. RESULTS: Incubation of membrane fractions isolated from endothelial cells with the enzyme and ^32p-NAD resulted in the ribosylation of the proteins with molecular weight of 24-25 kDa. Radio labelling of these proteins was suppressed in the presence of guanosine 5‘-O-(3-thiotriphosphate) (GTP-γS), a hydrolysis-resistant analog of GTP. In the isolated arteries, ADP-ribosyltransferase C3 attenuated the relaxations to bradykinin during contractions with prostaglandin F2α in the presence of tween 80 (non ionic detergent), but not in the absence of tween 80. CONCLUSION: Low molecular weight G-proteins of the Rho family contribute to the mechanism of relaxation induced by bradykinin.     

Studies on the action mechanism of the antidepressant effect of [quercetin 3 - O - apiosyl （1→2） ] - rhamnosyl （ 1→6 ） - glucoside

Aim To study the action mechanism of a novel antidepressant [ quercetin 3 -O- apiosyl （1→2） ] - rhamnosyl （1→6） - glucoside（ CTN - 986）, a flavonoid monosomer extracted from cottonseed . Methods （ 1 ） Firstly, using 5 - HTP induced head - twitches model and a special 5 - HT1A receptor agonist 8 - OH - DPAT induced hypothermia in mice, the antidepressant effect of CTN-986 was observed. （2） With immunohistochemistry method, we observed the effect of CTN-986 on neurogenesis and on the level of brain- derived neurotrophic factor （BDNF） in the dentate gyrus of hippocampus in chronically stressed mice. （3） We isolated and cultured neural progenitor cells from neonatal rat hippocampus, and identified the cells with immunocytochemistry method. By using MTT assay and ^3H-thymidine incorporation assay, the effect of CTN -986 on neural progenitor ceils proliferation was observed.     

β肾上腺素受体不可逆阻断剂BAlpM的合成

A highly potent beta-adrenergic irreversible antagonist——Bromoacetylalprenololmenthane (BAlpM)was synthesized by a Six step method with phenol and allychloride as the starting materials. Some improvement on purification of the product was described. The final product is identified by melting point, elemental analysis, UV and IR spectral analysis and mass spetrometry as well as β-adrenergic receptor bindingassays. [¹²⁵I]±IODOPINDOLOL binding assay of mouse lung cell membrane preparations treated with BAlpM in vitro or in vivo showed that there was a dosedependant decrease in the density of specific binding sites with no change in the Kd values. This result confirms that BAlpM is a β-adrenergic irreversible antagonist.     

Involvement of different Ca2+ pools during the canine bronchial sustained contraction in Ca2+-free medium: lack of effect of PKC inhibition

We evaluated the role of protein kinase C (PKC) in the sustained bronchial contraction (SBC) induced by carbachol (Cch) or histamine in a Ca²⁺-free medium and the possibility that each agonist uses a different Ca²⁺ store for this response. We studied third-order bronchi and airway smooth muscle (ASM) from first-order bronchi dissected free of cartilage and epithelium. Bronchial and ASM responsiveness to Cch or histamine were evaluated in Krebs solution (2.5 mM Ca²⁺) and in Ca²⁺-free medium. Cch and histamine induced an SBC in bronchial tissues in Ca²⁺-free medium. In ASM each agonist produced a transient contraction, but the response to histamine was much smaller. Cch induced a concentration-dependent accumulation of inositol phosphates (IPs) in both bronchi and ASM; however, histamine did not induce significant accumulation of IPs. Repeated exposure to histamine in bronchial rings abolished contractile responses in Ca²⁺-free media, but Cch added afterwards still produced a sustained contraction. This response was blocked when bronchial tissues were preincubated with 10 μM cyclopiazonic acid (CPA). Brief incubation of these preparations with a high EGTA concentration (1 mM) abolished the histamine-induced SBC. The SBC induced by Cch or histamine in Ca²⁺-free medium was not affected by the preincubation of the tissues with calphostin C, chelerythrine or staurosporine. We concluded that Cch mobilizes Ca²⁺ from two different sources during the SBC in Ca²⁺-free medium: from a CPA-sensitive one from sarcoplasmic reticulum (SR) and from a putative extracellular membrane Ca²⁺ pool sensitive to 1 mM EGTA, and neither process involved PKC activation. Histamine appeared to utilize the extracellular membrane pool only. Received: 12 March 1998 / Accepted: 2 September 1998     

Effects of SKF83959 on the excitability of hippocampal CA1 pyramidal neurons： a modeling study

Aim： 3-Methyl-6-chloro-7,8-hydroxy-1-（3-methylphenyl）-2,3,4,5-tetrahydro-1H-3-benzazepine （SKF83959） have been shown to affect several types of voltage-dependent channels in hippocampal pyramidal neurons. The aim of this study was to determine how modulation of a individual type of the channels by SKF83959 contributes to the overall excitability of CA1 pyramidal neurons during either direct current injections or synaptic activation.
Methods： Rat hippocampal slices were prepared. The kinetics of voltage-dependent Na^＋ channels and neuronal excitability and depolarization block in CA1 pyramidal neurons were examined using whole-cell recording. A realistic mathematical model of hippocampal CA1 pyramidal neuron was used to simulate the effects of SKF83959 on neuronal excitability.
Results： SKF83959 （50 μmol/L） shifted the inactivation curve of Na^＋ current by 10.3 mV but had no effect on the activation curve in CA1 pyramidal neurons. The effects of SKF83959 on passive membrane properties, including a decreased input resistance and depolarized resting potential, predicted by our simulations were in agreement with the experimental data. The simulations showed that decreased excitability of the soma by SKF83959 （examined with current injection at the soma） was only observed when the membrane potential was compensated to the control levels, whereas the decreased dendritic excitability （examined with current injection at the dendrite） was found even without membrane potential compensation, which led to a decreased number of action potentials initiated at the soma. Moreover, SKF83959 significantly facilitated depolarization block in CA1 pyramidal neurons. SKF83959 decreased EPSP temporal summation and, of physiologically greater relevance, the synaptic-driven firing frequency.
Conclusion： SKF83959 decreased the excitability of CA1 pyramidal neurons even though the drug caused the membrane potential depolarization. The results may reveal a partial mechanism for the drug’s anti-Parkinsonian effects and may also suggest that SKF83959 has a potential antiepileptic effect.     

Effect of telmisartan on expression of protein kinase C-α in kidneys of diabetic mice

Aim： To investigate the effects of angiotensin receptor blocker （ARB） telmisartan on the expression and distribution of protein kinase C （PKC）-α in the kidneys of diabetic mice. Methods： Diabetic mice were induced with streptozotocin and a group of them were randomly selected for treatment with telmisartan. After 6 weeks, the expression and localization of PKC-α in the renal cortex, and the outer and inner medulla were assessed by immunohistochemistry and semiquantitative Western blotting. In addition, expressions of PKC-α, transforming growth factor- β1 （TGF-β1）, and vascular endothelial growth factor （VEGF） in glomeruli were measured by semiquantitative immunohistochemistry. Results： Diabetic and normal mice showed similar distributions of PKC-α in the kidneys. The expression of PKC-α was found in glomeruli, epithelial cells of proximal tubules, and medullary- collecting duct, while not in the medullary and cortical thick ascending limb, and was different in the epithelial cells of proximal tubules of diabetic nephropathy （DN） mice, PKC-α was mostly translocated from the basement membrane to the apical membrane, whereas it was largely translocated from the apical membrane to the basement membrane in epithelial cells of the inner medullary-collecting duct. Western blotting detected increased expression of PKC-α in the renal cortex and outer medulla, but not in the inner medulla of DN mice. Enhanced expressions of PKC-α, TGF-β1, and VEGF were shown in the glomeruli of DN mice, where PKC-α exhibited a correlation to VEGF, but no correlation to TGF-β1. ARB telmisartan attenuated alterations of PKC-α as mentioned earlier in the DN mice. Conclusion： Our findings suggest that PKC-α may play a role in the pathogenesis of DN, and that the nephroprotective effects of ARB telmisartan may be partly associated with its influence on PKC-α.     

Hydrogen peroxide mobilizes Ca^2＋ through two distinct mechanisms in rat hepatocytes

Aim： Hydrogen peroxide （H2O2） is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca^2＋ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes. Methods： Hepatocytes were extracted from rats. Intracellular Ca^2＋ concentrations （[Ca^2＋]i）, inner mitochondrial membrane potentials and NAD（P）H levels were measured using fluorescence imaging. Phospholipase C （PLC） activity was detected using exogenous PIP2. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.
Results： H2O2 increased intracellular Ca^2＋ concentrations （[Ca^2＋]i） across two kinetic phases. A low concentration （400 μmol/L） of H2O2 induced a sustained elevation of [Ca^2＋]i that was reversed by removing extracellular Ca^2＋. H2O2 increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited HRO2-induced membrane current increases and [Ca^2＋]i elevation. A high concentration （1 mmol/L） of H2O2 induced an additional transient elevation of [Ca^2＋]i, which was abolished by the specific PLC blocker U73122 but was not eliminated by removal of extracellular Ca^2＋. PLC activity was increased by 1 mmol/L H2O2but not by 400 μmol/L H2O2.
Conclusions： H2O2 mobilizes Ca^2＋ through two distinct mechanisms. In one, 400 μmol/L H2O2-induced sustained [Ca^2＋]i elevation is mediated via a Ca^2＋ influx mechanism, under which H2O2 impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca^2＋ influx. In contrast, 1 mmol/L H2O2-induced transient elevation of [Ca^2＋]i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca^2＋ from intracellular Ca^2＋ stores.     

Inhibition of ATP-induced calcium influx in HT4 cells by glucocorticoids： involvement of protein kinase A

Aim: In our previous observations, adenosine triphosphate (ATP) was found to evoke immediate elevations in intracellular free calcium concentration ([Ca^2 ]i) in HT4 neuroblastoma cells of mice. We tried to see if a brief pretreatment of glucocorticoids could inhibit the Ca^2 response and reveal the underlying signaling mechanism. Methods: Measurement of [Ca^2 ]i was carried out using the dual-wavelength fluorescence method with Fura-2 as the indicator. Results: Preincubation of HT4 cells for 5 min with corticosterone (B) or bovine serum albumin conjugated corticosterone (B-BSA) inhibited the peak [Ca^2 ]i increments in a concentration-dependent manner. Cortisol and dexamethasone had a similar action, while deoxycorticosterone and cholesterol were ineffective. Both extracellular Ca^2 influx and internal Ca^2 release contributed to ATP-induced [Ca^2 ]i elevation. The brief treatment with only B attenuated Ca^2 influx. Furthermore, the [Ca^2 ]i elevation induced by the P2X receptor agonist adenosine 5‘-(β,γ-methylene) triphosphate (β,γ-meATP) was also suppressed. The rapid inhibitory effect of B can be reproduced by forskolin 1 mmol/L and blocked by H89 20 mmol/L. Neither nuclear glucocorticoid receptor antagonist mifepristone nor protein kinase C inhibitors influenced the rapid action of B. Conclusion: Our results suggest that glucocorticoids modulate P2X receptor-medicated Ca^2 influx through a membrane-initiated, non-genomic and PKA-dependent pathway in HT4 cells.     

Pharmacokinetics and tissue distribution of a novel PDE5 inhibitor, SK-3530, in rats

Aim： To investigate the pharmacokinetic profile and tissue distribution of a novel phosphodiesterase type 5 inhibitor, 5-ethyl-2-{5-[4-（2-hydroxy-ethyl）-piperazine- 1-sulfonyl]-2-propoxy-phenyl }-7-propyl-3,5-dihydro-pyrrolo（3,2-d）pyrimidin-4- one （SK-3530）, in rats after administration of the ^14C-labeled compound. Methods： The pharmacokinetic parameters of SK-3530 were measured based on the total radioactivity and parent SK-3530 concentration in rat plasma after intravenous and oral administration. The tissue distribution of total radioactivity after a single oral administration of [^14C]SK-3530 at a dose of 40 mg/kg was assayed. The plasma protein binding rates of SK-3530 were assessed by in vitro and ex vivo assay. Results： The total radioactivity profiles showed linear pharmacokinetics. The maximum plasma concentration and area under the curve of the parent SK3530 were 10%-20% compared to those of the total radioactivity. After the oral administration of [^14C]SK-3530, the radioactivity was widely distributed in all tissues, and the tissue/plasma ratio of the radioactivity 1 h after administration was calculated as 0.5-2.6 with the exception of excretory organs. A relatively high penetration was shown in the adrenal glands, liver, and lung. In vitro and ex vivo plasma protein binding assay by ultrafiltration showed a considerably high binding rate of more than 97%. Condusion： SK-3530 was relatively well absorbed in the gastrointestinal tract and showed linear pharmacokinetics over the investigated dose range. SK-3530 had low oral bioavailability due to a high, first-pass metabolism.     

Discovery of a novel inhibitor of NAD（P）＋-dependent malic enzyme （ME2） by high-throughput screening

Aim： Malic enzymes are oxidative decarboxylases with NAD or NAD（P） as cofactor that catalyze the conversion of L-malate to pyruvate and CO2. The aim of this study was to discover and characterize a potent inhibitor of human NAD（P）＋-dependent malic enzyme 2 （ME2）. Methods： Recombinant human ME2-His-Tag fusion protein was overexpressed in E coil and purified with Ni-NTA resin. A high- throughput screening （HTS） assay was developed to find ME2 inhibitors. Detergent Brij-35 was used to exclude false positives. The characteristics of the inhibitor were analyzed with enzyme kinetics analysis. A thermal shift assay for ME2 was carried out to verify the binding of the inhibitor with the enzyme. Results： An HTS system for discovering ME2 inhibitors was established with a Z＇ factor value of 0.775 and a signal-to-noise ratio （S/N） of 9.80. A library containing 12 683 natural products was screened. From 47 hits, NPD387 was identified as an inhibitor of ME2. The primary structure-activity relationship study on NPD387 derivatives showed that one derivative NPD389 was more potent than the parent compound NPD387 （the ICso of NPD389 was 4.63±0.36 pmoVL or 5.59±0.38 pmoVL, respectively, in the absence or presence of 0.01% Brij-35 in the assay system）. The enzyme kinetics analysis showed that NPD389 was a fast-binding uncompetitive inhibitor with respect to the substrate NAD~ and a mixed-type inhibitor with respect to the substrate L-malate. Conclusion： NPD389 is a potent ME2 inhibitor that binds to the enzyme in a fast-binding mode, acting as an uncompetitive inhibitor with respect to the substrate NAD~ and a mixed-type inhibitor with respect to the substrate L-malate.     

Mechanism underlying blockade of voltage-gated calcium channels by agmatine in cultured rat hippocampal neurons

INTRODUCTION Agmatine [4-(aminobutyl) guanidine] was firstidentified in 1910 by Kossel in herring sperm and wasknown as an intermediate in the polyamine metabolismof various bacteria, fungi, parasites and marine fanna[1, . 2]Now, accumulating evidence has revealed that agma-tine meets most criteria for a central neurotrans-mitter.It is synthesized in brain, stored in synaptic vesicles inheterogeneously distributed neurons, inactivated byreuptake, degraded by a specific enzyme, agmatinase,…     

Effects of huperzine A on secretion of nerve growth factor in cultured rat cortical astrocytes and neurite outgrowth in rat PC12 cells

Aim: To study the effects of huperzine A (HupA) on neuritogenic activity and the expression of nerve growth factor (NGF). Methods: After being treated with 10μmol/L HupA, neurite outgrowth of PC 12 cells was observed and counted under phase-contrast microscopy. Mitogenic activity was assayed by [^3H]thymidine incorporation. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. AChE activity, mRNA and protein expression were measured by the Ellman‘s method, RT-PCR, and Western blot, respectively. NGF mRNA and protein levels were determined by RT-PCR and ELISA assays. Results: Treatment of PC 12 cells with 10μmol/L HupA for 48 h markedly increased the number of neuritebearing cells, but caused no significant alteration in cell viability or other signs of cytotoxicity. In addition to inhibiting AChE activity, 10μmol/L HupA also increased the mRNA and protein levels of this enzyme. In addition, following 2h exposure of the astrocytes to 10μmol/L HupA, there was a significant up-regulation of mRNA for NGF and P75 low-affinity NGF receptor. The protein level of NGF was also increased after 24 h treatment with HupA. Conclusion: Our findings demonstrate for the first time that HupA has a direct or indirect neurotrophic activity, which might be beneficial in treatment of neurodegenerative disorders such as Alzheimer disease.     

锌7-金属硫蛋白对严重烫伤大鼠肝脏氧化应激反应的作用

AIM: Using the model of burned animal with delayed resuscitation to study antagonistic effect of Zn7 - metallothionein(Zn7-MT) on oxidative stress in the liver of rats suffered from severe thermal injury on skin. METHODS: Tocompare the changes in antioxidant concentrations and antioxidative enzyme activities in the liver or plasma ofburned rats with or without Zn7-MT in resuscitation fluid by biochemical assay. RESULTS: After injury, glutathione concentration was progressively decreased with time, At24 h after injury, activities of glutathione reductase and glutathione peroxidase in the liver of burned rats were increased and then decreased at 48 h postburn, αTocopherol in plasma was reduced at 24 h and malondialdehyde in the liver was increased significantly postburn.MT and MT-1 mRNA expression in burned rats were activated. Taken together, oxidative stress in the liver ofburned rats occurred. Exogenous Zn7-MT attenuated the changes in antioxidant concentrations and antioxidativeenzyme activities in the liver or plasma of burned rats. The effect of Zn7-MT was in a concentration-dependentmanner and the concentration of 10μtmol/L was the most effective. Exogenous Zn7-MT also inhibited MT-1 mRNA overexpression and increased MT protein concentration. CONCLUSION: Zn7-MT effectively antagonizedoxidative stress in the liver of rats with severe thermal injury.