五味子酚对大鼠中性粒细胞呼吸爆发的影响

目的　研究五味子酚对大鼠中性粒细胞呼吸爆发时自由基生成和溶酶体酶释放的影响。方法　分别用紫外分光法、荧光分光法和同位素法测定该细胞自由基生成、溶酶体酶释放、胞浆钙离子和环磷酸腺苷(cAMP)含量。结果　五味子酚能抑制中性粒细胞超氧阴离子、过氧化氢、脂质过氧化产物丙二醛（MDA）以及一氧化氮的生成,减少溶菌酶和β-葡糖醛酸苷酶的释放,而且,还能拮抗FMLP引起的细胞胞浆钙离子的增加,进一步增强FMLP引起的胞浆cAMP的增加。结论　五味子酚可能通过降低细胞内钙离子浓度和/或升高胞浆cAMP的含量而抑制中性粒细胞呼吸爆发所导致的上述变化。     

蝙蝠葛酚性碱抗氧化和降脂作用的实验研究

目的:研究蝙蝠葛酚性碱(PAMD)抗氧化和降脂作用。方法:通过TBA法测定大鼠肝匀浆自发和诱导的脂质过氧化产物MDA的量,研究PAMD对脂质过氧化的作用;利用Fenton反应和Marklund反应研究PAMD对羟自由基和超氧阴离子的清除作用;通过建立大鼠高脂血症模型,研究PAMD对高脂模型动物的降脂及抗氧化保护作用。结果:PAMD能抑制自发的和诱导的脂质过氧化产物MDA的生成,对化学体系产生的羟自由基和超氧阴离子有清除作用;能降低高脂模型动物血清及肝中TC、TG、LDL、MDA含量,对血清SOD活性和HDL含量有一定的升高作用。结论:PAMD具有抑制体外脂质过氧化和清除自由基作用,能调节高脂模型动物血脂水平。     

川芎嗪对创伤家兔的抗脂质过氧化作用

在家兔创伤后,观察了动物组织损伤和机体内脂质过氧化反应情况,及川芎嗪的防治效应。结果发现,动物创伤后逐渐陷入休克状态,胞浆酶和溶酶体酶漏出。血浆和组织中脂质过氧化产物丙二醇（ＭＤＡ）含量明显增加。而应用川芎嗪防治的动物,脂质过氧化反应被有效地抑制,组织损伤表现轻微。提示,川芎嗪通过抗氧自由基损伤而对创伤动物起保护作用。     

叶下珠对肝细胞损伤的保护作用

实验证实叶下珠能明显降低ＣＣｌ_４引起的小鼠血清丙氨酸转氨酶（ＡＬＴ）的升高；抑制肝脏脂质过氧化产物丙二醛（ＭＤＡ）的生成。叶下珠体外与大鼠肝细胞共同孵育，能抑制ＣＣｌ_４所致的肝细胞膜流动性降低，降低肝细胞内钙离子浓度。结果提示，叶下珠的保护肝脏损伤作用可能与其抗脂质过氧化和膜保护作用有关。     

氯胺酮抑制人离体中性粒细胞释放超氧阴离子的机制

目的研究氯胺酮的异构体S(+)氯胺酮[S(+)-Ket]和R(-)氯胺酮[R(-)-Ket]对中性粒细胞释放超氧阴离子的影响及机制。方法细胞色素C还原法测定3种刺激剂介导的中性粒细胞超氧阴离子释放量。用荧光分光法检测中性粒细胞内钙的水平。用Western Blot法检测中性粒细胞蛋白质p47phox磷酸化水平。结果S(+)-Ket和R(-)-Ket可抑制N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸(N-formyl-methionyl-leucyl-phenylalanine,fMLP)介导的中性粒细胞释放超氧阴离子(P<0.01)。S(+)-Ket可抑制佛波豆蔻醚乙酸盐(phorbol12-myristate13-acetate,PMA)介导的中性粒细胞释放超氧阴离子和钙离子的增加,而R(-)-Ket可轻度增加PMA介导的中性粒细胞释放超氧阴离子和促进钙离子的增加。S(+)-Ket抑制PMA介导中性粒细胞蛋白质p47phox磷酸化,而R(-)-Ket增强上述作用;钙螯合剂EGTA可取消R(-)-Ket对PMA介导中性粒细胞蛋白质p47phox磷酸化的抑制作用。结论S(+)-Ket通过PKC-钙信息通道,抑制NADPH氧化酶p47phox,抑制超氧阴离子的产生。     

川芎嗪嗪对创伤家兔的抗脂质过氧化作用

在家兔创伤后，观察了动物组织损伤和机体内脂质过氧化化应情况，及川芎嗪的防治效应。结果发现，动物创伤后逐渐陷入休克状态，胞浆酶和溶酶体酶漏出。血浆和组织中脂质氧化产丙二醛含量明显增加，。而应用川芎嗪防治的动物，脂质过氧化反应被有效地抑制，组织损伤表现轻微。提示，川芎嗪通过抗氧自由基损伤而对创伤动物起保护作用。     

灯盏花乙素对超氧阴离子引起的大鼠脑突触体氧化应激的保护作用

目的：研究灯盏花乙素对超氧阴离子引起的大鼠脑突触体氧化应激的保护作用．方法：采用与黄嘌呤(0．3mmol／L)和黄嘌呤氧化酶(0．02U)体系在37℃下孵育30min,建立大鼠脑突触体超氧阴离子氧化损伤模型．通过测定脂质过氧化产物丙二醛评价脂质过氧化程度．通过脂溶性荧光探针DPH的各向异性判断突触体膜的流动性．胞内钙离子的测定采用荧光光度法,以Fura2-AM为荧光探针．测定ATP酶解释放的无机磷确定Na~ ／K~ -ATP酶的活性．结果：超氧阴离子使大鼠脑突触体的脂质过氧化产物丙二醛及胞内钙离子浓度显著上升,突触体的膜流动性和Na~ ／K~ -ATP酶的活性则显著下降,预先加入灯盏花乙素(25-100μmol／L)则能显著缓解超氧阴离子引起的氧化性损伤,表现为丙二醛的水平和胞内钙离子浓度下降,膜流动性增加及Na~ ／K~ -ATP酶活性的恢复．结论：灯盏花乙素对超氧阴离子引起的大鼠脑突触体氧化应激具有良好的保护作用．     

茶多酚对脑缺血再灌注损伤的保护作用及在体外抗脂质过氧化和清除自由基作用

目的 :研究茶多酚对脑缺血再灌注损伤的保护作用及抗脂质过氧化和清除自由基的作用。方法 :结扎大鼠双侧颈总动脉及迷走神经后 ,再灌注 4 5min ,建立脑缺血再灌注模型。由自由基诱导的脑线粒体脂质过氧化 ,用硫代巴比妥酸法测定。由Xan XO诱导的O 2 and由Fe2 + H2 O2 诱导的·OH ,用紫外分光光度仪测定。结果 :在脑缺血再灌注损伤时 ,茶多酚能改善SOD、GSH Px、CAT活性 (P <0 0 1) ,同时降低脑水肿和MDA含量 (P <0 0 1和P <0 0 5 )。茶多酚具有明显的清除·OH和O 2 的作用 (IC50 分别为 2 2mmol·L-1和 1 9mmol·L-1) ,茶多酚明显抑制由·OH诱导脑线粒体脂质过氧化 ,并呈浓度依赖性。结论 :结果提示茶多酚能保护脑缺血再灌注损伤 ,与其清除自由基和抗脂质过氧化作用有关     

祁州漏芦对组织脂质过氧化作用的影响

本文报导了祁州漏芦根及地上部分水煎剂体外实验能显著抑制大鼠心、脑、肝、肾组织中过氧化脂质的生成,连续灌胃15天,能显著抑制小鼠血清及肝、脑中过氧化脂质的生成,证明漏芦根及地上部分均有抗脂质过氧化作用。     

安吉白茶黄酮抗氧化活性的体外实验研究

目的通过研究安吉白茶黄酮对自由基的清除作用及抗脂质过氧化作用来系统阐明安吉白茶黄酮的抗氧化活性。方法应用化学发光法和分光光度法系统评价了安吉白茶黄酮对超氧阴离子自由基(O-2·)、羟基自由基(·OH)、二苯代苦味酰基自由基(DPPH·)和H2O2的清除作用。采用小鼠肝脏为研究对象,评价了安吉白茶黄酮对自发及诱导性脂质过氧化模型的抑制作用。结果安吉白茶黄酮对O-2·、·OH、DPPH·和H2O2均有较强的清除能力,其清除作用与黄酮浓度呈明显的剂量依赖关系。安吉白茶黄酮可以以剂量依赖方式抑制小鼠肝脏脂质过氧化。结论安吉白茶黄酮在体外具有高效的自由基清除作用,并具有较强的抗脂质过氧化活性。     

Role of enhanced inositol phospholipid metabolism in neutrophil activation

When guinea pig neutrophils were stimulated with chemotactic peptide [formylmethionyl-leucyl-phenylalanine (fMLP)], a marked release of lysosomal enzyme and production of Superoxide anion were detected. The breakdown of phosphatidylinositol 4,5-bisphosphate (TPI) and the subsequent formation of diacylglycerol, phosphatidic acid and free arachidonic acid also occurred during the processes. Ca²⁺ ionophore A23187 caused an evident secretion of lysosomal enzyme but no Superoxide anion production. Ca²⁺ ionophore also caused TPI breakdown to diacylglycerol although this breakdown was not as significant as that detected by fMLP. The tumor promotor tetradecanoylphorbol acetate (TPA), which is a strong activator of Superoxide anion production but not a good stimulator of lysosomal enzyme secretion, did not cause a significant decrease of TPI or arachidonic acid release. Since TPA is known not to increase the intracellular Ca²⁺ level, these results suggest that lysosomal enzyme secretion is correlated closely with enhanced inositol phospholipid metabolism and Ca²⁺-dependent processes. On the other hand, Superoxide anion production seemed to be caused mainly by Ca²⁺-independent processes, perhaps by protein kinase-C activation through newly formed diacylglycerol, when neutrophils were activated by chemotactic peptide.     

Inhibitory effects of phenyltin compounds on stimulus-induced changes in cytosolic free calcium and plasma membrane potential of human neutrophils

To clarify the inhibitory mechanisms by triphenyltin chloride (TPTCl) on Superoxide anion formation in neutrophils, the effects of phenyltin compounds [TPTCl, diphenyltin dichloride (DPTCl₂) and phenyltin trichloride (MPTCl₃)] on the increase of cytosolic free calcium and the changes in membrane potential in neutrophils stimulated byn-formyl-methionyl-leucyl-phenylalanine (FMLP) were examined. TPTCl and DPTCh concentration dependently inhibited the increase of fluorescence intensity of the dye 3,3-dipropyl-thiodicarbocyanine iodide [diS-C₃-(5)] (membrane potential probe) in neutrophils induced by 0.1M FMLP in the presence or absence of extracellular calcium (1.26 mM). TPTCl had a greater inhibitory effect on FMLP-mediated membrane potential change than that of DPTCl₂. In the presence of extracellular calcium, TPTCl and DPTCl₂ increased intracellular free calcium ([Ca²⁺]i) of unstimulated fura-2-loaded neutrophils at concentrations from 1.0 to 10 M TPTCl and from 2.5 to 10 M DPTCl₂. TPTCl and DPTCl₂ also increased slightly, in the absence of extracellular calcium, [Ca²⁺]i without stimulation of FMLP in neutrophils. However, TPTCl and DPTCl₂ significantly inhibited the rise of [Ca²⁺]i in neutrophils stimulated by FMLP at concentrations from 2.5 M to 10 M TPTCl and at a concentration of 10 M DPTCl₂ in the absence of extracellular calcium. TPTCl and DPTCl₂ significantly inhibited the Superoxide anion production by FMLP at concentrations over 2.5 M in the presence of extracellular calcium. In the absence of extracellular calcium, TPTCl and DPTCl₂ also inhibited the Superoxide anion production by FMLP at concentrations over 1.5 M TPTCl and over 5.0 M DPTCl₂. Our results suggest that TPTCl and DPTCl₂ are potent inhibitors of membrane potential change and intracellular Ca²⁺ signal transduction in FMLP stimulation, in association with superoxide production in neutrophils.     

Daphnoretin-induced respiratory burst in rat neutrophils is,probably, mainly through protein kinase C activation

Daphnoretin, a dicoumarin isolated from Wikstroemia indica C.A. Mey. (Thymelaceae), induced superoxide anion (O₂⁻) formation in rat neutrophils in a concentration-dependent manner. Addition of staurosporine reduced daphnoretin-induced respiratory burst. Removal of extracellular free Ca²⁺ by EGTA did not affect the respiratory burst of neutrophils in response to daphnoretin. Prior exposure of neutrophils to phorbol 12-myristate 13-acetate (PMA) or daphnoretin reduced the O₂⁻ formation caused by a subsequent challenge with PMA and daphnoretin, but potentiated the response caused by a subsequent addition of formyl-Met-Leu-Phe (fMLP). Like PMA, daphnoretin did not increase the [Ca²⁺]_i during cell activation. In neutrophil suspension, daphnoretin increased the membrane associated protein kinase C activity. In the presence of Ca²⁺ and phosphatidylserine, daphnoretin also activated protein kinase C isolated from cytosolic fraction of resting neutrophils. Staurosporine inhibited the direct activation of protein kinase C caused by daphnoretin as well as by PMA. Daphnoretin reduced the [³H]Phorbol-12,13-dibutyrate ([³H]PDB) binding to the neutrophil cytosolic protein kinase C in a concentration-dependent manner with an IC₅₀ value of 1.77±0.37 μM. These results indicate that daphnoretin, like PMA, may direct activation of protein kinase C which in turn activated NADPH oxidase and elicited respiratory burst.     

Melatonin Counteracts Alterations in Oxidative Metabolism and Cell Viability Induced by Intracellular Calcium Overload in Human Leucocytes: Changes with Age

Abstract: Ageing is associated with an increased production of free radicals and alterations in the mechanisms of adaptation to oxidative stress. In fact, the free radical theory of ageing proposes that deleterious actions of free radicals are responsible for the functional deterioration associated with ageing. Moreover, a close relationship exists between calcium homeostasis and oxidative stress. The current work was aimed at proving that intracellular calcium overload induced by N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and/or thapsigargin leads to oxidative stress. We additionally examined the effect of melatonin on the levels of reactive oxygen species (ROS) and cell viability in human leucocytes collected from young (20–30‐year‐old) and elderly (65–75‐year‐old) individuals under both basal and oxidative stress‐induced conditions. Treatments with 10 nM FMLP and/or 1 μM thapsigargin induced a transient increase in cytosolic free‐calcium concentration ([Ca^{2 +} ]_c) in human leucocytes due to calcium release from internal stores, and led in turn to oxidative stress, as assessed by intracellular ROS measurement. Non‐treated leucocytes from aged individuals exhibited higher ROS levels and lower rates of cell survival when compared to leucocytes from young individuals. Similar results were obtained in FMLP and/or thapsigargin‐treated leucocytes from elderly individuals when compared to those from the young individuals. Melatonin treatment significantly reduced both hydrogen peroxide (H₂O₂) and superoxide anion levels, likely due to its free‐radical scavenging properties, and enhanced leucocyte viability in both age groups. Therefore, melatonin may be a useful tool for the treatment of disease states and processes where an excessive production of oxidative damage occurs.     

Influence of Diphenylhydantoin on Lysosomal Enzyme Release during Bone Resorption in Vitro

Abstract: The effect of diphenylhydantoin (DPH) on the release of lysosomal enzymes during resorption of cultured mouse calvarial bone was studied. The enzyme activities of β-glucuronidase and β-galactosidase in the culture medium was taken as indicators for lysosomal enzyme release. In concentrations 50 μg/ml or higher, DPH inhibited the release of β-glucuronidase and β-galactosidase in parallel with bone resorption as indicated by reduced release of ⁴⁵Ca, Ca²⁺, P_i and hydroxyproline. The release of the cytosolic enzyme lactate dehydrogenase was not influenced by concentrations of DPH up to 50 μg/ml but higher concentrations caused an increased release indicating cell injury. When bone resorption was stimulated by prostaglandin E₂, DPH (50 μg/ml) also reduced the mobilization of bone mineral and the release of β- glucuronidase without influencing the release of lactate dehydrogenase. It is suggested that DPH by interfering with cellular release processes reduces the resorption of bone.     

Inhibition of human polymorphonuclear leukocyte functions by ibuprofen

We have found that pretreatment of human neutrophils with ibuprofen (0.10-1.0 mg/ml) results in an irreversible, concentration-dependent inhibition of superoxide anion generation and release of lysosomal enzymes (myeloperoxidase, lysozyme) stimulated by the synthetic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), the complement fragment C5a, and to a lesser extent by serum opsonized zymosan. Inhibition of granule exocytosis and oxygen radical generation at ibuprofen concentrations less than 5 mg/ml was not due to drug cytotoxicity since release of the cytoplasmic enzyme lactate dehydrogenase was not affected by ibuprofen. In contrast to neutrophil responses mediated by C5a or FMLP, ibuprofen did not inhibit either enzyme release or superoxide anion generation by neutrophils stimulated with phorbol myristate acetate. Ibuprofen did not function as an oxygen radical scavenger in a cell-free system in which superoxide anion was generated by the aerobic action of xanthine oxidase on hypoxanthine. Ibuprofen also inhibited in a concentration-dependent fashion both directed migration (chemotaxis) and stimulated random migration (chemokinesis) of neutrophils exposed to either FMLP or C5a. Inhibition of neutrophil adherence to plastic surfaces and bovine pulmonary artery endothelial cells was equally effective when the neutrophils were treated with ibuprofen before stimulation with FMLP or phorbol myristate acetate. The inhibitory effects of ibuprofen pretreatment of neutrophils could not be overcome by addition of prostaglandins E1 or E2 (0.3-300 nM). These results demonstrate that ibuprofen is capable of suppressing many functions thought to be important in neutrophil-mediated acute pulmonary inflammatory processes. Results of these experiments further suggest that ibuprofen may inhibit neutrophil functions by acting on cellular components separate from membrane receptors or by blockade of cyclo-oxygenase products which may be involved in these neutrophil functions.     

Manganese promotes increased formation of hydrogen peroxide by activated human macrophages and neutrophils in vitro

Although pro-inflammatory mechanisms have been implicated in the pathogenesis of manganese (Mn²⁺)-related neurological and respiratory disorders, relatively little is known about the potential of this metal to interact pro-oxidatively with human phagocytes. The primary objective of the current study was to investigate the effects of Mn²⁺ as MnCl₂ (0.5–100 µM) on the generation of the reactive oxygen species (ROS), superoxide, hydrogen peroxide (H₂O₂), and hypohalous acids by isolated human blood neutrophils and monocyte-derived macrophages following activation of these cells with the chemotactic tripeptide, FMLP (1 µM), or the phorbol ester, PMA (25?ng/mL). Generation of ROS was measured using the combination of oxygen consumption, lucigenin/luminol-enhanced chemiluminescence, spectrofluorimetric detection of oxidation of 2,7-dichlorodihydrofluorescein, radiometric assessment of myeloperoxidase (MPO)-mediated protein iodination, release of MPO by ELISA, and spectrophotometric measurement of nitrite formation. Treatment of activated neutrophils with either FMLP or PMA resulted in significantly decreased reactivity of superoxide in the setting of increased formation of H₂O₂ and MPO-mediated iodination, with no detectable effects on either oxygen consumption or MPO release. Similar effects of the metal with respect to superoxide reactivity and H₂O₂ formation were observed with activated macrophages, while generation of NO was unaffected. Taken together with the findings of experiments using cell-free ROS-generating systems, these observations are compatible with a mechanism whereby Mn²⁺, by acting as a superoxide dismutase mimetic, increases the formation of H₂O₂ by activated phagocytes. If operative in vivo, this mechanism may contribute to the toxicity of Mn²⁺.     

Stimulation of histamine H2- (and H1)-receptors activates Ca2+ influx in all-atrans-retinoic acid-differentiated HL-60 cells independently of phospholipase C or adenylyl cyclase

In human neutrophils, histamine H₂-receptors mediate activation of adenylyl cyclase (AC) and inhibition of N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-induced superoxide anion (0 _inf2 ^sup– ) formation, and in HL-60 promyelocytes, H₂-receptors mediate parallel activation of AC, phospholipase C (PLC) and non-selective cation (NSC) channels. As all-trans-retinoic acid (RA) is successfully used in the differentiation therapy of acute promyelocytic leukaemia, we studied signal transduction in RA-differentiated HL-60 cells. Histamine and the H₂-receptor agonist, impromidine, induced both rises in cAMP levels and cytosolic Ca²⁺ ([Ca²⁺]_i). Substances acting at post-receptor sites to increase cAMP did not increase [Ca²⁺]_i. H₂-but not H₁-receptor antagonists inhibited histamine-induced cAMP accumulation and rises in [Ca²⁺]_i were more effectively inhibited by H₂- than by H₁-receptor antagonists. Histamine-induced rises in [Ca²⁺]_i were completely dependent on the presence of extracellular Ca²⁺ and were abolished by the blocker of NSC channels, Gd³⁺, but were resistant to inhibition by pertussis toxin. Unlike FMLP, histamine did not activate PLC. The effects of FMLP on [Ca²⁺]_i were less sensitive to blockade by Gd³⁺ than those of histamine, and there was no cross-desensitization between the two stimuli. FMLP, but not histamine, inhibited transiently thapsigargin-induced rises in [Ca²⁺]₁. Taken together, our results show that histamine activates AC-mediated cAMP accumulation in RA-differentiated HL-60 cells via H₂-receptors and NSC channel-mediated Ca²⁺ influx via H₂- (and H₁)-receptors. Histamine-induced NSC channel activation is not the consequence of AC- or PLC stimulation and occurs, directly or indirectly, via pertussis toxin-insensitive guanine nucleotide-binding proteins. FMLP and histamine activate Ca²⁺ influx by different mechanisms. There are similarities in H₂-receptor-mediated signal transduction between RA-differentiated HL-60 cells and HL-60 promyelocytes and differences between the former cells and neutrophils, indicating that RA-differentiated HL-60 cells must be considered as partially immature.     

Effect of capsazepine on [Ca2+]i in MDCK renal tubular cells

The current study explored whether capsazepine changed basal cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) levels in suspended Madin Darby canine kidney (MDCK) cells cells by using fura‐2 as a Ca²⁺‐selective fluorescent dye. At concentrations of 10–200 µM, capsazepine increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was partially reduced by 40% by removing extracellular Ca²⁺. Capsazepine induced Mn²⁺ quench of fura‐2 fluorescence, indirectly implicating Ca²⁺ entry. Capsazepine‐induced Ca²⁺ influx was unchanged by L‐type Ca²⁺ entry inhibitors and protein kinase C modulators [phorbol 12‐myristate 13‐acetate (PMA) and GF109203X]. In Ca²⁺‐free medium, 100 µM capsazepine‐induced Ca²⁺ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin‐induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change capsazepine‐induced [Ca²⁺]_i rises. Collectively, in MDCK cells, capsazepine induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via non‐L‐type Ca²⁺ channels. Drug Dev Res 72: 323–329, 2011. © 2010 Wiley‐Liss, Inc.