Activation of phosphatidylinositol 3-kinase/Akt-mammalian target of Rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine-induced place preference in rats.

Hippocampus is a critical structure for the acquisition of morphine-induced conditioned place preference (CPP), which is a usual learning paradigm for assessing drug reward. However, the precise mechanisms remain largely unknown. Phosphatidylinositol 3-kinase (PI3K) and its downstream targets, including Akt, mammalian target of Rapamycin (mTOR) and 70-kDa ribosomal S6 kinase (p70S6K), are critical molecules implicated in learning and memory. Here, we tested the role of PI3K/Akt-mTOR-p70S6K signaling pathway in morphine-induced CPP in the hippocampus. Our results showed that the acquisition of morphine CPP increased phosphorylation of Akt in the hippocampal CA3, but not in the nucleus accumbens (NAc), the ventral tegmental area (VTA) or the CA1. Moreover, the phosphorylated Akt exclusively expressed in the CA3 neurons. Likewise, levels of phosphorylated mTOR and p70S6K were significantly enhanced in the CA3 following morphine CPP. The alterations of these phosphorylated proteins are positively correlated with the acquisition of morphine CPP. More importantly, microinjection of PI3K inhibitor (LY294002) or mTOR inhibitor (Rapamycin) into the CA3 prevented the acquisition of CPP and inhibited the activation of PI3K-Akt signaling pathway. In addition, pre-infusion of ß-FNA (ß-funaltrexamine hydrochloride), a selective irreversible µ opioid receptor antagonist, into CA3 significantly prevented the acquisition of CPP and impaired Akt phosphorylation. All these results strongly implied that the PI3K-Akt signaling pathway activated by µ opioid receptor in hippocampal CA3 plays an important role in acquisition of morphine-induced CPP.

Protective effects of sodium tanshinone IIA sulphonate against adriamycin-induced lipid peroxidation in mice hearts in vivo and in vitro.

Protective effects of sodium tanshinone IIA sulphonate against adriamycin-induced lipid peroxidation were investigated. Data showed that treatment with sodium tanshinone IIA sulphonate could prevent mice from decrease in body weight caused by adriamycin. It was found that myocardial lipid peroxidation in sodium tanshinone IIA sulphonate-treated mice was lower compared with that in adriamycin-treated ones. The activities of some endogenous antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase and catalase, were higher in the sodium tanshinone IIA sulphonate group than that in the adriamycin group. In vitro experiments showed that sodium tanshinone IIA sulphonate could inhibit adriamycin-induced mitochondrial lipid peroxidation and swelling. Sodium tanshinone IIA sulphonate could scavenge adriamycin semiquinone free radical in heart homogenate dose-dependently. Thus, protective effects of sodium tanshinone IIA sulphonate may not only be related to its antioxidant activity but also to its regulation of antioxidant enzyme activities in the heart.

Two-peak kinetic curve of the chemiluminescence in phorbol-induced macrophage.

Phorbol-1,2-myristate-1,3-acetate can induce rat macrophage to emit chemiluminescence. The kinetic curve of the chemiluminescence clearly showed two peaks when a proper ratio of phorbol-1,2-myristate-1,3-acetate and macrophage were mixed. It was proved that the first one mainly came from the oxygen free radicals and the second one from NO generated in the phorbol-1,2-myristate-1,3-acetate stimulated cells by using the substitute and inhibitor of NO synthase.

Scavenging effects of salvia miltiorrhiza on free radicals and its protection for myocardial mitochondrial membranes from ischemia-reperfusion injury.

Using low temperature electron spin resonance (ESR) technique, we found that Salvia miltiorrhiza injection could scavenge the oxygen free radicals generated from ischemia-reperfusion injury in the myocardium as effectively as SOD. Using ESR spin trapping technique we found that one of its effective components, Danshensu, could scavenge superoxide anion free radicals generated from the reaction system of xanthine and xanthine oxidase, and that lipid free radicals generated from lipid peroxidation of myocardial mitochondrial membranes could be scavenged by another effective component, Tanshinone. The membrane fluidity of the mitochondria isolated from the ischemia-reperfused hearts was studied with the ESR spin labelling technique, and the TBA-method was used to detect the lipid peroxidation. It was found that Danshensu could protect the mitochondrial membrane from the ischemia-reperfusion injury and lipid peroxidation.

Studies on nitric oxide free radicals generated from polymorphonuclear leukocytes (PMN) stimulated by phorbol myristate acetate (PMA).

The interaction of NO and O2- free radicals generated from PMA (phorbol myristate acetate)-stimulated PMN (polymorphonuclear leukocytes) was studied by a nitroxide spin trap, DMPO (5,5-dimethyl-1-pyrroline-1-oxide). It was found that addition of L-arginine to the system would significantly decrease the trapped O2- by DMPO and addition of NG-monomethyl-arginine (NGMA) would significantly increase the trapped O2- by DMPO. It was proved that the formation of ONOO- by the reaction of NO and O2- was the main reason for the decrease of trapped O2- in the experiment with xanthine/xanthine oxidase and irradiation of riboflavin systems. The yield of NO during this process was calculated. The generation dynamic of NO was studied by a luminol-dependent chemiluminescence technique and it was found that after stimulation of PMN by PMA, there would be an immediate, significant chemiluminescence, which came mainly from the active oxygen free radicals generated by PMN. If L-arginine was added to this system, the chemiluminescence would increase about 100-fold, but NGMA inhibited the increase of the chemiluminescence. Ten minutes after addition of L-arginine, this increase did not change, the chemiluminescence peak decreased gradually, but the half life increased. The ESR and chemiluminescence properties of NO and ONOO- synthesized were also studied in model systems.

[Mechanism of scavenging effects of (-)-epigallocatechin gallate on active oxygen free radicals].

The concentration of 50% scavenging ratio (SC50) and the scavenging rate constants (k) and stiochiometric factor (n) were determined when (-)-epigallocatechin gallate [(-)-EGCG] scavenging superoxide anion free radical (O2.-) and hydroxyl radical (.OH). The mechanism of scavenging active free radicals of (-)-EGCG and the promotion of (-)-EGCG free radical and its structure were analyzed in vitro. Our results suggest that the centers of scavenging reaction of (-)-EGCG are B, D, and A cycles, and each (-)-EGCG traps six O2.- or .OH. It is in accord with the value of stiochiometric factor n = 6.

Scavenging effects of tetramethylpyrazine on active oxygen free radicals.

The scavenging effects of tetramethylpyrazine (ligustrazine, Lig) isolated from Ligusticum wallichii Franch on active oxygen free radicals were studied using spin trapping technic and chemiluminescence methods. The scavenging rate of superoxide anion (O2-.) by Lig (25 mg.ml-1) was 100% in xanthine/xanthine oxidase (Xan/XO) system, and that of hydroxyl radical (OH-.) was 44% in Fenton's reaction. The scavenging rate of lipid peroxyl radical (LOO-.) by Lig (25 mg.ml-1) was 80% in peroxidizing microsome system. It is possible that Lig scavenged O2-. by catalyzing its spontaneous dismutation. The results showed that Lig had strong effects of scavenging cyotoxic oxygen free radicals (O2-., LOO-., OH-.).

Scavenging effect of catechin on free radicals studied by molecular orbital calculation.

The eigenvectors, net charges, and electron population on catechin were calculated with molecular orbital method MNDO. It was found that the 2 hydroxyls on the benzene ring of the chromane were more active than the others on the other benzene ring when it reacted with a free radical.

Effects of oxygen radicals on the conformation of sulfhydryl groups on human polymorphonuclear leukocyte membranes.

The healthy intact polymorphonuclear leukocytes (PMNs) were labeled with 4-maleimide-TEMPO spin labeling compound (MAL) to study the effects of oxygen radicals produced by phorbol myristate acetate (PMA)-stimulated PMNs on the conformation of sulfhydryl (SH) groups of PMN membrane proteins. The lipid peroxidation induced by PMA-stimulated PMNs was detected by evaluating the formation of malonaldehyde (MDA) with the thiobarbituric acid (TBA) test. From the experiments of luminol-dependent chemiluminescence (CL) and fluorometry, it was found that Chinese herbs schizandrin B (Sin B) and quercetin (Q) possessed scavenging properties for oxygen radicals produced during the PMN respiratory burst. These two herbs can also inhibit the conformation changes in SH binding sites on the PMN membrane proteins caused by oxygen radicals produced by the PMNs themselves. They also decreased the amount of MDA, which was a final product formed during lipid peroxidation.

ESR spin trapping studies on the free radicals in gasphase of cigarette smoking.

Free radicals in the gasphase of cigarette smoke were determined by spin trapping agent PBN (N-tert-butyl-2-phenyl nitrone) and DMPO (5,5-dimethyl-1-pyrroline-oxide) on electron spin resonance spectrometer. They were identified according to the spectrum parameters calculated from the spectra. The free radicals mainly consist of alkoxyl and alkyl free radicals, of which alkoxyl free radicals make up 60-70% of the total spectral components. These free radicals are very active and reactive. They can damage cells and lead to some diseases. This work points out the importance of scavenging the free radicals of cigarette smoke to prevent diseases caused by them.

[Scavenging of probimane on semiquinone free radical formation by doxorubicin in rat heart].

Probimane, dl-bis (4-morpholin-methyl 3,5-dioxopipweazin-1-yl) propane first synthesized in China, is a dioxopiperazin compound with antineoplastic, antimetastatic and radiopotentiating activities. In order to evaluate the mechanisms of cardiotoxicity protective action of probimane, the free radical induced by doxorubicin were analysed by electron spin resonance (ESR) techniques. Our studies showed that doxorubicin stimulated the formation of semiquinone free radicals in the rat heart homogenate and heart cell mitochondria systems, and probimane inhibited the free radical formation in both systems, with the dose-dependent and time-dependent responses. The inhibitory rates of doxorubicin free radical formation in rat heart homogenate system by probimane 0.6 mmol.L-1 at time of 3, 5, 10, 15, 30, 45 and 60 min were 44.6%, 43.0%, 51.5%, 74.3%, 68.1%, 56.1% and 39.9% respectively. The inhibitory rates of semiquinone free radical formation in mitochondria system by probimane at the concentration of 0.02, 0.06, 0.6, 1.2 and 2.4 mmol.L-1 were 17.07%, 29.87%, 63.95%, 64.62% and 83.64%, respectively. Probimane had no effect on NADH2, but inhibited NADH dehydrogenase activity at higher concentration.

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes.

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.

Active oxygen radicals produced by leukocytes of malignant lymphoma.

The electron spin resonance (ESR) spectra of active oxygen radicals produced during the respiratory burst of PMA-stimulated leukocytes and oxygen consumption are studied by ESR spin trapping and spin probe oxymetry for 31 times in 17 cases of malignant lymphoma. The results showed that the spectra produced from PMA-stimulated patients' leukocytes were predominantly spin adducts of DMPO-hydroxyl radicals (DMPO-OH). The decrease in oxygen consumption during respiratory burst suggested that respiratory burst function was inhibited. Kinetic observations of patient condition showed that respiratory burst and oxygen consumption were improved and even approached normal, when the patient was in remission or the condition was ameliorated. This paper is the first of its type and helps clarify the mechanism of malignant proliferation and metastasis.

Detection of free radical scavenging activity of schisanhenol by electron spin resonance.

It was reported that Schisanhenol (Sal) isolated from Schisandrae Rubriflora inhibited lipid peroxidation induced by ferrous-cysteine and NADPH-ascorbic acid. In this studies the oxygen radical scavenging activity of Sal was detected by electron spin resonance (ESR) and spin trapping. Sal was shown to scavenge oxygen radicals produced by human neutrophils (Neu) stimulated by tetradecanoylphorbol acetate (TDPA). But no effect of Sal was seen on oxygen consumption measured by spin label oximetry in Neu during respiratory burst. In Fenton reaction system, the inhibitory rate of hydroxyl radical by Sal was 34.4%. In xanthine-xanthine oxidase and uv-irradiation of riboflavin systems, Sal scavenged superoxide anion radical by 26.1% and 21.9%, respectively. In all these systems the action of Sal was more potent than that of vitamin E. It may be concluded that Sal possesses a free radical-scavenging activity.

[Scavenging effects of sodium ferulate and 18 beta-glycyrrhetic acid on oxygen free radicals].

The scavenging effects of sodium ferulate (SF) and glycyrrhetic acid (GA) on oxygen free radicals were studied by using spin trapping technique and chemiluminescence (CL) methods. SF 0.3 mmol/L and GA 0.3 mmol/L effectively scavenged oxygen free radicals produced by phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMN), but had no effect on the oxygen consumption by PMN, measured with spin probe oxymetry. In 3 cell-free oxygen free radical generating systems, both SF and GA showed a remarkably scavenging effects on O2- and OH.. SF 30 mumol/L and GA 30 mumol/L prominently inhibited the CL response of PMA-stimulated PMN. The results demonstrated that SF and GA are oxygen free radical scavengers.

Scavenging effect of berbamine on active oxygen radicals in phorbol ester-stimulated human polymorphonuclear leukocytes.

The scavenging effect of berbamine (Ber) on active oxygen radicals was studied, using a spin-trapping technique and a chemiluminescence (CL) method in phorbol myristate acetate (PMA) stimulated polymorphonuclear leukocytes (PMN) and in four cell-free superoxide (O2-.) or hydroxyl radical (OH.) generating systems. Ber (0.1 to 0.3 mM) effectively reduced active oxygen radicals in PMN stimulated with PMA, but had no obvious effect on oxygen consumption during the respiratory burst of PMN, measured with spin probe oxymetry. Ber (0.3 mM) prominently inhibited the CL response of PMA-stimulated PMN. The agent remarkably quenched O2-. in xanthine/xanthine oxidase and irradiation riboflavin systems and OH. in the Fenton reaction. Its scavenging action on O2-. was stronger than that of Vitamin E in the xanthine/xanthine oxidase system but the same as Vitamin E in the riboflavin system, and its action on OH. was similar to that of Vitamin E.

Scavenging effect of schizandrins on active oxygen radicals.

The reactive oxygen radicals produced from human polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate), hydroxyl radicals generated by a Fenton reaction, and superoxide anion radicals produced by irradiating solutions of riboflavin in the presence of EDTA have been taken as the models for production of oxygen radicals. With the use of the electron spin resonance spin trapping method, the scavenging effects of schizandrol A (solA) (5 x 10(-4) M) and schizandrin B (sinB) (5 x 10(-4) M) have been studied and compared with the effects of vitamin E (5 x 10(-4) M) and vitamin C (5 x 10(-4) M). It has been found that in cell system the scavenging effects of sinB and solA, as judged by ESR spin trappings, on hydrpxyl radicals (.OH) are greater than vitamin E and vitamin C and the scavenging effects on superoxide anion (O2) are greater than vitamin E but lower than vitamin C. With respect to the Fenton reaction, sinB has the strogest scavenging effect on .OH (77%) and solA has strong scavenging effect on .OH (63%), both of them larger than that of vitamin E (35%) and vitamin C (56%). In the riboflavin/EDTA system, the scavenging effect of sinB (46%) is smaller than that of vitamin C (96%) but larger than that of vitamin E (23%); the scavenging effect of solA is not obvious (14%). With the use of spin probe oximetry, the oxygen consumption during the respiratory burst of stimulated PMN has been measured when exposed to schizandrins. The experiment results demonstrated that they do not affect the activity of production of active oxygen radicals in the respiratory burst of PMN stimulated with PMA.

Scavenging effects on active oxygen radicals by schizandrins with different structures and configurations.

We have studied the scavenging effects of different structures and configurations of schizandrins isolated from Fructus Schizandrae, a traditional Chinese herb, on active oxygen radicals with the method of spin-trapping technique. The active oxygen radicals were produced from human polymorphonuclear leukocytes (PMN) stimulated with phorbol myristate acetate (PMA). In addition, the scavenging effects of schizandrins on hydroxyl radicals (.OH) in Fenton's reaction and the scavenging effects on superoxide anions (O2-.) in both riboflavin/EDTA and xanthine/xanthine oxidase systems have also been studied. They are compared with the scavenging effects of both Vitamin C (Vc) and Vitamin E (VE). The experimental results have shown that the scavenging effect of schizandrin B (Sin B) on the active oxygen radicals is stronger than that of S(-) Sin B and R(+) Sin B. For schizandrins of the same molecular structures with different stereoconfigurations the scavenging effects of S type of the benzene ring on active oxygen radicals are stronger than those of R type and for schizandrins of the same stereoconfigurations with different structures the scavenging effects of schizandrin C (Sin C) on the active oxygen radicals are stronger than those of Sin B.

[Scavenging effects of probimane on active oxygen free radicals by electron spin resonance].

Probimane, dl-1,2-bis (4-morpholine-methyl-3, 5-dioxopiperazin-1-yl) propane, is a new antitumor agent synthesized by Shanghai Institute of Materia Medica, Chinese Academy of Sciences. The scavenging effects of probimane on active oxygen radicals produced in 3 different systems were studied with the ESR spin trapping methods. In Fenton's reaction, probimane remarkably scavenged hydroxyl radicals (.OH) and the rate of scavenging .OH by probimane 0.05 mmol/L was 47%, compared to 5% by vitamin E (VE) and 30% by ascorbic acid (AA). In irradiation riboflavin system, in which superoxide (O2-.) was produced, the agent also had the scavenging effects on O2(-.). The rate of scavenging O2-. by probimane 0.05 mmol/L was 13%, higher than that by VE (7%) but lower than that by AA (90%). In cell system where the active oxygen radicals were produced during the respiratory burst of human neutrophils (Neu) stimulated by TPA (tetradecanoylphorbol acetate), probimane exhibited a dose-dependent scavenging action on the radicals. The rate of the radical scavenging by probimane 0.05 mmol/L was 37%, much higher than that by VE (9%) but lower than that by AA (68%). Probimane had no effect on the rate of oxygen consumption by human Neu, measured with spin probe oxymetry.

ESR studies on active oxygen radicals produced in the respiratory burst of human polymorphonuclear leukocytes.

The mechanism and process of production of active oxygen radicals in the respiratory burst of polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate) was studied in this paper. The experimental results indicate that when the PMA was dilute enough or at the beginning of stimulation even when the PMA concentration was high, the spectrum of hydroxyl radical spin adducts, DMPO-OH, was dominant in the ESR spectra. However, at the maximum level of the respiratory burst, the spectrum of superoxide anion spin adducts, DMPO-OOH, was dominant.