Gossypol, a component in cottonseed, induced increases in cytosolic Ca2+ levels in Chang liver cells.

The effect of gossypol, a compound found in cottonseed, on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. Gossypol (0.2-5microM) increased [Ca2+](i) in a concentration-dependent manner with an EC(50) value of 1.5microM. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase within 5min after drug application. Removal of extracellular Ca2+ markedly reduced the [Ca2+](i) signals by 80+/-2%. Preincubation with 0.1mM La3+ or 10microM nimodipine abolished the Ca2+ influx. Gossypol (5microM)-induced release of intracellular Ca2+ was reduced by 75% by pretreatment with 1microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+. Conversely, pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. After pretreatment with 5microM gossypol in Ca2+-free medium for several min, addition of 3mM Ca2+ induced a [Ca2+](i) increase of a magnitude nine-fold greater than control. Gossypol (5microM)-induced Ca2+ release was not affected by inhibiting phospholipase C with 2microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Together, this study shows that gossypol induced significant [Ca2+](i) increases in Chang liver cells by releasing Ca2+ from intracellular pools in a phospholipase C-dissociated fashion and by causing La3+- and nimodipine-sensitive Ca2+ influx.

Melatonin might be one possible medium of electroacupuncture anti-seizures.

To explore the alteration of melatonin (MT) levels in pineal, hippocampus and serum during seizure crises and electroacupuncture (EA) anti-seizures, we established a rat seizure model by microinjecting benzylpenicillin into hippocampus. EA was performed on "Fengu" (DU 16) and "Jinsuo" (DU 8) acupoints in rats. Electroencephalogram (EEG) of rats was recorded and the relative power (RP) of 1 approximately 30 HZ band of EEG was analyzed. A capillary electrophoresis-electrochemical detection method was used to determine MT contents. Our results indicated that MT level was elevated in pineal and hippocampus, and first had no change then significantly evaluated in serum during seizure crisis. The elevation of MT level was greatly potentiated with 30 min EA treatment (P<0.05). Meanwhile, the degree of seizures and the increases of EEG RP induced by seizures were significantly reduced (P<0.05). Because MT was considered as an antistressor and a natural downregulator of epileptiform activity, we postulate that the elevation in MT level during seizures may be one endogenous mechanism that counteracts convulsions and seizure-induced stress. A further elevation of MT levels with EA treatment suggests that MT might be one of the possible mediums of EA anti-seizures.

Novel effects of 5,8,11-eicosatriynoic acid, a lipoxygenase inhibitor, on Ca2+ mobilization in Madin Darby canine kidney cells.

The effect of 5,8,11-eicosatriynoic acid, a widely used lipoxygenase inhibitor, on Ca2+ fate in Madin Darby canine kidney cells was examined by using fura-2 as a Ca2+ probe. At concentrations between 2-100 microM 5,8,11-eicosatriynoic acid increased [Ca2+]i concentration-dependently with an EC50 of 20 microM . Extracellular Ca2+ removal decreased the Ca2+ signals, indicating that 5,8,11-eicosatriynoic acid triggered Ca2+ release and Ca2+ influx. 5,8,11 -Eicosatriynoic acid (30 microM) induced a [Ca2+]i increase in Ca2+-free medium after pretreatment with carbonylcyanide m-chlorophenylhydrazone (2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor for 20 min. Conversely, 5,8,11-eicosatriynoic acid pretreatment almost abolished the Ca2+ release induced by carbonylcyanide m-chlorophenylhydrazone and thapsigargin. These results suggest that 30 microM 5,8,11-eicosatriynoic acid released Ca2+ from the endoplasmic reticulum, mitochondria and other stores. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 2-50 microM 5,8,11-eicosatriynoic acid for 10 min. in Ca2+-free medium concentration-dependently. Pretreatment with 10 microM La3+ abolished 30 microM 5,8,11-eicosatriynoic acid -induced [Ca2+]i increases, but adding La3+ during the decay phase had no effect. 5,8,11-Eicosatriynoic acid-induced Ca2+ release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), but was decreased by 60% by 40 microM aristolochic acid. Several other lipoxygenase inhibitors such as baicalein (50 microM), 5.8.11.14-eicosatetraynoic acid (ETYA; 0.1-0.2 mM), caffeic acid (5-50 microM), esculetin (5-50 microM), alpha-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV-5901; 0.1-0.2 mM) and alpha-pentyl-4-(2-quinolinylmethoxy)-benzenemethanol (L-655238; 80-100 microM) had no effect on [Ca2+]i. Collectively, the data suggest that the lipoxygenase inhibitor 5,8,11-eicosatriynoic acid induced a [Ca2+]i increase in renal tubular cells concentration-dependently, by releasing intracellular Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing extracellular Ca2+ influx in a La3+-sensitive manner.

Influence of acupuncture upon expressing levels of basic fibroblast growth factor in rat brain following focal cerebral ischemia--evaluated by time-resolved fluorescence immunoassay.

We investigate the expression of basic fibroblast growth factor (bFGF) during ischemia-reperfusion with or without electroacupuncture (EA) treatment, and observe the effect of EA on ischemic cerebral injury. In the present study, a sensitive sandwich time-resolved fluoroimmunoassay (TR-FIA) method was developed to quantitatively analyze the levels of bFGF in rat brain. The results indicated that the obvious cerebral infarction and swelling were observed after ischemia-reperfusion, and the opening amount of cerebral blood micrangium was increased. In the meantime, the expression of bFGF was also improved in striatum and frontoparietal cortex. EA alleviated the ischemic injuries induced by MCAO and markedly upregulated the opening amount of the micrangium. Owing to application of EA, the expression of bFGF was notably enhanced in striatum and cortex. The results give us some hints for the neuroprotective mechanism of EA, that is, EA may partially exert protective effects on neurons through regulating the blood dynamics and the endogenous expression of bFGF.

Electro-acupuncture attenuates nitric oxide release from rat striatum after transient middle cerebral artery occlusion.

Nitric oxide (NO) is an important diffusible neurotransmitter, which also has neurotoxicity when it is overproduced. To investigate whether electro-acupuncture (EA) could inhibit the excessive NO release during cerebral ischemia, we detected NO directly by our self-made NO sensitive electrode. The electrode was placed into rat striatum after transient middle cerebral artery occlusion. NO level was significantly increased upon the onset of ischemia and reperfusion. EA apparently antagonized the ischemia-elicited rise of NO, although it could not suppress the NO level to baseline. The results indicated that EA might inhibit directly the elevation of NO following cerebral ischemia.

Anticonvulsion effect of acupuncture might be related to the decrease of neuronal and inducible nitric oxide synthases.

To measure the levels of hippocampal nitric oxide synthase isoforms in penicillin induced epilepsy and to test the effect of electroacupuncture (EA) on changes of theses levels during epilepsy, we injected penicillin into rat hippocampus to make an epilepsy model and performed electroacupuncture treatment on "Feng Fu" (DU 16) and "Jin Suo" (DU 8) points in Wistar rats. Nitric Oxide synthase (NOS) mRNA levels of rat hippocampus were determined by reverse transcription-polymerase chain reaction (RT-PCR). The neuronal nitric oxide synthase (nNOS) mRNA markedly increased (P<0.01) and inducible nitric oxide synthase (iNOS) mRNA significantly emerged during epilepsy, whereas no significant change in epithelial nitric oxide synthase (eNOS) mRNA was observed. EA inhibited the epilepsy and decreased nNOS (P<0.01) and iNOS (P<0.01) correspondingly but had no effect on the amount of eNOS mRNA. The data suggest that penicillin-induced epilepsy caused an increase in nNOS and iNOS, and the EA anticonvulsant effect might be related to the decrease of these nitric oxide synthases.

Electroacupuncture reduces rat's neuronal ischemic injury and enhances the expression of basic fibroblast growth factor.

The present study was designed to investigate whether the Electroacupuncture (EA) is beneficial to extenuate cerebral injuries following transient Middle Cerebral Artery Occlusion (MCAO), as well as to observe the effect of EA on expression of Basic Fibroblast Growth Factor (bFGF) -like Immunoreactivity (IR) in rat brains. The results indicate that gross neuronal damages include infarction, swelling and neuron loss, accompanied by increased bFGF-like IR expression following MCAO. In peri-infarct striatum, bFGF-like IR was mainly located in astrocytes except some neurons also showed an upregulation of the IR; in frontoparietal cortex, strong induction of bFGF-like IR was mostly seen in neurons. Both the EA applied during ischemia and reperfusion could evidently alleviate cerebral lesion extent, notably upregulate the expression of bFGF-like IR in striatum and cortex, but there was no significant difference between the effects of EA applied during ischemia and reperfusion, except EA applied during reperfusion seems to be more effective in reducing the cerebral swelling. The results implied that, in striatum, astrocytes might play an important role in the protection of neuron via the expression of bFGF; whereas in cortex, neurons may exert autoprotection through secreting bFGF themselves. One possible protective effect of EA lies in regulating the endogenous expression of bFGF.

[Effect of electroacupuncture and 7-NI on penicillin-induced epilepsy and their relation with intrahippocampal NO changes].

In order to investigate the effect of 7-nitroindazole (7-NI) on penicillin-induced epilepsy and the relationship between NO and anti-epileptic effect of electro-acupuncture, computerized physiological polygraph and NO-sensitive electrode with potentiostat were respectively used to record the total power spectrum (TPS) of EEG and the concentration of NO in rat hippocampus pretreated with electro-acupuncture or 7-NI for 30 min before penicillin. The results showed that the TPS of EEG and concentration of NO in hippocampus increased greatly after the epilepsy induction. Treatment with electroacupuncture inhibited the TPS of EEG sharply (P < 0.01). The latency and threshold of epilepsy induction were decreased by 7-NI, but paroxysm abated and the TPS was inhibited (P < 0.05) as compared with the control group. Both pretreatments with 7-NI and electroacupuncture decreased the NO concentration (P < 0.01). The above results suggest that the propagation of penicillin-induced seizures is facilitated by nNOS, but with some increase in the latency and threshold of induction. Electroacupunture may inhibit seizure through decreasing nNOS transcription in hippocampus.

Anticonvulsion effect of acupuncture might be related to the decrease of neuronal and inducible nitric oxide synthases.

To measure the levels of hippocampal nitric oxide synthase isoforms in penicillin induced epilepsy and to test the effect of electroacupuncture (EA) on changes of these levels during epilepsy, we injected penicillin into rat hippocampus to make an epilepsy model and performed electroacupuncture treatment on "Feng Fu" (DU 16) and "Jin Suo" (DU 8) points in Wistar rats. Nitric oxide synthase (NOS) mRNA levels of rat hippocampus were determined by reverse transcription-polymerase chain reaction (RT-PCR). The neuronal nitric oxide synthase (nNOS) mRNA markedly increased (p<0.01) and inducible nitric oxide synthase (iNOS) mRNA significantly emerged during epilepsy, whereas no significant change in epithelial nitric oxide synthase (eNOS) mRNA was observed. EA inhibited the epilepsy and decreased nNOS (p<0.01) and iNOS (p<0.01) correspondingly but had no effect on the amount of eNOS mRNA. The data suggest that penicillin-induced epilepsy caused an increase in nNOS and iNOS, and the EA anticonvulsant effect might be related to the decrease of these nitric oxide synthases.

[Hippocampal non-NMDA and GABA-A receptors in benzylpenicillin-induced epilepsy and electro-acupuncture antiepilepsy].

AIM: To study the effect of 6,7-dinitroquinoxaline-2,3-dione (DNQX) and bicuculline (Bic) on benzyl-penicillin-induced-epilepsy (PIE) and electroacupuncture (EA) antiepilepsy in rats. METHODS: Epilepsy was elicited by intra-hippocampal microinjection of benzylpenicillin in rats. The analysis of electroencephalogram (EEG) and power spectrum was used to measure the extent of convulsion. RESULTS: EA or DNQX (1 microgram) showed partial inhibitory effect on PIE. EA + DNQX caused further inhibition of PIE, and Bic attenuated EA antiepileptic effect. CONCLUSION: Antagonization of GABA-A receptor attenuated EA antiepileptic effect, and EA acted synergistically with the antagonists of non-N-methyl-D-aspartate (non-NMDA) receptors.

Effects of electro-acupuncture on C-FOS expression in gerbil hippocampus during transient global ischemia.

We studied the effects of electro-acupuncture (EA) on C-FOS expression as well as on the histological changes in various regions of hippocampus in the gerbil acute global ischemia model. EA was administered at points of 'Feng-fu' and 'Jin-suo' with a frequency of 7 Hz and an intensity of 6 mA for 30 minutes. EA can substantially potentiate the induction of C-FOS protein like immunoreactivity (CFPLI) in neurons of various regions in hippocampus following transient global ischemia, especially in the CA1 subfield. At the same time EA can prevent most of the CA1 cells from delayed degeneration after ischemia. These results indicate that EA has the protective effect on neurons of hippocampus after cerebral ischemia and C-FOS may be involved in this process.

Effect of electroacupuncture on the level of preproenkephalin mRNA in rat during penicillin-induced epilepsy.

Our previous studies have shown that seizure induced by injecting penicillin (0.24 mg/2 microliters) into hippocampus could be inhibited by electroacupuncture (EA) probably via decreasing enkephalin content in hippocampus. To determine whether this change reflected the peptide synthesis, preproenkephalin (PPE) mRNA was detected in hippocampus and some other limbic structures during seizure and after EA treatment by in situ hybridization. Four hours after injecting penicillin into hippocampus, PPE mRNA levels were significantly increased by 10 folds in entorhinal cortex, subiculum, CA1 area of hippocampus, amygdaloid nucleus and piriform cortex, whereas EA treatment apparently attenuated the seizure-induced increase of PPE mRNA in the areas mentioned above. The results indicated that EA may regulate the biosynthesis of PPE in hippocampus during seizure by an alteration in gene transcription.

Endoscopic injection for the treatment of bleeding ulcers: local tamponade or drug effect?

Fifty-two patients with Forrest Ia or Ib bleeding ulcers were randomized to receive endoscopic injection therapy with either 1:10,000 epinephrine in water (Group I) or distilled water (Group II). Twenty-five out of 27 patients in group I, versus 22 out of 25 patients in group II, achieved initial hemostasis after endoscopic injection therapy (p > 0.05). Five patients who did not respond to local injection had bleeding controlled by heater probe thermocoagulation or surgical intervention. Three patients in each group developed rebleeding after initial hemostasis. Four of these patients had bleeding controlled by surgical intervention, while the other two died of underlying diseases. No change in systemic blood pressure, but a significant drop in the pulse rate were noted in both groups after injection therapy. Patients with shock at admission or ulcer size greater than 2 cm had a significantly higher rebleeding rate after initial hemostasis than patients with normal blood pressure and ulcers under 2 cm (p < 0.05). No serious complications were observed after injection therapy, and no significant difference in the amounts of solution required for successful hemostasis was noted between the two groups. We conclude that a local tamponade with distilled water is as effective and safe as diluted epinephrine solution for endoscopic injection therapy.

[Alteration of dynorphin1-8 and leu-enkephalin in rat hippocampus during seizure and electroacupuncture].

Immunocytochemical technics were used to evaluate the influence of penicillin-induced seizure and electroacupuncture treatment on dynorphin1-8 and leu-enkephalin immunoreactivity in hippocampus. It was found that 3 h after beginning of seizure there started a dramatic decrease in dynorphin1-8 in hilus, mossy fiber of hippocampus but an increase in hilus, mossy fiber of hippocampus but an increase in leu-enkephalin in subiculum, CA1 area of hippocampus and some other limbic structures. Electroacupuncture treatment decreased the leu-enkephalin immunoreactivity in the nuclei mentioned above and increased dynorphin1-8 immunoreactivity in hippocampus. The results show that epileptiform activity and electroacupuncture inhibitory effect on seizure may be related to the alteration of dynorphin1-8 and leu-enkephalin in the brain.

[C-fos expression in rat brain during seizure and electroacupuncture].

C-fos proteins were visualized immunohistochemically in the brain of rats after seizures induced by injecting penicillin into hippocampus and by penicillin+electroacupuncture treatment. Three hours following seizures there was an evident expression of c-fos proteins in the hippocampus (CA1 area), dentate gyrus, piriform cortex, dorsal part of entorhinal cortex, and amygdaloid nucleus, and there was a dramatic increase of c-fos proteins in CA3 area and the areas mentioned above except the CA1 area where c-fos proteins apparently decreased after electroacupuncture treatment. The results showed that seizures can induce c-fos proteins in some nuclei related with seizure and that electroacupuncture can also regulates the c-fos expression after seizure.