cytochrome-c-t and dauricine

Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine.

Topics: Animals; Apoptosis; bcl-Associated Death Protein; Benzylisoquinolines; Blotting, Western; Bronchi; Buthionine Sulfoximine; Caspase 3; Cell Survival; Cells, Cultured; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Cytochromes c; Enzyme Inhibitors; Epithelial Cells; Flow Cytometry; Glutathione; Humans; In Situ Nick-End Labeling; Ketoconazole; Lung; Male; Mice; Tetrahydroisoquinolines

To investigate the effect of dauricine on the apoptosis of neuronal cells and the expression of apoptosis-related proteins in the brain penumbra of rats induced by transient focal cerebral ischemia-reperfusion injury.. Male SD rats were randomly divided into five groups: sham group (Sham), model group (Model), and Dauricine groups of low, middle and high doses. To make the transient focal cerebral ischemia-reperfusion injury model, the middle cerebral artery on the right side of rat was occluded by inserting a nylon suture through the internal carotid artery for 1 h, followed by reperfusion for 24 h after withdrawing the suture. Dauricine groups, different doses of Dauricine (2.5, 5, 10 mg x kg(-1) as low, middle and high dose respectively) were administered intraperitoneally at the beginning of the cerebral ischemia, and at 11 h and 23 h after reperfusion. At the same time, Sham group and Model group was administered saline as controls. Brain samples of rats were treated with paraformaldehyde perfusion fixation 24 h after blood reperfusion and then collected for making pathological sections. Apoptotic changes of neuronal cells in the brain penumbra of rat were evaluated in situ by terminal deoxyribonucleotidyl transferasemediated dUTP-digoxigenin nick end-labelling (TUNEL). Cytochrome C (Cyt-C) release and the expression of caspase -3 and caspase -9 proteins of the ischemic-reperfusion brain tissue were determined by immunohistochemistry assay.. TUNEL-positive cells in groups of middle and high doses of dauricine (18.9 +/- 2.02 and 15.9 +/- 2.9 cells/mm2 respectively) decreased significantly compared with model group (25.5 +/- 3.3 cells/mm2, P<0.05). Cyt-C release and the expression of caspase-3 and caspase-9 proteins in groups of middle and high doses of dauricine were also inhibited compared with Model group (P<0.01).. The mechanism of the neuroprotective effect of dauricine after cerebral ischemia-reperfusion injury may parly, related with an inhibition of neuronal cells apoptosis in the penumbra.

Topics: Animals; Apoptosis; Benzylisoquinolines; Caspases; Cytochromes c; Dose-Response Relationship, Drug; Gene Expression Regulation; Ischemic Attack, Transient; Male; Neuroprotective Agents; Rats; Reperfusion Injury; Tetrahydroisoquinolines

1. Previous experimental studies have shown that dauricine can protect the brain against ischaemic damage, but the underlying mechanisms remain unknown. In the present study, we examined whether dauricine inhibits neuronal apoptosis in the penumbra in a rat model of transient focal cerebral ischaemia. 2. Male Wistar rats underwent a 90 min temporary occlusion of the middle cerebral artery. Dauricine (21, 42 and 84 mg/kg) was administered by intragastric gavage twice a day for 3 days before ischaemia. Rats were killed and brain samples were collected 24 h after ischaemia. Histopathological outcome was evaluated by haematoxylin-eosin staining. Apoptotic changes were evaluated by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) for DNA fragmentation. The mitochondrial pathway was explored using immunohistochemistry for cytochrome c release, caspase 9 and caspase 3 activation, as well as by reverse transcription-polymerase chain reaction for determination of caspase 9 and caspase 3 mRNA expression. 3. Cytochrome c release, activation of caspase 9 and caspase 3 and DNA fragmentation were detected 24 h after ischaemia. Dauricine (42 and 84 mg/kg) pretreatment improved histopathological recovery, diminished DNA fragmentation and reduced cytochrome c release and activation of caspase 9 and caspase 3 in the penumbra at 24 h. 4. These findings suggest that dauricine attenuates apoptosis in the penumbra after transient focal cerebral ischaemia. The infarct-reducing effects of dauricine may be due, in part, to the inhibition of apoptotic cell death via a mitochondrial pathway in the penumbra.

Topics: Alkaloids; Animals; Apoptosis; Benzylisoquinolines; Caspase 3; Caspase 9; Cytochromes c; DNA Fragmentation; Enzyme Activation; Immunohistochemistry; In Situ Nick-End Labeling; Ischemic Attack, Transient; Male; Mitochondria; Neuroprotective Agents; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Tetrahydroisoquinolines