Neuroprotection of phenolic alkaloids from Menispermum dauricum versus melatonin against oxygen-glucose-serum-deprivation-mediated injury

BACKGROUND： Recent studies have demonstrated that phenolic alkaloids from Menispermum dauricum （PAMD） can protect the heart and brain from ischemia/reperfusion injury, and promote neuron survival by inhibiting neuronal Bax and upregulating Bcl-2 expression following ischemia/reperfusion. OBJECTIVE： To investigate the neuroprotective effects of PAMD versus exogenous melatonin against ischemia/reperfusion injury. DESIGN, TIME AND SETTING： Observation and comparison experiments at a cellular level were performed at the Department of Biochemistry and Molecular Biology, Tongji Medical College of Huazhong University of Science and Technology between February 2007 and February 2008. MATERIALS： PAMD （95% purity） was provided by Kunming Institute of Botany, Chinese Academy of Sciences; melatonin was provided by Sigma, USA. METHODS： N2a mouse neuroblastoma cells were cultured in vitro deprived of glucose, serum and oxygen for 90 minutes, then cultured in normal medium containing different concentrations of PAMD （0.1, 1.0, 10 mg/L） or melatonin （1, 10, and 100 μmol/L）. Cells cultured in normal conditions served as a control. MAIN OUTCOME MEASURES： The culture solution was collected to determine the content of ex- citatory neurotransmitters such as glutamic acid and aspartic acid; cell viability was detected by MTT methods; reactive oxygen species production was determined by fluorescence spectroscopy; mito- chondrial transmembrane potential （？Ψm） was detected by laser confocal scanning; cytochrome C was measured by western blotting; and caspase-3 activity was determined by visible spectropho- tometry. RESULTS： Melatonin and PAMD both promoted oxygen-glucose-serum deprivation-mediated N2a cell survival （P 〈 0.01） and inhibited glutamic acid release （P 〈 0.01）, but melatonin did not inhibit aspartic acid production. The protective effects were the strongest using melatonin 100 μmol/L and PAMD 10 mg/L, so subsequent experiments were the performed at those doses. Although PAMD could no longer maintain mitochondrial transmembrane potential 6 hours after reperfusion, its in- hibitory effects on cytochrome C release from mitochondria and scavengers of reactive oxygen species were stronger than those of melatonin （P 〈 0.01）. However, its inhibitory effect on caspase-3 activity was weaker than that of melatonin： PAMD could inhibit caspase-3 activity 12 hours after reperfusion （P 〈 0.01）, but melatonin inhibited caspase-3 activity 28 hours after reperfusion （P 〈 0.01）. CONCLUSION： The results show that melatonin and PAMD have neuroprotective effects, but that the mechanisms are varied. Melatonin can maintain mitochondrial transmembrane potential, but its inhibitory effects on cytochrome C release, caspase-3 activity, and reactive oxygen species scav-enging are different from those of PAMD.

Melatonin combined with exercise cannot alleviate cerebral injury in a rat model of focal cerebral ischemia/reperfusion injury

Previous studies have demonstrated that melatonin combined with exercise can alleviate secondary damage after spinal cord injury in rats. Therefore, it is hypothesized that melatonin combined with exercise can also alleviate ischemic brain damage. In this study, adult rats were subjected to right middle cerebral artery occlusion after receiving 10 mg/kg melatonin or vehicle subcutaneously twice daily for 14 days. Forced exercise using an animal treadmill was performed at 20 m/min for 30 minutes per day for 6 days prior to middle cerebral artery occlusion. After middle cerebral artery occlusion, each rat received melatonin combined with exercise, melatonin or exercise alone equally for 7 days until sacrifice. Interestingly, rats receiving melatonin combined with exercise exhibited more severe neurological deficits than those receiving melatonin or exercise alone. Hypoxia-inducible factor la mRNA in the brain tissue was upregulated in rats receiving melatonin combined with exercise. Similarly, microtubule associated protein-2 mRNA expression was significantly upregulated in rats receiving melatonin alone. Chondroitin sulfate proteoglycan 4 （NG2） mRNA expression was significantly decreased in rats receiving melatonin combined with exercise as well as in rats receiving exercise alone. Furthermore, neural cell loss in the primary motor cortex was significantly reduced in rats receiving melatonin or exercise alone, but the change was not observed in rats receiving melatonin combined with exercise. These findings suggest that excessive intervention with melatonin, exercise or their combination may lead to negative effects on ischemia/reperfusion-induced brain damage.

Curcumin reduces inflammatory reactions following transient cerebral ischemia/reperfusion injury

Inflammatory reactions are important pathophysiological mechanisms of ischemic brain injury. The present study analyzed the anti-inflammatory characteristics of curcumin via myeloperoxidase activity and nitric oxide content after 2-hour ischemia/24-hour reperfusion in Sprague Dawley rats. In addition, expressions of nuclear factor kappa B, tumor necrosis factor-α and interleukin-lβ protein were measured. Curcumin significantly reduced myeloperoxidase and nitric oxide synthase activities and suppressed expressions of nuclear factor kappa B, tumor necrosis factor-α, and interleukin-lβ in ischemia/reperfusion brain tissue. Results suggested that the neuroprotective effect of curcumin following cerebral ischemia/reperfusion injury could be associated with inhibition of inflammatory reactions.

Side effects of different doses of ephedrine in rats with cerebral ischemia/reperfusion injury

Ephedrine has a protective effect against cerebral ischemia,but its side effects limit its clinical application.Results from a previous study showed that 1.5 mg/kg per day ephedrine can promote motion recovery in rats following cerebral ischemia/reperfusion without significant side effects.In the present study,ephedrine at doses of 3.0,2.5 and 2.0 mg/kg was used to treat rats with cerebral ischemia/reperfusion and the effects of ephedrine on the heart,liver,kidney and cerebrum were observed.Results showed that the blood pressure of rats with cerebral ischemia/reperfusion injury following ephedrine treatment was lower than in rats that recovered naturally from cerebral ischemia/reperfusion,but the pressure decreased with increasing doses of ephedrine.In addition,serum aspartate transaminase,alkaline phosphatase and creatinine concentration in rats with cerebral ischemia/reperfusion injury following ephedrine treatment were greater than in rats that recovered naturally from cerebral ischemia/reperfusion.The concentrations of these enzymes were decreased with increasing doses of ephedrine.Ephedrine-treated rats displayed hyperemia,degeneration and edema in the cerebrum,liver,heart and kidney.Results demonstrated that ephedrine exhibited side effects on the cerebrum,heart,liver and kidney in rats following cerebral ischemia/reperfusion in a dose-dependent manner.

Dingxin Recipe(定心方) Prevents Ischemia/Reperfusion-Induced Arrhythmias via Up-regulating Prohibitin and Suppressing Inflammatory Responses

Objective： To identify the underlying mechanisms of the protective effects of Dingxin Recipe （定心方, DXR）, a Chinese compound prescription that has been used clinically in China for more than 20 years, on ischemia/reperfusion （I/R）-induced arrhythmias in rat model. Methods： A total of 30 rats were randomly divided into three groups： sham group, I/R group, and DXR-pretreated I/R （DXR-I/R） group. Rats in the DXR- I/R group were intragastrically administrated with DXR （12.5 g/kg per day） for consecutive 7 days, while rats in the sham and I/R groups were administrated with normal saline. Arrhythmias were introduced by I/R and electrocardiograms （ECG） were recorded. Two-dimensional （2-D） polyacrylamide gel electrophoresis and matrix- assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF MS） were used to identify differentially expressed proteins. Immunohistochemistry, real-time quantitative polymerase chain reaction （RQ- PCR）, Western blot, and enzyme-linked immunosorbent assay （ELISA） were performed to analyze proteins obtained in the above experiments. Results： DXR significantly reduced the incidence and mean duration of ventricular tachycardia and ventricular fibrillation and dramatically decreased the mortality, as well as arrhythmia score, compared with those of the I/R group. Among successfully identified proteins, prohibitin （PHB） and heart fatty acid binding protein （hFABP） were up-regulated in DXR-pretreated I/R rats compared with those of the I/R rats. In addition, compared with the I/R group, the level of glutathione （GSH） was elevated accompanied by reduced expressions of interleukin-6 （IL-6） and neutrophil infiltration in I/R rats with DXR pretreatment. Conclusions： DXR could alleviate I/R-induced arrhythmias, which might be related to increased expression of PHB. The enhanced expression of PHB prevented against the depletion of GSH and consequently inhibited apoptosis of cardiomyocytes. Furthermore, up-regulation of PHB might ameliorate I/R-induced cell death and leakage of hFABP by suppressing neutrophil infiltration and IL-6 expressions.