The Effects Of Berberine On Adult Mouse Neurogenesis And On Injure Induced By Ischemia And Reperfusion

Berberine(BR)is an isoquinoline alkaloid present in the roots, rhizome and stem bark of a number of important medicinal plant species (e.g.Berberis aquifolium,Berberis vulgaris,Berberis aristata and Tinospora cordifolia etc.).The potential importance of BR is indicated by its use in the traditional Chinese medicine since time immemorial.In recent years,a number of beneficial therapeutic effects of BR have been revealed such as anti-cardiac failure and anti-cardiac arrhythmia, anti-proliferation effect on vascular smooth muscle cells, anti-hyperlipemia and anti-hyperglycemia.Some researches demonstrated that BR has potential in the prevention of atherosclerosis and restenosis. And there is increasing evidence that BR exhibited neuroprotective effects against ischemic brain damage,still little is known about the mechanism.In studies on the pharmacokinetics of BR in rat (intravenous administration),Wang et al.found that intra-hippocampus and intra-thalamus BR concentration increased rapidly and eliminated tardily compared with that in plasma.These data implie that BR might accumulate in hippocampus and thalamus.Whereas hippocampus is one of the most important brain regions for neurogenesis,thus the question is raised whether neurogenesis is affected by BR accumulated in hippocampus.And another question is whether BR can protect ischemia stroke via regulating neurogenesis?Acute ischemic stroke(AIS)caused by cerebral artery occlusion leading to infarction of brain tissue remains a leading cause of death and disability worldwide.Over two-thirds of stroke deaths worldwide are in developing countries.Neuronal loss during ischemia is due to a reduction in the oxygen and glucose supply,i.e.oxygen and glucose deprivation (OGD).Cerebral ischemia triggers a complex series of biochemical and molecular mechanisms that impair the neurologic functions through breakdown of cellular integrity mediated by excitotoxic glutamatergic signalling,ionic imbalance,free-radical reactions,etc.Traditionally,the cell death following cerebral ischemia is considered to be necrotic in nature,but research in the past decade have revealed that during the first few hours after a stroke,neurons in the ischemic penumbra or peri-infarct zone,suffer transiently reversible damage and then ultimately undergo death by apoptosis,an energy-dependent process which contributes equally to the pathogenesis.The complexity of the ischemic cascade and the variability of mechanisms leading to tissue infarction at different time points after stroke onset suggest that targeting one mechanism may be of limited therapeutic value.Until recently,neuroprotective drugs have not lived up to their promise predominantly because of side effects that were caused in large part by their interference with normal brain function.To date, intravenous thrombolysis using rt-PA remains the most effective and widely available acute stroke treatment in patients who present within 3 h of onset.So the concept that drugs preferentially inhibit pathological activation of their targets while preserving normal function,and of therapeutic alliance to optimize the extent of neuronal survival and recovery is an important path for future drug development.In the present work,we first investigated the effect of BR on the neurogenesis in vitro and vivo,aiming at providing experimental data for the safety evaluation of BR.Next,we demonstrated the protective effecs of BR on temporary middle cerebral artery occlusion(tMCAO)mice and it's effect on neurogenesis triggerred by ischemic insults.Last,we investigated the effect of BR on OGD-induced injury in PC12 cell,and elucidated the mechanism involved in the protective action of BR.Partâ… Effects of BR on the adult mouse neurogenesisAIM:To investigate the effects of BR on the adult neurogenesis in vitro and vivo.METHODS:1)Normal ICR mice were administered with BR(20 mg·kg.(-1)·day^-1,i.p.)or fluoxetine(10 mg·kg^-1·day^-1,i.p.)for 28 or 56 days. The proliferation of the newborn cells were determined using BrdU immunohistochemistry and the differentiation of newly formed cells were analyzed by double labelling for mature neurons with neuron specific unclear protein(NeuN)and for glia cells with glial fibrillary acid protein (GFAP);2)[³H]-thymidine incorporation was used to assess the cell proliferation rate of the primary cultured adult neural stem cells(NSCs);RESULTS:1)Chronic BR treatment failed to affect the number of proliferating cells in either SVZ or DG in vivo.Treatment with BR increased the number of surviving BrdU-posotive cells in DG.2)Lower concentrations of BR(＜10 nM)did not affect the proliferation of adult neural stem cells,but higher concentrations of BR(＞100 nM)suppressed the proliferation of stem cells.CONCLUSION:These results indicate that chronic BR treatment stimulates neurogenesis by increasing the number of newly generated cells.Partâ…¢Effects of BR on tMCAO-induced injury in miceAIM:To investigate the neuroprotective effects of BR in a tMCAO mice model and the involved mechanisms.METHODS:Transient focal cerebral ischemia was induced by right middle cerebral artery(MCA)occlusion(MCAO)with a modified intralumenal filament technique;cerebral infarction was stained by using 2%2,3,5-triphenyltetrazolium chloride(TTC);cresyl violet staining was used for determination of positive cells in the hippocampal CA1 region;3 days after achieved tMCAO,ICR mice were administered with BR(20 mg·kg^-1·day^-1,i.p.)for 7 days then the proliferation of the newborn cells were determined using BrdU immunohistochemistry.RESULTS:1)BR decreased the score of the neurological outcome of mice injured by cerebral ischemia;2)BR reduced cortical and subcortical infarct size in the tMCAO mice;3)BR protected CA1 pyramidal cells against ischemia;4)Chronic administration of BR failed to improve neurogenesis triggerred by ischemia.CONCLUSION:BR protects against neuronal cells death during I/R, and fails to promote brain repair after ischemia.Partâ…¢Effects of BR on OGD-induced injury in PC12 cellsAIM:To investigate the protective effects of BR on PC12 cells injured by OGD and the involved mechanisms.METHODS:Oxygen-glucose deprivation followed by reoxygenation was used to mimic ischemia and reperfusion injury.PC12 cell survival was measured by MTT method,apoptosis was determined by staining with Hoechst33342(Sigma,USA).Formation of ROS was evaluated using 2',7'-dichlorofluorescin diacetate(DCFH-DA,Sigma, USA),and we introduced western blotting for the analyses of AIF, Cytochrome c.RESULTS:1)BR concentration-dependently induced an increase of cell survival,which was decreased by OGD injury;2)1μM BR decreased the number of apoptotic PC12 cells compared with OGD group;3)inhibited the OGD-induced increase in ROS production;4) significantly depressed the OGD induced the decrease of mitochondrial cytochrome c and AIF levels. CONCLUSION:BR protects against OGD-induced injury in PC12 cells,via decreasing intracellular ROS level and subsequently inhibiting mitochondrial apoptotic pathway.In summary,the present study reports for the first time that 1)BR stimulates neurogenesis by increasing the number of newly generated cells;2)BR inhibits OGD-induced increase of ROS and subsequent release of proapoptotic protein(cytochrome c and AIF)from mitochondria in PC12 cells.The results of the present study provide a new mechanism involved in the neuroprotective effects of BR against ischemic brain damage.