berberine is an isoquinoline alkaloid and often isolated from goldenthread , coptidis rhizoma , and goldenseal , hydrastis canadensis ( mirska et al . , 1972 ) .
previous reports have shown that berberine has several pharmacological and biological properties including antibiotic ( mirska et al . , 1972 ) , anti - inflammatory ( marinova et al . , 2000 ; zhou and mineshita , 2000 ; yoo et al . , 2008 ) and anti - hypolipidemic ( kong et al . , 2004 ) effects .
it has been reported that berberine attenuated neuronal damage in ischemia / reperfusion model ( yoo et al . , 2006 ) , and in autoimmune encephalomyelitis model mice ( ma et al . , 2010 ) .
berberine showed neuroprotective effects on stroke models ( zhou et al . , 2008 ) and focal cerebral ischemia injury ( xiao et al . , 2007 ) .
previously , we reported that berberine enhances neuronal cell survival and differentiation in hippocampal precursor cells and neurons in the rat brains ( lim , 2008 ) .
tan and his colleges ( 2007 ) showed that berberine has an antioxidant action on corpus cavenosum smooth muscle cells in which oxidative stress were induced ( tan et al . , 2007 ) .
to examine whether berberine has survival promoting effect on damaged neuronal cells , we generated a degenerating brain disease model by injecting neurotoxin to developing rats .
mk-801 [ ( 5s,10r)-(+)-5-methyl-10,11-dihydro-5h - dibenzo [ a , d ] cyclohepten-5,10-imine maleate , dizocilpine ] is a noncompetitive antagonist of n - methyl - d - aspartate ( nmda ) receptors ( wong et al . , 1986 ; javitt and zukin , 1991 ) . in rodents
, mk-801 induces a behavioral syndrome , including hyperlocomotion , head weaving , body rolling , and ataxia ( clineschmidt et al . , 1982 ; tricklebank et al .
1991 ) , which represents certain aspects of schizophrenia ( carlsson and carlsson , 1990 ; tiedtke et al . , 1990 ) .
mk-801 is also known to induce neurodegeneration of hippocampal ca1 and entorhinal cortex in adult animals when administrated with high concentrations ( 10 mg / kg ) of mk-801 ( wohrl et al . , 2007 ) .
in the developing rat brain , blockade of nmda receptors with low dose of mk801 during late fetal or early neonatal life triggers widespread apoptotic neurodegeneration ( 12~26% of cells ) ( ikonomidou et al . , 1999 ) .
this suggests the transient blockade of nmda receptors can trigger neuronal cell death in the immature mammalian brain during a period of rapid axonal growth and synaptogenesis , and the excitatory neurotransmitter glutamate , acting at nmda receptors , controls neuronal survival .
thus , neurodegenerative mk-801 model of the developing rat has relevance to human neurodevelopmental disorders involving postnatal exposure to drugs that block nmda receptors such as pediatric anesthesia .
berberine has been reported to increase action potential by inhibition of voltage dependent potassium current in cat ventricular myocytes ( huang , 1990 ; sanchez - chapula , 1996 ) and in human myeloma cells ( wu et al . , 1998 ) and hepatocytes ( wang et al . , 2003 ) .
berberine suppresses dopamine - induced potassium current and acetylcholine induced potassium current in acutely dissociated ca1 pyramidal neurons ( wu and jin , 1996 ; 1997 ) .
it is also suggested that berberine contributes to its blockades of potassium currents in damaged ischemic brain ( wang et al . , 2004 ) .
this leads us a question whether berberine reduces cell death on damaged brain of developing animal model rats induced by mk801 .
we tested first the cell survival promoting effect of berberine on sh - sy5y neuronal cells damaged by oxidative stress and then examined whether berberine blocks cell death in vivo developing rat model induced by mk801 .
human neuroblastoma sh - sy5y cells were acquired from atcc . as previously described , shsy5y cells were maintained in dulbecco 's modified eagle 's medium ( dmem , invitrogen , usa ) supplemented with 10% fetal bovine serum ( fbs , thermo , usa ) , penicillin , and streptomycin at 37 ( heo et al . , 2009 ) .
for the sh - sy5y cells 0.1 mm mem non - essential amino acids were also added . to estimate damage of cultured cells which caused by oxidative stress , we performed cell viability assay by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt , sigma - aldrich , usa ) .
sh - sy5y cells were seeded at a density of 7.510 cells in 96-well plate .
cells were treated with different concentration ( 0.25 , 1.5 , 3 and 3.5g / ml ) of berberine chloride ( sigma - aldrich , usa ) .
4 mg / ml of mtt tetrazolium salt was added to each well and incubated for 4 hrs at 37. after that , 100l of solubilization buffer was added and then incubated for 24 hrs .
sprague dawley rats were maintained under standard housing conditions and a 12:12 light / dark cycle ( lights on at 06:30 am ) with free access to water and standard food .
all procedures were performed in accordance with the guidelines of the national institutes of health ( nih ) for the care and use of laboratory animals .
the neonatal rat model of developmental disorder was constructed as described previously ( ikonomidou et al . , 1999 ) . in brief , postnatal day 7 animals were randomly divided into a control group ( n=3 ) and a group injected with mk-801 ( dizocilpine 0.5 mg / kg of body weight ; sigma , st .
berberine chloride were dissolved in 0.9% saline and injected intraperitoneally every 24 hours for 5 days and control rats received saline again .
the degenerating brains were examined at 5 day by tunel ( terminal deoxynucleotidyl transferase - mediated dutp nickend labeling ) assay to detect apoptotic cells . to visualize nuclei with dna cleavage , brain were cut using a vibratome and brain slices were incubated with 0.1% triton x-100 in 0.1% sodium citrate ( permeable solution ) for 2 min on 4. subsequently brain slices were incubated with nucleotide - labeling mixture and terminal deoxynucleotidyl transferase ( roche , switzerland ) for 1 hr at 37 to catalytically add peroxidase - labeled digoxigenin nucleotide to dna fragments .
we counted the number of tunel positive cells in the microscophic fields ( n=12 ) .
human neuroblastoma sh - sy5y cells were acquired from atcc . as previously described , shsy5y cells were maintained in dulbecco 's modified eagle 's medium ( dmem , invitrogen , usa ) supplemented with 10% fetal bovine serum ( fbs , thermo , usa ) , penicillin , and streptomycin at 37 ( heo et al . , 2009 ) .
for the sh - sy5y cells 0.1 mm mem non - essential amino acids were also added .
to estimate damage of cultured cells which caused by oxidative stress , we performed cell viability assay by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt , sigma - aldrich , usa ) .
sh - sy5y cells were seeded at a density of 7.510 cells in 96-well plate .
cells were treated with different concentration ( 0.25 , 1.5 , 3 and 3.5g / ml ) of berberine chloride ( sigma - aldrich , usa ) .
4 mg / ml of mtt tetrazolium salt was added to each well and incubated for 4 hrs at 37. after that , 100l of solubilization buffer was added and then incubated for 24 hrs .
sprague dawley rats were maintained under standard housing conditions and a 12:12 light / dark cycle ( lights on at 06:30 am ) with free access to water and standard food .
all procedures were performed in accordance with the guidelines of the national institutes of health ( nih ) for the care and use of laboratory animals .
the neonatal rat model of developmental disorder was constructed as described previously ( ikonomidou et al . , 1999 ) . in brief , postnatal day 7 animals were randomly divided into a control group ( n=3 ) and a group injected with mk-801 ( dizocilpine 0.5 mg / kg of body weight ; sigma , st .
berberine chloride were dissolved in 0.9% saline and injected intraperitoneally every 24 hours for 5 days and control rats received saline again .
the degenerating brains were examined at 5 day by tunel ( terminal deoxynucleotidyl transferase - mediated dutp nickend labeling ) assay to detect apoptotic cells .
to visualize nuclei with dna cleavage , brain were cut using a vibratome and brain slices were incubated with 0.1% triton x-100 in 0.1% sodium citrate ( permeable solution ) for 2 min on 4. subsequently brain slices were incubated with nucleotide - labeling mixture and terminal deoxynucleotidyl transferase ( roche , switzerland ) for 1 hr at 37 to catalytically add peroxidase - labeled digoxigenin nucleotide to dna fragments .
we counted the number of tunel positive cells in the microscophic fields ( n=12 ) .
in the previous study , we have demonstrated that berberine promotes cell survival in neuronal stem cells . to investigate the cell survival effect of berberine on damaged neurons
, we examined berberine effect in the cultured cellular model under the condition of oxidative stress .
sh - sy5y cells were exposed under oxidative stress by adding 150 m h2o2 for 30 min after being pretreated with 0.25~3.5g / ml of berberine for 3 hrs . when serum is removed from media and changed to chemically defined n2 media survival of sh - sy5y cells are reduced and initiates neurite outgrowth . as shown in fig .
1 , the survival rate of the groups treated by 1.5 and 3.0g / ml berberine was increased by 287% and 344% , respectively compared to the n2 media control .
however , the survival rate of the groups treated by 3.5g / ml berberine was reduced to about 200% , the similar level of 0.25 g / ml concentration , suggesting that higher concentration is not more effective . to determine the protective effect of berberine in the damaged neuronal cells of developing animal model , we generated neonatal animal model of degenerating brain by injecting intraperitoneally to postnatal day 7 aged rats 0.5 mg / kg of mk-801 , which is a non - competitive antagonist of the nmethyl - d - aspartate ( nmda ) receptor , as described previously ( ikonomidou et al . , 1999 ) . as described previously , we also observed changes of cell shape including intracytoplasmatic vacuoles in the cortex of rats treated with a low dose of mk-801 ( 0.5 mg / kg body weight ) when stained with haematoxylin and eosin ( olney et al . , 1989 ; fix et al . , 1994 ) .
we intraperitoneally injected 20 mg / kg of berberine , or vehicle , to the model animals for 5 days . after sacrificing the animals ,
tunel positive apoptotic cells were outspread in entire brain of the developing rats injected by mk801 .
however , the cell death was decreased more than 3 fold in the brains of berberine administrated animals .
the number of tunel positive cells of the control group were 2710 cells per microscophic field ( 200 ; vehicle n=3 , ber n=3 ) , on the other hand , the number of tunel positive cells of the berberine group were 710 cells per microscophic field ( 200 ; vehicle n=3 , ber n=3 ) .
recently , berberine is shown to enhance cell survival by reducing reactive oxygen species ( ros ) , the release of cytochrome c and apoptosis - inducing factors ( aifs ) in pc12 cells damaged by oxygenglucose deprivation ( zhou et al . , 2008 ) .
in this report we have demonstrated that survival promoting effect of berberine in both damaged neuronal cell model and degenerating brain of neonatal animal model .
sh - sy5y cell culture model induced by adding h2o2 are well established as a damaged neuronal cell model for ischemic studies .
ischemic conditions induce apoptosis by generating excessive ros ( zhang et al . , 2009 ) .
we found berberine increases cell viability of sh - sy5y under oxidative stress about 3 fold .
we also found the anti - apoptotic effect of berberine in the mk801 induced animal model . to make the model animals
, we injected low dose of mk-801 into the developing postnatal day 7 rats , and mk801 induced tunel positive apoptotic cells were found in entire brain of the developing rats as ikonomidou and his colleges previously described ( 1999 ) .
although half life of mk-801 , nmda receptor anatagoist is only for a few hours ( vezzani et al . , 1989 ) , neuronal apoptosis in the immature mammalian brain can be triggered by the transient blockade of glutamate nmda receptors .
this is probably caused by mechanism of activity dependant cell survival which is induced by release of neurotransmitter , glutamate and activation of nmda receptor on target cells .
activation of the nmda receptor by glutamate on post - synaptic neurons releases retrograde signal which is required for cell survival of presynaptic neurons . during a period of rapid brain growth or synaptogenesis period , such target derived survival signals from post - synaptic neurons regulate strengthening of the synapses as well as cell survival of pre and post - synaptic neurons , thereby forming neuronal networks
. blockage of nmda receptors inhibits to produce target derived survival factors and cause apoptosis of pre and post - synaptic neurons .
when berberine was injected to the mk801 model rats , apoptotic cells were decreased more than 3 fold .
other researcher group also reported neuroprotective action of berberine in organotypic hippocampal slice culture induced by oxygen and glucose deprivation ( cui et al . , 2009 ) .
berberine has also been reported to block transient outward potassium current ( ia ) and delayed rectifier potassium current ( ik ) in acutely isolated ca1 pyramidal neurons of rat hippocampus by using the whole - cell patch - clamp techniques .
these biological functions were suggested as a protective mechanism against ischemic brain damage ( wang et al . , 2004 ) . in our animal model of neonatal brain damaged by nmda receptor antagonist , mk801
, berberine probably stimulates cell survival of neuronal cells expressing nmda receptors by blocking potassium current or lowering the threshold of the action potential .
this may elevate synaptic depolarization and activate channel opening of nmda receptors and calcium influx .
thus , we suggest that these anti - apoptotic effect of berberine on the neurodegenerating brain of neonatal animal model caused by promoting activity dependent cell survival . in the immature mammalian brain during a period of rapid synaptogenesis , the transient blockade of glutamate nmda receptors , or the excessive activation of gamma - aminobutyric acid ( gaba(a ) ) receptors trigger neuronal apoptosis .
apoptogenic agents include anesthetics and drugs of abuse including phencyclidine , ketamine , and ethanol . in humans ,
the brain growth period are between the sixth month of pregnancy and the third year after birth .
agents used in pediatric and obstetrical medicine for purposes of sedation , anesthesia , and seizure management may cause apoptotic neuronal degeneration in the developing human brain such as dysmorphogenic changes in the fetal brain and consequent neurobehavioral disturbances . in conclusion , berberine has cell survival promoting effect on damaged neuronal cells under oxidative stress and degenerating brains of developing animal model induced by mk801 .
 berberine is an isoquinoline alkaloid isolated from goldenthread , coptidis rhizoma and shown to have many biological and pharmacological effects . 
 we previously reported that berberine promotes cell survival and differentiation of neural stem cells . to examine whether berberine has survival promoting effect on damaged neuronal cells 
 , we generated a cellular model under oxidative stress and an neonatal animal model of degenerating brain disease by injecting mk-801 . 
 mk801 , a noncompetitive antagonist of n - methyl - d - aspartate ( nmda ) receptors , acts as a neurotoxin in developing rats by inhibiting nmda receptors and induce neuronal cell death . 
 we found that the survival rate of the sh - sy5y cells under oxidative stress was increased by 287% and 344% , when treated with 1.5 and 3.0g / ml berberine , respectively . in the developing rats injected by mk801 
 , we observed that tunel positive apoptotic cells were outspread in entire brain . 
 the cell death was decreased more than 3 fold in the brains of the mk-801-induced neurodegenerative animal model when berberine was treated to the model animals . 
 this suggests that berberine promotes activity dependent cell survival mediated by nmda receptor because berberine is known to activate neurons by blocking k+ current or lowering the threshold of the action potential . 
 taken together , berberine has neuroprotective effect on damaged neurons and neurodegenerating brains of neonatal animal model induced by mk-801 administration . 
INTRODUCTION
MATERIALS AND METHODS
Cell culture
Cell viability assay
Neonatal animal model of neuronal cell damaged by MK-801 injection
TUNEL assay
RESULTS
DISCUSSION