| Stroke is one of the common diseases of serious threat to mankind’s health and life,and it has the characteristics of high prevalence,high recurrence rate,high disability rate and high mortality rate,stroke has now recognized as the third most deadly disease in the world.Stroke can lead to body,behavior,cognitive and emotional disorders.Stroke can be divided into two categories:ischemic stroke and hemorrhagic stroke,the ratio of ischemic stroke in China is 60%～80%,even about three-quarters of the ischemic stroke patients after treatment have different level of nerve dysfunction,clinical manifestations are limb weakness,hemiplegia,sensory disorder,language barriers,visual disturbance,ataxia,etc.Ischemic stroke is not only cause sensory,motor dysfunction,but also can lead to cognitive dysfunction.The influence of cognitive impairment in ischemic stroke patients even have more than physical dysfunction in daily life activities,the influence of cognitive impairment affect the comprehensive recovery of neural function in ischemic stroke patients,therefore,in order to strengthen prevention and control of research of cognitive dysfunction after stroke is particularly important.Catalpol is iridoid glycoside compounds,mainly exist in traditional Chinese medicine radix rehmanniae,it has many pharmacological activities,such as reduce blood sugar,anti-inflammatory,anti-tumor,liver protective effect,anti-Alzheimer’s disease,anti-cerebral ischemia and nerve protective effect,etc,especially anti-cerebral ischemia and nerve protective effect are clear,they were received more attention in medicine.Catalpol can inhibit nerve cells apoptosis,anti-oxidative damage,anti-inflammatory,promot angiogenesis,promot injured nerve recovery of cerebral ischemia animal,and so on.The early research have been confirmed:catalpol can obviously improve nerve dysfunction of cerebral ischemia rats,permanent middle cerebral artery ischemia and ischemia-reperfusion animal model were adopted,catalpol can reduce rat neurological behavior scores,optimize integration and coordination of the injured limb,enhance motor ability and tactile sensitivity enhancement of the injured limb,promote neurological function recovery,reduce cerebral cortex neuron damage,increase full number of neurons,improve pathological ultrastructure of brain tissue.Its main mechanism may be related to inhibiting inflammation of cerebral ischemic injury,improvingt energy metabolism disorder and oxidative stress damage,inhibiting nerve cell apoptosis.This research is based on previous research.This research mainly divided into two parts,in the first part,because cerebral ischemia can cause cognitive dysfunction,study the cognitive impairment improvement on catalpol of cerebral ischemic injury rats;Neural functional recovery in cerebral ischemia not only relies on gray matter protection,but also depends on white matter protection.Cerebral ischemia not only affects gray matter,but also affects white matter.Cerebral ischemia can easily lead to white matter damage,corpus callosum is one part of white matter,due to its cerebral vascular anatomical characteristics,corpus callosum is one of the vulnerable areas of cerebral ischemia in white matter,study the protective effect of catalpol on white matter in cerebral ischemia injury.In the second part,potassium channels are main ion channels in neurons.Potassium channel function have obvious changes in cerebral ischemia,triggered a series of cascade reaction,then results in the decrease of the activity of neurons and function,cause neuronal damage;Excessive K+outflow and intracellular potassium loss is the main mechanism of ischemic cell injury and apoptosis.Potassium channels are abnormal activation and open are closely linked neurons apoptosis,inflammatory reaction,energy metabolism,and oxidative stress,so pharmacological against cerebral ischemia of catalpol and potassium channels were paid much attention.This research in order to explore whether catalpol anti-cerebral ischemia injury mechanism is related to potassium channels and possible potassium channel subtypes,respectively from three respects:the computer molecular docking,the animal experiment,cell experiments in vitro,study the interaction of catalpol and potassium channel proteins,in order to provide experimental basis for anti-cerebral ischemia targets of catalpol in the next step.1 Protective effect of catalpol in cerebral ischemic injury rats1.1 Improvement of catalpol on cognitive impairment caused by cerebral ischemic injury ratsObjective:Cerebral ischemia can cause cognitive dysfunction,in order to examine improvemen effect of catalpol on cognitive dysfunction caused by cerebral ischemia.Methods:The cerebral ischemic injury rats were induced by ligation of bilateral common carotid artery.After 30 days,the rats were divided into model group,catalpol 15mg/kg,30mg/kg,60mg/kg group,nicergoline 7mg/kg group,drugs were givens thirty days.Learning and memory was tested by Morris water maze,morphology in hippocampus was observed by HE staining,Bax and Bcl-2 protein expression in hippocampus were detected by immunehistochemistry,the content of Nitric oxide（NO）and the activity of nitric oxide synthase（NOS）in hippocampus were determined by colorimetric.activity of Acetylcholinesterase（AchE）and acetylcholine transferase（ChAT）in hippocampus were determined by elisa.Results:① Morris water maze,compared with the sham operation group,escape latency of model group rats significantly prolonged.Times across the platform of model group is zero,compared with model group,on the third and fourth day,escape latency of catalpol 30mg/kg and 60mg/kg significantly shortened.Times across the platform of catalpol 15mg/kg,30mg/kg and 60mg/kg obviously increased.② HE staining,hippocampus morphological of the model group is abnormal,pyramidal cellsin hippocampus arranged disorder,some neurons depigmentation,size is differ,morphological changes,cell nucleus narrow,condensation,in deep blue,ambiguous,nerve nuclei is cut few in catalpol group,neuronal degeneration degree alleviated,complete pyramidal cells were markedly increased.③Compared with model group,Bax protein expression of catalpol 30mg/kg and 60mg/kg in hippocampus significantly reduced,Bcl-2 protein expression increased significantly,Bcl-2/Bax ratio increased significantly.④Compared with model group,the content of NO and the activity of NOS in hippocampus of catalpol 60mg/kg reduced,the content of NO in hippocampus of catalpol 30mg/kg reduced.⑤Compared with model group,the content of AchE of catalpol 30mg/kg and 60mg/kg reduced.Conclusion:Catalpol has the improvement of cognitive impairment in ischemic injury rats,catalpol can inhibit cell apoptosis,reduce the content of NO,inhibit the activity of NOS and improve cholinergic system.1.2 Protective effect of catalpaol on white matter（corpus callosum）in cerebral ischemic injury ratsObjective:Earlier research has proved catalpol has protective effect in cerebral ischemia rats,it can reduce damage of cortex（gray matter）neuron pathology and ultrastructure of cerebral cortex damage,it has the good protection of neurons.Cerebral ischemia affects not only gray matter,but also cwhite matter.Neural function restore after cerebral ischemia not only relies on the protection of neurons and gray matter but also depends on the protection of the white matter.Cerebral ischemia can easily lead to white matter damage,the corpus callosum is one of the vulnerable areas of white matter in cerebral ischemia,this research study protection of white matter（corpus callosum）injury in cerebral ischemia rats of catalpol.Methods:The rat model was adopted by permanent middle cerebral artery ischemia ischemia.Rats were divided into model group,catalpol 15mg/kg,30mg/kg,60mg/kg group,butylphthalide 70 mg/kg,edaravone 7mg/kg group and sham operation group.Drugs were given for the third day to fourteenth day.Neurological behavior scores is adopted by Longa method;Pathology changes in corpus callosum were observed by HE staining and luxol fast blue（LFB）staining;Myelin basic protein（MBP）expression in corpus callosum were detected immunohistochemistry.Results:①Neurological behavior scores of catalpol 30mg/kg and 60mg/kg in cerebral ischemia rats decreased obviously in the tenth and fourteenth day;②HE staining,sham operation group can be seen white matter fiber order,glial cells form normal;model group can be seen white matter loose,glial cells vacuolation and hyperchromatic,interstitial edema;catalpol group can be seen white matter in corpus callosum has a little vacuolation,glial cells form returns to normal;③LFB staining,the corpus callosum can be seen the deep blue in sham operation.the corpus callosum dyed color becomes shallow blue myelin,myelin becomes sparse in model group,compared with sham operation group,model group significantly decreased integral optical density value.Compared with model group,catalpol 30mg/kg and 60mg/kg significantly increased integral optical density.④Myelin basic protein（MBP）expression in sham operation group is stronger,color is yellow brown;Expression decreased significantly in the model group,color becomes shallow,and uneven distribution.Integral optical density of model group were significantly lower than the sham operation group,compared with model group,catalpol 30mg/kg and 60mg/kg significantly increased integral optical density.Conclusion:Catalpol can reduce neurological behavior scores in ischemic rats,improve symptoms of neurological function defect.Catalpol has protective effect on white matter of cerebral ischemic injury.2 Explore mechanism against cerebral ischemia of catalpol based on voltage dependent potassium channels2.1 Molecular docking studies of catalpol and potassium channelsObjective:Study catalpol combined with Kir3.2,Kir6.2,K2P10.1,Kv1.4,Kv1.5,Kv2.1,Kv4.2,Kv7.1,Kv7.2 and Kv11.1 by adopting molecular docking method.Methods:Catalpol and potassium channels were calculated by using homology modeling and molecular docking method.Three-dimensional structure of receptor was obtained from the crystal structure of the database（www.rcsb.org）or homologous model built for structure.Homologous model was built using the building is the Prime of Schrodinger software modules.In the Schrodinger software,used protein preparation wizard tool complete hydrogenation,to crystallize water,plus the end group,such as preparation,choose OPLS2005 force field.Then use receptor docking generated lattice grid generation file,selected residues or crystal structure as docking centre in the original body,set the box size,lattice generated file.Ligand processing in Schrodinger software rendering small molecular compound structure,and then the Schrodinger software using LigPrep for processing,the force field for OPLS2005,generate possible conformation PH 7.0±2.0,with ion state handling way Epik,keep the specified chiral,get the ligand structure used for docking.Molecular docking is the Glide of Schrodinger software methods,the last is the receptor and the ligand docking.The receptor and the ligand docking module were calculated.By using Schrodinger software Glide.Results:Three-dimensional structure of Kir6.2,Kv1.4,Kv1.5,Kv2.1,Kv4.2,Kv7.2 and Kv11.1 have been obtained respectively with the homologous model building method,catalpol with these potassium channels and the known structure of Kir3.2,K2P10,Kv7.1 potassium channels for molecular docking,results showed that catalpol can enter into ten activity of potassium channels cavity,its docking score were respectively-4.22,-5.41,-5.72,-5.97,-5.70,-4.63,-5.49,-3.96,-6.33.Conclusion:According to molecular docking score and the combination pattern of catalpol and potassium channels,comparing with controlled drugs docking results,speculate that catalpol is in good combination with the eight potassium channel subtypes Kir3.2,Kir6.2,Kv1.4,Kv1.5,Kv2.1,Kv4.2,Kv7.1 and Kv7.2,catalpol is in bad combination with potassium channels K2P10.1 and Kv11.1.2.2 Influence on voltage dependent potassium channel subtypes expression of catalpol in hippocampus in cerebral ischemic injury rats2.2.1 Changes of voltage dependent potassium channel subtypes Kv1.4,Kv1.5,Kv2.1 and Kv4.2 mRNA expression in hippocampus in cerebral ischemic injury ratsObjective:Molecular docking results showed that the catalpol is in good combination with the eight potassium channel subtypes,reading a large number of papers at home and abroad shows voltage dependent potassium channel Kv1.4,Kv1.5,Kv2.1 and Kv4.2 have obvious increased after cerebral ischemia,so in animal experiment,using animal model of cerebral ischemia reperfusion injury,in order to examine changes of voltage dependent potassium channel subtypes Kv1.4,Kv1.5,Kv2.1 and Kv4.2 mRNA expression in hippocampus of cerebral ischemic injury rats for ten days.Methods:Using rats model of middle cerebral artery cerebral ischemia-reperfusion injury,in order to determine Kv1.4,Kv1.5,Kv2.1,Kv4.2 mRNA expression in hippocampus by quantitative polymerase chain reaction（q-PCR）in the first day,the third day,the fifth day,the seventh day and the tenth day after cerebral ischemia,in order to find out the changes law.Results:Compared with sham operation group,Kv1.5 mRNA expression in hippocampus obviously increases in the first day and the tenth day in the cerebral ischemia reperfusion rats;Kv1.4 mRNA and Kv4.2 mRNA expression in Hippocampus compared with sham operation group without difference in each time point;Kv2.1 mRNA in hippocampus expression significantly raised on the first day,the third day,the fifth day,the seventh day and the tenth day in the cerebral ischemia reperfusion rats.Conclusion:Four voltage dependent potassium channel Kv1.4,Kv1.5,Kv2.1 and Kv4.2 mRNA expression were raised to different extent after cerebral ischemic injury in hippocampus,among them Kv2.1 mRNA expression is raised obviously.2.2.2 Influence of catalpol on voltage dependent potassium channel Kv2.1 expression in hippocampus in cerebral ischemic injury ratObjectiv:Kv2.1 mRNA expression in hippocampus of cerebral ischemia-reperfusion rats has obvious increase,so focus on Kv2.1 mRNA expression of catalpol in hippocampus in cerebral ischemia-reperfusion injury rats.Methods:Using rats model of middle cerebral artery cerebral ischemia reperfusion injury,the model rats were divided into model group,catalpol 60mg/kg group.In order to detemine Kv2.1 mRNA expression in hippocampus by quantitative polymerase chain reaction in the first day,the third day,the seventh day and the tenth day after cerebral ischemia.Results:Compared with sham operation group,Kv2.1 mRNA expression of model group in hippocampus significantly rised in the first day,the third day,the seventh day and the tenth day after cerebral ischemia.Compared with model group,Kv2.1 mRNA expression in hippocampus of catalpol group significantly lowered.Catalpol can reduce Kv2.1 mRNA rises in hippocampus of ischemia rats.Conclusion:Catalpol has protective effect of cerebral ischemia injury,Catalpol may be against cerebral ischemia by suppressesing voltage dependent potassium channel Kv2.1.3 Protective effects of catalpol on PC12 cell induced by oxygen and sugar deprivation injury and influence on voltage dependent potassium channel subtypes expressionObjective:In order to study protective effects of catalpol on PC 12 cell induced by oxygen and sugar deprivation injury and the influence of voltage dependent potassium channel subtypes expression.Methods:Oxygen and sugar deprivation model of PC 12 cell was reduced by using sodium dithionite（Na2S2O4）combined oxygen deprivation sugar sugar-free culture medium,catalpol were given 24 hours before oxygen and sugar deprivation,cell morphology was observed by inverted microscope;cell survival was determined by MTT;the activity of lactate dehydrogenase（LDH）on PC 12 cell cell in cultures was determined;the activity of superoxide dismutase（SOD）,the content of malondialdehyde（MDA）and reduced glutathione（GSH）were determined;Expression of voltage dependent potassium channels Kv1.4,Kv1.5,Kv2.1 and Kv4.2 protein in PC 12 cell by western blot.Results:①Volume of PC 12 cell after oxygen and sugar deprivation was smaller,cell become spherical,cell swelled,some cell swelling;cell broke into pieces,damage of catalpol PC 12 cell relief,form a certain improvement,cell was close to normal.②Cell survival rate of PC 12 cell after oxygen and glucose deprivation decreased,10mmol/L,100mmol/L of catalpol can obviously against oxygen and glucose deprivation damage,cell survival rate significantly increased.③PC 12 cell after oxygen and glucose deprivation damage,the activity of LDH release increased,the activity of SOD activity decreased,the content of MDA increased,the content of GSH decreased significantly,catalpol can against oxidative stress,inhibit LDH release,increase the activity of SOD and the content of GSH,reduce the content of MDA,remove excess free radicals,catalpol has certain protective effect on PC 12 cell after oxygen and glucose deprivation.④After oxygen and glucose deprivation,voltage dependent potassium channels Kv1.5 and Kv2.1 protein expression significantly raised,catalpol can decreased Kv1.5 and Kv2.1 protein expression in PC 12 cell after oxygen and glucose deprivation.Conclusion:Catalpol has protective fffect of PC 12 cell after oxygen and glucose deprivation,it has resistance to oxidative stress damage,decreased voltage dependent potassium channels Kv1.5 and Kv2.1 protein expression.ConclusionCombined with previous research and this research results fully showed that catalpol has significant anti-cerebral ischemia.Catalpol can obviously improve neurological dysfunction in cerebral ischemic rats,permanent middle cerebral artery cerebral ischemia and cerebral ischemia reperfusion animal model were adopted,catalpol can reduce rat neural functional behavior scores,optimize integration and coordination of the injured limb,enhance motor ability and tactile sensitivity enhancement of the injured limb,promo recovery of neurological function,reduce cerebral cortex neuron damage,increase full number of neurons,improve pathological ultrastructure of brain tissue.Its main mechanism may be related to inhibit cerebral cerebral ischemia injury of inflammation,improve energy metabolism disorder and oxidative stress damage,inhibit nerve cell apoptosis.Catalpol can obviously improve cognitive impairment in cerebral ischemic rats,Catalpol can inhibit cell apoptosis,reduce the content of NO,inhibit the activity of NOS and improve cholinergic system.Catalpol has protective effect on white matter of cerebral ischemia injury.Catalpol can make callosity area a little vacuolation,glial cells form return to normal,improve structure of the disorder myelin sheath,enhence myelin LFB staining and myelin basic protein（MBP）expression.According molecular docking score and combination pattern of catalpol and potassium channels,speculate that catalpol is in good combination with the eight potassium channel subtypes.in the animal experiment,catalpol may be against cerebral ischemia by suppressesing voltage dependent potassium channel Kv2.1.Catalpol can decreased Kv1.5 and Kv2.1 protein expression.As a result,the mechanism of catalpol against cerebral ischemic injury associated with potassium channels,may inhibit voltage dependent potassium channel subtypes Kv1.5 and Kv2.1.The effect of catalpol on potassium ion current need to further explore in later research.The innovation of the research1 Ischemic stroke can lead to cognitive disorder.Cerebral artery stenosis or occlusion in ischemic stroke which reduced the volume of brain tissue perfusion,nerve cell excitability,cerebral metabolic rate decreased,leading to slow process of thought,cognitive decline.This study first discovered that catalpol on the improvement of cognitive dysfunction caused by cerebral ischemia injury.Catalpol can inhibit cell apoptosis,reduce the content of NO,inhibit the activity of NOS and improve cholinergic system.2 Potassium channels significantly change in cerebral ischemia,results in the activity of neurons and function decreased,caused the neuron injury.Inhibit the excessive open of voltage dependent potassium channel may protect cerebral ischemic cell damage.This study first discovered the mechanism of of catalpol against cerebral ischemia injury associated with potassium channels,may inhibit voltage dependent potassium channel subtypes Kv1.5 and Kv2.1. |
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