| Objective: Cereral stroke is a common and frequently-occurring disease,which is one of the three fatal diseases and the leading cause of disability.Cerebral stroke consists of ischemic stroke and hemorrhagic stroke,among them,cerebral ischemic stroke which threatens human health seriously accounts for about 60%~80% of all cerebrovascular diseases,and it has become a hot research topic in the field of pharmacy.The nosogenesis of ischemic stroke has not been fully defined yet,and there is no effective therapeutic drug.Rats with focal cerebral ischemia reperfusion were employed in this study,and the neurological scores,brain water content,infarct volume,pathological changes of the brain tissue,the ultrastructure of neuron,microvascular morphology and leakage degrees were detected to observe the protective effect of picroside II on brain tissue and blood brain barrier(BBB).In order to explore the mechanism of the protective effect of picroside II on the brain tissue and injured BBB,we detected the reactive oxygen species(ROS)content,nicotinamide adenine dinucleotide phosphate(NADPH)oxidase activity and the m RNA and protein levels of Rac-1,Nox2,ROCK,MLCK,MMP2 and Claudin-5,which provided theoretical basis for the clinical application of picroside II.Methods: Typical models of brain ischemia-reperfusion injury were established by middle cerebral artery occlusion(MCAO)in rats.All animals were randomly divided into five groups: sham group,model group,picroside II-treated group(i.p.),apocynin-treated group(i.p.)and picroside II+apocynin-treated group(i.p.).Drugs were dissolved in normal saline,and administrated after the MCAO and reperfusion.Normal saline was given to model and sham groups in the same administration.The present article is composed of four parts as follows:1.Protective effects of picroside II on focal cerebral ischemia-reperfusion injury:(1)Neurological function and body weight examination: modified neurological severity score points(mNSS)was performed ischemia 2h and 22 h after induction of reperfusion and scored on a 18-point scale.After the examination,the rats were weighed and the decreased percentage of body weight was calculated.(2)Measurement of brain water content: brain water content was measured by dry-wet weighing method.(3)Measurement of infarction volume: 2,3,5-triphenyl tetrazolium chloride(TTC)stainingmethod was used to assess the percentage of infarct volume.(4)Histopathological examination: After 22 h of reperfusion,rats were perfused with physiological normal saline and then with 4% paraformaldehyde(PFA)solution in 0.1mol/L phosphate buffered saline(PBS).Brains were removed,post-fixed in 4% PFA overnight.The specimens were dehydrated with gradient alcohol,made transparent by dimethylbenzene,and were embedded in paraffin routinely to prepare serial sections of 4?m.Sections were stained by hematoxylin and eosin(HE).Pathologic histological changes were observed by optical microscope.(5)The morphological changes of neurons in ischemic cortex were observed by electron microscopy: The 1mm×1mm×1mm brain tissue was prepared from the ischemic side cortex and fixed with glutaraldehyde.After 24 h,it was fixed with osmic acid and then cut into 50 nm ultrathin sections,and was observed under electron microscope.2.Protective mechanism of picroside II against oxidative damage following cerebral ischemia reperfusion:(1)Detection of ROS content and NADPH oxidase activity: ROS content and NADPH oxidase activity were detected by ELISA kit.(2)Examination of Rac-1 and Nox2 protein: The expression of Rac-1 and Nox2 protein was assessed by western blot.The monoclonal antibody of mouse anti rat and rabbit anti rat were adopted as first antibody differentialy.The IgG of rabbit anti mouse and goat anti rabbit were adopted as second antibody differentialy.The ?-actin was used as the internal reference.The gray level of bands were determined by Gel imaging analysis system(Biospectrum 810 Imaging system,UVP,USA).(3)Measurement of Rac-1 and Nox2 mRNA expression: The ischemic cerebral cortical tissue was isolated from the brain.Total RNA was extracted using Trizol reagent.2?g of total RNA was used for detecting the mRNA level.b-actin was used as reference.3.Protective effects of picroside II on blood brain barrier after cerebral ischemia reperfusion:(1)The leakage degree of lanthanum nitrate in capillary was observed by electron microscope: after modeling,2% 0.5ml lanthanum nitrate-saline was injected intravenously,and then it was fixed by lanthanum aldehyde stationary solution.And then the brain tissue of ischemia side was cut,and after fixation,slicing and drying,the ultrastructure of BBB was observed by electron microscope.(2)After cerebral ischemia/reperfusion injury,the BBB leakage degree was determined: Evans blue leakage rate was determined by fluorescence method.4.Protective mechanism of picroside II on blood-brain barrier after cerebral ischemia reperfusion in rats:(1)Measurement of mRNA levels of ROCK,MLCK,MMP-2 and Claudin-5: The ischemic brain tissue was isolated.Total RNA was extracted using trizol reagent.2?g of total RNA was used for detecting the mRNA level.b-actin was used as control.(2)Measurement of protein levels of ROCK,MLCK,MMP-2 and Claudin-5: The protein levels of ROCK,MLCK,MMP-2 and Claudin-5 were assessed by western blot.The monoclonal antibodies of rabbit anti rat and mouse anti rat were adopted as first antibody differently.The IgG of goat anti rabbit was adopted as second antibody.The ?-actin was used as the internal reference.The gray level of bands were determined by Gel imaging analysis system(Biospectrum 810 Imaging system,UVP,USA).Results1.Protective effects of picroside II on focal cerebral ischemia-reperfusion injury:(1)Modified neurological severity scores: neurological deficit scores of model group were higher than those of the sham group significantly.Compared with the model group,picroside II,apocynin and picroside II+apocynin groups decreased neurological deficit scores significantly(P values are all less than 0.05),and there was no significant difference in mNSS scores between the three groups(P>0.05).(2)Brain water content and infarction volume: The brain tissue of sham group showed rose red,while the model group showed great white infarct area in cortex,striatum and hippocampus(P<0.01).The two hemispheres were not symmetric and the injured side was obviously swollen.Treatment with picroside II reduced the infarct volume significantly(P<0.01).Compared with the model group,picroside II + apocynin can reduce the volume of cerebral infarction significantly,which is prior to picroside II group and apocynin group(P<0.05).Compared with the model group,the three groups had obvious effects on reducing brain edema(P<0.01).The 20mg/kg dose of picroside II was comparable to the 50mg/kg dose of apocynin.To reduce the volume and edema degree of cerebral infarction,two drugs synergy has more significant effect.(3)Histopathological examination: The sham group revealed healthy neuronal cells.The structure of neuron in model group was wrinkled and had obvious edema,and the cytoplasm presented acidophilic staining,while the nucleus dyed deeply or disappeared.The arrangement of pyramidal neurons was not in order and the amount was decreased.The shape of neurons of Picroside II,apocynin and picroside II+apocynin group were almost normal and the pathological changes of pyramidal neurons relieved significantly,and there was no significant difference in the DCI(denatured cells index,DCI)indices between the three groups(P>0.05).(4)Ultrastructure of neurons in the cerebral cortex: The morphology of the neurons in the sham operatedgroup was clear and complete,and the structures in the model group were all wrinkled and loose,and the damage was obvious.In the group of picroside II,apocynin and picroside II+apocynin,the shrinkage of cell and the damage of organelle were alleviated obviously.2.Protective mechanism of picroside II against oxidative damage following cerebral ischemia reperfusion:(1)ROS content and NADPH oxidase activity: Compared with the sham operation group,the production of ROS and the activity of NADPH oxidase increased significantly in the cerebral cortex of rats(P<0.001),while the picroside II,apocynin and picroside II+apocynin decreased the content of ROS and the activity of NADPH oxidase significantly(P<0.01).(2)The protein expression of Rac-1 and Nox2:Compared with the sham group,the expression of Rac-1 and Nox2 protein increased significantly in cerebral cortex of the model group(P<0.01),while picroside II,apocynin and Picroside II+ apocynin decreased the protein levels of Rac-1 and Nox2 significantly(P<0.01),and there was no significant difference between the three groups in reducing the protein expression of Rac-1 and Nox2(P>0.05).(3)mRNA expression of Rac-1 and Nox2: The mRNA expression of Rac-1 and Nox2 of the model group was much higher than that in the sham group(P<0.01).The treatment with picroside II,apocynin and picroside II+apocynin decreased the expression significantly(P<0.05),and there was no significant difference between the three groups in reducing the mRNA expression of Rac-1 and Nox2(P>0.05).3.Protective effects of picroside II on blood brain barrier after cerebral ischemia reperfusion:(1)The leakage of lanthanum nitrate: In the sham group,the vascular structure was intact,and lanthanum nitrate was in the lumen without leaking to the outside of the vessel.In the model group,the capillary structure was destroyed and the permeability increased,at the same time,lanthanum nitrate seep from the capillaries to the interstitial space.In the picroside II,apocynin and picroside II+apocynin group,lanthanum nitrate gathered in the tight junction of the lumen,but did not leak out into the interstitial space of the lateral vessels,which indicated that the damage of capillary capillaries in the three groups was alleviated obviously.(2)The leakage rate of evans blue:There was no EB exudation in the rat brains of the sham group.Compared with the sham group,the content of EB in the brain tissue of the model group increased significantly(P<0.001),which indicated that the blood-brain barrier was damaged seriously.At the same time,the levels of EB of the three medication groups were decreased significantly(P<0.01),and there was no significant difference in EB content between the three groups(P>0.05).4.Protective mechanism of picroside II on blood-brain barrier after cerebral ischemia reperfusion in rats:(1)ROCK,MLCK,MMP-2 and Claudin-5 mRNA expression: The mRNA expression of ROCK,MLCK,MMP-2 was much higher in the model group than that in the sham group(P<0.05).Picroside II and apocynin decreased the expression significantly(P<0.05),and there was no significant difference between the three groups in reducing the mRNA expression of ROCK,MLCK and MMP-2(P>0.05).The mRNA expression of Claudin-5 decreased significantly in the model group(P<0.01).After treatment with picroside II and apocynin,the mRNA level of Claudin-5 increased obviously(P<0.05),and there was no significant difference between the three groups in improving the m RNA expression of Claudin-5(P>0.05).(2)The protein levels of ROCK,MLCK,MMP-2 and Claudin-5: The protein expression of ROCK,MLCK,MMP-2 was much higher in the model group than that in the sham group(P<0.01).The treatment with picroside II and apocynin decreased the expression significantly(P<0.05),and there was no significant difference between the three groups in reducing the expression of ROCK,MLCK and MMP-2 protein(P>0.05).The protein expression of Claudin-5 decreased significantly in the model group compared with the sham group(P<0.01).After treatment with picroside II or apocynin,the protein level of Claudin-5 increased obviously(P<0.05),and there was no significant difference between the three groups in improving the expression of Claudin-5 protein(P>0.05). |
PDF Full Text Request