| Objective:Cerebrovascular disease is one of the main causes of death and disability.Ischemic cerebrovascular disease is called " stroke " in traditional Chinese medicine that has good curative effect on it.Scholars generally believe that blood stasis block is the main cause and pathogenesis of ischemic stroke,so the method of promoting blood circulation and removing blood stasis is the basic treatment.However,it was found that the most traditional Chinese medicines for promoting blood circulation and removing blood stasis could not pass blood-brain barrier(BBB),thereby affecting the plasma concentration in the brain and the therapeutic effect on stroke.Kaiqiao herbs are easy to pass through BBB and can promote other drugs to enter the tissue through BBB.Can the combination of Kaiqiao herbs and blood activating herbs promote the transportation of blood activating herbs into brain tissue to play a synergistic effect? In this study,the rat model of middle cerebral artery occlusion(MCAO)was established by using borneol,an orifices commonly used in clinic,and safflower,a blood activating herb commonly used in stroke.It was to explore the effect of borneol with safflower on cerebral ischemia injury and its mechanism,to provide new therapeutic thinking and theoretical experimental basis for the clinical treatment of cerebral ischemia injury.Method:132 male SD rats with were randomly divided into sham-operated group,model group,borneol group(0.03 g/kg),safflower group(5 g/kg),borneol with safflower group(0.03 g/kg +5 g/kg)and nimodipine group(0.01 g/kg).The model of MCAO rats was established by using the method of internal carotid artery with suppository.The rats were administered by gavage after 2 hours of the operation,which were intragastric gavage once a day for 7 days;The sham group and the model group were given isodose normal saline once a day for 7 days.The following indicators were detected:1)The neurological function score: After the modelling rats were awake 1 hour,,the neurobehavioral scores were scored in rats referring to scoring criteria of Zea Longa's 5th level 4 points..And the scores of 1-3 were included in subsequent experiments;After 7 days of medication,the neurological score was scored again.2)Cerebral cortical blood flow(CBF): The cerebral blood flow in the cerebral cortex of rats were detected by dual wavelength laser Doppler flow meter after anesthesia.3)The detection of cerebral infarction scope: The rats were decapitated to take the brain,which were taken for continuous coronary section.After TTC staining was performed,the area of cerebral infarction was measured by image acquisition and image analysis system.4)Histopathological observation of brain tissue: The paraffin sections of brain tissue were made by routine method,and HE staining was used to observe the morphological changes of nerve cells in rats,and to collect and analyze the images.5)The expression of MMP-2,MMP-9 and ZO-1 in ischemic brain tissue were detected by immunohistochemical method: Paraffin sections of brain tissue were made and the expression of the genes about MMP-2MMP-9 and ZO-1 related to blood-brain barrier in brain tissue was detected by immunohistochemical method.6)Reverse transcriptase polymerase chain reaction(RT-PCR)was used to detect mRNA expression of MMP-9 and Claudin-5 which were in the ischemic brain tissue of rats: After anesthesia,the rats were decapitated and taken the brains quickly.And then the cerebral cortex of the ischemic side was taken.The total RNA in cerebral cortex was extracted by routine methods,and mRNA expression of MMP-9 and Claudin-5 were detected by RT-PCR.Result:1)The effects on neurological function scores and cerebral infarct scopeThe neural function score of the model group was significantly higher than that of the sham operation group(P<0.01).Compared with the model group,each medication administration team could significantly reduce the neurological function scores and decrease the cerebral infarction scope(P<0.01 or P<0.05).The improvement of the above indexes in the borneol combined with safflower group was better than that in the single borneol group and the single safflower group.2)The effects on cerebral cortical blood flowCompared with the sham operation group,cerebral cortical blood flow was blocked obviously in model group.Compared with the model group,the block of cerebral cortex blood flow were significantly improved in the borneol with safflower group(P<0.01),which was superior to the borneol group or the safflower group.3)The effects on pathological morphology of brain tissueThe numbers of rat brain cells in the model group were decreased significantly,the neurons were appeared deformation,necrosis,nuclear pyknosis,some nucleoli disappeared,and the cell body deformation.Compared with the model group,the pathological morphology of the brain tissue in the herb teams were improved in different degree,and the morphology of the borneol combined with safflower group was the most obvious.4)The effects of protein expression of MMP-2,MMP-9 and ZO-1 on brain tissueThe expression of MMP-2 and MMP-9 in brain tissue of the model group were significantly increased(P<0.01),while the expression of ZO-1 was significantly decreased(P<0.01).Compared with the model group,the expression of MMP-2 and MMP-9 were significantly decreased and the expression of ZO-1 was significantly increased in each herb team,especially in the borneol combined with safflower group(P<0.01).5)The effects on mRNA expression of MMP-9 and Claudin-5 in brain tissueCompared with the sham operation group,the expression of MMP-9 mRNA in the model group was significantly increased,while the expression of Claudin-5 mRNA was significantly decreased(P<0.01).Compared with the model group,the expression of MMP-9 mRNA was significantly decreased and the expression of Claudin-5 mRNA was significantly increased in each herb team(P<0.05).Conclusion:1)The group of the borneol with the safflower which can not only reduce neurological score,increase cerebral cortical cerebral blood flow,reduce the scope of cerebral infarction in MCAO rats,but also can improve the pathological morphology of brain tissue,which indicated it has the brain protective on cerebral ischemia injury in rats.2)The protective mechanism of the borneol combined with safflower may be related to down-regulating the protein expression of MMP-2 and MMP-9,enhancing the protein expression of ZO-1,and down-regulating mRNA expression of MMP-9 and enhancing the mRNA expression of Claudin-5 in brain,which affected the permeability of blood-brain barrier and reduce brain edema. |
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