Research Of The Mechanism Of Anti-Cerebral Ischemia Reperfusion Injury Of Traditional Medicine Monomer Tetramethylpyrazi NE

Purpose:Cerebral vascular diseases are common and frequent health-threatening illnesses of human beings,especially for the aged and the middle-aged people.Among them,about 80%is composed of Ischemic Cerebral Vascular Disease（ICVD）.Brain is the most sensitive organ to hypoxia that ultimately results in the injuries of local cerebral tissues and their functions.Rehabilitating blood reperfusion in time is beneficial to the reduction of the ischemic cerebral injury.However,the recovered blood sometimes causes further tissue injuries and dysfunction under certain circumstances,which is called cerebral ischemia and reperfusion injury（CIRI）.Inhibiting CIRI has become the key step in ICVD treatment. Currently treatment medicines are usually deficient in function mechanism and toxic, whereas Chinese medicines treat more efficiently in multiple factors.Thus research of the treatment mechanism of ischemic cerebral disease by Chinese medicine is greatly valuable for its importance of theoretical and clinical applications.Cerebral diseases are closely related to the blood stasis syndrome in Chinese Medicine,for which methods of promoting blood circulation and removing blood stasis are usually used, incorporated with medicine of regulating Inspirit and promoting blood.Ligusticum chuanxiong is widely used clinical treatment for promoting blood circulation and removing blood stasis,it is active in promoting blood and Inspirit circulation.In previous study,we have demonstrated that tetramethylpyrazine can penetrate the blood-brain barrier of normal and ischemia and reperfusion conditions.The active mechanism of tetramethylpyrazine hydrochloride from CIRI is closely related to several factors,such as reducing cerebral edema,adjusting the injury reaction of astrocytes,anti-free radical injury,preventing vasospasm,decreasing NO toxicity,anti-cellular apoptosis,reducing neurons denaturation necrosis and even gene regulations.Nevertheless,considering of the multiple central steps of CIRI,it is necessary for us to further explore the neural protection mechanism of tetramethylpyrazine.The research contents includes whether tetramethylpyrazine can regulate increase of excitatory amino acid,the overload of Ca²⁺ ion in cells,the generation of toxic free-radical,cellular apoptosis and the damage from inflammatory cells.We hope that this research will provide theoretical basis for the clinical use of ligusticum chuanxiong andtetramethylpyrazine in treatments for CIRI,and also a better experimental methodology platform for researching and screening Chinese medicines treating CIRI.Methods:Firstly we developed an in vivo animal reperfusion model for Middle Cerebral Artery Occlusion（MCAO）,examine the effect of tetramethylpyrazine on regulating Bax/Bc1-2 protein and mRNA by use of immunohistochemistry and real-time fluorescence quantitative PCR detection.Then we derived glutamate acid model,hydrogen peroxide model,caffein model and dicarboamide model based on PC 12 cells from the pathology and physiology of CIRI.Afterwards,a series of researches are applied to the above mentioned models,including:（1） Test whether tetramethylpyrazine has neuroprotective effects in these models by use of CCK-8 technique;（2） In order to investigate the function mechanism of tetramethylpyrazine and whether it can regulate apoptosis in various types of PC12 CIRI, we observe apoptosis cells with a Hoechst-33342 fluorescent microscope,measure the rate of apoptosis and Caspase activities with flow cytometry,measure the mitochondrial membrane potential with Rhodamine stains,and measure the mRNA level of apoptosis regulating gene Bax and Bc12 by real-time fluorescence quantitative PCR;（3） Discuss the changes of inflammation and the protection mechanism of tetramethylpyrazine by measuring the inflammatory cytokine（IL-1β、IL-6 and TNF-α） by ELISA,and measuring HMGB1 by Western-blot;（4） Test the activity of NF-KB by EMSA and further investigate whether tetramethylpyrazine relieves CIRI by regulating the activity of NF-KB;（5） Test the oxidation agent index SOD、MDA、LDH and GSH/GSSG in all these models and discuss the mechanism of generating toxic free radicals by use of tetramethylpyrazine.Results:In the animal model of MCAO,Bax/Bc1-2 increases enormously on protein and mRNA levels.However,preprocessed by large dose of tetramethylpyrazine decreases this ratio.（2） It is shown by CCK-8 that glutamate acid,hydrogen peroxide,caffein and dicarboamide have cytoxicity.After the preprocessing of tetramethylpyrazine,cellular injuries are decreased dose-dependently.（3） It is observed that after the preprocess by glutamate acid,hydrogen peroxide,caffein and dicarboamide,PC12 cell apoptosis occurs; after 1mM tetramethylpyrazine preprocess,PC12 cell appears karyopyknosis and apoptosis body are decreased.（4） AV/PI flow cytometry indicates that once processed by glutamate acid,hydrogen peroxide,caffein and dicarboamide,PC12 cell apoptosis rate rises dramatically.After being preprocessed by tetramethylpyrazine,the apoptosis rate of PC12 cells decreases dose-dependently.（5） The test of Caspase activity shows that the activities of Caspase-3 and Caspase-9 increase once processed by glutamate acid,hydrogen peroxide, caffein and dicarboamide.After being preprocessed by tetramethylpyrazine,the activities of Caspase-3 and Caspase-9 decrease dose-dependently.In glutamate acid,hydrogen peroxide and dicarboamide models,the activity of shows little change while in caffein model,the activity of Caspase-8 increases considerably.Preprocessed by tetramethylpyrazine,the activity of Caspase-8 decreases dose-dependently.（6） Rhodamine stain mitochondrial membrane potential detection indicates that the PC12 cell mitochondrial membrane potential decreases after being processed by glutamate acid,hydrogen peroxide,caffein and dicarboamide.The preprocessing of tetramethylpyrazine dose-dependently increases the ratio of Bax/Bc-12.（7） real-time fluorescence quantitative PCR detection shows that the ratio of Bax/Bc-12 increases after being preprocessed by glutamate acid,hydrogen peroxide, caffein and dicarboamide.The preprocessing of tetramethylpyrazine dose-dependently decreases the ratio of Bax/Bc-12.（8） ELISA technique indicates that in different models the excrete levels of IL-1β,IL-6 and TNF-αincrease considerably.After the preprocess of tetramethylpyrazine,the excrete level of IL-1βdramatically in glutamate acid and caffein models,while in hydrogen peroxide and dicarboamide models decrease dramatically.The excrete level of 1L-6 shows little change in glutamate acid and dicarboamide models shows little change,while in caffein model it decreases enormously.The excrete level of TNF-αshows a dramatic decrease in glutamate acid and caffein model,while in hydrogen peroxide and dicarboamide models shows little change.（9） Western-blot indicates that HMGB1 level increases dramatically in distinct models.After the preprocess of tetramethylpyrazine,the level of HMGB1 dramatically declines in glutamate acid and caffein models,while in hydrogen peroxide and dicarboamide models barely changes.（10） As indicated by EMSA, the activities of NF-KB decline dramatically in glutamate acid,caffein and dicarboamide models.After being preprocessed of tetramethylpyrazine,the activities of NF-KB increase greatly in glutamate acid,caffein and dicarboamide models.In contrast,in hydrogen peroxide model,the activities of NF-KB increase greatly.After the treatment of tetramethylpyrazine,the activities of NF-KB decrease considerably.（11）In oxidative stress index display group,the transformation trend of all indexes are generally the same,i.e. glutamate acid,caffein,hydrogen eroxide and dicarboamide all decrease SOD and GSH/GSSG,increase LDH and MDA;after the treatment of tetramethylpyrazine,SOD increases,LDH and MDA decline,while GSH/GSSG increases.Conclusions:The pathological and physiological changes of CIRI are various in aspects and levels.Tetramethylpyrazine improves the symptoms of CIRI by regulating the central generating and developing steps of CIRI,such as increase of excitatory amino acid,the overload of Ca²⁺ ion in cells,the generation of toxic free-radical,cellular apoptosis and the damage from inflammatory cells.The original aspects in our research rely on:（1） although some researchers have developed models on Ischemic Cerebral Vascular Disease（ICVD） based on PC12 cells,there are few studies of mechanism of tetramethylpyrazine by use of PC12 cells.In this study,by referred to the pathological mechanism of ICVD,we derived four models based on PC12 cells,i.e.glutamate acid model,hydrogen peroxide model, caffein model and dicarboamide model;and applied these models to the study of mechanism of tetramethylpyrazine.Especially,the processing of hydrogen peroxide disturbed the balance of oxidation and anti-oxidation without generating free radicals with toxicity,which is an improvement of toxic free radical model（hydrogen peroxide model）. （2） In the animal experiment,we have proved the protection mechanism of tetramethylpyrazine,and the mechanism is related to anti-cellular apoptosis and anti-apoanti-oxidation free radicals.（3） We adopted various measured techniques,among which the real-time fluorescence quantitative PCR technics and gel mobility testing EMSA are original ideas.（4） The experiments demonstrated that in different models, tetramethylpyrazine can protect PC12 cells through different mechanisms,and thus complete the active mechanism of tetramethylpyrazine.（5） Our research has established an experimental methodology platform for researching and screening Chinese medicines treating CIRI,and is also of great importance to the modernization of Chinese Medicine.