| Objective:Cerebrovascular disease is one of the major diseases that seriously harm human health currently.In China,there are more patients who die of cerebrovascular disease every year than in patients with heart disease and cancer,ranking first among the three causes of death.Ischemic cerebrovascular disease is a common and frequently-occurring disease that threatens human health,with high mortality,high disability and low cure rate.Therefore,the purpose of this study is to find an effective treatment and clarifying its mechanism of action to provide a new idea and drug target for the clinical treatment of ischemic cerebrovascular disease.Moreover,what is important is that we would improve the cure rate of ischemic cerebrovascular disease,reduce mortality and disability,and improve the cognitive function of patients with ischemic cerebrovascular disease.Methods:In this study,the effects of Gastrodin on microglia with oxygen-glucose deprivation(OGD)and cognitive function in mice with the model of ischemic cerebrovascular disease were elucidated,including two parts:in vitro cell experiments and in vivo animal experiments.Part I of the study:The microglia cell line(BV2 cell line)was cultured in vitro.Prepare an hypoxia-ischemia cell model by OGD and simulate the pathophysiological process of ischemic cerebrovascular disease in vivo.According to the techniques of LDH Cytotoxicity Assay Kit,flow cytometry of Annexin V-FITC Apoptosis Detection Kit,Laser Confocal Fluorescence Immunostaining(Double staining method),enzyme-linked immunosorbent assay(ELISA),and Western blotting,BV2 cells are observed through morphology and function changes induced by OGD.Moreover,these techniques were used to analyze changes in key proteins expression of signal transduction pathway in ischemic cerebrovascular disease,to explore the mechanism of gastrodin on ischemic cerebrovascular disease,and to elucidate the available ways for cell protection following ischemia and hypoxia.Part II of the study:An animal model of middle cerebral artery occlusion in mice was prepared by a suture method.By given with Gastrodin,changes in cognitive function in mice were observed and evaluated through observing neural function score,cerebral infarction rate,degree of brain edema,macroscopic morphology of the mouse brain,and Morris water maze,etc.These proved a more accurate theoretical basis for better clinical improvement of cognitive function recovery after ischemia.Results:1.In vitro cell experiments:① Different concentrations of Gastrodin acted on BV-2 cells.After 24 hours,LDH cytotoxicity test indicated that there was no obvious toxic lethal effect on the growth of BV-2 cells under different concentrations of Gastrodin,and there was no statistical difference in cell mortality(P>0.05)between different groups.②The activity of BV-2 cells changed by OGD for different lengths of time.Compared with non-OGD cells,the mortality rate of OGD cells was dramatically increased with statistically significant differences(P<0.05).There was a statistically significant difference in cell death rate between 4h and 8h,12h and 16h after OGD,but there was no obvious difference in cell mortality between 8h,12h and 16h(P>0.05).③ Compared with BV-2 cells cultured under normal condition,BV-2 cells was induced by OGD for 12h,following these results with statistical difference(P<0.05):the cell death rate increased significantly;the apoptosis increased significantly;the concentration of IL-1β and TNF-a increased significantly;the concentration of IL-10 decreased;the expression of BDNF decreased;the expression of TNF-a increased;the expression of JNK and P-P38 increased;the expression of JNK,ERK and P-ERK decreased.In the BV-2 cells with different concentrations of Gastrodin(low-dose group,middle-dose group and high-dose group)after pretreatment for 4h,following these results with statistical difference(P<0.05)compared to OGD introduced cells:cell death rate declined;the concentrations of IL-1β and TNF-a were significantly decreased;the concentration of IL-10 was increased;the expression of BDNF increased;the expression of JNK,P-P38 and P-38 decreased;and the expression of JNK,ERK and P-ERK increased.The effects of different concentrations of Gastrodin on microglia were not statistically different(P>0.05).Summary:In vitro cell experiments indicated that:① Gastrodin has no obvious toxic effect on BV-2 cells under physiological conditions.② The death rate of BV-2 cells increases as the time of hypoxia increases.③ In the absence of oxygen,Gastrodin has a protective effect on the survival of BV-2 cells.This protective effect is related to the reduction of apoptosis rate.It can also improve the hypoxic tolerance of BV-2 cells,and there is no obvious drug dose dependence.④ Gastrodin has dual effects on BV-2 cells.On one hand,it can inhibit the secretion of harmful cytokines.On the other hand,it can promote the secretion of protective cytokines.⑤ The dual role of Gastrodin is closely related to the expression of several proteins that can affect the MAPK signal transduction pathway.2.In vivo experiments in mice:①MCAO modeling can lead to different degrees of neurological impairment in mice;② The sham operation did not do harm to neurological impairment in mice.After modeling of MCAO successfully,the mice showed obvious neurological dysfunction.There were statistical differences between MCAO group and sham operation group(P<0.05).After treatment with Gastrodin,the degree of neurological deficits in mice was improved,which had statistic differences compared to MCAO group(P<0.05).And Gastrodin can exert brain protection in ischemic cerebrovascular diseases.③ The sham operation did not cause cerebral insufficiency in mice.But MCAO led to large area infarction in the brain tissue.There were statistical differences between MCAO group and sham operation group(P<0.05).After treatment of Gastrodin,the area of cerebral infarction in mice was significantly reduces,which had statistic differences compared to MCAO group(P<0.05).And Gastrodin can play a role in brain protection by reducing the area of cerebral infarction in ischemic cerebrovascular diseases.④The sham operation did not cause cerebral edema in mice After successfully modeling of MCAO,the mouse brain tissue was significantly swollen.There were statistical differences between MCAO group and sham operation group(P<0.05).After treatment of Gastrodin,cerebral edema in mice was dramatically reduced,which had statistic differences compared to MCAO group(P<0.05).And Gastrodin can play a role in brain protection by reducing cerebral edema in ischemic cerebrovascular diseases.⑤ The sham operation did not cause cognitive impairment in mice.After successfully MCAO modeling,mice’s learning and memory ability were significantly impaired.There were statistical differences between MCAO group and sham operation group(P<0.05).Gastrodin could significantly improve the learning and memory abilities of mice,which had statistic differences compared to MCAO group(P<0.05).And Gastrodin can improve cognitive dysfunction in ischemic cerebrovascular disease.Summary:In vivo experiments in mice indicated that:① MCAO model by the suture method is used to establish an animal model of ischemic cerebrovascular disease with good repeatability and stable cerebral infarction.It is a good tool for the study of ischemic cerebrovascular disease.② In the MCAO model group,the neurological function was significantly impaired,and Gastrodin could reduce the neurological impairment.The protective effect of Gastrodin on ischemic cerebrovascular disease is related to reducing the area of cerebral infarction and reducing cerebral edema.③The learning and memory abilities of mice with MCAO were significantly impaired.Gastrodin can improve cognitive dysfunction and significantly improve learning and memory after MCAO.④Gastrodin is a safe and non-toxic drug that can be used to improve cognitive dysfunction following ischemic cerebrovascular disease.Conclusion:1.Gastrodin has a dual effect on microglia with OGD.On the one hand,Gastrodin can inhibit the inflammatory cytokines secreted by microglia and aggravate the inflammatory response;on the other hand,Gastrodin can promote the secretion of protective cytokines from microglia to reduce the inflammatory response.2.Gastrodin can improve the learning and memory ability of mice with the model of ischemic cerebrovascular disease.It can be used as a selective drug for improving cognitive dysfunction after ischemic cerebrovascular disease. |
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