Study On The Protective Effect Of Total Flavonoids Of Fagopyrum Cymosum On Excitatory Amino Acid Toxicity In Rats With Cerebral Ischemia-reperfusion

Objective:To investigate the protective effects of total flavonoids of Fagopyrum cymosum on excitatory amino acid toxicity after cerebral ischemia reperfusion in rats from the perspective of astrocyte activation and glutamate uptake and release,this study provided a pharmacological basis for the application of total flavonoids of Fagopyrum Cymosum in cerebral ischemia injury.Methods:A rat model of middle cerebral artery occlusion and reperfusion（MCAO/R）was established by suture occlusion.252 male SD rats were divided into Sham operation group（Sham）,Cerebral ischemia-reperfusion group（CIR）,Naoxintong capsule group(NXT,0.8 g·kg^-1),High-dose total flavonoids of Fagopyrum cymosum(JQM HD,0.1 g·kg^-1),Medium-dose(JQM MD,0.05 g·kg^-1),Low-dose(JQM LD,0.025 g·kg^-1)groups,each group is divided into three groups:6h,24h,48h Subgroup.Except for the Sham group and CIR group,the remaining groups were given Naoxintong capsules and total flavonoids of Fagopyrum cymosum suspension for 7 days,and the MCAO/R model was replicated on the 7th day after gavage 1 hour.The Sham group is not plugged,and other operations are the same as the CIR group.（1）Rat neurological deficit score:each group of rats in the three subgroups of 6h,24h and 48h were scored to observe the nerve damage in MCAO/R rats.（2）Morphological observation:take materials at various time points after modeling,observe the brain tissue edema,calculate the brain index of each group;detect the cerebral infarct volume by TTC staining method;observe the neuronal cell morphology of brain tissue by HE staining.（3）Detection of related factors:biochemical method to detect the levels of Glu,GS,Gln and ATP enzymes in brain tissue,and ELISA to detect the levels of S-100βand NSE in serum.（4）Detection of glutamate metabolism-related proteins:Immunohistochemical method to observe the expression of GFAP,VGLUT-1 and GLT-1 in the hippocampal CA1 area and cortical area of the ischemic brain tissue of rats in each group.Western Blot to detect the brain tissue GFAP,VGLUT-1,GLT-1,GS expression.Results:（1）Improve neurological deficits in CIR rats.The results of nerve scoring show that CIR has severe nerve defect symptoms at 6h,and the degree of defect is further aggravated at CIR 24h and 48h.Fagopyrum cymosum total flavonoids can significantly reduce the rat nerve score after 6h and 48h of reperfusion,and the nerve score of each administration group also decreased at CIR 24h.（2）Reduce brain index and reduce cerebral infarction volume.In the CIR group,there is obvious infarction in the brain at 6 hours of reperfusion,and the infarct area is further enlarged and pale in color at 24h.Compared with the CIR group at the same time,the white area of the JQM HD and JQM MD groups is significantly reduced,and the volume of cerebral infarction and brain index are significantly reduced.（3）Improve the biochemical indexes of CIR rats.CIR 6h-48h rat serum NSE,S-100β,brain tissue Glu,Gln,GS levels are significantly increased（P<0.01 or P<0.05）,and GS levels gradually increased with the extension of CIR time;Compared with the CIR group at the same time point,the levels of NSE,S-100β,and Glu in the JQM HD and JQM MD groups are significantly reduced（P<0.05）,and the levels of Gln and GS in the JQM HD group are higher than those in the CIR group.（4）Improve the pathological damage of CIR rat brain tissue and increase the activity of ATPase in brain tissue.After CIR 6h-48h,there are obvious pathological damages in hippocampus and cortical neurons,neuron cell arrangement disorder,edema,intercellular space enlargement,cell vacuole-like degeneration,nucleus shrinkage obviously,a large number of cell necrosis;In the NXT and JQM HD groups,there are more neurons with a slightly complete structure,which can significantly reduce cell edema and improve the pathological damage in the hippocampus and cortex.The ATPase activity of brain tissue decreased significantly at CIR 6h and the lowest activity at CIR 24h.Compared with the CIR group at the same time point,the ATPase activity of the JQM HD group increased significantly（P<0.01）.（5）Regulate the expression of GFAP in CIR rat brain tissue.At CIR 6h,the number of AS with positive GFAP expression in hippocampal CA1 area and cortical area is significantly increased,the protrusion is prolonged,and the coloration become darker.With the extension of CIR time,its expression in the hippocampal CA1 area gradually increased;The positive expression of CIR is highest in the cortical area at24h.The total flavonoids of Fagopyrum cymosum can up-regulate the expression of GFAP at CIR 6h,and down-regulate the expression of GFAP at CIR 24h and CIR 48h.（6）Regulate the expression of VGLUT-1 and GLT-1 in the brain tissue of CIR rats.The expression of VGLUT-1 in the hippocampal CA1 area of normal brain tissue is very low,and the expression of VGLUT-1 increases after CIR,and it increases with the extension of CIR time;The positive expression in cortical area is the highest at CIR 24h;Fagopyrum cymosum total flavonoids can significantly down-regulate the expression of VGLUT-1 after CIR.In normal brain tissue,the expression of GLT-1 in the hippocampal CA1 area and cortical area tightly surrounds the neurons,and the expression is high,while the expression of GLT-1 is significantly decreased at CIR 6h,significantly decreased at CIR 24h,and increased at CIR 48h is still low;total flavonoids of Fagopyrum cymosum can significantly increase the expression of GLT-1.Conclusion:VGLUT-1 is involved in the excitatory toxicity injury of Glu after cerebral ischemia reperfusion.Total flavonoids of Fagopyrum cymosum can reduce the excitatory toxicity of Glu and have a protective effect on cerebral ischemia reperfusion injury.The mechanism may be related to improving energy metabolism,regulating astrocyte activation,inhibiting the expression of VGLUT-1 and promoting the expression of GLT-1.