| Objective:The mechanism of anti-focal cerebral ischemia of THSWD was initially elucidated by studying the effects of THSWD on hippocampal neurotrophic factor in cerebral ischemia reperfusion injury rats,which providing theoretical basis of the effects of THSWD for cerebral ischemia in the further study.Method:Part IThe healthy male SD rats were treated with middle cerebral artery occlusion/reperfusion?MCAO/R?using modified Longa method.After successful modeling,they were randomly divided into model group,Nimodipine group?20 mg/kg?and THSWD group?9 g/kg?.Rats without fishing line was inserted as sham operation group.Then administered 12 h,24 h,7 d,14 d.After the successive oral administration,TTC staining was used to detect the cerebral infarction in each group at different time points.The contents of BDNF,NGF and NT-3 in the hippocampus were detected by ELISA.Part IIThe rats were treated with middle cerebral artery occlusion/reperfusion.After successful modeling,they were randomly divided into model group,nimodipine group?20 mg/kg?,THSWD treated group at doses of 4.5,9,18 g·kg-1.Rats without fishing line was inserted as sham operation group.After the successive oral administration for 14 d.The expression and distribution of BDNF and proBDNF in rat were detected by immunohistochemistry.ELISA was used to detect the levels of BDNF-producing key enzymes in the hippocampus of rats.Part IIIpre-animal treatment with the second part.Neurological scores?balance beam test and Bederson score?of rats in each group were evaluated.Transmission electron microscopy was used to observe the morphological characteristics of neurons in the hippocampus of rats.The protein expression of BDNF,PSD-95,Synapyophysin,TrkB,ERK1/2 and pERK1/2 were detected by Western blot.Results:Part I1 The effects of THSWD on cerebral ischemic injury at different time points in MCAO/R ratsA large pale area appeared on the ischemic side of the rats in the model group.No pale appeared on the brain slices in the sham operation group.The volume of cerebral infarction reached a maximum at 24 h after operation.After 24 h,the infarct volume tended to decrease as time went by.Compared with the model group at the same time point,the pale tissues in the rats in the THSWD group at 7 d and 14 d after cerebral ischemia/reperfusion were significantly less than those in the model group?P<0.01?,which could effectively reduce the cerebral infarction volume,while nimodipine group rats at each time point infarct area were significantly reduced after operation?P<0.05 or P<0.01?.2 The effect of THSWD on the expression of BDNF,NGF and NT3 in hippocampus of MCAO/R ratsIn the model group,BDNF showed a decreasing trend as time went by.At 12 h after operation,the expression of BDNF in hippocampus in model group was significantly higher than that in sham operation group at the same time point?P<0.01?.However,the level of BDNF in hippocampus of THSWD group and nimodipine group were significantly higher than those in model group at 7 d and 14 d after ischemia/reperfusion?P<0.01?.Compared with sham group,the expression of NGF protein in hippocampus in model group increased at 12 h after cerebral ischemia reperfusion,at reached the maximum at 24 h after cerebral ischemia,and there was significant difference.After 7days of reperfusion,the expression of NGF decreased.The trend of expression of NGF in hippocampus of rats in administration group was consistent with that in model group.The expression of NGF in hippocampus of nimodipine group was significantly higher than that in model group at 24 h and 7 d after cerebral ischemia/reperfusion?P<0.05?.There was no significant difference in the expression levels of NT-3 in hippocampus between groups at all time points after cerebral ischemia/reperfusion.Part II1 The effects of THSWD on BDNF and proBDNF expression in hippocampal of MCAO/R ratsAs immunohistochemistry results,BDNF-positive neurons positive staining was brown,and it was densely distributed in the hippocampus.The mean optical density of BDNF in hippocampus of rats in nimodipine group and THSWD group?9 g/kg,18 g/kg?was significantly higher than that in model group?P<0.05 or P<0.01?.There was no significant difference in the expression level of proBDNF between groups.2 The effect of THSWD on expressions of key enzymes of BDNF production and degradation in hippocampal tissues of MCAO/R ratsChanges of plasmin and MMP-7 in brain hippocampal region related to brain derived neurotrophic factor synthesis in MCAO/R Rats.Compared with the sham group,the content of plasmin in the hippocampus of the model group was significantly decreased?P<0.01?.The content of plasmin in hippocampal tissue of rats in THSWD?9g/kg,18 g/kg?and nimodipine were significantly higher than that in model group?P<0.05 or P<0.01?.There was no obvious change of MMP-7 in each group.Changes of enzymes in brain hippocampal region related to brain derived neurotrophic factor degradation pathway in MCAO/R Rats.Compared with sham group,the content of TIMP-1 in hippocampus of model group was significantly decreased?P<0.01?.While the content of MMP-9 in the model group increased significantly.Compared with the model group,the content of TIMP-1 in the hippocampus of the treated group was significantly increased?P<0.05 or P<0.01?.The content of MMP-9 in hippocampus of THSWD?9 g/kg,18 g/kg?and nimodipine were significantly lower than that in model group?P<0.01?.Part III1 The effects of THSWD on the neurological function in MCAO/R ratsThe effect of THSWD on balance beam test in MCAO/R rats.There was no change in walking ability of rats in sham group,and the walking ability of crossbar in each ischemic group decreased obviously,showing an improvement trend with the change of time,but still not returned to normal levels.Compared with the model group,THSWD?18 g/kg,9 g/kg?and nimodipine could promote the recovery of walking ability of rats.The difference was statistically significant after 7 days of ischemia/reperfusion?P<0.05or P<0.01?.The effect of THSWD on Berderson Score in MCAO/R Rats.There was no obvious neurological deficit in sham rats.The neurological deficits were significant in the model group rats,but they showing an improvement trend with the change of time.After the successive oral administration for 7 days,the Berderson score of THSWD?18g/kg,9 g/kg?and nimodipine group was significantly lower than that of model group?P<0.05?,and the neurological function improved obviously.2 The transmission electron microscope was used to observe the neuronal damage in hippocampus of MCAO/R ratsSham group neurons have intact nuclei.Cytoplasm visible lysosomes.In the model group,nucleus vacuolization,and a few mitochondria also have vacuolar degeneration,fewer neurons.Neurons in the THSWD group had a more intact nucleus,slight enlargement of a few rough endoplasmic reticulum,and mild mitochondria vacuolization.Neuronal injury has significantly improved the role.Nimodipine group more complete nucleus,mitochondrial spine mild dilatation,neuronal damage also significantly improve the role.3 The effects of THSWD on the expressions of BDNF,TrkB,ERK1/2,pERK1/2,Synapyophysin and PSD-95 in hippocampus of MCAO/R ratsThe expression of TrkB protein in hippocampus in model group was significantly higher than that in sham group?P<0.01?.Compared with model group,BDNF,TrkB,pERK1/2/ERK1/2 expression up-regulated in THSWD.The expression of PSD-95 and Synapyophysin in hippocampus of model group was significantly lower than that in sham group?P<0.01?.Compared with model group,the content of PSD-95,Synapyophysin expression up-regulated in THSWD?4.5 g/kg,9g/kg,18 g/kg?.Conclusion:?1?THSWD had a clear modulatory effect on BDNF among the three neurotrophic factors?BDNF,NT3,and NGF?,and which had the strongest regulatory effect at 14days.?2?The expression of BDNF was affected by THSWD through down-regulating the expression of neurotrophin synthase plasmin,increasing the expression of MMP-9and reducing the expression of BDNF degradation inhibitor TIMP-1.?3?THSWD may upregulate the expression of BDNF and activate the BDNF-TrkB-ERK1/2 signaling pathway to regulate the survival of neurons,thereby promoting the recovery of cerebral ischemic injury.Furthermore,The expression of BDNF was affected by THSWD,which influencing the expression of PSD-95 and synaptophysin,even involving in axonal growth and synaptic plasticity. |
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