Study On The Effect And Mechanism Of Four Compounds Of Astragalus Isoflavones In Promoting Nerve Function Recovery

Backgrounds and ObjectivesAt present,most of ischemic stroke patients will leave neurological deficit,resulting in different degrees of functional activities loss or disability,such as hemiplegia,blurred vision,aphasia,memory loss and other sequelae.So it is particularly important to develop nerve restorer after stroke.Astragalus isoflavones are one of the important components of Astragalus membranaceus,mainly including calycosin,formononetin,calycosin-7-glucoside and ononin.It is not clear that the effect of astragalus isoflavones on the recovery of nerve function after cerebral ischemia.This will be discussed in this study,and provide the possibility for the development of drugs to improve or restore the damaged neurological function after stroke.MethodsIn vitro:The viability of calycosin,formononetin,calycosin-7-glucoside and ononin on PC 12 cells were detected by CCK-8.In addition,in the presence of 0.3 ng/mL NGF,PC 12 cells were co-cultured with calycosin,formononetin,calycosin-7-glucoside and ononin for 5 days respectively to observe whether they could promote the neurite outgrowth of PC12 cells and the expression of GAP-43 andβIII-tubulin.In vivo:Male SD rats(n=28)were randomly divided into sham,model and formononetin groups.Middle cerebral artery occlusion(MCAO)ischemia-reperfusion was established except sham group.The rats in the formononetin group were administered 30 mg/kg formononetin,sham group and model group were treated with equal volume of 0.5%CMC-Na,once a day for 14 d by intragastrical gavage(i.g.).Neurobehavioral tests and weight changes were performed before and at 1,3,7 and 14 d after operation,respectively.After 14 days,brain tissue was obtained after cardiac perfusion or direct execution in rats to complete the following experiments:(1)The number of neuronal dendritic spines around M1 region of brain tissue were measured by golgi staining.(2)The expression ofβIII-tubulin,GAP-43,NGF,BDNF,Trk A,p-Trk A,Trk B,p-Trk B,AKT,p-AKT,ERK 1/2 and p-ERK 1/2 in the border of infarct area was detected by western blot.(3)The number of NeuN~+/BrdU~+double-labeled neurons in the cerebral cortex was measured by immunofluorescence staining.ResultsIn vitro:(1)CCK-8 results showed that four isoflavones from Astragalus enhanced the viability of PC12 cells.(2)Compared with the vehicle group,four isoflavones from Astragalus significantly promoted neurite outgrowth of PC 12 cells in the presence of 0.3 ng/mL NGF.(3)Compared with the vehicle group,four isoflavones from Astragalus significantly increased the number ofβIII-tubulin positive cells in the presence of 0.3 ng/mL NGF.(4)Compared with the vehicle group,the expressions of GAP-43 andβIII-tubulin were significantly up-regulated under the treatment of four isoflavones from Astragalus(0.1μM)in the presence of 0.3 ng/mL NGF.In vivo:(1)Compared with the sham group,the mNSS were significantly increased,the residence time on the rotating drum and the rat weights were significantly decreased after operation.Compared with the model group,the rats in the formononetin group,the mNSS were significantly decreased on day 7 and day 14,the residence time on the rotating drum was significantly increased on day 14and the weights were significantly increased on day 7 and day 14.(2)Compared with the sham group,the number of neuronal dendritic spines of the rats after operation significantly decreased.Compared with the model group,the number of neuronal dendritic spines in formononetin group(30mg/kg)significantly increased.(3)Compared with the sham group,the expression ofβIII-tubulin,GAP-43,NGF,BDNF,Trk A,p-Trk A,AKT and p-AKT significantly decreased,and the expression of ERK 1 and p-ERK1 significantly increased.Compared with the model group,formononetin significantly upregulated the expression ofβIII-tubulin,GAP-43,NGF,BDNF,Trk A,p-Trk A,p-Trk B,p-AKT and p-ERK1/2.(4)The BrdU~+/NeuN~+positive cells were not detected in the cortex of sham rats.Compared with the model group,the number of BrdU~+/NeuN~+positive cells in ischemic penumbra of rats in formononetin group significantly increased.ConclusionIn vitro,in the presence of 0.3 ng/mL NGF,calycosin,formononetin,calycosin-7-glucoside and ononin could induce the differentiation of PC 12 cells.In vivo,formononetin restored the injured nerve functions after ischemic stroke by promoting neuronal differentiation and synaptic plasticity.The phosphorylation of PI3K/AKT/ERK pathway might be involved in this process.