Effects And Mechanisms Of Trillium Saponins On Synaptic Remodeling In Rats With Post-stroke Cognitive Impairment

Objective To investigate the effects and mechanisms of trillium saponins on synaptic remodeling in rats with post-stroke cognitive impairment(PSCI)based on the Sonic hedgehog signaling pathway.Methods: One hundred,healthy male Sprague Dawley(SD)rats were randomly divided into a model group(82 rats)and a sham operation group(18 rats).Rats with ischemic stroke models were prepared by middle cerebral artery occlusion(MCAO),and evaluated with the use of Zea-longa scores,and the scores ranging from 1 to 3were randomly divided into model group(MCAO),total Trillium Saponins group(TTM,100 mg/kg),and donepezil hydrochloride as a positive control group(DON,0.45 mg/kg).Rats in Sham group(Sham)were only isolated from the left common carotid artery without embolization.Twelve hours after operation,the rats in the Sham group and the MCAO group were intragastrically administrated with the equal volume of normal saline once a day for 28 days.Then,the following tests were carried out: 1.Score of neurological deficit.2.Morris water maze and new object recognition experiment.3.Cerebral ischemia was observed using Magnetic resonance(MRI)imaging.4.Changes of cerebral ischemia volume was tested using2,3,5-Triphenyltetrazolium chloride(TTC)staining.5.The activity of superoxide dismutase(superoxide dismutase,SOD)and glutathione peroxidase(glutathione peroxidase,GSH-PX)and the content of malondialdehyde(malondialdehyde,MDA)in rat hippocampal tissue were determined using the enzyme kit.6.The pathological damage of brain tissue was observed with the using of Hematoxylin-eosin(HE)and Nissl(Nissl)staining.7.Apoptosis was detected using TUNEL staining.8.The dendritic spine density using Golgi staining.9.The location and expression of vascular endothelial growth factor(VEGF)protein was detected using immunohistochemical(IHC)method.10.The expression of PSD-95,SYN,and GAP-43 was tested using immunofluorescence(IF)staining.10.The co-localization and expression of Shh and Gli1 was tested using IF double-labeled staining.11.The expression levels of Bcl-2,Bax,Caspase-3,cl-Caspase-3,PSD-95,SYN,GAP-43,BDNF,Shh,Ptch1,Smo and Gli1 proteins in rat hippocampal tissue were tested using Western blot(WB)analysis.Results: 1.The scores of neurological function evaluation showed that the neurological deficit of the rats in the TTM group and the DON group were significantly lower than those in the MCAO group(P<0.01).2.The results of the Morris water maze and new object recognition experiments showed that compared with the MCAO group,escape latency lowed,the times of crossing the platform increased,the residence time in the target quadrant increased,swimming distance shortened and target orientation was cleared(P<0.01)of the rats in the TTM group and the DON group;Compared with the MCAO group,the time to explore new objects and familiar objects increased in the rats in the TTM group and the DON group.At the same time,the activity and the relative recognition index of the rats were significantly higher than those in the MCAO group(P<0.01).3.Results of MRI imaging: Compared with Sham group,there was obvious high-density shadow ischemic lesion area in the left hemisphere in MCAO group.Compared with MCAO group,the area of ischemic lesion in the left hemisphere in TTM group and DON group was all significantly reduced.4.TTC staining results showed that the volume of cerebral infarction in TTM group and DON group was significantly smaller than that in MCAO group(P<0.01).5.Oxidative stress detection results showed that compared with the Sham group,the SOD and GSH-PX activities in the brain tissue of the rats in the MCAO group were significantly reduced(P<0.01),while the MDA content increased(P<0.01).Compared with MCAO group,the activity of SOD and GSH-PX in TTM group and DON group was significantly increased(P<0.05 or P<0.01),while MDA content decreased(P<0.01).6.Compared with the Sham group,the hippocampus of the rats in the MCAO group showed significantly atrophy,the cells in the ischemic cortical area were necrotic,and the nerve cells in the CA1 area were significantly reduced by HE staining.Compared with the MCAO group,the hippocampal atrophy improved,cell necrosis was relatively improved,and nerve cells in the CA1 area increased significantly in the TTM group and the DON group.7.Nissl staining results showed that compared with the Sham group,the arrangement of nerve cells in the hippocampal CA1 area was loose and disordered,nuclear pyknosis appeared,and the area of Nissl bodies decreased in the MCAO group(P<0.01).Compared with the MCAO group,the nerve cells were closely arranged and the area of Nissl bodies increased in the TTM group and the DON group(P<0.01),and the nerve cell damage was alleviated.8.TUNEL staining showed that apoptotic neurons in hippocampus CA1 region in TTM group and DON group significantly decreased compared with MCAO group(P<0.01).9.Golgi staining results showed that compared with Sham group,dendritic spine density in MCAO group decreased significantly(P<0.01).And compared with MCAO group,dendritic spine density in TTM group and DON group increased significantly(P<0.01).10.Immunohistochemical results showed that compared with the Sham group,the expression of VEGF protein in the hippocampus of rats in the MCAO group significantly reduced(P<0.01).compared with the MCAO group,the expression in the TTM and DON groups significantly increased(P<0.01).11.IF staining results: Compared with Sham group,the expression of PSD-95,SYN and GAP-43 positive cells in hippocampus of MCAO group was significantly decreased(P<0.01).Compared with MCAO group,the expression of PSD-95,SYN and GAP-43 positive cells in hippocampus of TTM group and DON group increased significantly(P<0.01).12.The colocalization of Shh and Gli1 was detected by IF double labeling.Compared with the Sham group,the Shh and Gli1 colocalization in the MCAO group increased(P<0.01).Compared with the MCAO group,the colocalization in the TTM group and the DON group significantly increased(P<0.01).13.Western blot analysis showed that compared with the Sham group,the protein expressions of Bcl-2,PSD-95,SYN,GAP-43,and BDNF in the hippocampus of rats in the MCAO group significantly reduced(P<0.01),and Bax,cl-Caspase-3/Caspase-3,Shh,Ptch1,Smo and Gli1 levels increased(P<0.05 or P<0.01).Compared with the MCAO group,the expressions of Bcl-2,PSD-95,SYN,GAP-43,BDNF,Shh,Ptch1,Smo,and Gli1 proteins in the TTM group and the DON group increased(P<0.05 or P<0.01),and Bax,cl-Caspase-3/ Caspase-3 protein expressions decreased significantly(P<0.01).Conclusion: 1.TTM significantly improved the neurological deficit in rats with PSCI,reduced the volume of cerebral infarction,significantly improved the spatial learning and memory ability and new object recognition ability.2.TTM significantly reduced the oxidative stress response and neuron apoptosis caused by cerebral ischemia-reperfusion,promoted synaptic remodeling,and repaired the damaged brain.3.TTM significantly increased the protein expression level of VEGF,promoted angiogenesis and the establishment of collateral circulation,restored blood flow recanalization in time,and rescued neurons in the ischemic penumbra.4.TTM may activate the Sonic hedgehog signaling pathway,promote neuronal recovery and synaptic remodeling in the hippocampus and ischemic penumbra,and play a neuroprotective role in PSCI rats.