Effects And Mechanisms Of Steroidal Saponin TSA On Neurological Function Recorvery After Cerebral Ischemia In Rats

Ischemic stroke is extremely harmful because of its high morbidity,mortality and disability incidence.The effective treatments of ischemic stroke so far are thrombolysis with recombinant tissue plasminogen activator(rtPA) in 6h of onset.The restrictions on time and potential adverse effects have limited the use of rtPA to approximately 2%-5%of stroke patients in the United States.Meanwhile the ischemia-reperfuion lesion is the main issue of thrombolysis with rtPA. How to improve the neurological function recovery after stroke is the important therapeutic items in early recovery stage for most patients which can not receive the therapy of thrombolysis in time.Cerebral ischemia can activate the neurogenesis in adult mammal brain,which induce neuroal progenitor cells to divide,proliferate and migrate to the lesion region and differentiate to be mature neurons that partly take place of the missing specific neuron after ischemia. Neurogenesis is close related to angiogenesis therefore the key therapeutic phase of ischemic cerebrovascular disease is to reconstruct the blood supply in ischemic area and to retrieve injured neurons,neuroglia cells and endothelia cells as soon as possible.Motivating endogenous neurogenesis and angiogenesis following cerebral ischemia could be an original clinical therapeutic tool.Vascular endothelial growth factor(VEGF) and angiopoietin(Ang) are valued angiogenesis factor which have the role to protect neurons and stimulate neurogenesis.The effect of Ang1 and Ang2 relies on the role of VEGF.VEGF is probably one of the key factors of regulation mechanism linking both neurogenesis and angiogenesis.Wnt/β-catenin is a classic signal pathway to regulate the proliferation and differentiation of neuroal stem cells on the nerves system development.β-catenin promote the proliferation of NSC and determine its differentiation fate.What the study mainly concerns is the level of Ang1,Ang2, VEGF/VEGFR2 and the expression of several key factorsβ-catenin,GSK3β,c-myc of Wnt/β-catenin signal pathway as well as the relationship between the cytokines and neurogenesis & angiogenesis following ischemic stroke.Steroidal Saponin TSA is the major active components of anemarrhenae.The activation of anemarrhenae has been reported in the neuroprotective manifestations on acute cerebral ischemia rats model.Regulating the blood vessel endothelium function of ischemic brain, anti-thrombopoiesis and improving blood supply are the effective mechanisms of TSA.While the researches to TSA on the neurogenesis and angiogenesis after cerebral ischemia haven't been reported so far.Here,in the study we were interested in the effect of TSA on the recovery of neurological function after cerebral ischemia,using middle cerebral artery occlusion model during a relatively long term of 14 days.We investigated the effect of TSA on the pathogenicchange and neurogenesis and angiogenesis in the early reparative period post ischemia in brain.So that we might provide experimental evidence for TSA clinical application and explore the rationality of optimal therapeutic window with Chinese medicine intervention on cerebral infarction.1.Effects of TSA on the recovery of neurological function after cerebral ischemia in rats and its primary mechanisms1.1 Effects of TSA on the recovery of neurological function and angiogenesis after cerebral ischemia in ratsAim To investigate the effect of TSA on the recovery of neurological function after cerebral ischemia in rats and whether it is involved in the procedure of angiogenesisMethods Cerebral ischemia was induced by middle cerebral artery occlusion in rats which were departed into 6 groups:sham-operated,vehicle-treated,TSA 15mg/kg,30mg/kg,60mg/kg, angongniuhuang 400mg/kg group,Ischemic medication Animals received intragastric administration once a day 3-14day post-ischemia.At the same time rats in sham operation and vehicle group were lavaged with 5%CMC which was used to suspend TSA.Neurological functionwas evaluated with beam-walking,adhesivetape-exposing and bar-grasping performance at 3,7,10,and 14day post ischemia.Animals were killed at 14day post ischemia. HE staining and nissl staining to observe the injury of neuron of sensorimotor region of cortex.Immunohistochemistry methods were used to evaluate the localization and expression of VEGF and VEGFR2 and C D34-labeled MVD of the ischemic boundary zone.Result Vehicle-treated rats showed severe neurological disfuction compared with sham-operated group.Rats receiving TSA at a dose of 30 mg/kg and 60mg/kg had a more rapid recovery of beam-walking and adhesivetape-exposing performance than vehicle-treated rats, and the improvement became significant at 14 days after ischemia(P＜0.05,P＜0.01).There is no significance between TSA treated and vehicle-treated rats on the bar-grasping evaluating. Treatment with TSA 30mg/kg,60mg/kg also significantly improved the expression of the VEGF and VEGFR2 and the micrvascular density(MVD) of the ischemic boundary zone 14days post ischemia.(P＜0.05,P＜0.01).Conclusion The results demonstrate that TSA can improve the recovery of sensorimotor function follwing focal cerebral ischemia in rats.The abitity of TSA might result from its promotion of angiogenesis mediated by VEGF and VEGFR2 after ischemia.1.2.Effects of TSA on the proliferation of neuronal progenitor cell after cerebral ischemia in ratsAim To investigate the effect of TSA on the proliferation of neural progenitor cell after cerebral ischemia in rats and whether it is involved in the procedure of n eurogenesis.Methods Cerebral ischemia was induced by middle cerebral artery occlusion in rats which were departed into 6 groups:sham-operated,vehicle-treated,TSA 15mg/kg,30mg/kg,60mg/kg and angongniuhuang 400mg/kg group,Ischemic medication Animals received intragastric administration once a day 3-14day post-ischemia at the same time rats in sham operation and vehicle group were lavaged with 5%CMC which was used to suspend TSA.BrdU i.p 50mg/kg.d,at 2-7day to label the proliferated cells post ischemia.Animals were killed on day7 and day14.Brains were made into brain sections.Imunofluorescence and immunohistochemistry methods were used to evaluate the proliferation of neuronal progenitor cell in SVZ by Brdu-labeled cells number and the expression of nestin protein of the ischemic boundary zone.Result:Vehicle-treated rats showed increase of Brdu positive cells in the SVZ and the expression of nestin protein compared with sham-operated group.TSA 30mg/kg,60mg/kg increased the number of Brdu positive cells in the SVZ and the expression of nestin protein at day7 and day14 post ischemia(P＜0.05,P＜0.01).Conclusion The results demonstrate that TSA can improve the proliferation of neural progenitor cell follwing focal cerebral ischemia in rats.The ability of TSA might result from its promotion of neurogenesis by upgrading VEGF and VEGFR2 after ischemia.And the promotion of neurological function induced by TSA might come from its effect of neurogenesis promotion.2 Effects of TSA on the recovery of neurological function after permanent cerebral ischemia in rats and its primary mechanismsAim To investigate the effect of TSA o n the recovery of neurological function after permanent cerebral ischemia in rats and whether it is involved in the procedure of angiogenesis and neurogenesis.Methods Permanent Cerebral ischemia was induced by permanent middle cerebral artery occlusion(pMCAO) in rats which were departed into7groups:sham-operated,vehicle-treated, TSA 15mg/kg,30mg/kg,60mg/kg and angongniuhuang 400mg/kg and bicun 7mg/kg group, Ischemic medication Animals received intragastric administration except bicun i.p.once a day 3-14day post-ischemia at the same time rats in sham operated and vehicle group were lavaged with 5%CMC which was used to suspend TSA.BrdU i.p 50mg/kg.d,at 1-7day to label the proliferated cells post ischemia.Neurological functionwas evaluated with beam-walking, adhesivetape-exposing and bar-grasping performance at 3,7 and 14day post ischemia.Animals were partly killed 14days post ischemia.Brains were made into ice brain sections. Imunofluorescence methods were used to evaluate the proliferation,migration and differentiation of neuronal progenitor cell in cortex and stritum of ischemic boundary zone by Brdu/Dcx, Brdu/Neun,Brdu/GFAP labeled cells.Angiogenesis was valued with microvascular density (MVD) labeled by Brdu/factorⅧin cortex and stritum of ischemic boundary zone.Result:Vehicle-treated rats showed severe neurological disfuction compared with sham-operated group.Rats receiving TSA at a dose of 30 mg/kg and 60mg/kg had a more rapid recovery of beam-walking,adhesivetape-exposing and bar-grasping performance than vehicle -treated rats,and the improvement became significant at 14 days after ischemia(P＜0.05,P＜0.01).Vehicle-treated rats showed increase number of Brdu/Dcx,Brdu/Neun,Brdu/GFAP positive cells and MVD labeled by Brdu/factorⅧin the ischemic boundary zone compared with sham-operated group.TSA 30mg/kg,60mg/kg increased the number of Brdu/Dcx positive cells and MVD in ischemic boundary zone of cortex 14days(P＜0.05,P＜0.01) and the number of Brdu/Neun cell in ischemic boundary zone of cortex 28days after ischemia (P＜0.05).There is no significance on Brdu/GFAP labeled cells in cortex and stritum compared with vehicle treated. Conclusion The results demonstrate that TSA can improve the neurological function follwing focal permanent cerebral ischemia in rats.Which might related to the promotion of neurogenesis and angiogenesis and the reestablishing of function of neuron-vascular system improved by TSA.3 Effects of TSA on the signaling pathway of neurogenesis and angiogenesis after permanent cerebral ischemia in rats3.1 Effect of TSA on the regulating pathway of Ang1/Ang2 and VEGF/VEGFR2 in angiogenesis after permanent cerebral ischemia in ratsAim To investigate the effect of TSA on the expression of Ang1/Ang2 and VEGF/VEGFR2 in angiogenesis after permanent cerebral ischemia in ratsMethods Permanent Cerebral ischemia was induced by permanent middle cerebral artery occlusion(pMCAO) in rats which were departed into7groups:sham-operated,vehicle-treated, TSA 15mg/kg,30mg/kg,60mg/kg and angongniuhuang 400mg/kg and bicun 7mg/kg group, Ischemic medication Animals received intragastric administration except bicun by i.p.once a day 3-14day post-ischemia at the same time rats in sham operated and vehicle group were lavaged with 5%CMC which was used to suspend TSA.Animals were killed at 3,7 and 14 days post ischemia.ELISA methods were used to evaluate the expression of Ang 1 and/Ang2 of ischemic cortex.3,7 and 14 days post ischemia.Western blot methods were used to evaluate the expression of VEGF and real time PCR methods were used to evaluate the expression of VEGF mRNA and VEGFR2 mRNA of ischemic cortex 7 and 14 days post ischemia.Result:the expression of Ang1 of Vehicle-treated rats decreased compared with sham-operated group 3,7 and 14 days post ischemia.Rats receiving TSA at a dose of 30 mg/kg and 60mg/kg and angongniuhuang group showed a significant increase of expression of Ang1 at 7days post ischemia compared with vehicle-treated rats(P＜0.05,P＜0.01),and the expression of Ang2 was not significant difference within all groups at 3,7 and 14 days post ischemia.TSA at a dose of 60mg/kg significantly increased the expression of VEGFR2mRNA but had no effect on the expression of VEGF and VEGFmRNA. Conclusion The results demonstrate that TSA can improve the neurological function follwing focal permanent cerebral ischemia in rats.Which might related to the upregulating of the expression of Ang1 and VEGFR2mRNA and the angiogenesis induced by Ang1.3.2 Effect of TSA on the Wnt/β-catenin signaling pathway after permanent cerebral ischemia in ratsAim To investigate the effect of TSA on the expression ofβ-catenin,c-myc,GSK3βin the Wnt/β-catenin signaling pathway after permanent cerebral ischemia in rats.Methods Permanent Cerebral ischemia was induced by permanent middle cerebral artery occlusion(pMCAO) in rats which were departed into7groups:sham-operated,vehicle-treated, TSA 15mg/kg,30mg/kg,60mg/kg and angongniuhuang 400mg/kg and bicun 7mg/kg group, Ischemic medication Animals received intragastric administration except bicun by i.p.once a day 3-14day post-ischemia at the same time rats in sham operated and vehicle group were lavaged with 5%CMC which was used to suspend TSA.Animals were killed at 3,7 and 14 days post ischemia.ELISA methods were used to evaluate the expression of GSK3βof ischemic cortex.3,7 and 14 days post ischemia.Western blot methods were used to evaluate the expression ofβ-catenin and c-myc and real time PCR methods were used to evaluate the expression ofβ-catenin mRNA and c-myc mRNA of ischemic cortex 7 and 14 days post ischemia.Result:The expression of GSK3βof Vehicle-treated rats increased in 3-7day and decreased in 14days post ischemia compared with sham-operated group.Rats receiving TSA group showed no significant of expression of GSK3βat 3-14days post ischemia compared with vehicle-treated rats.The expression of c-myc of vehicle-treated rats significantly increased 7days post ischemia compared with sham-operated group(p＜0.01).TSA 60mg/kg significantly increased the expression of c-myc(p＜0.01) and TSA 30mg/kg significantly increased the expression of c-mycmRNA7days post ischemia compared with vehicle-treated.TSA30mg/kg,60mg/kg significantly increased the expression ofβ-cateninmRNA (p＜0.05) 7days post ischemia compared with vehicle-treated.The expression of c-myc, c-mycmRNA andβ-catenin,β-catenin mRNA of vehicle-treated decreased compared with sham-operated group 14days post ischemia.TSA at all dose group significantly increased the expression ofβ-catenin mRNA and c-mycmRNA compared with vehicle-treated(P＜0.05) 14days post ischemia and the increased trend is same betweenβ-catenin mRNA and c-mycmRNA.Besides TSA 60mg/kg group significantly increased the expression ofβ-catenin and c-myc compared with vehicle-treated(P＜0.05) and the increased trend is same betweenβ-catenin and c-myc 14days post ischemia.Conclusion The results demonstrate that TSA can stimulatethe the Wnt/β-catenin signaling pathway after permanent cerebral ischemia in rats by upregulating the the expression ofβ-cateninmRNA andβ-catenin protein.Which might related to the promotion of angiogenesis and neurogenesis procedure and neurological function recovery follwing focal permanent cerebral ischemia in rats.The innovation of the research1.Thinking of the situation that most stroke patients miss the best theropetic window and leave Sequelae,in the study,TSA was adopted to intervene in the early recovery stage of neurological function in animal models after cerebral ischemia to explore the effect of TSA on the reconstruction of brain function.The timepoint of the intervention is a novelty and also is a positive complement of drug intervention on the subacute and early stage of recovery of cerebral ischemia.2.The researcher observed the behavioral performance of neurological deficit and detect the level of VEGF,VEGFR2 and Angiopoietin in angiogenesis-regulated path way andβ-catenin,GSK3-β,c-myc,in neurogenesis-regulated classic Wnt/β-catenin signal pathway to explore the effect and mechanism of TSA on neurogenesis and angiogenesis after cerebral ischemia in rats.