Effects And Mechanisms Of Saponins From Aralia Taibaiensis And Its Components In The Treatment Of Stroke

Stroke is one of the three major threats to human health and has been the leading cause of death and disability among adults in China.To date,more and more young people are seized with strokes in which ischemia stroke accounts for 60-80%.In clinics,thrombolytic therapy is mainly used for revascularization in the treatment of ischemic stroke,but the narrow therapeutic time window limits the benefits at 2.4% in Chinese patients.Although some therapeutic effects can be achieved through the thrombolysis performed in an extended therapeutic time window,it may result in reperfusion injury called as cerebral ischemia/reperfusion injury(CI/RI).In general,ischemic stroke causes cell death in the ischemic central zone,whereas cells damaged in the ischemic penumbra can be rescued.Thus,it is great important in the treatment of stroke to protect the ischemic penumbra tissue and cells and to prevent development of the damage.Aralia taibaiensis is a special herb which is mainly growing in Taibai mountain,one of the “Three major medicine mountains” in China.The main components in Aralia taibaiensis are saponins which are named as saponins from Aralia taibaiensis(sAT).It has been reported to have various functions such as antioxidant,anti-inflammation and anti-apoptosis,et al.However,the neuroprotective effects of s AT against CI/RI have not been clarified.In addition,the possible mechanisms and effective components have not been reported.Objective: 1.To investigate the effects of sAT against CI/RI,especially,to reveal the effects on two key pathological mechanisms of CI/RI(oxidative stress and mitochondrial dysfunction);2.To clarify the possible mechanisms of s AT against CI/RI,and to further identify the effective saponin monomers in sAT based on rapid screening assays.These results will provide theoretical and experimental basis for the sAT to be used in the treatment of stroke.Methods: To investigate the protective effects of sAT against CI/RI,we performed in vivo experiments by using rat middle cerebral artery occlusion/reperfusion(MCAO/R)model,and in vitro assays by using HT22 mouse hippocampal neuron cells under oxygen and glucose deprivation/reoxygenafion(OGD/R).To predict the potential targets of sAT,network pharmacology was explored.Based on the results obtained from the network pharmacology,we investigated exprssions of the proteins involved in oxidative stress and mitochondrial function.Western blot was performed to detect expressions of the acetylation or phosphorylation levels of AMP-activatedprotein kinase(AMPK),protein kinase B(Akt),peroxisome proliferator-activated receptor ? coactivator-1?(PGC-1?),forkhead box protein O 3a(FOXO3a)and their downstream proteins.SiRNA and specific agonists or inhibitors were used to further verify the crosstalk relationship among the proteins.To identify the upstream protein of AMPK/Akt,we performed siRNA and Western blotting to study the effects of sAT on Apelin 13.To identify the upstream proteins of Apelin 13,we focused on the signaling pathways of P38 MAPK/ATF4 and HIF-1? pathways.Firstly,the effects of sAT on the expression levels of P38 MAPK,ATF4 and HIF-1? were investigated.Secondly,the specific inhibitor(SB203580)and activator(anisomycin)of P38 MAPK,and siRNA for HIF-1? or ATF4 were used to demonstrate the up-regulation effects on Apelin 13 and neroprotecitve effects of sAT.To understand the possible effective substances of sAT,we carried out a rapid screening assays on the saponin monomers involved in sAT.RT-PCR and fluorescent reporter cell screening models were used to measure the effects of different saponins on the transcriptional activity of HRE and C/EBP?.Finally,HIF-1? siRNA and the specific activator or inhibitor of P38 MAPK was used to verify whether the saponins screened could up-regulate Apelin 13 through regulating P38 MAPK/ATF4 or HIF-1?.(Part VI)Results 1.Pharmacological studies.In vivo,MCAO/R increased the cerebral infarction(46.6 ± 2.86% vs 0,P<0.01),cerebral edema(85.13 ± 1.36% vs 77.6 ± 4.03%,P<0.01),neurological dysfunction(4.33 ± 0.82 vs 0,P<0.01),while sAT inhibited the cerebral infarction(34.27 ± 3.69%,26.97 ± 2.15%,17.57 ± 2.72% vs 46.6 ± 2.86%,P<0.01),cerebral edema(81.97 ± 1.36,79.9 ± 2.29,77.97 ± 2.46 vs 85.13 ± 1.36,P<0.01),neurological dysfunction(3.5 ± 1.05,2.67 ± 1.03,1.0 ± 0.89 vs 4.33 ± 0.82,P<0.01),oxidative stress(P<0.01)and cell apoptosis(P<0.01).In vitro,OGD/R inhibited cell viability(0.46 ± 0.09 vs 1 ± 0,P<0.01),increased LDH release(149.6 ± 14.19 vs 39.63 ± 8.89,P<0.01),induced cell apoptosis,mitochondrial dysfunction and ROS accumulation,and sAT inhibited cell injuries,increased the antioxidant proteins levels and ATP levels,and improved the mitochondrial function.These results indicated that sAT had protective effects against CI/RI and the possible mechanisms might involve reducing oxidative stress and improving mitochondrial function.2.31 saponins monomers were collected from sAT,and 415 targets were predicted bansed on the structure.389 targets were screened for sroke.72 conmmon targets were screnned based on the Venny.KEGG analysis obtained 248 biological processes,32 cellular components and 63 molecular functions.According to the 104 KEGG pathways,the possible targets of sAT,including TNF,PI3K/Akt,HIF-1,MAPK,FoxO,Toll-like receptor,AMPK and calcium related signalling pathway,et al.Among these,PI3K/Akt,AMPK,MAPK,HIF-1 and FoxO pathways played important roles in modulating oxidative stress and mitochondrial function.Thus,these pathways were studied further in the next chapter.3.s AT could inhibite oxidative stress and mitochondrial dysfunction through regulating SIRT1 by AMPK/Akt.sAT could increase the expressions of phosphorylated AMPK and Akt,the expression level of SIRT1,as well as the deacetylation and phosphorylated FOXO3 a and PGC-1?.By using siRNA interference experiments,sAT was demonstrated to increase SIRT1 expression through activating AMPK/Akt,then regulate FOXO3 a and PGC-1? activities and thus inhibited oxidative stress and mitochondrial dysfunction.4.s AT could modulate Apelin 13/AMPK/Akt through regulating P38MAPK/ATF4 and HIF-1?.Compared with model group,sAT increased Apln gene expression levels significantly(2.26,2.73 and 5.31 fold of model)in different groups,meantime,Aplnr gene expression levels were increased significantly(1.5,2.75 and 3.27 fold of model)in different groups.We also found sAT increased the proteins expression levels of Apelin 13 in brain cells.The results also showed that Apelin 13 was involved in the neuroprotective effects of sAT by regulating AMPK/Akt and its downstream protein expression.To study the upstream pathways of Apelin 13,P38MAPK/ATF4 and HIF-1? signaling pathway were studied in the further studies.sAT inhibited P38 MAPK phosphorylation and ATF4 expression in an dose dependent manner.OGD/R inhibited Apelin 13 expression by increasing P38 MAPK phosphorylation and ATF4 expression,and s AT reversed these effects.sAT increased the expression of HIF-1?,while siHIF-1? reduced the effects of sAT on Apelin 13 expression,and the protective effects were also abolished by siHIF-1? treatment.These results showed that sAT up-regulated Apelin 13 expression in brain cells through increasing HIF-1? expression and inhibiting P38 MAPK/ATF4 activation.5.Screening results of sAT.Results of RT-PCR experiments showed that 12 saponin monomers had significant regulation effects on Apln gene expression.Among these,6 saponin monomers(No.10,14,15,15,19,20,and 21)had significant effects on HRE transcriptional activity.These 6 saponins induced the expression of Apelin 13 through up-regulating HIF-1?,which was demonstrated in Western blotting and siRNA interference experiments.5 saponin monomers(No.10,11,14,19,and 21)showed significant effects on C/EBP? transcriptional activity.The inhibitor and activator treatments revealed that No.10,14 and 19 saponin monomer regulated Apelin 13 expression through targeting P38 MAPK/ATF4.No.10(araloside A),No.14(deglucose-chikusetsu sapon Iva)and No.19(chikusetsu sapon Iva)increased the expression of Apelin 13 through modulating both P38 MAPK/ATF4 and HIF-1a.No.15(chikusetsu sapon Ib),No.20(elatoside F),No.21(pseudoginsenoside RT1)increased Apelin 13 expression through regulating HIF-1?.No.11(aralia saponin D)and No.21(ginsenoside RT1)showed significant effects on C/EBP? transcription activity and upregulating effects on Apelin 13 expression,but no effect on P38 MAPK/ATF4.This suggested that they might regulate C/EBP? transcription activity in direct way or in other pathways.Conclusion In this study,the protective effect of sAT against CI/RI was firstly systematically studied,and the possible neuroprotective mechanisms of sAT were elucidated by network pharmacology and molecular biology experiments.Based on this,the saponin monomer components involved in sAT were screened and studied further.The results showed that saponin monomer components regulated the expression of Apelin 13 through different mechanisms to regulate AMPK/Akt/SIRT1 pathway,to inhibit oxidative stress and mitochondrial dysfunction,thus resulting in protection effects against the brain injury.These results revealed the possible mechanisms and effective components of sAT,and provided theoretical and experimental basis for sAT in the treatment of stroke.