Study On The Anti-ischemic Stroke Effects And The Underlying Mechanisms Of The Active Substances In Polygonum Multiflorum Thunb.

Stroke is a leading cause of death and disability worldwide.Ischemic stroke,accounting for almost 87% of stroke cases,is caused by occlusion of a major cerebral artery.The pathophysiology of ischemic stroke involves a series of detrimental signaling cascades.Oxygen and glucose deprivation leaded to immediate failure of energy-dependent ion pumps and channels,resulting in the release of excitatory neurotransmitters and subsequent vulnerable neurons death.The oxidative and nitrosative stress are further accelerated following reperfusion,inducing ischemia and reperfusion(I/R)injury.Despite advances in understanding the stroke pathophysiology,successful treatment remains a major challenge in clinic.Reperfusion with recombinant tissue plasminogen activator(rt-PA)remains the only pharmacological therapy.Drugs/strategy targeting individual gene or protein have been proved limited efficacy or ineffective clinically.Discovering multiple-target/pathway candidates from natural products of Traditional Chinese medicine to treat ischemic stroke is therefore important and urgent.Dating back to AD 812 years ago,Polygonum multiflorum Thunb.(PMT)was recorded as an extremely tonic medicine that could “benefit for the essence,strong your spirits,protect your beauty,black your hair,and extend your life”(Polygonum multiflorum Biography).Modern pharmacological studies showed that PMT and its isolated bioactive compounds exerted a wide spectrum of pharmacological effects,including anti-aging,anti-oxidation,anti-inflammatory.In our previous study,the crude extract of PMT exerted excellent anti-stroke activity.However,there are still insufficient data on the active substances of PMT and the underlying molecular mechanisms for treating ischemic stroke.Thus,our purpose aims at elucidating the active substances and corresponding mechanisms to provide theoretical basis for its application.Firstly,network pharmacology was applied to reveal PMT active substances against ischemic stroke.PMT compounds displayed multiple-target characteristics and the key 25 active substances mainly included flavones,anthraquinones and stilbene glycosides.The“active compound–target” network implicated in disclosing active compounds and providing targeted proteins.Molecular docking results verified the binding capacity between polydatin/stilbene glycoside(TSG)and adenosine A1 receptor(adora1),7.96 and 7.71 kcal/mol respectively,which is worthy of further study.PMT extracts and compounds were screened on models of oxygen-glucose deprivation/recovery(OGD/R)and excitatory amino acid(glutamate)toxicity tests.The 50%ethanol extract of PMT had the best anti-OGD/R activity,probably due to the main ingredient TSG.Stilbene glycosides E-TSG,Z-TSG and polygonimitin C and flavone tricin showed good potential of anti-OGD/R and anti-glutamate toxicity by significantly increasing cell viability to80% in treatment groups compared to OGD/R group.Z-TSG was an isomer of E-TSG(TSG namely)which accounted for 90%;polygonimitin C was structurally similar to TSG with adding one more glycoside.Thus,TSG was considered as the core active substance of PMT in treating ischemic stroke.An I/R model of middle cerebral artery occlusion(MCAO)in SD rats was used to characterize the neuroprotective effects of TSG in vivo.The pharmacokinetics of TSG in healthy/MCAO rats were analyzed on LC-MS instrument.TSG markedly decreased mortality,neurological deficit score,cerebral infarct size and brain water content of MCAO rats,ameliorated I/R induced oxidative stress injury,and improved blood supply dring ischemia.Moreover,the pharmacokinetic results revealed the material basis of TSG as a neuroprotective agent: the concentration of TSG in ischemic hemisphere reached 123.0 ng/m L in MCAO rats,significantly higher than that in control/non-ischemic hemisphere.The mechinisms underlying TSG against ischemic stroke were scarce till now.1H-NMR based metabolomics analysis gave rich information and clues on TSG targets.TSG markedly ameliorated disorders in purine metabolism,energy metabolism and antioxidative defense system in MCAO rats.Adenosine A1 receptor(A1R)is a purine receptor significantly associated with purine metabolism and energy metabolism,and is one of the high-scoring targets of TSG verified in network pharmacology.In vivo/vitro,adenosine A1 receptor(A1R)antagonist DPCPX abolished TSG neuroprotective effects;TSG and adenosine(an endogenous A1 R agonist)could stimulate p ERK1/2,while DPCPX decreased p ERK1/2 and inhibited its upregulation by TSG.Thus,TSG might be an A1 R agonist.Docking and binding assays confirmed the binding of TSG with A1 R.In addition,TSG upregulated the A1 R level lowered by I/R.The downstream signals of A1 R activation,ERK1/2,HIF-1α and NF-κB contributed to the neuroprotection of TSG.Moreover,void of “well-known” cardiovascular side effects of classical A1 R agonists,TSG showcased its great potential for stroke treatment.