Effects And Mechanisms Of Tetrahydroxystilbene Glucoside On Cerebral Ischemia Injury And Learning-memory

Partâ… Protection by tetrahydroxystilbene glucoside against cerebralischemia injuryBackgroud: Ischemic brain injury is considered as one of the leading causes of death andadult disability for its high mortality rate in many countries. Reactive oxygen/nitrogenspecies (ROS/RNS) have been considered important mediators of the brain damage afterischemia/reperfusion injury. Oxidative stress generation during ischemia/reperfusioncontributes to a disturbed membrane function, results in a critical intracellular calciumaccumulation and leads to cell apoptosis and death by triggering various critical cellularsignal transduction pathways such as c-Jun N-terminal kinase (JNK), an importantsubgroup of the mitogen-activated protein kinase (MAPK) superfamily which is activatedby oxidative stress. Oxidative stress generation during ischemia/reperfusion also leads toactivation of nuclear transcription factor-κB (NF-κB), which upregulats expression ofinductible nitric oxide synthase (iNOS) and results in the increase of nitric oxide (NO)production and subsequently the increase of peroxynitrite formation. Tetrahydroxystilbeneglucoside (TSG), an active component of the rhizome extract from Polygonum Multiflorum,exhibits anti-oxidative and scavenges free radical effects. However, whether TSG hasneuroprotecive effects on cerebral ischemia injury remains unkown. Therefore, in this study,we investigated the neuroprotective effects of TSG on ischemia/reperfusion brain injury aswell as the underlying mechanisms.Methods: An in vitro ischemic model of oxygen-glucose deprivation followed byreperfusion (OGD-R) was established. Colorimetric assay methods of 3-(4, 5-Dimeth-ylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) and lactate dehydrogenase (LDH)and staining method of Hoechst were used to measure the cell viability and apoptosis. Cytoimmunity fluorescence and flow cytometry were employed to evaluate the levels ofROS and mitochondrial membrane potential (MMP) and activation of NF-κB. Intracellularcalcium concentration ([Ca²⁺]_i) was monitored using the fluorescent Ca²⁺ indicatorfura-2/AM by calcium imaging technique. The expressions of apoptosis-related proteins(Bcl-2 and Bax), stress-activated protein of JNK and anti-aging protein of SIRT1 weremeasured by western blotting and real-time PCR analysis. An in vivo ischemic model ofmiddle cerebral artery occlusion (MCAO) was established. Staining method of2,3,5-triphenyltetrazolium chloride (TTC) was used to assay the area and volume ofcerebral infarct. Tunel assay was used to measure the extent of apoptosis in cerebral cortex.Results: The viability of the cells exposed to OGD for 1 h, 2 h and 4 h followed by 24-hreoxygenation decreased time-dependently compared with the control, respectively. Thus,the protocol of 2-h OGD followed by 24-h reoxygenation was selected for furtherexperiments in the present study. When compared with the control, OGD-R inducedneuronal injury determined by MTT, LDH and Hoechst staining, intracellular ROSgeneration and mitochondrial membrane potential dissipation, which were reversed by TSG.The elevation of H₂O₂-induced [Ca²⁺]_i was also attenuated by TSG. The inhibition of JNKand Bcl-2 family related apoptotic signaling pathway was involved in the neuroprotectionof TSG. Meanwhile, TSG inhibited iNOS mRNA expression induced by OGD-R, whichmay be mediated by the activation of SIRT1 and inhibition of NF-κB activation. In vivostudies further demonstrated that TSG significantly reduced the brain infarct volume andthe number of positive cells for Tunel staining in the cerebral cortex when compared toMCAO group.Conclusion: Our study indicated that TSG protected against cerebral ischemia/reperfusioninjury through multifunctional cytoprotective pathways including decrease of ROSgeneration, attenuation of intracellular calcium overload, downregulation of apoptoticprotein Bax expression and stress-activated protein phospho-JNK level, activation ofanti-aging protein SIRT1 and inhibition of NF-κB activation. Partâ…¡Tetrahydroxystilbene glucoside promotes hippocampal synapticplasticity and learning-memory of miceBackgroud: Impaired cognition and memory associated with aging-related neurode-generativediseases such as Alzheimer's disease (AD), vascular dementia (VD) andParkinson's disease (PD) have become a large public issue with the increasing elderlypopulation. Tetrahydroxystilbene Glucoside (TSG), an active component of the rhizomeextract with polyphenolic structure from Polygonum Multiflorum, which has been widelyused in the Orient as a tonic, anti-oxidative and anti-aging agent since ancient times. Recentstudies have demonstrated that TSG has the ability of changing the ultrastructure ofhippocampal synapses and enhancing learning-memory in both APP transgenic mice andaged rats. However, whether TSG can affect physiological synaptic transmission orimprove learning and memory as well as the related mechanisms in normal animal remainsunknown. In the present study, we investigated the roles of TSG on long-term potentiation(LTP) in the CA1 region of the hippocampal slices and cognitive behavioral performancesin intact adult mice as well as the underlying mechanisms.Methods: Acute isolation of hippocampal slice in mouse, field potentials recordings andwestern blotting analysis were used to evaluate the effects and mechanisms of TSG on LTPin CA1 region of hippocampal slices. Behavioral tests of Morris water maze and step-downfear conditioning were applied to investigate the effects of TSG on learning and memory.Results: TSG enhanced the N-Methyl-D-Aspartate (NMDA) receptor dependent CA1-LTPin a dose-dependent manner, with a maximal effective dose at 1μM. The concentrations ofTSG used in this study did not affect the basic synaptic transmission, PPF and input-outputcurves. The enhancement of TSG-induced LTP required calcium/calmodulin-dependent protein kinaseâ…¡(CaMKâ…¡) and extracellular signal-regulated kinases (ERKs) activation.Behaviorally, TSG-treated mice performed significantly better than that in the controlgroup in Morris water maze and in step-down fear conditioning. Furthermore, increasedpostsynaptic density was found in TSG-treated group by electron microscopy.Conlusion: Our data demonstrate that TSG promotes LTP in CA1 region of hippocampalslices and enhances memory in mice, which positively correlates with the increase of theactivities of plasticity-related proteins such as CaMKâ…¡and ERK1/2 and change theultrastructure of hippocampal synapses.