| Among all diseases of dementia, vascular dementia represents the first cause indeveloping countries. To date, mankind has yet found an effective treatment to preventvascular dementia, and good means of prevention and treatment for it is little. Chroniccerebral ischemia (CCI) is an important component in the study of the pathogenesis ofvascular dementia. And it was a common pathological process of vascular dementia,Alzheimer’s disease and other neurodegenerative diseases, Binswanger disease, which couldlead to progressive cognitive impairment. The pathophysiological mechanisms for process ofchronic cerebral ischemia lead to cognitive impairment remain unclear, which need to beaddressed urgently.Role of cell signaling pathway dysfunction and oxidative stress in brain ischemia,dementia caused by Alzheimer’s disease and other degenerative diseases have beenrecognized. Treatments of some antioxidants have benefited patients suffered fromdementia. Cellular signal transduction systems control growth, differentiation and death ofcells, and take part in regulation of cell activity. It is well known that many diseases arecaused by the defects of the signaling pathways. Reconstruction of signaling pathwaysunder pathological state will help people learn more about the etiology and pathogenesis ofthe diseases, and provide opportunities to find new ways for disease treatment meanwhile.As a NAD+-dependent deacetylase, SIRT1(silent information regulator1)impactspotentially in different important biological processes, such as cell differentiation,senescence, apoptosis, circadian rhythm, metabolic control, transcriptional regulation, signaltransduction, oxidative stress. Recently studies have revealed that SIRT1played aneuroprotective effect through anti-oxidation, anti-inflammatory, reducing apoptosis andother mechanisms in oxygen and glucose deprivation experiments in vitro and in acutecerebral ischemia and ischemic preconditioning animal models. Evidence also showed SIRT1was a critical regulator of the expression of genes involved in postnatal angiogenesisin endotheliocyte, which suggesting that SIRT1may play an important role under conditionsof chronic cerebral ischemia.This study was to investigate the role of SIRT1signaling pathway on cognitiveimpairment caused by chronic cerebral hypoperfusion. For this, a rat model of chroniccerebral ischemic dementia induced by permanent bilateral common carotid arteryocclusions(2-VO) was established. Using the Morris water maze, learning and memoryabilities of the rats were evaluated. Serial changes of SIRT1expression and activity wasmeasured by immunohistochemistry and Western blot. After that, resveratrol and EX-527were used to regulate activity of SIRT1in animal model, and expression of SIRT1togetherwith its target genes including phospho-FOXO3α(Ser253) and PGC-1α were detected byimmunohistochemistry, real-time PCR and Western blot. Impact on Oxidative stress ofSIRT1signaling pathway was also observed in the present study. The study was to provide atheoretical basis for SIRT1signaling pathway on role of anti-vascular cognitive impairment.1Establishment rat model of chronic cerebral ischemia and assessment of cognitivefunctionHealthy Male Wistar rats(aged2-3months, weighing260-300g) were randomlydivided into the sham operated group and chronic cerebral ischemia group for6weeks. Ratmodel of spatial learning and memory impairment was induced by permanent bilateralcommon carotid artery ligation (2-VO). Morris water maze was used to evaluate learningand memory abilities of rats. Place navigation test was carried out twice with an interval of15minutes for5consecutive days, and the escape latency was measured to evaluateanimals’ ability of learning and memory. Spatial probe test was performed on day5afterplace navigation test. Total time for rats spent in crossing the original quadrant where thehidden platform located was determined. Six weeks after2-VO rats were decapitated, andthe frontal cortex and hippocampus were collected for pathological assessment byhematoxylin-eosin(HE) staining. Experimental results showed that mortality rate of2-VOrats was22.7%, similar to ones reported in the literature. In place navigation test the escapelatency of sham operated rats decreased rapidly and leveled off and spatial localization for hidden platform occurred mainly in the first two trial days. CCI rats spent four days inlearning to find the hidden platform and escape latency on day5was prolonged again. Inspatial probe test time spent in crossing the original quadrant of2-VO rats was significantlyless than the sham operated ones. HE staining of2-VO rats showed scattered ischemicchanges in the frontal cortex and hippocampus, such as decrease in the number of normalneurons, loss of neural cells, disorder of organization arrangement, irregular cellmorphology and disorganized cell arrangements, degeneration and necrosis of neurons,scattered eosinophils, uneven vacuoles in the cytoplasm, nuclear pyknosis, and unclearnucleoli. In sham operated group structure of organization was intact, cells arranged orderly,no apoptosis and degeneration and necrosis of neurons was found, and cytoplasm andnucleolus normal. The results above suggested that cognitive dysfunction of rat modelinduced by chronic cerebral ischemia was established successfully, and2-VO rat modelcould well display the behavioral and pathological process of chronic cerebral ischemia,which could cause significant cognitive impairment reliably.2Changes of SIRT1gene expression and level of oxidative stress in rat model ofchronic cerebral ischemiaHealthy male Wistar rats were grouped as follows:(1)sham operated for6weeks(2)sham operated for9weeks (3)sham operated for12weeks (4)chronic cerebral ischemiafor6weeks (5)chronic cerebral ischemia for9weeks (6)chronic cerebral ischemia for12weeks. Levels of SIRT1protein expression were examined by western blot andimmunohistochemical staining via SP method. SOD and MDA levels in hippocampus andfrontal lobe were examined according to the corresponding kit instructions.In Morris water maze cognitive impairment of2-VO rats showed dynamic changes withthe ischemia time prolonged.6weeks after operation escape latency of2-VO rats lengthenedsignificantly on day2,3, and5compared to sham operated ones.9weeks after operationescape latency of2-VO rats extended significantly on day2to5. In the whole trial escapelatencies of2-VO rats were longer than sham operated ones significantly12weeks afteroperation. time spent in crossing the original quadrant in CCI group gradually reduced as theischemia time extended and which was less significantly than the sham group. HE staining from brains of the consecutive three months2-VO rats demonstrated that loss of neuron anddegeneration and necrosis gradually increased with the ischemia time extended, proliferationof astrocytes and vessels gradually increased, organizational structure was disordered andirregularly arranged. The cells were severely deformed, uneven vacuoles gradually increasedin cytoplasm, karyopyknosis increased, and small liquefaction lesion could be seen in whitematter areas. Results of immunohistochemistry showed that SIRT1protein was locatedmainly in neulei. After2-VO the ratio of SIRT1-positive cells was significantly reduced,especially six weeks after operation. Western blot showed that SIRT1protein expressionlevels decreased significantly, which was lowest6weeks after operation. Reduction of SODas an antioxaditive substance and increase of MDA as a markers of lipid oxidation wereobserved at the three time point after operation, but the significant alteration occurred only at6weeks after2-VO. These results suggested that2-VO could cause progressive cognitiveimpairment in rats, effectively demonstrated the clinical course of vascular dementia, anddownregulation of SIRT1was involved in the development and progression of cognitiveimpairment induced by chronic cerebral ischemia. Oxidative stress in chronic cerebralischemia occurred mainly in the early stages of ischemia six weeks, which provided a basisfor selection of time point in the follow-up experiment of oxidative stress intervention.3Impact of SIRT1signaling pathway on cognitive function and oxidative stress of ratssubjected to chronic cerebral ischemiaHealthy male Wistar rats were grouped as follows:(1)sham operated(SH) group(2)chronic cerebral ischemia(CCI) group (3)resveratrol+chronic cerebral ischemia(Res)group (4)resveratrol+EX-527+chronic cerebral ischemia(Res+EX-527) group (5)EX-527+chronic cerebral ischemia(EX-527) group. Resveratrol was administered by intraperitonealinjections at a dose of50mg/kg daily for6weeks. EX-527was was injected to the lateralventricle at a dose of5μg.6weeks after2-VO learning and memory ability of all rats weredetected via the Morris water maze. Expression levels of SIRT1, FOXO3α and PGC-1α wereexamined through real-time quantitative PCR and western blot analysis.10%homogenatesupernatant of hippocampus and frontal lobe were centrifuged, and obtained supernatantfluid was used to measure SOD and MDA levels according to the corresponding kit instructions.Behavioral test of water maze showed that the rats’ learning and memory abilities ofRes group were significantly higher than CCI group, expressed as shorter escape latency onday2,3,5, and more time in crossing the original quadrant. In EX-527group rats showedlonger escape latencies on day2to5and less time in crossing the original quadrant than CCIgroup. In Res+EX-527group rats demonstrated longer escape latency and less time incrossing the original platform too. Results of real-time quantitative PCR showed differencesof mRNA levels of SIRT1and PGC-1α among the five groups were not significant.FOXO3αmRNA level was lower in2-VO rats compared to sham operated ones, which couldbe elevated by resveratrol and reduced by EX-527significantly. Immunohistochemicalresults of frontal cortex and hippocampal CA1region showed SIRT1protein was locatedmainly in neulei. A certain mount of SIRT1protein was observed in sham operated group,which was lower in2-VO group. SIRT1protein level could be elevated by resveratrol butcould not be altered by EX-527in2-VO group. Phospho-FOXO3α（Ser253）protein wasobserved mainly in cytoblast. In sham operated group amount of phospho-FOXO3α wasabundant in in the nuclei of the neurons, and little in cytoplasm. Phospho-FOXO3α（Ser253）protein in neurons were reduced significantly in2-VO group, but could be seen in bothcytoplasm and nuclei, and markedly in nuclei. Level of phospho-FOXO3α（Ser253）proteinwas increased by resveratrol in2-VO group which mainly seen in nuclei. In frontal cortex,phospho-FOXO3α（Ser253）protein levels of EX-527and Res+EX-527group were notsignificant different to CCI group，but lower than Res group significantly. In hippocampalCA1region, difference of phospho-FOXO3α（Ser253）protein levels between EX-527andCCI group was significant, and phospho-FOXO3α（Ser253）protein level of Res+EX-527group were not significant different to CCI group，but lower than Res group significantly.Rich expression of PGC-1α protein was detected in sham operated group, which was more incytoplasm than that of nulei. Level of PGC-1α protein was reduced both in nucleus andcytoplasm in2-VO group accompanied by less number of neurons and PGC-1α protein wasseen near the cytomambrane. PGC-1α protein level was increased in Res group and locatedmainly in nuclei. Contrarily PGC-1α protein level was decreased in EX-527and Res+ EX-527group and located mainly in cytoplasm. Western blot results demonstratedresveratrol could strengthen expression of SIRT1protein in2-VO rat brain. Resveratrolcould elevate inhibited phospho-FOXO3α（Ser253）protein level in2-VO rat brain. Althoughphospho-FOXO3α（Ser253）protein levels of EX-527group and Res+EX-527group were notreduced than that of CCI group significantly but lower than that of Res group significantly.The reduced PGC-1α protein level in2-VO rat brain was increased by resveratrol and lowerafter EX-527treatment. Reduced level of SOD and elevated level of MDA in CCI groupwere significantly different to sham operated group6weeks after operation. These changescould be lessened by resveratrol treatment and enlarged by EX-527significantly. Levels ofSOD and MDA were different significantly between Res group and CCI group, EX-527group and CCI group.The results above suggested that SIRT1-FOXO3α/PGC-1α signaling pathway wasinhibited in chronic cerebral ischemic brain. SIRT1could regulate transcription ofFOXO3α but its regulation of PGC-1α expression was mainly on protein level in chroniccerebral ischemic brain. Activity of SIRT1-FOXO3α/PGC-1α signaling pathway could alteroxidative stress reaction in chronic cerebral ischemic brain. Enhanced activity ofSIRT1-FOXO3α/PGC-1α signaling pathway could increase the ability of anti-oxidativestress for rat in chronic cerebral ischemic brain, and effectively improved cognitiveimpairment of CCI rats. Improvement of resveratrol on cognitive dysfunction caused bychronic cerebral ischemia in rats was associated with enhanced ability of anti-oxidativestress mediated by SIRT1signaling pathway. The present study provided laboratoryevidences for its application to the prevention and treatment of vascular cognitiveimpairment, and it was expected to become one of the new drugs in vascular cognitiveimpairment. |
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