L-3-n-butylphthalide Improves Cognitive Function Deficits Through Activation Of PI3K/Akt Cell Signaling Pathway In The Hippocampus Of VaD Mice

As an acquired syndrome of cognitive impairment, vascular dementia(VaD), which is caused by various kinds of cerebral vascular disease,demonstrates mainly as learning and memory dysfunction, accompanied withthe possible disorder of motion, tongue, direction and personality. VaD, as thesecond most common form of dementia after Alzheimer’s disease in the elderpeople, has attracted much attention in recent years. Recently, there isemerging epidemiological evidence that VaD is likely the leading cause ofdementia in Asian population. At present, the pathogenesis of VaD remainsunclear, but increasing evidence suggests that VaD is associated with a groupof diverse pathologies affecting the cerebrovascular circulation. One ofimportant mechanisms in VaD is the pathophysiology of ischemia/reperfusion(I/R) including excitotoxicity, oxidative stress and inflammatory reactions.Cerebral ischemia can induce the death of cerebral infarction or selectiveneurons in the brain, when ischemia occurred in associated with learning,memory areas, can lead to the occurrence of VaD. Post-IR neurons exhibit twoobvious forms of cell death: necrosis and programmed cell death whichinclude apoptosis and autophagy. Previous researches showed that there mightbe a continuum between autophagy and apoptosis. Hence the extent of thesalvage of apoptosis and autophagy might contribute to the outcome of thepathological process. Recently, accumulating evidence has shown that thephosphatidylinositol-3kinase/Akt (PI3K/Akt) cell signaling pathway takespart in the apoptosis after IR. The PI3K/Akt signal transduction pathway is aclassic anti-apoptosis pathway, promoting the survival signal transduction. Itsactivation in the nerve cells, especially in hypoxic ischemic neuronal injurycells, plays an important role, and has received extensive attention. In recentyears, it found to be closely related with the regulation of autophagy. The neuroprotective role of PI3K/Akt in cerebral ischemia has been widely studied.PI3K/Akt survival signaling pathway regulate cell survival and are importantin the pathogenesis of degenerative diseases, activation of the Akt/proteinkinase B (PKB) kinase pathway can be neuroprotective after stroke throughblocking neuronal death after stroke. Akt/mTOR signal pathway inducedNF-κB (nuclear factor kappa B) activation pathway and Bcl-2and Baxbalance adjustment play an important role in nerve cells inflammatoryresponses and apoptosis of ischemic cerebrovascular disease. Akt may rescuecells from apoptosis by inhibiting the Bax-dependent apoptosis pathwaythrough a forkhead box transcription factor.3-n-butylphthalide is successfully synthesized to treating ischemic strokewith independent intellectual property rights of a class of chemical drugs inour country. The first is the L-isomer extracted originally from celery seed byYANG Jun-shan, after the artificial synthesis of racemes, also known asbutylphthalide. As the yellow oily liquid, butylphthalide has a celery flavor.3-n-butylphthalide (NBP) has three different stereo isomers known as l-, dl-,and d-NBP. A large number of studies have demonstrated that dl-NBP is ableto be a neuroprotective drug for the treatment of ischemic stroke throughmultiple mechanisms such as improving energy metabolism, reducingoxidative damage, improving microcirculation in arterioles, decreasingneuronal apoptosis, improving mitochondrial function, and inhibitinginflammation. Although the positive effects of NBP on cerebral infarct andAlzheimer’s disease have been verified in clinic trials and animal researches,few studies has investigated whether NBP could be beneficial as a treatmentfor VaD. A previous study discovered that only l-NBP ameliorated thecognitive impairment induced by chronic cerebral hypoperfusion while d-anddl-NBP did not show significant beneficial effects. Furthermore, l-NBPreduces β-amyloid-induced cell death in neuronal cell cultures, and improvescognitive performance in an animal model of Alzheimer’s disease. However,the effects of l-NBP in this area are still not clear.Therefore, this study set out to examine whether treating with l-NBP and pretreating with l-NBP have the ability to improve cognitive impairment inVaD mice induced by cerebral repetitive IR. Furthermore this study explorewhether activating PI3K/Akt cell signaling pathway is one of the mechanismsof l-NBP providing the protective effect on VaD mice.Part1: The establishment of the vascular dementia mice model and theeffects of l-3-n-butylphthalideObjectives: To observe the behavioral changes and hippocampalpathology changes4weeks after cerebral repetitive ischemia/reperfusion inC57BL/6mice, and to observe if l-NBP can improve learning and memoryimpairment induced by cerebral repetitive ischemia/reperfusion.Methods: C57BL/6mice were randomly divided into five groups: thesham group, IR group(vegetable oil was given daily for28consecutive daysafter IR), Pre-l-NBP group (30mg/kg l-NBP was administered daily for7daysprior to IR), Low-l-NBP group (30mg/kg l-NBP was given daily for28consecutive days after IR) and High-l-NBP group (60mg/kg l-NBP was givendaily for28consecutive days after IR). Mice, except the sham group, weresubjected for continuously three repeated times ischemia-reperfusion throughthe ligation of the bilateral common carotid arteries. In the sham group,protocols were the same as the model group but bilateral common carotidswere only exposed. The capability of learning and memory of mice wereinvestigated by the Morris water maze test and the step-down test, thepathological change of hippocampus in each group was observed by thioninestaining.Results:1Learning and memory ability of Morris water maze test:1.1L-NBP significantly ameliorated learning impairment induced by cerebralrepetitive IR.For the purpose of evaluating whether the mice cognitive function wasaffected after the oral administration of l-NBP, we used the MWM test toevaluate the mice abilities of spatial learning acquisition and memoryretention. There was a marked treatment effect [F(3,50)=10.315，P<0.01] on escape latency, suggesting that l-NBP was protective against impaired spatiallearning in the cerebral repetitive IR mice. The escape latency of IR group wasevidently longer than that of the sham group (P<0.01), suggesting that cerebralrepetitive IR successfully induced learning deficit. Compared with IR group,after treated with30mg/kg l-NBP or60mg/kg l-NBP, the escape latency wasmarkedly decreased from day3to day5(P<0.01, P<0.05). And comparedwith IR group, after pretreated with30mg/kg l-NBP, the escape latency wasmarkedly decreased from day2to day5(P<0.01). These results indicated thatoral administration of l-NBP, especially pretreatment with l-NBP significantlyrescues learning impairment caused by cerebral repetitive IR1.2L-NBP significantly rescued memory deficits induced by cerebralrepetitive IR.To investigate the effects of l-NBP on memory deficits caused bycerebral repetitive IR, we conducted a spatial probe test after the5days oftraining in the water maze. In the test, we discovered that the sham group, thePre-l-NBP group and the High-l-NBP mice were able to show significantpreference for the target quadrant compared with IR group mice (P<0.05,P<0.01).2Learning ability of step-down test:2.1Latency timeThe latency time of IR group are significant shorter than those of shamgroup (P<0.01), that suggest there are impairment of memory formation in IRgroup mice. The latency time of Low-l-NBP, High-l-NBP and Pre-l-NBPgroup are marked longer than those of IR group (P<0.05), that suggest l-NBPcan improve impairment of memory formation.2.2The number of errorThe number of error of IR group were much more than those ofsham-operated group (P<0.05). The number of error of l-NBP-treated groupwere lower than those of model group, but without statistical significance (P>0.05). 3Neuropathological evaluationIn sham mice, the pyramidal neurons in the CA1hippocampus werearranged in sequence with4-5cell layers, intact lineament, full nuclei andclear nucleoli. In the IR group, part neurons died4weeks after IR, which wererepresented by increases of HG (P<0.05) and by significant decreases of ND(P<0.01) compared with sham group. And, there was significant improvementin l-NBP groups as compared to those in the IR group (P<0.05).Conclusions:1This study has successfully established C57BL/6VaD mouse model bythree repeated times ischemia-reperfusion through the ligation of the bilateralcommon carotid arteries. Mice, treated with l-NBP, especially pre-treated withl-NBP significantly attenuated learning and memory impairment induced bycerebral repetitive IR.2L-NBP might improve learning and memory ability of mice byattenuating the injury of hippocampus neurons.Part2: The effects of PI3K agonists and inhibitors on cognitive functionin mice with vascular dementiaObjectives：To observe the effects of PI3K agonists and inhibitors oncognitive function in VaD mice.Methods: C57BL/6mice were equally and randomly divided into fivegroups: the sham group, IR+IGF-1group, IR+DW group, IR+LY294002group and IR+DMSO group. Mice, except the sham group, were subjected forcontinuously three repeated times ischemia-reperfusion through the ligation ofthe bilateral common carotid arteries. In the sham group, protocols were thesame as the model group but bilateral common carotids were only exposed.The capability of learning and memory of mice were investigated by theMorris water maze test at4weeks after operation. The expression of Akt andp-Akt in hippocampus were studied by immunohistochemistry and WesternBlot technique.Results:1Learning and memory ability of Morris water maze testThe escape latency of IR+DW group, IR+LY294002group and IR+DMSO group was evidently longer than that of the sham group andIR+IGF-1group[F(4,45)=9.702，P<0.01].Compared with IR+DW, IR+LY294002and IR+DMSO group mice, thesham and IR+IGF-1group mice were able to show significant preference forthe target quadrant (P<0.01).2Protein expression of LC3B in hippocampus by Western blotThere is no marked change in total Akt among five groups (P>0.05) at4weeks after operation. However, compared with the sham group, p-Akt weresignificantly increased in the IR+DW、 IR+IGF-1、 IR+DMSO andIR+LY294002group(P<0.01). Moreover, IR+IGF-1group mice showed astrong expression of p-Akt compared with the IR+DW、IR+DMSO andIR+LY294002group mice (P<0.05). P-Akt were significantly decreased in theIR+LY294002group compared with those of IR+DMSO group (P<0.05)Conclusions:PI3K agonist IGF-1could alleviate the learning and memory impairmentand promote the p-Akt protein expression in hippocampus in VaD mice, andits possible mechanism of improving cognitive function may be activating ofPI3K/Akt signal transduction pathway and regulating apoptosis and autophagyof nerve cells.Part3: The expression level of Akt, p-Akt, mTOR, p-mTOR protein inhippocampus of VaD mice and the effects of l-NBP.Objectives： To observe the Akt, p-Akt, mTOR, p-mTOR proteinexpression in hippocampus of VaD mice and the effects of l-NBP.Methods: The expressions of Akt, p-Akt, mTOR and p-mTOR inhippocampus were studied by measure of Western Blot and the expression ofp-Akt were studied by immunohistochemistry at4weeks after operation.Results:1L-NBP induce expression of p-Akt Ser473in the hippocampus of VaD mice.There is no marked change in total Akt among five groups (P>0.05).However, p-Akt Ser473were significantly increased in the IR group micecompared with those in the sham group (P<0.01). Moreover, Low-l-NBP, High-l-NBP and Pre-l-NBP group mice showed a strong expression of p-Aktcompared with the IR group mice (P<0.01).2L-NBP induce expression of p-mTOR Ser2448in the hippocampus ofcerebral repetitive IR mice.There is no marked change in total mTOR among five groups (P>0.05).However, p-mTOR Ser2448were significantly increased in the IR group micecompared with those in the sham group (P<0.01). Moreover, Low-l-NBP,High-l-NBP and Pre-l-NBP group mice showed a strong expression ofp-mTOR compared with the IR group mice (P<0.01, P<0.05).Conclusions:1There is significant increase in the expression of p-Akt Ser473andp-mTOR Ser2448protein in hippocampal tissue of VaD mice at4weeks postsurgery. It is suggest that activation of the PI3K/Akt signal transductionpathway might play a protect role in resisting neuronal damage after repeatedcerebral ischemia and reperfusion.2L-NBP can promote the expression of p-Akt Ser473and p-mTORSer2448in hippocampal tissue of VaD mice. The result suggest that l-NBPmay play a neuroprotective role by promoting the expression of p-Akt Ser473and p-mTOR Ser2448, thereby improving cognitive dysfunction and neuronaldamage in mice.Part4: The expression level of LC3B�'�Beclin1in hippocampus of VaDmice and the effects of l-NBPObjectives：To observe the LC3B�'�Beclin1expression in hippocampusof VaD mice and the effects of l-NBP.Methods: The expression of LC3B�'�Beclin1in hippocampus werestudied by Western-Blot technique or FQ RT-PCR technique at4weeks afteroperation.Results:1The expression level of LC3B protein in hippocampus by Western blot.The expression level of LC3B protein were significantly increased in theIR group mice compared with those in the sham group (P<0.01). Howover, in High-l-NBP and Pre-l-NBP group, the protein expression of LC3B were lowerthan those of IR group (P<0.05).2LC3B and Beclin1mRNA expression in hippocampus by FQ RT-PCRLC3B and Beclin1mRNA expression were significantly increased in theIR group mice compared with those in the sham group (P<0.05). There are nosignificant difference among Low-l-NBP, High-l-NBP, Pre-l-NBP group andsham group. Howover, LC3B and Beclin1mRNA expression of l-NBPgroups were marked lower than that of IR group (P<0.05).Conclusions:1There are significant increase in the expression of LC3B protein, LC3BmRNA and Beclin1mRNA in hippocampal tissue of VaD mice at4weekspost surgery. It suggests that increase in the expressions of LC3B and Beclin1was related to cognitive damage and neuronal damage in VaD mice.2L-NBP can reduce the expression level of LC3B and Beclin1inhippocampal tissue of VaD mice, thereby improving cognitive dysfunction andneuronal damage in VaD mice.Part5: Features of ependyma of the lateral ventricle in VaD mice byscanning electron microscopy (SEM) and the effect of l-NBPObjectives：To observe the change of ependyma cells of the lateralventricle in the VaD mice and the effect of l-NBP.Methods: To observe the change of ependyma cell and choroid plexusepithelial cell of the lateral ventricle in the VaD mice by scanning electronmicroscopy (SEM) and the effect of l-NBP.Results:1Structure features of choroid plexus epithelial cell of the lateral ventricle inmiceThe choroid plexus epithelial cell of the lateral ventricle of sham groupare of regular shape, clear boundary between cells. On cell surface, there arefull of bushy and shorter microvilli, and kind of secretory vesicles withdifferent sizes. In IR group, the shape of choroids plexus epithelial cellbecame irregular, and some cells have raised, some are of gyrus shrinkage, some cell surface are smooth. There were a large number of secretory vesiclesand increased secretions. Sediment is accumulated on the cell surface andintercellular boundaries are not clear. Microvillus decreased significantly evendisappear. The arrangement of choroid plexus epithelial cell in High-l-NBPgroup was in order, with clear boundaries between cells.2Structure features of ependyma cell of the lateral ventricle in miceIn sham group, there were abundant cilia and microvillus on its unevensurface. The quantities of cilia and microvillus on the uneven surface of in IRgroup reduced obviously. Both of them became twisty and stiff or bent and fellon the surface of cell in lesion regions and ‘mud-like’ secretions flaky out onthe ependymal surface. In High-l-NBP group, the quantities of cilia andmicrovillus on the uneven surface increased obviously compared with IRgroup.Conclusions:1The ependyma cells of the lateral ventricle of VaD mice were abnormal,that suggests abnormal ependyma cells may be involved in the pathogenesis ofVaD.2L-NBP can improve the abnormal ependyma cells of the lateralventricle, thereby improving cognitive dysfunction.