Lipoxin A₄Methyl Ester Ameliorates Cognitive Deficits Through Activating ERK/NRF2and PI3K/Akt Signaling Pathway In Vascular Dementia Rats

Vascular dementia (VD) is an acquired syndrome of cognitiveimpairment which is caused by various kinds of cerebral vascular diseases. Itis incontrovertible that VD is the second most common cause of dementiaafter Alzheimer’s disease (AD) in the world. Up to now, the pathogenesis ofVD is not yet entirely clear and there are no especially effective drugs.Considerable studies characterize chronic cerebral hypoperfusion as acommon pathophysiological status contributing to VD. Thus, exploring themechanism by which chronic cerebral hypoperfusion leads to VD and seekingthe effective drugs for VD have become one of the focuses of currentmedicine.Oxidative injury and the apoptosis of neuron play an important role in thepathogenesis of VD induced by chronic cerebral hypoperfusion. So, inhabitingthe oxidative injury induced by chronic cerebral hypoperfusion and reduingthe apoptosis of neuron may be crucial for managing VD. It has beendemonstrated that the activation of extracellular signal-regulatedkinase/nuclear factor E2-related factor2(ERK/Nrf2) signaling pathway isvery important for neurons to resist the oxidative injury induced by cerebralischemia; the phosphatidylinositol-3kinase/Akt (PI3K/Akt) cell signalingpathway is a classic anti-apoptosis pathway, promoting the survival signaltransduction. In previous studies, our research team have explored that theactivation of PI3K/Akt cell signaling pathway can improve cognitive functiondeficits of VD mice induced by cerebral repetitive ischemia/reperfusion.Lipoxins (LXs), a class of endogenous anti-inflammatory lipid-basedautacoids, are generated from arachidonic acid via sequential actions oflipoxygenases during the onset of the inflammatory. Lipoxin A4(LXA4) is oneof the main physiologic forms of LXs. LXA4has been widely studied in various diseases related to inflammation. Until recent years, the novelneuroprotection of LXA4for neurons against injury induced by cerebralischemia is being recognized by researchers. several recent studies revealedthat LXA4methyl ester (LXA4ME), a stable synthetic analog of LXA4, whichwas administrated through intracerebroventricular injection (i.c.v.), couldsignificantly reduce infarct volumes by inhibiting inflammatory responses inrat models of cerebral ischemia/reperfusion (I/R) and permanent focalcerebral ischemia. Intriguingly, besides its classic anti-inflammatory effect,considerable attention has been attracted to the novel antioxidant effect ofLXA4in recent years.However, no information is available with regard to the possibleprotective effect of LXA4on neuronal injury and cognitive deficits induced bychronic cerebral hypoperfusion. So, in the present study, a widely acceptedmodel of chronic cerebral hypoperfusion induced by permanent bilateralcommon carotid artery occlusion (BCCAO) in rats was used to explore:(1)whether long-term administration of LXA4ME can protect neurons inhippocampus against oxidative injury, reduce neuronal apoptosis andameliorate cognitive deficits induced by chronic cerebral hypoperfusionthrough activating the ERK-Nrf2signaling pathway?(2) whetheradministration of LXA4ME during early phase of chronic cerebralhypoperfusion can provide long-term neuroprotection and improve cognitiveimpairments? And if so, whether the effects of LXA4ME is are associatedwith the activation of PI3K/Akt signaling pathway? Based on the abovestudies, we will further observe the effects of LXA4ME on the thickness ofpostsynaptic density (PSD）and the expression of PSD-95in CA1area ofhippocampus of rats underwent chronic cerebral hypoperfusion, and providemore insights into the neuroprotective mechanisms of LXA4ME.Part Ⅰ LXA4ME ameliorates cognitive deficits induced by chroniccerebral hypoperfusion through activating ERK/Nrf2signalingpathway in ratsObjective: To establish a VD rat model induced by chronic cerebral hypoperfusion through BCCAO and observe the effects of LXA4ME on thecognitive deficits of VD rats. At the same time, we will evaluate the effects ofLXA4ME on the expression of ERK/Nrf2signaling pathway,4-Hydroxynonenal (4-HNE) and apoptosis-associated proteins, and explorethe neuoprotective mechanisms of LXA4ME.Methods: Adult male Sprague–Dawley rats weighing250-300g wereprovided by the Laboratory Animal Center of Hebei Medical University.7days before BCCAO surgery, all rats received intracerebroventricular catheterimplantation. Rats were randomly assigned into four groups: Sham(sham-operated) group, vehicle (BCCAO+normal saline) group, LXA4ME10group and LXA4ME100group.10ng or100ng LXA4ME (purchased fromCayman Chemical, Ann Arbor, MI, USA) were dissolved in5μl normalsaline, respectively. Rats in LXA4ME10group and LXA4ME100groupreceived10ng or100ng LXA4ME (i.c.v.) immediately after BCCAO,respectively. Rats in sham group and vehicle group received5μl normal saline(i.c.v.). All rats received LXA4ME dissolved in normal saline or normalsaline according to the aforementioned experimental plan every24h up to14days afterwards.The next day after the final injection，all rats were evaluated spatiallearning and memory abilities by the Morris water maze test. Each rat receivedfour trails per day for five consecutive days，with an intertrial interval of60s.In every trail, each rat was gently placed the water at one starting position,facing the wall of water maze. The time to reach the platform (escape latency)was recorded in each trial. The rat was allowed to find the platform within120s. If the rats failed to find the hidden platform within120s, they were gentlyguided to find the platform by the experimenter and allowed to remain on theplatform for10s and their escape latencies were recorded as120s. Theprocedure was repeated for all the four start locations. On day6, the rats weretested on a spatial probe trial in which the platform was removed, and the ratswere placed in a quadrant which is opposite to the target quadrant and allowedto swim freely for120s. The percentage of rats spent in the target quadrant where the platform had been located and the times of rats crossing theplatform were recorded.Shortly after the behavioral tests, six rats chosen randomly from eachgroup were anesthetized with pentobarbital sodium (50mg/kg, i.p.) andperfused with normal saline rapidly through the left cardiac ventricle andascending aorta, then followed by4%paraformaldehyde. The brains were thenembedded in paraffin. Coronal brain sections were cut and underwent Nisslstaining with0.5%cresyl fast violet and immunohistochemically stained for4-Hydroxynonenal (4-HNE), respectively. IOD/area value was used toindicate the mean optical density for each slice. Furthermore, six rats fromeach group were randomly chosen to be decapitated under anesthesia, Thehippocampi were quickly dissected and homogenated to detect the proteinexpression of p-ERK、Nrf2、NQO1、Bcl-2、Bax and caspase-3in hippocampusby western blot.Results: In the Morris water maze, beginning on day2, rats in vehiclegroup took significantly longer time to find the platform (P<0.01) comparedwith sham group. Rats in LXA4ME100ng group showed shorter meanlatencies compared with vehicle group (P<0.01) on day3. Over the next twodays, rats treated with LXA4ME (10ng and100ng) showed significantshorter escape latencies compared with vehicle group (both P<0.01),meanwhile, administration of LXA4ME100ng resulted in shorter escapelatencies than LXA4ME10ng group (P<0.01). In the probe trial, rats invehicle group stayed in the target quadrant for significantly less time andpossessed fewer times of crossing the platform than sham group (both P<0.01).Compared with vehicle group, rats received LXA4ME (10ng and100ng)evidently increased the ratio of time spent in the target quadrant,(bothP<0.01). Similarly, administration of LXA4ME (10ng and100ng)significantly increased the times of rats crossed the platform relative to thevehicle group (P<0.05，P<0.01，respectively). In detail, rats in LXA4ME100ng group crossed the platform for more times than LXA4ME10ng group(P<0.01). The pyramidal neurons in the CA1region of hippocampus in sham groupwere tightly ranked in order, and the neurons were clear and moderate in sizewith normal microstructure. In vehicle group, obvious pathological changeswere exhibited with loosely arranged neurons, neuronal shrinkage, loss andlight color staining. Administration of LXA4ME，especially100ng per day,evidently reversed the morphologic changes.Extensively positive staining for4-HNE was seen in the neuronalperikarya and axons of neurons in hippocampus of rats in vehicle group(0.17±0.02). However, compared with vehicle group, the IOD/area value of4-HNE significantly decreased in group treated with LXA4ME (both P<0.01),which was lower in LXA4ME100ng group (0.08±0.02) than LXA4ME10nggroup(0.11±0.02)(P<0.05).There was no significant difference in the expression of t-ERK1/2amongall groups (P＞0.05). The expression of p-ERK1/2in vehicle group obviouslydecreased compared with sham group (P<0.01). However, this decrease wasremarkably reversed by LXA4ME at doses of10ng and100ng (P<0.05，P<0.01, respectively). Similarly, the expression of total Nrf2, nuclear Nrf2andNQO1in vehicle group were also significantly downregulaed (all P<0.01).Interestingly, LXA4ME (10ng and100ng) significantly elevated theexpression of total Nrf2, nuclear Nrf2and NQO1（P<0.01，P<0.01，P<0.05,respectively）. Furthermore, except that no significant difference was found inthe expression of total Nrf2between LXA4ME100ng group and LXA4ME10ng group(P＞0.05), there were higher expression of p-ERK1/2, nuclearNrf2and NQO1in LXA4ME100ng group than LXA4ME10ng group(P<0.01).Compared with sham group, chronic cerebral hypoperfusion significantlydecreased the expression of Bcl-2and increased the expression of Bax, thusmarkedly reduced the ratio of Bcl-2/Bax (P<0.05, P<0.01, P<0.01,respectively). However, administration of LXA4ME (10ng and100ng)significantly reversed the downregulation of Bcl-2(both P<0.01) and theupregulation of Bax (P<0.05, P<0.01, respectively) and elevated the expression ratio of Bcl-2/Bax (both P<0.01). Moreover, compared with LXA4ME10ng group, treatment with LXA4ME100ng more effectivelyupregulated the ratio of Bcl-2/Bax (P<0.01).Cleaved caspase-3protein was significantly upregulated in vehicle groupcompared with sham group (P<0.01). Adminstration of LXA4ME100ng perday significantly downregulated cleaved caspase-3protein expression(P<0.01).Conclusion: The present study indicates protective effects of LXA4MEon cognitive impairment induced by chronic cerebral hypoperfusion, andprovides further insight into the antioxidant and anti-apoptotic mechanisms ofLXA4ME. The activation of ERK/Nrf2signaling pathway contributes to theneuroprotection of LXA4ME against chronic cerebral hypoperfusion injury.PartⅡLXA4ME provides long-term neuroprotection for VD rats inducedby chronic cerebral hypoperfusion through activating PI3K/Aktsignaling pathwayObjective: To evaluate the possible long-term neuroprotection of LXA4ME administrated during early phase of chronic cerebral hypoperfusion andfurther explore the underlying mechanisms.Methods: Adult male Sprague–Dawley rats weighing250-300g wereprovided by the Laboratory Animal Center of Hebei Medical University.7days before BCCAO surgery, all rats received intracerebroventricular catheterimplantation. Rats were randomly assigned into four groups: Sham group,vehicle group, LXA4ME100group and LXA4ME100+LY294002group. Ratsin Sham group and vehicle group received2μl vehicle of LY294002（i.c.v.）shortly after BCCAO, and which was followed by administration of5μlnormal saline (i.c.v.)30min later; Rats in LXA4ME100group received2μlvehicle of LY294002（i.c.v.）shortly after BCCAO, and which was followedby administration of LXA4ME (100ng in5μl)(i.c.v.)30min later; Rats inLXA4ME100+LY294002group received2μl LY294002（i.c.v.） shortlyafter BCCAO, and which was followed by administration of LXA4ME (100ng in5μl)(i.c.v.)30min later. The aforementioned experimental plan was performed every24h up to14days afterwards.At day29following BCCAO, all rats received Morris water maze test toevaluate patial learning and memory abilities.Shortly after the Morris water maze test, six rats chosen randomly fromeach group were anesthetized with pentobarbital sodium (50mg/kg, i.p.) andperfused with4%paraformaldehyde. The brains were then embedded inparaffin. Coronal brain sections were cut and underwent Nissl staining with0.5%cresyl fast violet to observe the pathology changes in CA1region ofhippocampus. Moreover, six rats from each group were randomly chosen to bedecapitated under anesthesia. The hippocampi were quickly dissected andhomogenated to detect the protein expression of total Akt (t-Akt),phospho-Akt (p-Akt), total CREB (t-CREB), phospho-CREB (p-CREB),Bcl-2and Bax in hippocampus by western blot.Results: In the Morris water maze, all animals showed a progressivedecline in the escape latency with training. Beginning on day2, rats in vehiclegroup took significantly longer time to find the platform (P<0.01) comparedwith sham group. Rats in LXA4ME100ng group showed shorter meanlatencies compared with vehicle group （day2：P<0.05；day3-5：P<0.01）.Compared with LXA4ME100ng group, the escape latencies of rats in LXA4ME100+LY294002group significantly increased（day2：P<0.05；day3-5：P<0.01）. In the probe trial, rats in vehicle group stayed in the target quadrantfor significantly less time and possessed fewer times of crossing the platformthan sham group (both P<0.01). Compared with vehicle group, rats receivedLXA4ME (100ng/d) evidently increased the ratio of time spent in the targetquadrant and the times of crossing the platform (both P<0.01). However, ratsin LXA4ME100+LY294002group showed significantly decreased ratio oftime and the fewer times of crossing the platform (both P<0.01).In sham group, the neurons in the CA1region of hippocampus weretightly ranked in order, and the neurons were clear and moderate in size withnormal microstructure. In vehicle group, obvious pathological changes wereexhibited with loosely arranged neurons, neuronal shrinkage, loss and light color staining. Administration of LXA4ME (100ng/d), evidently reversed themorphologic changes. However, there are obvious phenomena of neuron lossand shrinkage of neurons.At day34following BCCAO, the expression of p-Akt in vehicle groupobviously decreased compared with sham group (P<0.01); The decrease wasremarkably reversed by LXA4ME (100ng/d)(P<0.01); However, theexpression of p-Akt in LXA4ME100+LY294002group obviously decreased(P<0.01). There was no significant difference in the expression of t-Aktamong all groups (P>0.05). The expression of p-CREB showed a tendencysimilar to p-Akt. There was no significant difference in the expression of t-CREB among all groups (P>0.05).Compared with sham group, at day34following BCCAO, chroniccerebral hypoperfusion significantly decreased the expression of Bcl-2andincreased the expression of Bax, thus markedly reduced the ratio of Bcl-2/Bax(all P<0.01). Administration of LXA4ME (100ng/d) significantly reversedthe downregulation of Bcl-2(P<0.01) and the upregulation of Bax (P<0.01)and increased the expression ratio of Bcl-2/Bax (P<0.01). Interestingly, thecombined use of LY294002and LXA4ME (100ng/d) blocked the increase ofBcl-2(P<0.01) and the decrease of Bax (P<0.01) induced by LXA4ME only,and decreased the ratio of Bcl-2/Bax (P<0.01).Conclusion: Administration of LXA4ME during early phase of chroniccerebral hypoperfusion can provide long-term neuroprotection, inhabit theapoptosis of neurons, and improve cognitive impairments induced by chroniccerebral hypoperfusion. The activation of PI3K/Akt signaling pathway may beinvolved in the long-term neuroprotection of LXA4ME.PartⅢ Effects of LXA4ME on postsynaptic density of rats with VDObjective: To observe the effects of LXA4ME on the thickness of PSDand the expression of PSD-95in CA1region of rats underwent chroniccerebral hypoperfusion, and provide more insights into the neuroprotectivemechanisms of LXA4ME.Methods: Adult male Sprague–Dawley rats weighing250-300g were provided by the Laboratory Animal Center of Hebei Medical University.7days before BCCAO surgery, all rats received intracerebroventricular catheterimplantation. Rats were randomly assigned into three groups: Sham group,vehicle group, LXA4ME100group.100ng LXA4ME was dissolved in5μlnormal saline. Rats in LXA4ME100group received100ng LXA4ME (i.c.v.)immediately after BCCAO. Rats in sham group and vehicle group received5μl normal saline (i.c.v.). All rats received LXA4ME dissolved in normalsaline or normal saline according to the aforementioned experimental planevery24h up to14days afterwards.At day34following BCCAO, three rats chosen randomly from eachgroup were anesthetized with pentobarbital sodium (50mg/kg, i.p.) andperfused with4%paraformaldehyde. The thickness of PSD in CA1area ofhippocampus was observed through the electron microscopy. Furthermore,another six rats chosen randomly from each group were fully anesthetized andperfused with4%paraformaldehyde through the left cardiac ventricle. Thebrains were then embedded in paraffin. Coronal brain sections were cut andimmunohistochemically stained for PSD-95. IOD/area value was used toindicate the mean optical density for each slice.Results: At day34following BCCAO, the thickness of PSD in CA1areaof hippocampus in vehicle group was34.61±5.66nm，which was significantlyless than that of sham group (49.65±4.84nm)(P<0.01); Meanwhile, thethickness of PSD of LXA4ME100group was47.53±6.10nm，which wasevidently more than that of vehicle group (P<0.01).Immunohistochemical results showed that extensively positive stainingfor PSD-95was seen in the cytoplasm of neurons in hippocampus CA1regionof rats in sham group. The IOD/area value of PSD-95markedly decreased invehicle group compared with sham group (P<0.01). However, compared withsham group, administration of LXA4ME (100ng/d) significantly increased thepositive expression of PSD-95(P<0.01).Conclusion: Administration of LXA4ME can promote the expression ofPSD-95and increase the thickness of PSD, which contribute to the neuroprotective effects of LXA4ME.