Effects Of Lycopene On Vascular Remodeling Mediated By LXR/PI3K/AKT Signaling Pathway In APP/PS1 Mice

Objective:Alzheimer’s disease（AD）is the most common degenerative disease in aging Society.AD mainly affects memory.AD is the result of the interaction of many factors,and the mechanism is unknown.Aβ_1-42-42 has been recognized to be at the center of many pathogenesis.Aβ_1-42-42 has been recognized to be at the center of many pathogenesis.Cerebral blood flow CBF is necessary for cerebral function.Mammalian brains have evolved a unique CBF control mechanism called neurovascular coupling.This mechanism ensures that rapidly increasing CBF and oxygen are delivered to the activated brain structures.The neurovascular unit is composed of neurons,astrocytes,vascular smooth muscle cells,pericytes and endothelial cells,which regulate the neural-vascular coupling.In recent years,more and more studies have shown that vascular lesions run through the whole process of AD onset and progression.Many vascular changes precede the pathological changes and clinical symptoms of AD,and vascular lesions and AD have many common risk factors.Understanding the relationship between vascular factors and the pathological process of AD is conducive to finding new prevention and treatment strategies and delaying the progress of AD.Lycopene has good antioxidant,neuroprotective and anticancer effects,but the molecular mechanism of its action is still unclear.Clinical studies have found that lycopene can reduce AD mortality.Liver X receptor（LXR）is an important member of the nuclear receptor superfamily with two subtypes,alpha and beta.In contrast,LXR is highly expressed in almost all tissues and organs,especially brain tissue.After activation,LXR can inhibit inflammation,participate in the regulation of lipid metabolism,play a neuroprotective role,and promote neurogenesis.LXR plays an important role in mammalian brain.It has been reported that the metabolism of cholesterol and lipoprotein in chronic ischemia rat model is disturbed,and LXR agonist treatment can prevent brain injury.However,little is known about the vascular aspects of LXR in AD.PI3K/AKT pathway is an important intracellular signal transduction pathway.Activated AKT activates eNOS by inducing eNOS phosphorylation and generates NO,thereby regulating the proliferation,migration and vasodilator of endothelial cells.bEnd.3 cell line is a kind of artificial transformation for internationally recognized vascular endothelial cell lines,with a series of microvascular endothelial cells（microvascular endothelial cell,MVEC）characteristics.Bend.3 cells are important cells for the study of angiogenesis and blood-brain barrier.This study in vivo as the AD model,using the APP/PS1 transgenic mice in vitro cultivation of bEnd.3 cells,using Western blot and immunofluorescence staining methods such as lycopene to LXR/PI3K/AKT signaling pathway activation to express,to explore the lycopene nerve vascular factors in alzheimer disease（AD）,and the prevention and treatment of AD pathogenesis of provides new theoretical basis and experimental evidence.Methods:In vitro:astrocytes were obtained from the brain cortical tissues of new born 1-3 days mice,the purity of cultured astrocytes was identified by immunocytochemical staining of GFAP protein.The astrocytes were stimulated with1 ng/ml IL-1βfor the tested time course.Cultures were pretreated with resveratrol or MAPKs pathway inhibitors and the control group was the same dose of DMSO.Immunocytochemical staining for morphological detection of astrocytes,GFAP,Sirt1,p-ERK,p-JNK and p-p38 protein expression were assessed by western blot,ELISA was applied to detect the expression of inflammatory cytokines IL-1β、IL-6 and TNFαin activated astrocytes.In vivo:Nigrostriatal pathway injury model was made and mice were pretreated with resveratrol and MAPKs pathway inhibitors such as U0126,SP600126和SB203580,DMSO treatment was as a control group.Western blot was applied to detect the expression of p-ERK,p-JNK and p-p38 proteins around the lesion site;Immunohistochemical double staining technique was used to detect the expression of Sirt1 and key proteins of MAPK signaling pathways such as p-ERK,p-JNK and p-p38 in activated astrocytes;The ELISA was to detect expression of inflammatory factor IL-1β,IL-6 and TNFα;Animal behavioral tests such as beam balance test,grip strength test and modified Neurological Severity Score（mNSS）were tested to explore the effects of resveratrol and MAPK signal pathway inhibitors U0126 SP600126 and SB203580 treatment on the recovery of neurological function after the injury.Statistical analysis was performed using GraphPad statistical software Prism5AND the data was showed as mean±standard error（mean±SD）.One way analysis of variance（ANOVA）was applied compared among groups and comparisons between two groups using Bonferroni test.P<0.05 was considered statistically significant.Results:in vivo experiments:1.Western blot results showed that compared with the normal control group,the expressions of GFAP,iNOS and A proteins in the prefrontal lobe and hippocampus of the model group were significantly increased,and the expressions of GFAP,iNOS and A proteins were decreased after the application of lycopene.Compared with the normal control group,the expressions of NEUN,VEGF,CD105,LXRβand P-AKT proteins in the prefrontal lobe and hippocampus of the model group were significantly decreased,and the expressions of NEUN,VEGF,CD105,LXRβand P-AKT proteins were increased after the application of lycopene.2.Immunofluorescence confocal results showed that,compared with the normal control group,there were significant activation expressions of astrocytes in the prefrontal lobe and hippocampus of the model group,and the application of lycopene could significantly improve the activation expressions of astrocytes.Compared with the control group,the expressions of NEUN,VEGF,CD105,LXRα,LXRβ,and P-AKT in the prefrontal lobe and hippocampus of the model group were significantly decreased,and the expressions of NEUN,VEGF,CD105,LXRα,LXRβ,and P-AKT in the prefrontal lobe and hippocampus of the lycopene group were significantly increased compared with the APP/PS1 group.3.The length of microvessels in the prefrontal lobe and hippocampus of the mice in the three groups were statistically analyzed.The results showed that compared with the normal control group,the length of blood vessels in the model group and the lycopene group was significantly reduced,and the application of lycopene could improve the reduction of the length of microvessels in APP/PS1 mice.4.Behavioral water maze test results showed that compared with the normal group,mice in the model group had a longer incubation period,fewer times of crossing over the platform per unit time,decreased spatial learning and memory ability,and decreased cognitive ability.After lycopene administration,the incubation period of mice in the treatment group was significantly shortened,the number of crossing over the platform was significantly increased,and the spatial learning behavior ability and cognition were significantly improved.In vitro experiments:1.The results of CCK8 experiment showed that the appropriate concentration and time of action were 24 hours after lycopene treatment of bEnd.3cells with 10 M as the reaction condition for subsequent experiments.2.Western blot results showed that LXR positive expression was significantly increased in bEnd.3cells treated with lycopene compared with the control group.LXR positive expression in bend.3 cells of Ly294002 group showed no significant change compared with that of the control group.LXR positive expression was significantly increased in Ly294002+lycopene treated bend.3 cells compared with the control group.Compared with the control group,P-AKT positive expression was significantly increased in the lycopene treated bend.3 cells.Compared with the control group,P-AKT positive expression was significantly decreased in bEnd.3 cells of Ly294002treatment group.Compared with the control group,P-AKT positive expression decreased in Ly294002+lycopene treatment group.Compared Ly294002 with Ly294002+lycopene the control group,In the lycopene treatment group,P-AKT positive expression was increased in bend.3 cells,but not statistically significant.There was no significant difference in total AKT protein expression among the groups.3.Immunofluorescence confocal results showed that LXR positive expression was significantly enhanced in the lycopene treated cells of bEnd.3compared with the control group.LXR positive expression in bEnd.3 cells of Ly294002 group showed no significant change compared with the control group.LXR positive expression in bEnd.3 cells and bEnd.3 cells of Ly294002+lycopene treatment group was significantly enhanced compared with the control group.No significant changes in cell morphology were observed between the groups.Compared with the control group,the P-AKT positive expression was significantly enhanced in the lycopene treated group.Compared with the control group,the P-AKT positive expression in bend.3 cells of Ly294002 treatment group was significantly weakened,cells were shed,cells that were not shed became round and retracted,and intercellular space increased.Compared with the control group,in the Ly294002+lycopene treatment group,the positive expression of P-AKT was significantly decreased,the cell morphology was not significantly improved,and cells that did not fall off became round and retractile,and the intercellular space increased.Conclusion:1.Lycopene can activate LXR/PI3K/AKT signaling pathway in APP/PS1 mice,thus affecting angiogenesis in the brain of mice.2.Lycopene can activate PI3K/AKT signaling pathway by activating LXR,and then regulate the proliferation of bEnd.3 cells.