The Improvement Effect And Mechanism Of Berberine On Alzheimer’s Disease Through The FOXO3a/BIM Signaling Pathway

Backgrounds:Alzheimer’s disease(AD)is a chronic neurodegenerative disease with insidious onset and progressive development,with memory impairment,language function and other cognitive decline as the main symptoms,which can lead to a decline in the patient’s daily life abilities,mental and behavioral abnormalities,and bring great burden to the family and society.The number of AD patients worldwide has reached 50 million and is expected to increase to 152 million by 2050.AD has become the fifth leading cause of death in the world,so finding drugs to improve AD is a major problem that researchers need to solve urgently.The FOXO protein family is closely related to the occurrence of AD.After stimulation of β amyloid(Aβ),it causes apoptosis of neuronal cells.When the FOXO3a protein is overexpressed,it leads to oxidative stress and apoptosis of neurons,eventually leading to the loss of neurons and causing a series of neurodegenerative diseases.Aβ can lead to a series of post-translational modifications and nuclear localization of FOXO3a and induce cell death through promote BIM expression.Therefore,FOXO3a may be a potential target for the treatment of Alzheimer’s disease.Berberine(molecular formula C20H19NO5,molecular weight 353.36 g/mol)is the main isoquinoline alkaloid component of the stems and roots of various berberine plants.Berberine has a wide range of applications in traditional Ayurvedic and Chinese medicine systems.It has good pharmacological effect,including analgesic,anti-inflammatory,anti cancer,hypoglycemic,antihyperlipidemia,cardioprotective,memory-enhancing and antidepressant,antioxidant,anti-harm,antibacterial,anti-HIV and cholesterol-lowering effects.In the previous research of our lab,we found that berberine can not only enhance the anti-apoptotic ability of hippocampal neurons,but also have an improving effect on cognitive dysfunction in diabetic encephalopathy.Therefore,the effect of berberine in improving cognitive dysfunction can be confirmed.At the same time,in bioinformatics analysis,after intersecting berberine and AD targets,a total of 92 common targets were found.Among them,the most relative proteins is the FOXO3a.Therefore,we can hypothesize that berberine may improve cognitive dysfunction and neuronal damage in AD through the FOXO3a pathway.Objective:This paper will use the constructed in vivo and in vitro AD models to investigate whether berberine can improve cognitive dysfunction and neuronal damage in Alzheimer’s disease through the FOXO3a pathway,and explore its related mechanisms.Methods and results:1.The therapeutic effect of berberine on AD cognitive dysfunction and neuronal damage on vivo and vitro.The AD mice model constructed by D-gal and AlCl3 was used to detect the improvement of cognitive function through the Morris water maze.Mice were sacrificed and brain tissues were extracted.The results showed that berberine could significantly reduce the escape latency of mice and improve cognitive dysfunction in mice.Then,HE,Nissel and TUNEL staining,were performed to detect apoptosis and FOXO3a/BIM pathway protein expression in mice,and we found the apoptosis level is decreased by BBR.Later,we used HT22 cells and Aβ25-35 to construct vitro cell model,and used MTT assay to detect safe and effective concentration of berberine.The results showed that 40μM concentration of Aβ25-35 could reduce the viability of HT22 cells to about 70%,which met the requirements of modeling.Berberine below 1μM concentration has no obvious killing effect on HT22 cells,berberine can improve the cell viability of HT22 cells in a dose-dependent manner within 1 μM,and berberine has a most effective therapeutic effect at the concentration of 0.2 μM,so 40μM concentration of Aβ25-35 was selected for modeling and 0.2 μM concentration of berberine administration was selected in subsequent experiments.2.Berberine improves AD through the FOXO3a/BIM signaling pathway.A total of 92 potential binding sites between berberine and AD were obtained through network pharmacological screening,and then through protein-protein interaction analysis,KEGG pathway enrichment and GO function enrichment screened the top 20 targets most relevant to AD,among which we found that PI3K/AKT pathway and FOXO3a had a high correlation when berberine and AD were crosslinked.In further literature reading,FOXO3a is inextricably linked to apoptosis of neuronal cells.Immunofluorescence results showed that the level of nuclear localization of FOXO3a was increased upon Aβ25-35 stimulation,while berberine improved the level of nuclear translocation.To verify whether the FOXO3a/BIM pathway is involved in the process of berberine treatment in AD,Firstly,we extracted brain tissue proteins from mouse animal models and examined the alteration of FOXO3a/BIM protein expression using WB method.The results found that the phosphorylation of FOXO3a decreased and BIM expression increased in the model group;the phosphorylation of FOXO3a increased and BIM expression decreased in berberine group,demonstrated that berberine can increase phosphorylation of FOXO3a and decrease BIM protein expression in AD animal models.Subsequently,we extracted cellular proteins from HT22 cell AD model.Similar to the in vivo results,the phosphorylation of FOXO3a was significantly suppressed in Aβ25-35 after stimulation.Berberine administration increased the phosphorylation level of FOXO3a and decreased the expression of BIM.FOXO3a After decreased phosphorylation leads to an increased degree of its nuclear translocation.After FOXO3a decreased phosphorylation leads to an increased level of its nuclear translocation.Subsequently,to observe the effect of the level of nuclear translocation of berberine FOXO3a,we used immunofluorescence experiments in the AD cell model and showed that the level of nuclear localization of FOXO3a increased upon Aβ25-35 stimulation,while berberine decreased the level of nuclear translocation.Through in vitro and in vivo experiments,we found that the FOXO3a/BIM pathway might be an effective pathway for AD treatment.3.Berberine can regulate FOXO3a/BIM signaling pathway by affecting the PI3K/AKT pathway.The PI3K/AKT pathway is directly upstream of the FOXO3a/BIM pathway,and dephosphorylation of PI3K causes dephosphorylation of both AKT and FOXO3a.Therefore,we used the PI3K inhibitor LY294002 and/or BBR and then extracted protein for WB assay.Subsequently,the expression levels of p-AKT/AKT,p-FOXO3a/FOXO3a,and BIM were detected.The results showed that in the model group,the levels of p-FOXO3a/FOXO3a and p-AKT/AKT were significantly reduced and the expression of BIM protein was significantly increased compared with the control group.In the berberine administration group,the decreases of p-AKT/AKT and p-FOXO3a/FOXO3a levels were significantly reversed,and BIM expression levels were significantly reduced.In the inhibitor group,the levels of p-FOXO3a/FOXO3a and p-AKT/AKT were downregulated again,the expression of BIM protein was also significantly increased,and the therapeutic effect of berberine was eliminated by LY294002,demonstrated that berberine regulates the FOXO3a/BIM signaling pathway by affecting the PI3K/AKT pathway.Conclusion:Berberine can improve cognitive dysfunction and neuronal damage in Alzheimer’s disease through the FOXO3a/BIM signaling pathway.