Neuroprotective Effects Of Bergamot Essential Oil On Alzheimer’s Disease Model Animals And Cells,and Its Molecular Mechanisms

Alzheimer’s disease（AD）is a neurodegenerative disease characterized by features such as memory impairment,anxiety,deposition of beta-amyloid protein,excessive phosphorylation of Tau protein,and decreased number of neurons.Oxidative stress and inflammatory reactions are involved in the development of AD.Bergamot essential oil（BEO）is a fresh and slightly floral plant essential oil extracted from Bergamot fruit.The biological activities of BEO are manifested in anti-anxiety,anti-depressive,anti-inflammatory,and antioxidant effects.BEO can significantly alleviate depressive and anxiety-like behaviors in animals and also has therapeutic effects on mild mental disorders such as anxiety and depression in humans.Based on the biological activities of BEO in antioxidant and anti-inflammatory,as well as the important role of oxidative stress and inflammatory reaction in the onset and development of AD,this study investigated the therapeutic effect of BEO on AD and its possible mechanism.In this paper,Al Cl₃was used to construct an AD animal model,and Aβ25-35 was used to construct an AD cell model.Behavioral,morphological,immunohistochemical,biochemical and other methods are used to conduct research from the three levels of behavior,tissue,cells,and molecules.The purpose of this study is to provide a theoretical basis for the application of BEO.（1）BEO component analysis.GC-MS analysis was performed on BEO,and a total of 38components were identified,including L-limonene（60.91%）,γ-terpinene（27.08%）,α-pinene（1.71%）,β-pinene（1.70%）,β-myrcene（1.58%）,cyclohexene（1.33%）,etc.（2）BEO improves the behavioral and pathological symptoms of AD model animals.The AD model in rats was established by intraperitoneal injection of Al Cl₃（50 mg/kg）for 60consecutive days.The effects of BEO on the behavioral and pathological symptoms of AD model animals were studied by oral gavage with three doses of 100 mg/kg,200 mg/kg,and400 mg/kg of BEO for 60 consecutive days,or by oral gavage with a dose of 400 mg/kg of BEO for 5 consecutive days,or by intranasal administration with three doses of 1%,2.5%,and 5%BEO for 60 consecutive days.The results showed that the AD model animals exhibited significant behavioral symptoms such as anxiety,depression,and cognitive impairment,accompanied by pathological symptoms such as abnormal mitochondrial structure in the hippocampus and excessive phosphorylation of Tau protein.Oral gavage of BEO for 60 consecutive days alleviated anxiety-like behavior observed in the open field test and elevated plus maze test,depressive-like behavior observed in the forced swimming test and sucrose preference test,and cognitive impairments observed in the Morris water maze test and novel object recognition test in the model animals.It reduced the levels of CDK5,p-Tau181,and p-Tau231 in the hippocampus of the model animals.It also alleviated the pathological symptoms such as excessive phosphorylation of Tau protein and abnormal mitochondrial structure in the hippocampus of the model animals.However,when the model rats were given BEO at a dose of 400 mg/kg for 5 consecutive days,it did not alleviate the behavioral symptoms of anxiety,depression,cognitive impairment,and the pathological symptoms of abnormal mitochondrial structure and excessive phosphorylation of Tau protein.When intranasally administered with three concentrations of 1%,2.5%,and 5%BEO for 60consecutive days,only the group treated with 5%BEO showed some alleviation of the behavioral symptoms of anxiety,depression,cognitive impairment,and the pathological symptoms of abnormal mitochondrial structure and excessive phosphorylation of Tau protein in the model animals.Both oral gavage and intranasal administration of BEO could alleviate the behavioral and pathological symptoms of the model rats to varying degrees,with the best effects observed with oral gavage of 200 mg/kg or 400 mg/kg of BEO for 60 consecutive days.The alleviating effect of BEO on the behavioral and pathological symptoms of AD model animals may be related to its regulation of CDK5 levels in the hippocampus.（3）BEO improves the inflammatory response and oxidative stress status in AD model animals and upregulates the GABA and CREB-BDNF signaling pathways.The AD model in rats was established by intraperitoneal injection of Al Cl₃（50 mg/kg）for 60 consecutive days.By oral gavage,the model rats were given BEO（200 mg/kg）for 60 consecutive days to investigate the effects of BEO on the inflammatory response,oxidative stress status,and the GABA and CREB-BDNF signaling pathways in AD model animals.The results showed that the model animals exhibited significant oxidative stress and inflammatory response.BEO increased the activities of antioxidant enzymes such as GPx,CAT,and SOD in the hippocampus and cortex of the animals.It reduced the levels of protein carbonylation,lipid peroxidation product MDA,and DNA oxidative damage product 8-OHd G.It also decreased the levels of inflammatory cytokines IL-1β,IL-6,and TNF-α,and increased the levels of GABA,BDNF,p-CREB,and p-ERK.BEO improved the oxidative stress and inflammatory response status in the model animals,showing neuroprotective effects.The GABA and BDNF/ERK/CREB signaling pathways were involved in mediating the neuroprotective effects of BEO.（4）Preparation of BEO Liposomes.BEO liposomes were prepared using cholesterol and lecithin at ratios of 1:4,1:5,and 1:6.The average particle sizes of the resulting BEO liposomes were 146±2.69,165.1±6.34,and 159.2±3.90,and their polydispersity index（PDI）was 0.192±0.017,0.134±0.074,and 0.119±0.029,respectively.The PDIs of each ratio were less than 0.2,indicating good stability.The encapsulation efficiencies of the essential oil liposomes prepared with cholesterol and lecithin at ratios of 1:4,1:5,and 1:6 were 79.4%,87.1%,and 96.3%,respectively,while the drug loading rates were 38.8%,36.7%,and 35.5%,respectively.Based on comprehensive analysis of particle size,PDI,Zeta potential,drug loading rate,encapsulation efficiency,and other parameters,the cholesterol-to-lecithin ratio of1:4 and 1:5 was more effective for preparing BEO liposomes.（5）BEO liposomes regulate the AKT/GSK-3β,Nrf2,and NF-κB signaling pathways in AD model cells.PC12 cells were treated with Aβ25-35 to establish an AD cell model,and the effects of BEO liposomes on the AKT/GSK-3β,Nrf2,and NF-κB signaling pathways in AD model cells were studied.The results showed that when cells were treated with BEO liposomes at concentrations ranging from 0.00375 to 0.96 mg/m L,the cell viability began to significantly decrease at a concentration of 0.12 mg/m L.Low concentrations of BEO（0.00375,0.0075,0.015,0.03,0.06 mg/m L）promoted cell proliferation,while high concentrations（0.12,0.24,0.48,0.96 mg/m L）inhibited cell proliferation.Aβ25-35 treatment（20μM）resulted in decreased cell viability,increased apoptosis rate,and cell cycle arrest in PC12 cells,successfully establishing an AD cell model.BEO liposomes at a concentration of0.03 mg/m L significantly improved the decreased cell viability,increased apoptosis rate,and cell cycle arrest induced by Aβ25-35 treatment in PC12 cells.ROS and inflammatory cytokine levels were elevated in the AD model cells,and BEO treatment at a concentration of0.03 mg/m L reduced ROS levels and the levels of NO,PGE2,i NOS,COX-2,TNF-α,and IL-1β,thereby improving the oxidative stress and inflammatory response status of the model cells.BEO increased the expression levels of p-GSK-3β,p-AKT,Nrf2,and HO-1 in AD model cells,while reducing the phosphorylation levels of p65 and IκB.The NF-κB and Nrf2pathways were involved in mediating the anti-inflammatory effects of BEO,while the AKT/GSK-3βand Nrf2 pathways were involved in mediating its antioxidant effects..The above research results indicate that BEO has neuroprotective effects and can alleviate the behavioral and pathological symptoms of AD model rats,and improve the oxidative stress and inflammatory response status of AD model rats.Pathways including CREB-BDNF,GABA,AKT/GSK-3β,Nrf2,and NF-κB collectively mediate the neuroprotective effects of BEO.