| Alzheimer’s disease(AD),a neurodegenerative disease and the main source of dementia,is mainly characterized by dementia symptoms caused by the decline of learning and memory ability.Though the pathogenesis of AD is complex,the deposition of amyloid beta peptide(Aβ)in brain tissue formed senile plaques(SP),which leads to a series of neuronal damage and lesions has been widely recognized.Therefore,reducing the production of Aβ in brain tissue,enhancing the clearance of Aβ,and reducing the deposition of Aβ in brain tissue has become a research hotspot.AD is divided into predromal stage,mild cognitive impairment(MCI)stage and dementia stage.In the past few years,new drugs targeting Aβ have failed in clinical trials of MCI and dementia.Before the symptom of dementia appears in AD,Aβ deposition in brain tissue was estimated to take17 years,12 years from the detectable to threshold concentration,4.2 years for the hippocampal atrophy,3.3 years for memory loss.It ws specu Lated that the Aβ content in MCI and dementia might be very high,and the neurons had been irreversibly damaged,reducing Aβ content in MCI and dementia is too late in the natural of the disease.The 17 years period from detectable Aβ to the appearance of memory decline provides an opportunity for early intervention in onset and progression of AD.The latest guidance issued by the US FDA in 2018 states showed that very early intervention,reducing Aβ deposition,and delaying AD progression will be supported.In2014,more scholars conceived the concept of subjective cognitive decline(SCD),which is earlier than the predromal stage.In addition to missing the optimal intervention phase,the other reason for clinical trials failure might be related to targeting only on Aβ.AD,as a neurodegenerative disease,its pathogenesis is extremely complex such as Aβ deposition,oxidative stress,immune inflammatory response,neuronal apoptosis,etc.Looking for mu Ltitarget agents to control the mu Ltiple risk factors of AD has become important.It was found that flavonoids has antioxidant and anti-inflammatory effects.Early intervention of curcumin and breviscapine in AD animal models cou Ld increase the activity of Aβmetabolic enzymes,reduce the content of Aβin vivo,and improve the ability of learning and memory.Hesperetin is a natural dihydroflavonoid compound with anti-inflammatory and antioxidant pharmacological effects.In this study,hesperetin was given to 3-month-old APPswe/PS1d E9 double transgenic mice for 6 months to observe the effects of hesperetin early intervention on Aβmetabolic enzyme activity,neuronal damage as well as learning and memory ability,which mihgt provide a theoretical basis for mu Ltitarget and early intervention of flavonoids on AD.Objective:To investigate the effects of early intervention of hesperetin on the clearance of Aβ metabolism,neuron loss and the improvement of learning and memory ability in APPswe/PS1 d E9 double transgenic mice.Methods:3-month-old C57BL/6J mice were divided into wild group,wild 10 and20 dose groups(0.5% CMC,10 and 20 mg/kg/d),and 3-month-old APPswe/PS1 d E9 double transgenic mice were divided into as model group,hesperetin low,medium and high dose groups(0,20,40 and 80 mg/kg/d),respectively,once a day for 6 months.After intragastric administration,3-month-old C57BL/6J mice were added as normal group.Morris water maze and Barnes maze behavior experiments were used to observe the learning and memory ability of mice.HE staining was used to observe the morphology of neurons in the hippocampal CA1 region.Immunohistochem-istry(IHC)and Western Blot were used to observe the expression of Aβ42,neprilysin(NEP)and insu Lin-degrading enzyme(IDE)in brain tissues of mice.The content of Aβ42 in the serum of mice was determined by ELISA.Results:1.Ethological experimentDirectional navigation experiment of Morris water maze : Compared with the wild group,latency finding the platform of the model group was significantly prolonged(P<0.01),compared with the model group,that of low,medium and high dose hesperetin groups was significantly shorter(P<0.01),compared with the wild group,the latency was significantly shortened in wild 10 and 20(P<0.01).Compared with the wild group,the number of mice crossing the platform in the model group was significantly reduced(P<0.05),compared with the model group,that in low,medium and high dose hesperetin groups was significantly increased(P<0.01),compared with the wild group,the crossing times of wild hesperetin 20 dose group was significantly increased(P<0.05).Barnes maze : Compared with the wild group,the escaping latency and the number of errors in finding the target box in the model group were significantly increased(P < 0.05),Compared with the model group,those were significantly reduced in the low,medium and high dose groups of hesperetin(P < 0.01),Compared with the wild group,those in the wild hespertin 20 group were significantly reduced(P<0.05).2.Hematoxylin-eosin stainingThere were 3-4 layers neurons in the hippocampal CA1 region of mice in wild group,neurons were closely arranged and the structure was relatively complete.In the wild hespertin 10 and 20 groups,there were 4-5 layers,neurons were more closely arranged,and the morphology of neurons was complete and clear.There were 5-6 layers neurons in the hippocampal CA1 region in normal group,and the neurons were densely arranged,with plump nuclei and clear boundaries.In the model group,there were 2-3 layers,the number of neurons was significantly reduced,the intracellu Lar space was significantly widened,neurons arranged disorderly with obvious cell body swelling,and the surrounding tissues of neurons were damaged.The low,medium and high dose groups of hesperetin had relatively regu Lar and compact arrangement of neurons,and the morphology of neurons were improved.3.Effect of hesperetin early intervention on the expression of Aβ42,NEP and IDE in brain tissueThe Results of IHC showed that compared with the wild group,the Aβ42 expression in brain tissue of the model group increased significantly(P<0.05),and that in the wild hespertin 10 and 20 groups and the normal group was significantly decreased(P<0.05).Compared with the model group,that in low,medium and high dose hesperetin groups was significantly decreased(P<0.05).Western blot Results showed that compared with the wild group,the Aβ42 expression in the brain tissue of the model group was significantly increased(P<0.01),and the wild hespertin 10,20 groups and the normal group significantly decreased(P<0.05).Compared with the model group,the Aβ42 expression in low,medium and high dose hesperetin groups was significantly decreased(P<0.01).The Results of IHC showed that compared with the wild group,there was no significant difference in the expression of NEP in the brain tissue of model group and normal group(P>0.05),the NEP expression in the wild hespertin 10 and 20 groups was significantly increased(P<0.01).compared with the model group,the NEP expression in the medium and high dose hesperetin groups increased significantly(P<0.01).Western blot Results showed that compared with the wild group,there was no significant difference in the expression of NEP in the brain tissue of model group and normal group(P>0.05),and the NEP expression in the wild hespertin 10 and 20 groups was significantly increased(P<0.01).Compared with the model group,that in the middle and high dose hesperetin groups was significantly increased(P<0.05).The Results of IHC showed that compared with the wild group,there was no significant difference in the expression of IDE in the brain tissue of model group and normal group(P>0.05),the IDE expression in the wild heperertin 10 and 20 groups was significantly increased(P<0.05).Compared with the model group,the IDE expression in the low,medium and high dose hesperetin groups was significantly increased(P<0.05).Western blot Results showed that compared with the wild group,there was no significant difference in the expression of IDE in the brain tissue of model group and normal group(P>0.05),the NEP expression in the wild heperetin 10 group was significantly increased(P<0.01).Compared with the model group,the IDE expression in the low and medium dose hesperetin groups was significantly increased(P<0.05).4.Effect of hesperetin early intervention on Aβ42 in serum of mice.Compared with the wild group,the Aβ42 content in serum of the model group was significantly increased(P<0.01).compared with the model group,that in the low,medium and high dose hesperetin groups was significantly decreased(P<0.01),and the middle dose hesperetin group was lower than that of the low dose hesperetin group(P<0.05).There was no significant difference among other groups(P>0.05).Conclusion:Early intervention with hesperetin significantly improved neuron damage in hippocampal CA1 region,learning and memory ability of APPswe/PS1 d E9 double transgenic mice,and the mechanism might be related to the enhancement of NEP and IDE activities,the enhancement of Aβ metabolism,and the reduction of Aβ content in vivo. |
