Effect And Mechanism Of Rice Bran Extract On Learning And Memory In Model Mice

Alzheimer's disease(AD)is a neurodegenerative disease with a high prevalence in the elderly,which main clinical manifestations are deterioration of learning and memory abilities.The pathogenesis of AD is not clear.According to the widely accepted hypothesis of ?-amyloid protein(A?)toxic aggregates(such as soluble oligomers,fibres,etc.)formed by A? misfolding can destroy the cell membrane of nerve cells and induce a series of cascades such as oxidative stress and the entanglement of phosphorylated Tau protein.It leads to neuronal apoptosis,which in turn leads to AD.In this thesis,rice bran,a by-product produced in the process of rice processing,was used as a raw material to investigate the effects of Rice Bran Ethanol Extract(RBEE)on the memory of neuronal cells.Specifically,this thesis will investigate the effects of Rice Bran Ethanol Extract(RBEE),Rice bran oil(RBO)and p-Hydroxycinnamic Acid(p-HCA)on the improvement of learning and memory in D-Galactose(D-Gal)model mice and its inhibitory effect on A ?-induced apoptosis of SH-SY5Y cells.The main research of this thesis is as follows:1.RBEE was found to have the effect of improving spatial learning,memory and new object recognition in mice through localization navigation,spatial exploration and new object recognition experiments;RBEE was found to reduce the expression of A? and Tau,which is the marker of AD pathogenesis in the hippocampal region of mouse brain tissue through immunohistochemical experiments.In addition,it could reduce the expression of Glial Fibrillary Acidic Protein(GFAP).The Western blotting(WB)experiments showed that RBEE could reduce D-gal-induced learning and memory impairment by upregulating p-AKT and downregulating the expression of Tau,Receptor for Advanced Glycation Endproducts(RAGE),Beta-site APP cleaving enzyme I(Bace-1),GFAP and apoptosis mediator Caspase-3(cleaved).It could achieve neuroprotective effects and function as a mitigating agent of AD.Moreover,to investigate the effect of RBEE on A?(1-42)toxic aggregates,firstly,the experiment of thiazolyl blue(3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide,MTT)indicates that RBEE could inhibit the A?(1-42)-induced neuronal cell SH-SY5Y apoptosis with certain neuroprotective effects;RBEE was found to inhibit A?(1-42)aggregation by Thioflavin T(ThT)and Atomic Force Microscope(AFM)experiments.Therefore,RBEE may up-regulate the expression of p-AKT by activating AKT,resulting in an increase in the phosphorylation level of its downstream substrate Glycogen synthase kinase-3 ?(GSK-3?),thus reducing the activity of GSK-3? and reducing the phosphorylation level of Tau protein,and a decrease in A? toxic aggregates through down-regulation of RAGE,Bace-1 and GFAP,thereby-reducing expression of apoptosis mediator Caspase-3(cleaved).Under the synergistic action of multi-targets and multi-angles,the learning and memory ability of the model mice will improve.Finally,from the analysis by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry,the result showed that RBEE might contain 1040 chemicals.We selected a kind of active ingredient-p-hydroxycinnamic acid,which may have inhibitory effect on AD,and did further research on it.2.Through localization navigation,spatial exploration and new object recognition experiments on mice,the result showed that RBO has the function of improving spatial learning,memory and new object recognition in mice;through immunohistochemical experiments on brain sections from the hippocampal region of mice,it was found that RBO can reduce the positive expression of A? and Tau which are the markers of AD onset;In addition,it could reduce the expression of Glial Fibrillary Acidic Protein(GFAP).through WB experiments,it was found that RBO has the function of improving spatial learning,memory and new object recognition in mice which might associated with the down-regulation of RAGE,Bace-1,GFAP,Tau and apoptosis mediator Caspase-3(cleaved)expression in the hippocampal region of brain tissue.Thus,RBO may improve learning memory function in mice by down-regulating RAGE and GFAP resulting in reduced phosphorylation levels of Tau protein,down-regulating RAGE,GFAP and Bace-1 and resulting in a reduction of A?toxic aggregates.Finally,we did gas chromatography-time-of-flight mass spectrometry experiments on RBO and analysed that it may contain 88 chemical substances.After screening these substances,we selected a component with possible anti-AD activity called p-hydroxycinnamic acid,for subsequent validation of its inhibitory effect on A?(1-42)-induced apoptosis in SH-SY5Y cells.3.The monomer p-HCA,which was selected from RBEE and RBO,was selected for the experiments to verify its inhibitory effect on A?(1-42)-induced apoptosis in SH-SY5Y cells.Firstly,the content of p-HCA in RBEE and RBO was quantified using ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry assay.The results demonstrated that both RBEE and RBO contained p-HCA and their contents were 664.09 ng/g and 13943.46 ng/L respectively.From MTT assay,p-HCA was found to have an inhibitory effect on A?(1-42)-induced apoptosis of SH-SY5Y;The WB experiment indicated that p-HCA could inhibit A?(1-42)-induced apoptosis of SH-SY5Y cells by down-regulating the expression of p-p38 and Caspase-3(cleaved);The ThT and AFM experiment showed that p-HCA also had a blocking effect on the aggregation of A?(1-42).Therefore,p-HCA may exert its protective effect on neuronal cells by inhibiting the production of toxic aggregates of A?.In conclusion,this thesis mainly investigated the effects of three rice bran extracts on the improvement of learning and memory abilities as well as the inhibition of A?-induced apoptosis in SH-SY5Y cells in D-galactose model mice,and tentatively explored their mechanisms,with a view to providing directions for the study of Alzheimer's disease and providing a basis for the development of rice bran-related products.