| Objective: Cognitive aging is a common clinical manifestation of elderly people,and also the main symptom of neurodegenerative diseases such as Alzheimer’s disease.Improving cognitive function is of great value and significance in "active aging" and Alzheimer’s disease.Zanthoxylum bungeanum Maxim.(Z.bungeanum)is a common traditional Chinese medicine that can be used as both medicine and food.In this study,D-galactose-induced fast-aging mouse model was used to reveal the mechanism and material basis of Z.bungeanum in improving cognitive aging,so as to provide a modern biological basis for the "mental enlightenment" effect of Z.bungeanum,expand its clinical application scope,and lay a foundation for its in-depth utilization and potential development.Methods:1.Effects of Z.bungeanum on cognitive function of D-galactose-induced cognitive aging mice and its mechanism.The mice were divided into five groups: control group,model group,Hydergine administration group,Z.bungeanum water decoction administration group,and volatile oil administration group,with 10 mice in each group.After 1h of intragastric administration,mice in the model group and the administration group were subcutaneously injected with a normal saline solution of 500 mg/kg D-galactose,and mice in the control group were injected with normal saline,once a day,for 48 consecutive days.On the 40 th day after administration,the passive avoidance experiment was used to investigate the effect of Z.bungeanum on fear memory impairment in mice,using the escape latency period and the number of mistakes as evaluation indexes.On the 42 nd days of the administration,the Morris water maze experiment was used to evaluate the effects of Z.bungeanum on long-term learning and memory and spatial memory of mice by using the escape latency in the platformseeking experiment,the swimming time in the target quadrant,and the number of crossing the platform in the space exploration experiment.HE pathological staining and Nissl staining were used to evaluate the effects of Z.bungeanum on the pathological morphological and functional damage of neurons in the hippocampus of mice.The contents of MDA and GSH,the activities of SOD and CAT in the brain tissues of mice were detected by the biochemical kit.Western blot was used to detect the expression levels of Nrf2 protein in cytoplasm and nucleus,the expression levels of HO-1,Bcl2 and Bax,and the phosphorylation levels of PI3 K and Akt in brain tissues of mice.2.Study on the pharmacodynamic material basis of Z.bungeanum to improve cognitive function of D-galactose-induced cognitive aging mice.Different polar parts of Z.bungeanum were obtained by the systematic solvent extraction method.Mice were divided into control group,model group,Hydergine group,a petroleum ether group,dichloromethane group,ethyl acetate group,and nbutanol group,a total of 7 groups with 6 mice in each group.After 1h of intragastric administration,mice in the model group and the administration group were subcutaneously injected with a normal saline solution of 500 mg/kg D-galactose,and mice in the control group were injected with normal saline,once a day,for 48 consecutive days.On the 40 th day after administration,the passive avoidance experiment was used to investigate the effects of different polar parts of Z.bungeanum on fear and memory impairment in mice,with the escape latency period and the number of errors as evaluation indexes.On the 42 d day after administration,the Morris water maze experiment was used to evaluate the effects of different polarity parts of Z.bungeanum on long-term learning and memory and spatial memory of mice by using the escape incubation period in the platform-seeking experiment,the swimming time in the target quadrant and the number of crossing the platform in the space exploration experiment were used to evaluate the effects of different polar parts of Z.bungeanum on neuronal damage in the hippocampus of mice.UHPLC-MS/MS was used to qualitatively analyze the main chemical components in the effective parts of Z.bungeanum to improve cognitive aging.3.Study on the mechanism of active fraction of Z.bungeanum improving cognitive function of D-galactose-induced cognitive aging mice.Biochemistry kit was used to detect the effects of the Z.bungeanum active fraction on MDA content and SOD activity in mouse brain tissue.Immunohistochemistry and Western blot were used to detect the effects of the fraction on the protein expression levels of Iba1 and CD11 b in brain tissues.Tunel staining was used to detect the Tunel positive rate in brain tissue.MTT assay was used to detect the effect of active site on survival rate of BV-2 cells induced by LPS+ATP.The protein expression levels of NLRP3,Caspase-1 p20,GSDMD-N,IL-1β and IL-18 were detected by Western blot.Results:1.Z.bungeanum could improve the cognitive function of cognitive aging mice induced by D-galactose by activating the PI3K/Akt/Nrf2 pathway to antagonize oxidative damage.Z.bungeanum water decoction and volatile oil could improve the escape latency of D-galactose-induced cognitive aging mice,reduce the number of errors,increase the time of swimming in the target quadrant and the number of crossing the platform.Both could alleviate the pathological changes and neurological function damage of hippocampal CA1 and CA3 region induced by D-galactose,reduce the generation of brain oxidation product MDA,and increase the levels of GSH,SOD,and CAT.The water decoction and volatile oil could also activate PI3K/Akt/Nrf2 signal,increase HO-1 and Bcl2 protein,and inhibit the expression of Bax protein.2.Ethyl acetate fraction was the main active fraction of Z.bungeanum improving the cognitive function of cognitive aging induced by D-galactose.Ethyl acetate and n-butanol extracts from several different polar extracts of Z.bungeanum could improve the escape latency of D-galactose-induced cognitive aging mice,reduce the number of errors,increase the time of swimming in the target quadrant and the number of crossing the platform.Both could also alleviate the pathological damage in the CA1 and CA3 regions of the hippocampus of model mice,and increase the number of Neu N-positive neurons in the cortex.However,the pharmacological activity of ethyl acetate fraction is stronger than that of n-butanol fraction,which is the main active fraction of Z.bungeanum against cognitive aging.UHPLC-MS/MS analysis of the ethyl acetate extract showed that it mainly contained phenolic acids,alkaloids and flavonoids,and 34 compounds were identified.3.The ethyl acetate fraction of Z.bungeanum could improve neuroinflammation in D-galactose-induced cognitive aging mice and inhibit the activation of NLRP3 /caspase-1 pathway.The ethyl acetate fraction of Z.bungeanum could decrease the production of MDA,increase the level of SOD,inhibit the expression of Iba1 and CD11 b and the positive rate of Tunel in the brain of D-galactose model mice,and increase the survival rate of BV-2 cells induced by LPS+ATP in a dose-dependent manner.And the active fraction of ethyl acetate of Z.bungeanum also could inhibit the expression of NLRP3,caspase-1 P20,GSDMD-N,IL-1β and IL-18 proteins.Conclusion: Z.bungeanum has a definite effect on cognitive aging,and its mechanism is related to the activation of PI3K/Akt/Nrf2 pathway to prevent oxidative stress injury and thereby inhibit the apoptosis of neuron.The ethyl acetate fraction is the main active fraction,which can inhibit the activation of NLRP3/Caspase-1 signal and thus inhibit neuroinflammatory injury. |
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