The Study Of Anti-inflammatory Mechanism Of Huperzine A And Its Effect On Plasma Metabolites In Alzheimer’s Disease Model Rats

Objective:Alzheimer’s disease（AD）is an irreversible and progressive neurodegenerative disease,which is mainly manifested in the loss of learning and memory ability,and eventually leads to death.AD frequently occurs in the elderly population.With the increasing level of population aging,the incidence rate of AD is also increasing.However,there is a lack of effective treatment of AD.Astrocyte（AS）is involved in many physiological and pathological processes.More and more evidence showed that the deposition of amyloid-β（Aβ）can induce the activation of peripheral as to mediate inflammation.Huperzine A（Hup A）is an acetylcholinesterase（ACh E）inhibitor,and has neuroprotective and anti-inflammatory effects.Recently,it has been found that cysteine aspartate specific protein-1（caspase-1）plays an important role in inflammatory response.It can cleave the precursor of inflammatory factors to make them mature and play an inflammatory role.Metabolomics can detect the metabolites in organisms through efficient,rapid and high-throughput screening methods.Many experiments have reported the use of metabolomics technology to detect the potential markers of AD.In addition,this technology can be used to explain the potential mechanism of drug treatment of AD from the perspective of metabolites.Therefore,this study explored the anti-inflammatory and cognitive dysfunction improving mechanisms of Hup A from the perspective of caspase-1,and used ultra-high performance liquid chromatography coupled with a triple quadrupole time-of-flight mass spectrometry to explore the effect of Hup A on plasma metabolites of AD rats.Methods:Male Wistar rats were randomly divided into three groups:Sham operation group（Sham group）,AD model group（AD group）and AD+Hup A group.The rats in AD group and AD+Hup A group were constructed by intraperitoneal injection of D-galactose（D-gal）,oral administration of Al Cl₃and bilateral ventricles injection of Aβ25-35 to establish AD model,and the Sham group was treated with the same dose of normal saline.1)Morris water maze test was used to test whether the establishment of AD rat model was successful and the improvement of symptoms in AD rats by Hup A;2)The expression and deposition of Aβin hippocampus were detected by Western blot and immunohistochemistry;3)The protein expression of glial fibrillary acidic protein（GFAP）was detected by Western blot and the co localization of GFAP and Aβwas detected by immunofluorescence confocal microscopy;4)The expression of caspase-1 protein was detected by Western blot,and GFAP and caspase-1 were co localized by immunofluorescence confocal microscopy;5)The protein expression and secretion levels of interleukin（IL）-1βand IL-18 were detected by Western blot and ELISA respectively,immunofluorescence confocal analysis was used to co localize IL-1β,IL-18 with GFAP;6)All experimental data were expressed by mean±standard deviation,analyzed by SPSS 20.0 software,and one way ANOVA was used for comparison among multiple groups.Each sample of each group was tested,P<0.05 showed significant difference;7)The information of plasma metabolites was collected by UPLC-Q-TOF/MS.After screening the different metabolites,endogenous metabolites identification and pathway enrichment analysis were performed.Results:1)In AD group,the escape latency was prolonged and the times of platform crossing and the time of target quadrant were decreased,which indicated that the learning and memory function of AD group was impaired and the AD model was successfully established.Hup A can reduce the escape latency of AD rats,increase the times of platform crossing and the time in the target quadrant,which indicates that Hup A can improve the learning and memory function of AD rats;2)Hup A can reduce the expression level of Aβprotein and Aβplaque in the hippocampus of AD rats,indicating that Hup A can alleviate the deposition of Aβin the brain of AD rats;3)Hup A can reduce the Aβplaque and the expression of GFAP around it,which indicates that Hup A can reduce the deposition of Aβand the activation of AS;4)Hup A can significantly reduce the protein expression of caspase-1 in hippocampus of AD rats,and reduce the expression of caspase-1 and GFAP in co localization,indicating that Hup A can reduce the protein expression of caspase-1 in activated AS;5)Hup A can reduce the protein expression and secretion levels of IL-1βand IL-18 in hippocampus,and simultaneously reduce the expression levels of IL-1β,IL-18 and GFAP in co localization,indicating that Hup A can alleviate the inflammatory response of AS;6)A total of 87 different metabolites were screened by rat plasma metabolomics,involving 28 related metabolic pathways.The main pathway Hup A effected were linoleic acid metabolism,glycerophospholipid metabolism and arachidonic acid metabolism,which were related to lipid metabolism.Conclusion:Hup A can alleviate the learning and memory dysfunction of AD rats by reducing the expression and deposition of Aβin the hippocampus of AD rats and inhibiting the activation and inflammatory response of AS,this effect may be related to the inhibition of caspase-1 protein expression in AS.The results showed that Hup A mainly affected the lipid metabolism in the plasma of AD rats by detecting the metabolomics of rat plasma.The metabolism of linoleic acid was closely related to inflammation.It was suggested that Hup A might have anti-inflammatory effect by affecting the concentration of linoleic acid.The study of anti-inflammatory mechanism of Hup A and its effect on plasma metabolites of AD rats will help to explore new targets and potential biomarkers of AD,and provide new ideas for drug research and treatment of AD.