Study On The Material Basis And Mechanism Of Longan Meat In Preventing And Treating Spleen Deficiency Type Dementia In Mice

Purpose:This project supported by the Open Fund of Key Laboratory(Liaoning University ofTraditional Chinese Medicine)of Ministry of Education for Traditional Chinese Medicine(TCM)Viscera-State Theory(ZYZX1709).Under the guidance of TCM theories and according to the medication habits of TCM,the project prepared the water extract of longan aril(LY)by water decocting method,used LC quadrupole time-of-flight MS(LC-Q-TOF/MS)and R software platform to conduct the full spectrum identification and analysis of LY,and used high performance liquid chromatography(HPLC)to determine the contents of partial components.The model of dementia with spleen deficiency in mice was established by D-galactose/Al Cl₃combining irregular diet and spleen injury,and the pharmacodynamics evaluation of longan aril against dementia with spleen deficiency was carried out by morris water maze test,kits,hematoxylin-eosin staining(HE),nissl and tunel analysis.Metabolomics method and western blot(WB)analysis were used to analyze the mechanism of longan aril against dementia with spleen deficiency,and the mechanism and material basis of longan aril in the treatment of dementia with spleen deficiency in mice were preliminarily discussed,which provided theoretical basis for the development of effective ingredients and drug development of longan aril against dementia with spleen deficiency.Method:1.The LY was prepared by water decocting.LC-Q-TOF-MS was used to analyze the full spectrum of LY.The contents of 10 kinds of carbohydrates,17 kinds of amino acids,12 kinds of nucleotides and 10 kinds of flavonoids were determined by HPLC.2.The model of dementia with spleen deficiency in mice was established by D-galactose/Al Cl₃combining irregular diet and spleen injury.Different doses of LY were used for treatment.The grouping and administration were as follows:80 KM mice were randomly divided into control group(ZC),dementia group(DC),spleen deficiency group(PX),spleen deficiency dementia group(PC),longan low,medium and high dose groups(LY1,LY2&LY4)and piracetam positive control group(YX).Except for ZC and DC groups,mice in each groups were induced to establish spleen deficiency model by irregular diet and coercive swimming method for 15 days continuously,after successfully preparing model of spleen deficiency,then the mice in each groups were given intragastric administration on the basis of spleen deficiency,respectively,once a day,for 60 days continuously.The mice of DC group were daily given 140 mg/kg D-galactose via hypodermic injection on the back and neck,and given 20 mg/kg Al Cl₃via intragastric administration;in PC group,the modeling method of DC group was implemented while the diet was irregular(mice were fed cabbages only on odd days,and mice received intragastrically 0.2 m L/10g lard on even days);the low,medium and high-dose LY groups were intragastrically administered with 1.0,2.0 and 4.0 g/kg LY crude drug on the basis of PC modeling,respectively;the mice of YX group were intragastrically given 0.6 g/kg piracetam on the basis of PC modeling;ZC group was given intragastric administration of distilled water of equal volume and hypodermic injection of normal saline of equal volume.The weight and swimming time of the mice were used to evaluate the status of spleen deficiency.Morris water maze test was used to detect the learning and memory abilities of the mice.The levels of oxidative stress,?-amyloid protein(A?)and p-tau of the mice were detected by the kits,and brain histopathology of mice was observed by HE and nissl staining.3.After the morris water maze experiment,brain tissues of mice in each groups were taken and homogenated with pre-cooled methanol:water(4:1),and then be dried with a nitrogen blower,and reconstituted with methanol:water(4:1)for LC-MS detection and analysis.HMDB,KEGG and Metaboanalysis databases were used in combination with literature retrieval to analyze the signaling pathways involved in anti-dementia with spleen deficiency of LY,and the expression of related proteins in the signaling pathways was detected by western blot.Results:1.The yield of LY was about 49.27%±3.98%.In LC-Q-TOF-MS experiment,a total of 105components with high reliability(?ppm?30)were identified,including organic acids,amino acids,vitamins,nucleosides,carbohydrates,phenols,purines,etc.The contents of LY were determined by HPLC,among which the content of glucose was the highest,accounting for about 24.62%.2.The morris water maze results of place navigation test showed that compared with PC group,the escape latency of the treatment group was shortened in different degrees,especially in LY2 and LY1 groups(P<0.001).The results of space exploration showed that compared with PC group,the crossing times of the original platform quadrant of LY1 group were extended(P<0.05).Compared with PC group,the percentages of time in residence in the original platform quadrant of LY2 and LY4 groups were significantly extended(P<0.05);enzyme-linked immunosorbent assays(ELISA)results showed that LY2 and LY1 could significantly reduce reactive oxygen species(ROS)activities in serum and brain tissue of PC mice,respectively(P<0.01);in serum and brain tissue,superoxide dismutase(SOD)activities in LY groups were significantly increased compared with PC group(P<0.05,P<0.01,P<0.001);LY2 and LY4 groups were significantly reduced serum and brain malondialdehyde(MDA)levels(P<0.01,P<0.001);LY of each dose reduced significantly A?and p-Tau protein expression levels of brain tissue in PC mice(P<0.05,P<0.01,P<0.001).HE staining showed that the conditions of hippocampus and cerebral cortex cells in LY1,2 and 4 groups were significantly improved and orderly compared with those in PC group,the cells were arranged neatly.Nissl staining showed that after treatment with LY,the cell morphology was improved and the numbers of tigroid body were increased.The tunel analysis showed that the apoptotic rates in the hippocampus and cortex of each LY groups were significantly lower than that in the PC group(P<0.01),but there was no significant difference among all treatment groups.3.According to the analysis of related differential compounds in HMDB database,24compounds were found to be different in the brain before and after treatment with LY.After through Metabo Analysis database,22 metabolic pathways were found to be related to dementia.Then KEGG database analysis showed that the signaling pathways highly correlated with LY anti-dementia of spleen deficiency were RAS/MEK/ERK(MAPK),NF-?B,AMPK,m TOR and PI3K/Akt signaling pathways,and several metabolites were associated with the MAPK pathway,which was further verified by western blot,and the results showed that LY decreased the expression levels of HRAS,MEK,ERK,p-MEK,p-ERK(P<0.01)and the ratio of p-ERK/ERK and p-MEK/MEK(P<0.05,P<0.01).Conclusion:1.LY could contain carbohydrates,organic acids,amino acids,vitamins,nucleosides,phenols,purines,etc.,in which the content of carbohydrates was the highest,and the content of glucose was about 24.62% of LY.2.LY improved the learning and memory abilities of PC mice,restrained oxidative stress in PC mice,reduced A?and p-Tau protein expression levels of PC mice,alleviated the pathological damage of brain tissue and alleviated dementia symptoms in mice.3.LY could affect the changes of metabolites in the brain of PC mice,and inhibiting the activities of proteins related to the RAS/MEK/ERK pathway could be one of the anti-dementia with spleen deficiency mechanisms of LY.