The Amelioration And Mechanisms Of Procyanidins Extracted From The Lotus Seedpod On Alzheimer’s Disease

Part 1 The protection effect of procyanidins from the lotus seedpod on the cells model of Alzheimer’s diseaseObjectionOur study aimed to explore the protection effect of procyanidins from the lotus seedpod on Alzheimer’s disease.This research designed an AD cell model by the intervention of Aβ in PC12 cells,and then to explore both the effect of LSPC on the apoptosis and the amelioration of cell morphology through the AD cell model.Materials and methods(1)Determination of intervention dosages and time of Aβ.To decide an intervention dose of Aβ25-35,PC12 cells were cultured in 96-wells plates with different doses of Aβ25-35(0,5,10,20,and 40μM)and five periods of incubation time(6,12,24,48,and 72 h),respectively.Cells with 0βM Aβ25-35 were as control group.The survival rate of cells was measured by CCK-8.The intervention dose and time of Aβ25-35 were verified through the survival rate of cells.(2)Determination of intervention dosages of LSPC.After determination of intervention dosages and time of Aβ,we decided an intervention dose of LSPC by CCK-8.PC12 cells were cultured in 96-wells plates with different doses of LSPC(1,2.5,5,10,20,and 40μg/mL)for 30 minutes before Aβ25-35 incubation,respectively.Cells with 0μM A325-35 were as control groups and cells with 20μM Aβ25－35 were as Aβ groups.The survival rate of cells was measured by CCK-8.The intervention dose of LSPC were verified through the survival rate of cells.(3)The protection effect of LSPC on an AD cells model.Cells were cultured as three groups(PC12 cells,PC12 cells with 20μM Aβ25-35,PC12 cells with 20μM Aβ25-35 and 10μg/mL LSPC).After intervention,the morphology of cells was observed through Hoechst staining and microscope.The apoptosis rates of cells were measured by flow cytometry.Results(1)According to the result of CCK-8,the dose of 5μ[M Aβ had no significant effect on the survive rate of cells.The survive rate of cells was decreased as the intervention time of 10μM Aβ being longer.After the dose of 20μM Aβ incubation with 24 h,the survive rate of cells was lessened,significantly.The survive rate of cells was under 50%after intervention with the dose of 40μM Aβ for 12 h.Thus,the dose of 20μM Aβ with 24 h was chosen as the intervention dose and time.(2)The Aβ induced apoptosis rate of cells was diminished by LSPC from 0 to 10μg/mL and it was minimum when the dose of LSPC reached 10μg/mL.LSPC had on effect on the survive rate of cells without Aβ Thus,we chose 10μg/mL LSPC as the intervention dosage.(3)According to Hoechst staining and microscope,PC12 cells in 20μM Aβ25-35 group suggested conspicuous karyopyknosis and cell apoptosis compared to control group while 10μg/mL LSPC prevented the damage from Aβ25-35 remarkably.Through flow cytometry analysis,we validated that the total apoptosis rates(TA)of PC12 cells in control was<5%while after 20μM Aβ25-35 treatment,total apoptosis rates(TA)were 33.36%(P<0.05);compared to Aβ group,10μg/mL LSPC significantly lessened apoptosis rates(P<0.05).ConclusionLSPC can reduce the apoptosis and ameliorate cell morphology of cells through the AD cell model,indicating LSPC as potential treatment for AD.Part 2 The distribution of procyanidins from the lotus seedpod in vivoObjectionOur study aimed to confirm the distribution of LSPC in vivo and explore the potential ingredients in LSPC with a protection effect on Alzheimer’s disease.Materials and methods(1)Animals treatmentFourteen SD rats were randomly divided into two groups(n=7/per).Control group was given 1 ml/kg physiological saline by oral gavage daily;for LSPC group was administered LSPC at dose of 200 mg/kg body weight by oral gavage daily.Rats were sacrificed after two weeks intervention.Tissues,intestine content,and plasma were harvested and stored at-80℃ until analysis.For the extraction of LSPC and its metabolites,tissues(60 mg)were homogenized and analyzed by LC-MS/MS.(2)Sample preparationThe standard sample was prepared as different concentrations(0.5,1,2,5,10,20,40,50,80,100 ng/mL).Samples(50μL,each)were hydrolyzed with a β-glucuronidase/sulfatase type H1(1500U/mL)from H.pomatia(Sigma,USA)for two hours at 37℃ Ethyl gallate(20μL)was as internal standard.For extraction of compounds in tissues,methanol(200μL)was added for each sample following by vibration and centrifugation;the supernatant was collected.The extraction was repeated once.The supernatant was dried at 37℃under vacuum and redissolved in 50μL solution(50%water and 50%methanol).(3)LC-MS/MSThe analysis was performed on a High-performance liquid chromatograghy-tandem mass spectroscopy(LC-MS/MS,AB Sciex Q-Trap 4500,Applied Biosystems,Foster City,CA,USA).5μL samples were injected for LC-MS and the analytes were separated by BETASIL Phenyl Column(2.1 mm×150 mm,3μm;Thermo Scientific,USA)at 35℃The mobile phases composed(A)water with 0.2%acetic acid and(B)methanol with 0.2%acetic acid.Ionization was carried out by electrospray in the negative mode.The transitions for each analyte were screened.The collision energy(CE)and declustering potential(DP)were optimized for each analyte.The calibration curves of the respective standards were utilized to quantify compounds.Results(1)In hippocampus of LSPC group,quercetin(P<0.01),epicatechin(P<0.001),vanillic acid(P<0.05),p-HPPA(P<0.05),gallic acid(P<0.01),and 3-HPAA(P<0.05)had significantly increased.In cortex,quercetin(P<0.01),epicatechin(P<0.001),vanillic acid(P<0.05),p-HPPA(P<0.05),ferulic acid(P<0.05),caffeic acid(P<0.01),m-coumaric acid(P<0.001),PCC(P<0.001)in LSPC group was significant more than control group.In cerebellum,the levels of quercetin(P<0.01),gallic acid(P<0.05),p-HPPA(P<0.001),m-coumaric acid(P<0.05),3-HPAA(P<0.01),and3-HBA(P<0.05))had significantly improved.(2)With LSPC intervention,the enhancement of three other compounds was verified after enzyme preprocess,these being caffeic acid,3-HPAA,and 3-HBA in cardiac and liver.Besides,in liver,homovanillic acid and p-HPPA were also augmented significantly.Diverse compounds in kidney indicated significant differences between control and LSPC groups through enzyme disposal,including epicatechin,homovanillic acid,caffeic acid,vanillic acid,3,4-DHPA,p-HPPA,3-HBA,and pyocatechol.Apart from quercetin,3,4-DHPA alone in spleen and gallic acid solely in pancreas were confirmed to significantly increase.In intestine content,as a primary metabolism pathway,most detected compounds showed discrepancies between control and LSPC samples,except 3-HBA.(3)LSPC intervene notably increased quercetin,epicatechin,homovanillic acid,caffeic acid,vanillic acid,p-HPPA,3-HBA,and pyocatechol in plasm.Several compounds in plasma were aggrandized,including syringic acid(1.98±0.90ng/mL),ferulic acid(31.77±11.23ng/mL),m-coumaric(10.95± 6.75ng/mL),and protocatechuic acid(29.00 ±8.08ng/mL).ConclusionLSPC intervention contributes to various compositions with different concentrations increasing in tissue and plasma of animals.Notably,quercetin was significant increased in brain and other tissues after LSPC treatment,which may be attributed to quercetin-3-o-glucuronide(Q3G)in LSPC.Hence,Q3G could be the potential composition in LSPC for the protection effect on AD.Part 3 The amelioration and mechanisms of procyanidins extracted from the lotus seedpod on Alzheimer’s diseaseObjectionWe aimed to explore the amelioration of learning and memory damage of AD model animals by Q3G in LSPC and verify the mechanism pathway of its effects.Materials and Methods(1)C57 mice were divided into three groups(n=12/per)as control group,AD group,and intervention group.Three groups were administrated physiological saline,Aβ1-42,and Aβ1-42 through intracerebroventricular injection,respectively.After injection,control group and Aβ group were gave sterile water at dose of 20 u1 mg/kg body weight by oral gavage daily while LSPC group was gave Q3G at dose of 50 mg/kg body weight by oral gavage daily for 30 days.The body weight and intake of food were recorded.(2)Mice were accepted i.p.GTT test after six hours fasting,and then,were accepted i.p.ITT test after a week recovery.After intervention,the behavior experiments including open-field test,elevated plus maze test,and Morris water maze were implemented orderly.Then mice were sacrificed and hippocampus and cortex were stored。(3)The concentrations of Aβ in tissue and the levels of insulin in plasm collected in an i.p.GTT test was measured by ELISA.HE staining was used to detect the cell apoptosis and death in mice brain.The expressions of different proteins were confirmed by western blot and immunohistochemical staining,including p-Tau/Tau,p-JNK/JNK,p-PKR/PKR,p-IRS-1/MRS-1,p-Akt/Akt,and p-ERK/ERK.Results(1)Through behavior experiments,we found that learning and memory abilities of mice in Aβ group decreased significantly(P<0.05)whereas Q3G improved learning and memory abilities(P<0.05).Moreover,open-field test and elevated plus maze test verified that Aβ and Q3G had not effects on spontaneous activity and anxiety of mice.The results of i.p.GTT test showed that Aβ group might have slight insulin resistance while Q3G ameliorated insulin resistance of AD model animals.There was no significant difference between three groups in i.p.ITT test and insulin secretion in i.p.GTT test.(2)Compared to control group,the levels of Aβ and the protein expression of p-Tau/Tau in mice brain tissue were increased significantly in AD group whereas Q3G reduced the level of Aβ(P<0.05).The death of cells in mice brain tissue was augmented in Aβ group significantly and Q3G diminished the death of cells(P<0.05)according to HE staining.In AD group,the levels of p-JNK/JNK and p-PKR/PKR were increased,and serine phosphorylation of IRS-1 was augmented.Phosphorylation of Akt and ERK,the downstream of insulin signaling,was lessened.The level of p-GSK/GSK was reduced due to the decrease of phosphorylation of Akt.However,Q3G downregulated the levels of p-JNK/JNK and p-PKR/PKR and serine phosphorylation of IRS-1,and then,upregulated the phosphorylation of Akt,GSK,and ERK significantly(P<0.05).ConclusionAs a composite of LSPC,Q3G could improve learning and memory capability of AD model animals through ameliorating of insulin resistance.Thus,Q3G could be applied in the treatment of AD.