| ObjectiveAlzheimer’s disease(AD)is a neurodegenerative diseasea affecting the health and life quality of the elderly.With the aging of population,AD is becoming a serious public health problem.At present,there is no cure for AD.Present drugs only alleviate the cognitive function of patients,cannot prevent the progression of AD,nor can they reverse the deterioration of AD,have different degrees of side effects.In recent years,some flavonoids have shown potential anti-AD activities in vivo and in vitro experiments.Myricetin and morin are two types flavonols.Previous studies have shown that myricetin and morin exhibit anti-β-amyloid(Aβ)activity in vitro,and can act as metal chelating agents and inhibit oxidative stress to alleviate neuronal damage.In this study,two natural compounds with potential anti-AD activity,myricetin and morin,were selected as intervention drugs.The AD rat model was established by microinjection of Aβ1-43 oligomer into the hippocampal CA1 region of rats and gavage of aluminum chloride hexahydrate(AlCl3·6H2O)solution.By determinating cholinergic related indexes in brains and serum of rats,and the contents of monoamine neurotransmitters and amino acid neurotransmitters in brains of rats,the effects of two flavonols on cognitive dysfunction in AD rats were estimated;By determinating oxidative/antioxidative system related indexes in brains and serum of rats,the activities of adenosine triphosphatase(ATPase)in brains of rats and the activities of nitric oxide synthase(NOS)in brains and serum of rats,the protective effects of two flavonols on oxidative damage in AD rats were evaluated.Methods1.Experimental animals and treatmentExperimental animals were SPF Sprague-Dawley(SD)rats.They were divided into control(Con,n=12)and experiment(Aβ/Al,n=84)group,and received microinjection of drugs into hippocampal CA1 region:Rats in Aβ/Al group were slowly injected with 5 μL Aβ1-42 oligomer solution(2μg/μL),and rats in Con group were injected with sterile phosphate buffered solution.After surgery,rats in the Aβ/Al group were gavaged with 281.40 mg/kg/d AlCl3·6H2O solution for 4 weeks.At the same time,normal saline was administrated to rats in Con group.After the 4-week intragastric administration period,in order to evaluate the spatial learning ability and memory function of rats in two groups,we performed Morris water maze experiment.After model evaluation,84 rats in the Aβ/Al group were divided into 7 groups:model group(Aβ/Al),low-,medium-and high-dose myricetin groups(Myr-L,Myr-M,Myr-H),as well as low,medium-and high-dose morin groups(Mor-L,Mor-M,Mor-H),with 12 rats in each group.The rats in these groups were gavaged with 0.5%sodium carboxymethylcellulose(CMC-Na)solution,50 mg/kg,100 mg/kg,200 mg/kg myricetin suspension and 50 mg/kg,100 mg/kg,200 mg/kg morin suspension,and the rats in Con group were gavaged the vehicle for 8 weeks.Eight weeks later,to evalutate the spatial learning ability and memory function of rats,we performed behavioral experiment.All rats were sacrificed by collecting abdominal aorta blood.Then serum and brain tissues were separated for subsequent indicators.Meanwhile,during the experiment,general situation of rats was recorded,the weekly body weight of rats was weighed,and the brain coefficients were calculated,finally,the growth situation of rats was evaluated.The dilution ratios of brain tissue homogenates and serum were determined according to the pre-experiment,then the contents of acetylcholine(ACh)and the activities of acetylcholinesterase(AChE)in brain tissues and serum were determined according to the instructions of the kit,by which the changes of cholinergic related indexes in brains and serum of rats was evaluated.High performance liquid chromatography(HPLC)with fluorescence detector was used to determine the levels of 4 monoamine neurotransmitters:noradrenaline(NE),epinephrine(E),dopamine(DA),and 5-hydroxytryptamine(5-HT).And HPLC with diode array detector was used to determine the levels of 5 amino acid neurotrasmitters:aspartic acid(Asp),glutamic acid(Glu),glycine(Gly),taurine(Taur),and y-aminobutyric acid(γ-GABA).The separation was performed on a reversed-phase C18 column.Retention time was used for qualitative determinnation.External standard combined with peak area were used for quantification.According to the pre-experiment,appropriate concentrations of brain tissue homogenates and serum were selected,and the optimal sampling volume was explored.The activities of ATPase in brains of rats,the contents of malondialdehyde(MDA)and glutathione(GSH),the activities of catalase(CAT),glutathione peroxidase(GSH-Px),total superoxide dismutase(TSOD),copper zinc-superoxide dismutase(CuZn-SOD),total nitric oxide synthase(TNOS)and inducible nitric oxide synthase(iNOS)in brains and serum of rats were determined.Detection of the above indicators was carried out according to the kit instructions,and they were used to evaluate the changes of oxidative stress levels in rats.Results1.Evaluation of rat AD modelIn the positioning navigation experiment,the escape latency and swimming distance of rats in the Aβ/Al group was longer than that in the Con group(P<0.05).In the spatial exploration experiment,the platform crossing numbers of the rats in the Aβ/Al group were 38.02%lower than those in the Con group(P<0.05).2.Effects of two flavonols on the growth situation of AD ratsDuring the experiment,the rats in each group were in good health,and there was no difference in general growth status.And the weight of rats in each group increased with time.Compared with the rats in the Con group,the brain coefficients of rats in the Aβ/Al group were increased(P<0.05).Compared with rats in Aβ/Al group,the brain coefficients of rats in MyrH group were decreased(P<0.05).3.Effects of two flavonols on the learning and memory function of AD ratsIn the positioning navigation experiment,the escape latency and swimming distance of rats decreased with training time.Compared with the rats in Con group,the escape latency of rats in the Aβ/Al group was longer on the second day(P<0.05).Compared with the rats in the Aβ/Al group,the escape latency of rats in the Mor-L group was shorter on the second day(P<0.05),the escape latency of rats in the Mor-H group was also shorter on the third day(P<0.05).In the spatial exploration experiment,the platform crossing numbers of rats in the Aβ/Al group were lower than those in the Con group(P<0.05).While the platform crossing numbers of rats in the intervention groups were higher than those in the Aβ/Al group,and the differences were statistically significant except Mor-L group(P<0.05).4.Effects of two flavonols on the cholinergic related indexes in brains and serum of AD ratsAfter 8-week intervention of myricetin and morin,compared with the rats in Con group,the contents of ACh in brains of rats in Aβ/Al group were lower(P<0.05).Compared with the rats in Aβ/Al group,the ACh contents in the brains of rats in each intervention group were not restored.Compared with the Con group,the AChE activities in the brains of rats in the Aβ/Al group were increased(P<0.05).Except Myr-M group,the AChE activities in brains of rats in each intervention group were lower than those in Aβ/Al group(P<0.05).The serum ACh contents of rats in each group had no obvious change,and the serum AChE activities of rats in each group did not show significant difference.5.Effects of two flavonols on the levels of 9 neurotransmitters in brains of AD ratsThere was no significant difference in the contents of DA and 5-HT in the brains of rats in each group.Compared with the rats in the Con group,the contents of NE in the brains of rats in the Aβ/Al group were increased(P<0.05).Compared with the rats in Aβ/Al group,the contents of NE in brains of rats in Myr-H,Mor-L and Mor-H groups decreased significantly(P<0.05),and returned to the same level as those of Con group.The contents of E in brains of rats in Myr-H,Mor-L and Mor-M groups increased significantly(P<0.05).Compared with the rats in the Con group,the Glu contents in the brains of rats in the Aβ/Al group were increased(P<0.05),while the Gly and y-GABA contents were decreased(P<0.05).Compared with the Aβ/Al group,the contents of Gly in the brains of rats in the 6 intervention groups were significantly increased(P<0.05).Except for the rats in Myr-M group,the contents of y-GABA in the brains of rats in the 5 intervention groups were significantly increased(P<0.05),while the contents of Glu in the brains of rats in the Myr-M and Mor-L groups were significantly decreased(P<0.05).8.Effects of two flavonols on the function of oxidative and antioxidant system in brains tissue and serum of AD ratsThere was no significant difference in TNOS activities in brains of rats in each group.The iNOS activities in brains of rats in Aβ/Al group were significantly higher than those in Con group(P<0.05),while the iNOS activities in brains of rats in Myr-H,Mor-L and Mor-M groups were significantly lower than those in Aβ/Al group(P<0.05).There was no significant difference in TNOS activities and iNOS activities in serum of rats in each group.Compared with the rats in the Con group,the Na+K+-ATPase activities in the brains of rats in the Aβ/Al group were decreased(P<0.05).Compared with the rats in Aβ/Al group,the activities of Na+K+-ATPase were increased in the rats of Myr-L and Myr-M group(P<0.05),the activities of Mg2+-ATPase were increased in the 5 intervention groups except the Mor-M group(P<0.05),and the activities of Ca2+-ATPase were increased in the 6 intervention groups(P<0.05).Compared with the rats in the Con group,the MDA contents in brains of rats in Aβ/Al group were higher(P<0.05),the activities of T-SOD,CAT and the contents of GSH in the brains of rats in the Aβ/Al group were decreased(P<0.05).Compared with rats in Aβ/Al group,the MDA contents in the brains of the rats in the Myr-M group were decreased(P<0.05),the activities of T-SOD and the contens of GSH in brains of rats in each intervention group were increased(P<0.05),the CAT activities in the brains of rats in the Myr-M and Myr-H groups were increased(P<0.05),and recovered to the same level as those in the Con group.However,there was no significant difference in the activities of CuZn-SOD and GSH-Px in the brains of rats in each group.There was no significant difference in MDA contents,T-SOD activities,GSH-Px activities,GSH contents and CAT activities in serum of rats in each group.The activities of CuZn-SOD in the serum of rats in the Aβ/Al group were lower than those in the Con group(P<0.05).The activities of CuZn-SOD in the serum of rats in three myricetin intervention groups and Mor-L group were higher than those in the Aβ/Al group(P<0.05).ConclusionBoth myricetin and morin improved the learning and memory function of AD rats to some extent,mainly by restoring the function of the central nervous cholinergic system,regulating the disorder of brain neurotransmitters,reducing the activity of iNOS in brain tissues,restoring brain energy metabolism of brain tissues,and reducing the level of oxidative stress in brain tissues by restoring the activities of antioxidant enzymes. |
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