Effects Of Novel GSK-3 Inhibitors On Cognitive Dysfunction And Peroxidative Damage In AD Rats

ObjectiveThe aggravation of Alzheimer's disease(AD)and the complications it caused have become the fourth leading cause of death in the elderly.The aging society is intensifying and the number of AD patients is gradually increasing,and the mortality rate is rising.Therefore,AD has become a worldwide problem that has received widespread attention.At present,the etiology and pathogenesis of AD are not completely clear,and there is a lack of ideal preventive and therapeutic measures.The existing therapeutic drugs cannot achieve the purpose of completely curing AD,and can only delay the development of the disease.Glycogen kinase synthase 3(GSK-3)is widely distributed in the body and is closely related to the occurrence and development of neurological diseases.GSK-3? is associated with various hypotheses in the pathogenesis of AD,is a linking molecule between various causes,and is a popular target for AD drug development.The novel GSK-3 inhibitor 9b has an innovative effect on metal ion imbalance and oxidative stress targets,and has achieved good effects in improving oxidative stress in in vitro cell experiments.In this experiment,SD rats were selected as experimental animals,and the AD model was constructed by microinjecting A? in the hippocampal CA1 area.Donepezil hydrochloride was used as a positive control drug,and a new GSK-3 inhibitor 9b was used to gavage the model rats.The effects of new GSK-3 inhibitor 9b on cognitive dysfunction and peroxidative damage in AD model rats and its mechanism were studied by the Morris water maze experiment,the measurement of the ATPase activity,the antioxidant system index level,the acetylcholinesterase(AChE)activity,the nitric oxide synthase(NOS)activity,and neurotransmitter level changes.Methods1.Experimental Animals50 SPF healthy male SD rats weighing 180?220 g were selected and randomly divided into 5 groups according to their weights after adapting to the new growth environment:negative control group,model group,positive control group,low-dose 9b group and high-dose 9b group,10 in each group.AD model was established by microinjection of A?42 into the hippocampal CA1 area after anesthesia with 10%chloral hydrate,and intramuscular injection of penicillin to prevent infection.After successful induction of the model,the method of intragastric administration was selected to intervene in the experimental rats by once a day for an intervention period of 28 days.The 9b low-dose group and 9b high-dose group were given 9b suspension 6 mg/(kg·d)and 18 mg/(kg·d),the positive control group was given donepezil suspension 12 mg/(kg·d),and the remaining two groups were given 1 mL of 0.5%sodium carboxymethyl cellulose.The Morris water maze experiment was carried out after the administration,the feed was removed,and normal drinking water was taken.After 24 hours,the mouse head was cut off for sacrifice,blood was collected,and brain tissue was isolated.2.Evaluation of animal growth and developmentRecord the growth rate,development degree,daily life status and weekly weight of the experimental animals.Weigh the brain tissue of the experimental animal to obtain the brain coefficient.3.Morris water maze experiment28 days after drug administration,the positioning navigation experiment and the space exploration experiment were carried out.The total distance and the escape latency of the experimental animals in the previous four days were recorded to evaluate their spatial learning ability.The number of experimental animals crossing the original platform on the 5th day was used to analyze their ability to maintain learning and memory.4.Determination of the changes of various indexes of antioxidant system in the cerebral cortex of AD model ratsThe animals' cerebral cortex was treated into 10%tissue homogenate with normal saline,pre-test according to the operation steps of the kit,and determine the levels of total protein(TP)and malondialdehyde(MDA)in the rat cerebral cortex Glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)activity values.5.Determination of the changes of various indexes of serum antioxidant system in AD model ratsRats were killed by decapitation,blood was collected,left to stand,and the serum was centrifuged after hemagglutination.According to the instructions of the kit,pre-test and determine the viability and MDA content of GSH-Px and SOD in the rat serum.6.Determination of ATPase activity in cerebral cortex of AD model ratsThe experimental animal cerebral cortex and physiological saline were mixed into 10%tissue homogenate,and the vitality values of Mg2+-ATPase,Na+K+-ATPase and Ca2+-ATPase were quantitatively analyzed according to the steps of the kit.7.Determination of the activity of acetylcholinesterase(AChE)and nitric oxide synthase(NOS)in cerebral cortex of AD model ratsThe animals' cerebral cortex was treated into 10%tissue homogenate with normal saline,and quantitatively analyze the activity values of AChE and NOS according to the steps.8.Determination of changes in monoamine neurotransmitters in the cerebral cortex of AD model ratsThe rat cerebral cortex was mixed with 0.1 mol/L perchloric acid solution to prepare a 10%brain tissue homogenate.The contents of norepinephrine(NE),Epinephrine(E),dopamine(DA)and serotonin(5-HT)in the rat cerebral cortex were measured using a high-performance liquid chromatography(fluorescence detector).9.Determination of amino acid neurotransmitters in the cerebral cortex of AD model rats10%brain tissue homogenate was prepared by mixing rat cerebral cortex with 50%acetonitrile solution,and derivatized with 0.5 mol/L sodium hydrogen carbonate solution and 0.5%2,4-dinitrofluorobenzene(DNFB)solution in dark,the contents of aspartate(Asp),glutamic acid(Glu),glycine(Gly),taurine(Tau),and y-aminobutyric acid(?-GABA)in the rat cerebral cortex were measured using a high-performance liquid chromatography(diode array detector).Results10.Effects of novel GSK-3 inhibitors 9b on general growth and development in AD model ratsThe rats in the negative control group grew rapidly,developed normally,and were mentally stable during the experimental intervention period.Rats in the model group developed symptoms such as dry stools,swollen abdomen,rough and dull hair,and severe hair loss in the late period of administration,accompanied by a reduction in exercise and diet.During the experiment,the body weight of rats showed a gradual increase trend,and there was no significant difference in body weight between groups(P>0.05),but the weight gain of the model group had a downward trend compared with other groups.No significant difference was found between the brain tissue coefficients of rats in each treatment group(P>0.05).11.Effects of novel GSK-3 inhibitors 9b on cognitive function in AD model ratsThe analysis of the results of the positioning navigation experiment shows that the total swimming distance and escape latency of the animals in each treatment group decreased overall.From the third day,the total distance and escape latency of the model group were significantly longer than those of the negative control group(P<0.01).The distance and escape latency in the positive control group,9b low-dose group and 9b high-dose group were significantly shortened by comparing with the model group(P<0.05).The analysis of the data from the space exploration experiment shows that the number of times the model group rats crossed the original platform position was significantly reduced(P<0.05);compared with the model group,the number of platform crossings in the positive control group and the high-dose 9b group was significantly increased(P<0.05).12.Effects of novel GSK-3 inhibitor 9b on the antioxidant system in the cerebral cortex of AD model ratsCompared with the negative control group,the GSH-Px and SOD activities in the cerebral cortex of the model group were significantly reduced(P<0.05),(P<0.01),and the MDA content was significantly increased(P<0.01).Compared with the model group,the activity of GSH-Px and SOD in the cerebral cortex of the positive control group and the high-dose 9b group was significantly increased(P<0.05),and the MDA content was significantly decreased(P<0.01).Compared with the model group,the low dose of 9b can significantly increase the activity of GSH-Px in the cerebral cortex of rats(P<0.05)and reduce the content of MDA(P<0.01).13.Effects of novel GSK-3 inhibitors 9b on serum antioxidant system in AD model ratsCompared with the negative control group,the GSH-Px and SOD activities in the serum of the model group rats were significantly reduced(P<0.05),(P<0.01),and the MDA content was significantly increased(P<0.05).Compared with the model group,the activity of GSH-Px and SOD in the serum of the positive control group and the high-dose 9b group was significantly increased(P<0.05),and the MDA content was significantly reduced(P<0.05).Compared with the model group,low dose of 9b can significantly increase the activity of GSH-Px(P<0.05)and SOD activity(P<0.01)and reduce the content of MDA(P<0.05)in rat serum.14.Effects of novel GSK-3 inhibitors 9b on ATPase activity in cerebral cortex of AD model ratsCompared with the negative control group,the Na+K+-ATPase activity(P<0.05)and the Ca2+-ATPase and Mg2+-ATPase activities(P<0.01)in the cerebral cortex of the model group were significantly reduced;Compared with the model group,In the high-dose 9b group,the Na+K+-ATPase activity(P<0.05),the Ca2+-ATPase and Mg2+-ATPase activities(P<0.01)in the cerebral cortex of rats were increased significantly.Compared with the model group,the low-dose 9b can significantly increase the activity of Mg2+-ATPase(P<0.05).15.Effects of novel GSK-3 inhibitors 9b on AChE and NOS activities in cerebral cortex of AD model ratsCompared with the negative control group,the AChE activity in the cerebral cortex of the model group was significantly reduced(P<0.05)and the NOS activity was significantly increased(P<0.01).Compared with the model group,in the positive control group and the high-dose 9b group the activity of AChE in rat cerebral cortex was significantly increased(P<0.05)and NOS activity was significantly reduced(P<0.05);while low dose of 9b had no significant effect on AChE and NOS activities in rat cerebral cortex(P>0.05).16.The effect of a new GSK-3 inhibitor 9b on the changes of neurotransmitters in the cerebral cortex of AD model rats7.1 The effect on the level of monoamine neurotransmittersCompared with the negative control group,the contents of NE and DA in the cerebral cortex of the model group were significantly reduced(P<0.05),(P<0.01),and the 5-HT content was significantly increased(P<0.01).Compared with the model group,the 5-HT content in the cerebral cortex of the high-dose 9b group was significantly reduced(P<0.01);there was no significant effect of low-dose 9b on the level of various monoamine neurotransmitters in the rat cerebral cortex(P>0.05).7.2 The effect on the level of amino acid neurotransmittersCompared with the negative control group,the Asp and Gly contents(P<0.05),the Tau and y-GABA contents(P<0.01)in the cerebral cortex of the model group were significantly increased,and the Glu content was significantly reduced(P<0.01).Compared with the model group,9b can significantly reduce the levels of Gly and y-GABA in the cerebral cortex(P<0.05).ConclusionsThe new GSK-3 inhibitor 9b can improve cholinergic nervous system disorders,regulate neurotransmitter levels,multi-target and multi-link to improve cognitive dysfunction and peroxidative damage in AD rats by targeting oxidative stress,thus exerting resistance AD effect.