The Protective Effect Of Novel GSK-3? Inhibitor Compound 9b On Nervous System Damage And Apoptosis In Rats Induced By A?

ObjectivesAlzheimer disease(AD)is a common neurodegenerative disease with progressive development of age in the elderly.In recent years,with the change of China's population structure,the aging problem is becoming more and more serious,and the incidence of AD is also on the rise year by year.Under the current medical background,due to its lack of effective prevention and treatment methods,AD patients have caused inconveniences in their lives,and have also brought huge economic and spiritual burdens to their families and society.Therefore,the pathogenesis,effective prevention,timely diagnosis and symptomatic treatment of AD have become the key points of multidisciplinary research in the world.At present,due to the complicated pathogenesis of AD,a variety of drugs on the market are only symptomatic treatment,but failed to fundamentally treat AD patients.Therefore,GSK-3 kinase molecules,which related to the pathogenesis of AD,have been explored.Small molecular inhibitors of GSK-3 have emerged,which can prevent the occurrence of AD,improve the symptoms of AD and cure the causes of AD through acting on multiple links of AD.Therefore,in this experiment,SD rats were used as the research object,and the AD rat model was established by the microinjection of A?1-42 into the lateral ventricle,and then treated with a novel GSK-3? inhibitor compound 9b.By measuring the content of related elements,A?1-42 protein and p-Tau protein,observing the morphological changes and apoptosis of hippocampus,we explored the protective effect of the novel GSK-3? inhibitor compound 9b on A?-induced nervous system damage and apoptosis in rats.Methods1.Animal grouping and modeling60 healthy SPF male SD rats(180?220g)were selected and housed in cages,with free access to water and food.After one week of adaptive feeding,they were randomly divided into 5 groups according to their body weight,which were the negative control group,the model group,the donepezil control group,the low-dose 9b group and the high-dose 9b group,with 12 animals in each group.In addition to the negative control group,the rats in the remaining groups were microinjected with 10?g A?1-42 in the lateral ventricle(the negative control group was injected with equal volume of sterile PBS)to establish the AD experimental rat model.After the model building was completed,the rats were subjected to a 28-day intervention by intragastric administration:1 mL of 0.5%sodium carboxymethyl cellulose was administered to the negative control group and the model group;the donepezil control group,the low-dose 9b group and the high-dose 9b group were given 12 mg/kg of donepezil,6 mg/kg compound 9b,18mg/kg compound 9b respectively,once a day.2.Animal handlingTwo rats were randomly selected from each group were perfused by 4%paraformaldehyde after the administration stage.Their brain tissue were quickly removed and fixed in 4%paraformaldehyde for paraffin section.The remaining rats were fasted for 24 hours,and after being decapitated and sacrificed,the brain hippocampus and cortex were quickly separated.Both the hippocampus and the cerebral cortex were saved in the refrigerator at-80? for future use.3.Determination of A?142 and p-Tau content in the hippocampusAppropriate amounts of hippocampal tissue were weighed and homogenized with 9 times homogenate medium to prepare a 10%tissue homogenate.The levels of A?1-42 and p-Tau in the hippocampal tissue of rats were determined using commercially available ELISA kits in accordance with the manufacturer's protocol.4.Determination of Cu?Al?Ca?Mg?Fe and Zn content in brain tissueWeigh 0.20g of brain tissue,and after microwave digestion,the content of Cu,Al,Ca,Mg,Fe and Zn in brain tissue of each group of rats was determined by inductively coupled plasma emission spectrometer(ICP).5.Histopathological observation of rat hippocampusThe paraffin sections were prepared and stained by hematoxylin-eosin staining.And the sections were observed under a microscope to observe the morphological changes of hippocampus in rats.6.Determination of apoptosis in hippocampal of ratThe prepared paraffin sections were taken out,and after dewaxing treatment,stained by TUNEL method to observe the overall apoptosis of hippocampus cells.Western blot was used to determine the protein content of Bax,Bcl-2 and Caspase3 in hippocampus.Results1.Effect of 9b on the content of AP142 and p-Tau in the hippocampus of rats induced by A?Compared with the negative control group,the content of A?1-42 and p-Tau in the model group were increased significantly(P<0.05),and the content of A?1-42 and p-Tau in the donepezil control group,the low-and high-dose 9b group were lower than that of model group(P<0.05).2.Effect of 9b on the content of Cu,Al Ca Mg,Fe and Zn in the brain tissue of rats induced byA?The results showed that the content of Cu and Al in the brain tissue of the compound 9b treatment group showed a downward trend.Under the conditions of this experiment,compound 9b has no obvious adjustment effect on the content of essential elements Ca,Mg,Fe and Zn(P>0.05).3.Morphological observation of neurons in the hippocampus of ratsThe hippocampal neurons in the negative control group were mostly oval and a few were round,with relatively complete structure,rich cytoplasm and uniform texture.While the number of hippocampal cells in the model group was significantly lower than that in the negative control group,and the neuron structure was loose,the arrangement between cells was disordereddisordered arrangement,the nucleus staining was fuzzy,and the morphology was incomplete.After the treatment of compound 9b,the morphology of neurons in hippocampus of rats was significantly improved,the number of cells was significantly increased,and the structure of neurons tended to be complete and orderly.4.Observation of neuronal apoptosis in the hippocampus of ratsThe results showed that the apoptosis rate of hippocampal neurons in the model group was significantly higher than that in the negative control group(P<0.01).And the apoptosis rate of hippocampal neurons in the donepezil control group,the low-and high-dose 9b group was significantly lower than that in the model group(P<0.05),and with the increase of the dose of 9b,the apoptosis of hippocampal neurons in the 9b intervention group was significantly reduced.5.Effect of 9b on the expressions of Bax,Bcl-2 and Caspase-3 ptotein in the brain tissue of rats induced byA?Compared with the negative control group,the protein expression of Bax and Caspase-3 in the hippocampus of the model group increased significantly,and the protein expression of Bcl-2 decreased significantly(P<0.05);Compared with the model group,the protein expression of Bax and Caspase-3 in the donepezil control group,the low-and high-dose 9b group decreased significantly(P<0.05),but the protein expression of Bcl-2 did not change significantly(P>0.05).ConclusionsCompound 9b can reduce the content of A?1-42 protein and p-Tau protein in the hippocampus of rats,reduce the protein expression of apoptosis related genes Bax and Caspase-3,which resulting in the morphological changes of rat hippocampus,and ultimately achieve the protective effect on nervous system damage and neuron apoptosis.In addition,compound 9b has a certain effect of excluding Cu and Al elements,but under this experimental condition,9b has no obvious regulating effect on the contents of essential elements Ca,Mg,Fe and Zn.