| ObjectiveAlzheimer’s disease（AD）mostly occurs in the elderly.With the development of an aging society,the number of patients with AD is increasing,and it has become one of the diseases that seriously affect the health of the elderly.The pathogenesis of AD is not yet clear,and the current drugs are mainly used to relieve symptoms but not to change or reverse the course of the disease.Glycogen synthase kinase-3（GSK-3）is an important protein kinase in the body,which can phosphorylates a variety of substrates.It has links with many AD-related targets,such as formation of β-amyloid（Aβ）,phosphorylation of Tau（Tubulin-associated unit）protein,and cell apoptosis et al,which is one of the important factors that promote the progress of AD.In addition,the excessive presence of copper and other elements promotes the accumulation of Aβand increases the risk of AD.（E）-N-（4-（（（2-Amino-5-phenylpyridin-3-yl）imino）methyl）pyridin-2-yl）cycloprop anecarboxamide（PIMPC）,a GSK-3 inhibitor with metal chelation,was designed and synthesized based on the crystal structure of GSK-3β and introduction of a metal chelation group.In this experiment,SD rats were used as experimental subjects.After Aβ42 intracerebroventricular injection combined with copper sulfate gavage was used to establish AD model in rats,PIMPC was administered orally for treatment.The evaluation of learning and memory function,Aβ and Tau levels,element levels,apoptosis,energy metabolism,cholinergic system,oxidative stress,neurotransmitters,etc.was discussed and analyzed from multi-angles to explore the multi-target effect of PIMPC on Aβ/Cu2+-induced AD model of rats.MethodsFifty SPF healthy and male SD rats were randomly divided into a negative control group（10）and a test group（40）.Rats in the test group were injected with Aβ42（2 μg/μL,5μL）into the lateral ventricle and given CuSO4 solution（2 mg/（kg·bw）/day,2 weeks）by oral gavage to establish AD rats model.At the same period,the rats in the control group were injected with PBS into the lateral ventricle and given the same volume of saline（2 weeks）by oral gavage.After the model was successfully established by evaluation of Morris water maze test,the rats in the test group were randomly divided into a model group,a donepezil group,a low-dose PIMPC group,and a high-dose PIMPC group,with 10 rats in each.Rats in the donepezil group were given 3 mg/（kg·bw）/day donepezil by gavage,and rats in the PIMPC low-dose,high-dose groups were given 10 mg/（kg·bw）/day,20 mg/（kg·bw）/day PIMPC respectively for treatment.The negative control group and the model group were given the same volume of 0.5%sodium carboxymethyl cellulose by gavage per day.The administration period was 4 weeks.After the administration,the Morris water maze test was used to conduct a behavioral test to evaluate the learning and memory function of rats in each group.After the behavioral test,2 rats in each group were randomly selected for cardiac perfusion,and the brain tissues were separated and fixed with paraformaldehyde for subsequent preparation of paraffin sections for HE staining and TUNEL staining to observe the change of morphology and overall apoptosis of rat hippocampal neurons.After the remaining rats were decapitated,the serum was collected and brain tissue and hippocampus were separated,which was stored at-80℃ for subsequent analysis.The mRNA expression levels of amyloid precursor protein（APP）,Tau,B cell lymphoma/leukemia-2（Bcl-2）,Bcl-2 associated X protein（Bax）,and Caspase-3 in hippocampaus were measured by RT-PCR.Western blot was used to determine the protein expression levels of Aβ,Tau,p-Tau,Bcl-2,Bax,Caspase-3,and cleaved Caspase-3.Inductively coupled plasma optical emission spectrometry（ICP-OES）was used to determine the content of Copper（Cu）,Aluminum（Al）,Calcium（Ca）,Magnesium（Mg）,Iron（Fe）,and Zinc（Zn）in the brain and serum.The corresponding kit was used to measure Adenosine triphosphatase（ATPase）,Acetylcholinesterase（AChE）,Nitric Oxide Synthase（NOS）activity in brain,and Copper Zinc-Superoxide dismutase（CuZn-SOD）,Manganese-Superoxide dismutase（Mn-SOD）and Glutathione peroxidase（GSH-Px）activity,and Malondialdehyde（MDA）content in brain and serum.The content of norepinephrine（NE）,epinephrine（E）,dopamine（DA）,5-hydroxytryptamine（5-HT）in brain of rats in groups was determined by high performance liquid chromatography-fluorescence detector.The content of aspartic acid（Asp）,glutamic acid（Glu）,glycine（Gly）,taurine（Taur）,γ-aminobutyric acid（GABA）in brain of rats in groups was determined by pre-column derivatization-high performance liquid chromatography-Photo diode array detector.Results1.Model evaluationThe results of the Morris water maze test for model evaluation suggested that compared with the negative control group,the latency of the rats in the test group was significantly longer on the third and fourth days in the training period,and the frequency of crossing the platform in probe period significantly reduced（p<0.05）.2.The effect of PIMPC on the learning and memory function in AD ratsThe results of Morris water maze test suggested that the latency of rats in the model group was significantly longer than that of the negative control group on the fourth day in the training period（p<0.05）;compared with the model group,the latency of rats in the low-dose and high-dose PIMPC groups was significantly shorter（p<0.05）.The frequency of crossing the platform of rats in the model group was significantly reduced compared with the negative control group（p<0.05）;compared with the model group,the frequency of crossing the platform of rats in the donepezil group,low-dose and high-dose PIMPC groups was significantly increased（p<0.05）.3.The effect of PIMPC on Aβ and Tau in hippocampus of AD ratsThe protein expression of Aβ,Tau and p-Tau in the hippocampus of the model group was significantly higher than that of the negative control group（p<0.05）.Compared with the model group,the protein expression of Aβ in the high-dose PIMPC group was significantly decreased（p<0.05）,and the protein expression of Tau and p-Tau in the the donepezil group,low-dose and high-dose PIMPC group was decreased significantly（p<0.05）.However,there was no statistical difference in the mRNA expression levels of APP and Tau in the hippocampus of rats among groups（p>0.05）.4.The effect of PIMPC on copper,aluminum,calcium,magnesium,iron and zinc in brain and serum of AD ratsThe content of Cu in the brain of rats in the model group was significantly higher than that in the negative control group（p<0.05）.The content of Cu in the brain tissue of rats in high-dose PIMPC group was significantly lower than that in the model group（p<0.05）.Compared with the negative control group and the model group,the Al content of brain in rats in the high-dose PIMPC group was appeared to have a downward trend.However,there was no significant difference in the content of Cu,Al in the serum of rats in each group（p>0.05）.Under the conditions in this study,the content of Ca,Mg,Fe,and Zn in the brain and serum of rats did not change significantly among groups,and the difference was not statistically significant（p>0.05）.5.The effect of PIMPC on the morphology of hippocampus in AD ratsThe results of HE staining suggested that the hippocampal neurons in the negative control group were neatly arranged and distributed tightly,and no pathological changes were observed.The hippocampal neuron cells in the model group were loosely distributed and cell vacuoles appeared.After the treatment,the hippocampal neuron cells in the donepezil group,low-dose and high-dose PIMPC groups were more densely distributed than the model group,and cell vacuoles were reduced apparently.6.The effect of PIMPC on the apoptosis of hippocampal neurons in AD rats6.1 The effect of PIMPC on overall apoptosis of hippocampal neurons in AD ratsApoptosis in the hippocampal neurons was observed in rats in the model group,and the proportion of positive apoptotic cells was significantly higher than that in the negative control group（p<0.05）.Compared with the model group,the proportion of positive apoptotic cells in hippocampus in the donepezil group,low-dose and high-dose PIMPC group was significantly reduced（p<0.05）.6.2 The effect of PIMPC on apoptosis-related mRNA and protein expression of hippocampus in AD ratsThe protein expression of cleaved Caspase-3（17kDa）in the hippocampus of rats in the model group was significantly higher than that of the negative control group（p<0.05）;compared with the model group,the protein expression the cleaved Caspase-3（17kDa）in the hippocampus of rats in the low-dose and high-dose PIMPC group was decreased significantly（p<0.05）.However,the protein expression levels of Bcl-2,Bax and Caspase-3（35kDa）in the hippocampus of rats did not significantly differ（p>0.05）among groups.However,there was no statistical difference in the mRNA expression levels of Bcl-2,Bax and Caspase-3 in the hippocampus of rats among groups（p>0.05）.7.The effect of PIMPC on ATPase of brain in AD ratsThe activity of Na+K+-ATPase and Mg2+-ATPase in the brain of rats in the model group was significantly lower than that in the negative control group（p<0.05）;compared with the model group,Na+K+-ATPase and Mg2+-ATPase activities in the brain of rats in low-dose and high-dose PIMPC group were significantly increased（p<0.05）.The activity of Na+K+-ATPase and Mg2+-ATPase in the brain of rats in the donepezil group was not different significantly compared to the model group（p>0.05）.Compared with the negative control group,the Ca2+-ATPase activity in the brain of rats in the model group has a tendency to decrease.The Ca2+-ATPase activity in the brain of rats in the donepezil group,low-dose and high-dose PIMPC group showed a tendency to increase compared with the model group（p>0.05）.8.The effect of PIMPC on anti-oxidation related indexes of brain and serum in AD ratsThe activity of CuZn-SOD and GSH-Px in the brain of rats in the model group was significantly lower than that in the negative control group（p<0.05）.Compared with the model group,the activity of CuZn-SOD in the brain of rats in high-dose PIMPC group was significantly increased（p<0.05）,and the activity of GSH-Px in the brain of rats in each therapy group was significantly increased（p<0.05）.The MDA content in the brain of rats in the model group was significantly higher than that in the negative control group（p<0.05）;the MDA content in the brain of rats in the low-dose and high-dose PIMPC group was significantly lower than that in the model group（p<0.05）.There was no significant difference in the activity of Mn-SOD in the brain of rats among groups.The activity of CuZn-SOD in serum of rats in the model group was significantly lower than that in the negative control group（p<0.05）;compared with the model group,donepezil and high-dose PIMPC could significantly increase the activity of CuZn-SOD in serum of rats（p<0.05）.The MDA level in serum of rats in the model group was significantly higher than that in the negative control group（p<0.05）;the MDA level in serum of rats in the donepezil group and high-dose PIMPC group was significantly lower than that in the model group（p<0.05）.There was no significant difference in the activities of Mn-SOD and GSH-Px in the serum of rats among groups.9.The effect of PIMPC on AChE and NOS of brain in AD ratsThere was no significant difference in AChE activity of brain in rats between the negative control group and the model group（p>0.05）.Compared with the model group,the AChE activity of brain in rats in the high-dose PIMPC group was significantly reduced（p<0.05）.The NOS activity of brain tissue in the model group was significantly lower than that in the negative control group（p<0.05）;compared with the model group,the NOS activity of brain in each therapy group was significantly increased（p<0.05）.10.The effect of PIMPC on neurotransmitter of brain in AD ratsRegarding monoamine neurotransmitter,the NE content of brain in the model group was significantly lower than that in the negative control group（p<0.05）;compared with the model group,the NE content of brain in the low-dose and high-dose PIMPC group was significantly increased（p<0.05）.There was no significant difference in the contents of E,DA and 5-HT of brain in the rats among groups（p>0.05）.The 5-HT content of brain in the model group appeared a downward trend compared with the negative control group,and that in the donepezil group,low-dose and high-dose PIMPC group appeared an upward trend compared with the model group.Regarding amino acid neurotransmitter,compared with the negative control group,the contents of Glu,Gly and Taur of brain tissue of the model group were significantly reduced（p<0.05）;compared with the model group,the contents of Gly and Taur of brain in the donepezil group and high-dose PIMPC group were significantly increased（p<0.05）.The Glu content in brain of rats in each therapy group had a tendency to increase compared with the model group（p>0.05）.Compared with the negative control group,the content of Asp and GABA of brain in the model group suggested a downward trend（p>0.05）;compared with the model group,the content of Asp and GABA of brain in the donepezil group and high-dose PIMPC group was increased significantly（p<0.05）.ConclusionsPIMPC could down-regulate the high expression levels of Aβ,Tau and p-Tau proteins of hippocampus in Aβ42/Cu2+-induced AD model rats,improve the morphological changes of hippocampal neurons,and down-regulate the protein expression of cleaved Caspase-3 to improve hippocampal neuronal apoptosis.It appeared to have a chealtion effect on Cu and Al of brain in Aβ/Cu2+-induced rats,but it had no obvious effect on the basic level of Ca,Mg,Fe,and Zn.It could regulate the changes of ATPase and NOS,oxidative stress and neurotransmitter disorders in AD model rats.Overall,PIMPC may play a role to improve the learning and memory impairment of Aβ/Cu2+-induced AD rats in multiple links and multiple targets. |
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