HDAC6 Inhibitor W5 Improves Learning And Memory Impairment In Aβ/Cu²⁺-Induced Alzheimer’s Disease Rats And Its Related Mechanism

ObjectiveWith the increase of population aging and the increase of disease burden,Alzheimer’s disease（AD）has become an increasingly serious global public health problem,caused great burden and pressure on patients,their families and society in economy,health,emotion and other aspects.At present,the situation of AD is becoming more and more serious.It is still an important topic to explore drug or non-drug means to treat AD.The level of histone deacetylase 6（HDAC6）in the brain tissue of AD patients was significantly higher than that of normal brains.Reducing or inhibiting HDAC6 has been shown to reduce cognitive deficits,reduce betaamyloid plaques,improve Tau pathology,and alleviate memory impairment in animal models of AD.W5 is a new selective HDAC6 inhibitor.Previous in vitro experiments have shown that W5,could directly target Aβ,Tau,and metal homeostasis disorder,which may have multi-target anti-AD effect.In this study,W5 was used to treat AD model rats induced by Aβ/Cu²⁺ to study the improving effect of W5 on learning and memory impairment in AD rats and its related mechanism,to provide the basis for the subsequent development of W5 as an anti-AD drug.MethodsAfter one week of adaptive feeding,50 SPF SD rats were randomly divided into the control group（n=10）and the model group（n=40）.Rats in the model group were injected with 5μL Aβ1-42（2 μg/μL）in the hippocampal CA1 region by using stereotaxic apparatus,and CuSO4 solution（2 mg/（kg·bw）/day）was given by intragastric administration for 4 weeks.Rats in the control group were injected with 5μL sterile PBS solution in the hippocampal CA1 region by using stereotaxic apparatus,and normal saline（2 mg/（kg·bw）/day）was given by intragastric administration for 4 weeks.Then water maze was used to evaluate the model.After model evaluation,rats in the model group were randomly divided into Aμ/Cu²⁺ group,donepezil group,low-dose W5 group and high-dose W5 group,with 10 rats in each group.Rats in the control and model group were given W5 solvent.Rats in donepezil group,low-and high-dose W5 group were given 3 mg/（kg·bw）/day of donepezil hydrochloride solution,15 mg/（kg·bw）/day and 30 mg/（kg·bw）/day of W5 solution,respectively.After administration for 8 weeks,the Morris water maze was used to evaluate the effect of intervention.When the evaluation finishing,two rats in each group were randomly selected for heart perfusion,brain tissue was isolated and fixed with paraformaldehyde.Paraffin sections were prepared for HE staining to observe the morphology of hippocampal neurons of rats.The blood and brain of the other animals were collected after anesthesia,and the hippocampi were separated and stored for later use.Real-time quantitative PCR was used to determine the mRNA expression of amyloid precursor protein（APP）,Tau,Bcl-2 associated X protein（Bax）,B-lymphoblastoma-2 gene（Bcl-2）,Caspase-3,tumor necrosis factor α（TNF-α）,interleukin-1β（IL-1β）,and interleukin-6（IL-6）in the hippocampus.Western blot was used to determine the protein expression of Aβ,Tau and phosphorylated Tau（p-Tau）in the hippocampus.Microplate reader was used to determine the activity of adenylate triphosphate（ATPase）in rat brain,the contents of acetylcholine（ACh）,glutathione（GSH）,malondialdehyde（MDA）and the activities of acetylcholinesterase（AChE）,superoxide dismutase（SOD）,glutathione peroxidase（GSH-PX）and catalase（CAT）in the tissue and serum.HPLC-PDAD was used to determine the contents of amino neurotransmitters,namely aspartic acid（Asp）,glutamic acid（Glu）,glycine（Gly）,taurine（Taur）,γ-aminobutyric acid（γGABA）in the brain.HPLC-FLD was used to determine the contents of monoamine neurotransmitters,namely norepinephrine（NE）,epinephrine（E）,dopamine（DA）,and 5hydroxytryptamine（5-HT）in the brain.Flame atomic absorption spectrometry was used to determine the contents of Cu、Ca、Fe、Zn.Results1.Effect of W5 on the learning and memory of AD ratsIn a four-day navigation experiment,the escape latency of rats in each group showed a downward trend.The escape latency of rats in Aβ/Cu²⁺ group was significantly higher than that in control group on the first day,the third day,and the fourth day（p<0.05）.Compared with Aβ/Cu²⁺ group,the escape latency of rats in donepezil group significantly reduced on the first day and the fourth day（p<0.05）,the escape latency of rats in low-dose W5 group significantly reduced on the fourth day（p<0.05）,the escape latency of rats in high-dose W5 group significantly reduced on the first day,the third day,and the fourth day（p<0.05）.The crossing platform number in Aβ/Cu²⁺group was significantly lower than that in control group（p<0.05）,and the crossing platform number in drug intervention group was significantly higher compared with Aβ/Cu²⁺group（p<0.05）.2.Effect of W5 on the morphology of hippocampal neurons of AD ratsIn the control group,the nerve cells were arranged neatly;the cell structure was intact,as well as the cell nucleus was clear;and there was no obvious swelling and necrosis.Nonnucleated cells in the Aβ/Cu²⁺ group are numerous and loosely arranged.After administration of donepezil and W5,the integrity of nerve cells was improved and there was no obvious necrosis of cells.3.Effect of W5 on the Aβ and Tau in the hippocampus of AD ratsIn the Aβ/Cu²⁺ group,the mRNA expression of APP and Tau in the hippocampus of rats was significantly higher than that in the control group（p<0.05）.Compared with Aβ/Cu²⁺ group,the APP mRNA level was significantly lower in donepezil and W5-administrated groups（p<0.05）,and there was no significant difference in the Tau mRNA expression（p>0.05）.The Aβ,Tau and p-Tau protein levels in the hippocampus of rats in Aβ/Cu²⁺group were significantly higher than that in control group（p<0.05）.Compared with Aβ/Cu²⁺ group,the Aβ protein level in high-dose W5 group decreased significantly（p<0.05）,Tau protein level in donepezil and high-dose W5 groups decreased significantly（p<0.05）,p-Tau protein level in donepezil and W5 treated groups decreased significantly（p<0.05）.4.Effect of W5 on apoptosis-related mRNA expression in the hippocampus of AD ratsIn the Aβ/Cu²⁺group,the mRNA expression of Bax and Caspase-3 in the hippocampus of rats was significantly higher than that in the control group（p<0.05）.Compared with Aβ/Cu²⁺group,the Bax mRNA level was significantly lower in donepezil and W5-administrated groups（p<0.05）,the Bcl-2 mRNA level was significantly higher in high-dose W5 group（p<0.05）,the Caspase-3 mRNA level was significantly lower in donepezil and high-dose W5 groups（p<0.05）.5.Effect of W5 on inflammatory cytokines in the hippocampus of AD ratsIn the Aβ/Cu²⁺group,the TNF-α mRNA expression in the hippocampus of rats was significantly higher than that in the control group（p<0.05）,the mRNA expression of IL-1βand IL-6 showed an upward trend.Compared with Aβ/Cu²⁺ group,the TNF-α and IL-6 mRNA levels were significantly lower in donepezil and W5-administrated groups（p<0.05）.There was no significant difference in the expression of IL-1β mRNA（p>0.05）,but there was a downward trend.6.Effects of W5 on antioxidant related indexes in the brain and serum of AD ratsIn the Aβ/Cu²⁺ group,the levels of SOD,CAT,GSH-Px,and GSH in the brain of rats were significantly lower than that in the control group（p<0.05）,MDA content in the brain of rats was significantly higher than that in the control group（p<0.05）.Compared with Aβ/Cu²⁺ group,the activities of T-SOD and GSH-Px were significantly increased and the MDA content was significantly decreased in donepezil group（p<0.05）,the activities of T-SOD and GSH-Px were significantly increased in W5 treated groups（p<0.05）.In the Aβ/Cu²⁺ group,the levels of CuZn-SOD,CAT,GSH-Px,and GSH in the serum of rats were significantly lower than that in the control group（p<0.05）,MDA content in the serum of rats was significantly higher than that in the control group（p<0.05）.Compared with Aβ/Cu²⁺group,the activities of T-SOD,CuZn-SOD,CAT,and GSH-Px were significantly increased in donepezil group（p<0.05）,the activity of GSH-Px was significantly increased in low-dose W5 group（p<0.05）,the activities of T-SOD,CAT,and GSH-Px were significantly increased in high-dose W5 group（p<0.05）.7.Effect of W5 on the ATPase activity in the brain of AD ratsIn the Aβ/Cu²⁺ group,the activities of Mg²⁺-ATPase and Ca²⁺-ATPase in the brain of rats were significantly lower than that in the control group（p<0.05）.Compared with Aβ/Cu²⁺group,the Na+K+-ATPase activity was significantly increased in donepezil and W5 treated groups（p<0.05）,the Mg²⁺-ATPase activity was significantly increased in donepezil and lowdose W5 groups（p<0.05）,the Ca²⁺-ATPase activity was significantly increased in donepezil and high-dose W5 groups（p<0.05）.8.Effect of W5 on cholinergic system in AD ratsThere were no significant differences in the levels of ACh and AChE in the brain of rats among all groups（p>0.05）.But the levels of ACh and AChE in the brain of rats in drug intervention groups showed a reverse trend compared with Aβ/Cu²⁺ group.The ACh content in the serum of rats in donepezil group was significantly higher than that in Aβ/Cu²⁺ group（p<0.05）,the AChE activity in the serum of rats in high-dose W5 group was significantly lower than that in Aβ/Cu²⁺ group（p<0.05）.9.Effect of W5 on the contents of neurotransmitters in the brain of AD ratsIn the Aβ/Cu²⁺ group,the levels of Glu,Gly,γ-GABA,NE,and E in the brain of rats were significantly lower than that in the control group（p<0.05）,there were no significant differences in the contents of Asp,Taur,DA,and 5-HT（p>0.05）.Compared with Aβ/Cu²⁺ group,the levels of Gly,Taur,y-GABA,NE,and E were significantly increased in donepezil group（p<0.05）,the levels of Gly,NE,and E were significantly increased in low-dose W5 group（p<0.05）,the levels of Gly,y-GABA,NE,and E were significantly increased in high-dose W5 group（p<0.05）,there were no significant differences in the contents of Asp,Glu,DA,and 5-HT（p>0.05）.10.Effects of W5 on Cu、Ca、Fe、Zn in the brain and serum of AD ratsIn the Aβ/Cu²⁺ group,the level of Cu in the brain of rats was significantly higher than that in the control group（p<0.05）.Compared with Aβ/Cu²⁺ group,the Cu level was significantly decreased in donepezil and high-dose W5 groups（p<0.05）.In the Aβ/Cu²⁺ group,the level of Zn in the brain of rats was significantly higher than that in the control group（p<0.05）.There were no significant differences in the contents of Ca and Fe（p>0.05）.In the Aβ/Cu²⁺ group,the level of Cu in the serum of rats was significantly higher than that in the control group（p<0.05）.Compared with Aβ/Cu²⁺ group,the Cu level was significantly decreased in donepezil and high-dose W5 groups（p<0.05）.In the Aβ/Cu²⁺ group,the level of Ca in the serum of rats was significantly lower than that in the control group（p<0.05）,and the level of Fe in the serum of rats was significantly higher（p<0.05）.There were no significant differences among the other groups（p>0.05）.ConclusionW5 could decrease the expression of Aβ,Tau and p-Tau proteins in the hippocampus of AD rats to inhibit the formation of senile plaques and neurofibrillary tangles,down-regulate the expression of Bax mRNA and Caspase-3 mRNA and up-regulate the expression of Bcl-2 mRNA to reduce the apoptosis of neuron cells,reverse the expression of TNF-α,IL-1β and IL-6 mRNA to regulate neuroinflammatory response in AD rat brain.W5 also could regulate the ATPase activity and oxidative stress state of AD rats,and balance the neurotransmitter and metal elements disorder in AD rats brain tissue.Overall,W5 has the potential as an anti-AD drug,which could recover the morphology of hippocampal neurons and improve the learning and memory dysfunction in AD rats through regulating multiple targets in AD rats.