Study On The Construction Of Au@TPM And The Mechanism Of Anti-Alzheimer’s Disease Activity

Alzheimer’s disease（AD）is a chronic progressive neurodegenerative disease.The pathogenesis is related to its own factors,genetic conditions and environmental influences.Current research shows that oxidative stress occurs before senile plaques and neurofibrillary tangles are produced.It is one of the earliest changes in the pathogenesis of AD.It is worth noting that a series of oxidative stress markers such as malondialdehyde,peroxynitrite and thiobarbituric acid will be detected in the brain of AD patients.This shows that the main cause of brain death in AD patients is related to the imbalance of the antioxidant system.Therefore,the development of antioxidants that inhibit oxidative stress has become an effective way to prevent AD.Gold nanoparticles（Au NPs）are nanocarriers for intracellular drug or gene delivery.They have the characteristics of low toxicity,large specific surface area,easy controllable surface assembly and high specificity.It is widely used in the field of biomedicine.In this paper,Au NPs of different particle sizes were synthesized by the classical method of reducing tetrachloroauric acid with sodium citrate.Then,the maize-derived monomer peptide TPM（Leu-Asp-Tyr-Glu）with excellent antioxidant properties was self-assembled with Au NPs.We combined the N-terminal of TPM leucine with a molecule of 3-mercaptopropionic acid to form s-TPM.In this way,the recombinant maize derived peptide could be stably loaded on the surface of Au NPs.At the same time,the activity of the antioxidant in vivo and in vitro was studied.Finally,it is verified that S-Au@TPM and L-Au@TPM It has certain feasibility and applicability in the field of anti-AD.The specific research contents are as follows:In this paper,the morphology of PC12 cells was observed after administration.It was found that 48 h after the administration of S-Au@TPM and L-Au@TPM,it can promote the differentiation of cells,and the axons of cells will elongate like synapses of neurons with the increase of dosages.It is preliminarily proved that the constructed nano-antioxidant has a certain neuroprotective effect on PC12 poorly differentiated cells.Therefore,we continue to explore the specific inhibitory effect of AD cell model in vitro.L-glutamic acid（L-Glu）was selected as the drug for establishing cell models.The optimal dose of 25 m M was selected by MTT method.In AD model cells,administration of 0.25 n M and 0.5 n M of S-Au@TPM and L-Au@TPM can significantly inhibited the rates of apoptosis induced by L-Glu.The survival rates of the cells were 90.02±13.89%and 86.89±13.24%,respectively.Donepezil hydrochloride（DH）was the positive control drug in this study.It is a reversible central acetylcholinesterase inhibitor,which is widely used to treat cognitive dysfunction in AD patients.We used Hochest 33342 fluorescent staining method to observe the morphology and number of apoptosis.It was found that nano-antioxidants could significantly inhibit the the number of apoptotic nuclei compared with DH and s-TPM.The results showed that in L-Glu-induced damaged cells,the administration of different concentrations of S-Au@TPM and L-Au@TPM pre-treatment for 3 h,could significantly reduced the amount of intracellular lactate dehydrogenase（LDH）leakage and the production of malondialdehyde（MDA）was significantly reduced.Nano-antioxidants inhibited the increases of intracellular Ca²⁺concentration,improved the abnormal mitochondrial trans-membrane potential and catalase（CAT）activity,thereby alleviating the excessive accumulation of intracellular reactive oxygen species（ROS）caused by modeling drugs.The above research showed that S-Au@TPM and L-Au@TPM could significantly inhibit the series of injury of AD cell models in vitro and resist the imbalance of oxidative system caused by oxidative stress.In the study of anti-AD activity in vivo,Al Cl3 and D-galactose were used in combination to establish an AD model in female KM mice.This model could fully replicate the main pathological features and clinical manifestations of AD.It can cause the imbalance of oxidation and antioxidant system,degeneration and necrosis of hippocampal and cerebral cortex neurons,abnormal cholinergic system,senile plaque lesion of cerebral cortex,and behavioral dysfunction in experimental mice.In this part of the experiment,the results showed that AD model mice were treated with S-Au@TPM and L-Au@TPM at concentrations of 0.5 mg/kg,0.7 mg/kg and 1.0mg/kg for 16 days.It will significantly improved a series of indicators in animal behavior:（1）In the water maze test,it can enhance the spatial learning and memory ability of model mice;（2）In the fatigue rotating rod behavior,it can improve the movement of model mice（3）In the autonomic activity test,it can increase the adaptability of the model mice in the unfamiliar dark environment,and make timely sensitive stress behavior to the environment.In summary,S-Au@TPM and L-Au@TPM could improved the behavior disorder caused by functional injury of nervous system in AD model mice.Furthermore,the cholinergic indexes were detected.It was found that AD model mice were treated with different doses of S-Au@TPM and L-Au@TPM,the levels of acetylcholine（ACh）and acetylcholine transferase（Ch AT）in the hypothalamus and serum were increased.The excessively released acetylcholinesterase（Ach E）content in neuronal synapses was inhibited,indicating that nano-antioxidants can effectively regulate the activity of the cholinergic system.In addition,we studied the effect of nano-antioxidants on the interference expression of Nrf-2/Keap-1 pathway proteins.The results showed that compared with AD model mice,S-Au@TPM and L-Au@TPM could up-regulated the expression of Nrf-2 and HO-1,increased superoxide dismutase（SOD）and glutathione peroxidation.It also reduced the activity of GSH-Px and CAT,and reduced MDA content and Keap-1 protein level.The above results indicated that S-Au@TPM and L-Au@TPM could reduced the damage induced by Al Cl3-and D-galactose through the Nrf-2 or Keap-1 pathway.At the same time,we analyzed by inductively coupled plasma mass spectrometry（ICP-MS）that the experimental mice were treated with S-Au@TPM and L-Au@TPM in tissues（small intestine,liver,spleen,lung,kidney,brain）has the distribution of nano-antioxidants.Among them,the small intestine is the most enriched.In the remaining organs,the enrichment of S-Au@TPM was lower than that of L-Au@TPM,indicating that the small size of S-Au@TPM could achieve long-term blood circulation,thereby achieving more effective biological utilization degree.The concentration and distribution of nano-antioxidants decreased with the time of administration.It was proved that it will be gradually discharged from the body to avoid excessive nano toxicity.In summary,the constructed S-Au@TPM and L-Au@TPM have great anti-oxidant protection.It could effectively inhibit the series of injuries caused by AD models in vivo and in vitro.The antioxidant activity of S-Au@TPM is stronger than that of L-Au@TPM.This may be due to the fact that Au NPs of small particle size will be more effectively conjugated with the loaded functional active peptides.Moreover,compared with large-diameter Au NPs,small-diameter Au NPs are more likely to penetrate cell membranes and enter patient tissues to exert therapeutic effects.Therefore,this research provides novel ideas for the development of new drugs for the treatment or prevention of AD.Thus,nano-antioxidants are promising as neuroprotective agents to be administered for the prevention of neurodegenerative diseases.