| Alzheimer's disease(AD)is a progressive neurodegenerative disease that mostly occurs in the elderly over 65 years old and is the most common type of dementia.With the acceleration of population aging,the number of AD patients is increasing every year,which brings a heavy burden to society and families.Therefore,the pathogenesis of AD has received extensive attention from researchers.However,the etiology of AD is extremely complex,and the?-amyloid(A?)cascade theory is considered to be one of the key mechanisms of AD pathogenesis.At present,many researches on AD drug development are based on the A?pathogenic mechanism.With the continuous development of biology,a variety of animal models have been established to study AD.Among them,nematodes have many advantages such as short life cycle,strong reproductive ability,easy cultivation and preservation,and conserved human disease pathways.Therefore,nematodes have been widely used as an excellent model organism in studying the occurrence and development of many diseases,including AD.Using genetic engineering technology,a variety of transgenic nematode strains expressing human A?was successfully constructed to study the pathogenesis of AD and screen anti-AD drugs.For example,human A?1-42peptide and smg-1 m RNA monitoring system were introduced into CL4176 transgenic nematode strains.In this way,the expression of A?protein and the related appearance of paralysis phenotype can be easily controlled by the culture temperature of nematodes.Ethanol,commonly known as alcohol,is widely used in the chemical industry,medical and health care,agricultural production,and many other fields all over the world.The main component of wine is ethanol,which is also one of the oldest foods for human beings.Many countries around the world have the habit of drinking alcohol.As the saying goes,a little drink can bring pleasure,much drink is harmful to health.Studies have shown that drinking alcohol can reduce the risk of dementia,but many researchers have also confirmed that drinking alcohol can cause damage to the liver,brain,and other organs.The researches on the benefits of low-concentration ethanol are still controversial.Therefore,this study focused on the role of low-concentration ethanol in anti-Alzheimer's disease and its related molecular mechanisms using the nematode and cell models of Alzheimer's disease.Firstly,the effects of ethanol on the general physiological indexes of normal nematodes,such as lifespan,swallowing frequency,and movement capacity,were explored in this paper.1%and 2%ethanol could significantly prolong the lifespan of nematodes,while 2%ethanol also increased the movement capacity of nematodes in the aging stage.After 1%ethanol treatment,the average paralysis time of the transgenic nematode strains CL4176,which were usually used to study AD was significantly prolonged(20.9%).The average paralysis time of nematodes treated with 2%ethanol was also prolonged,but their body length was smaller and the number of eggs laid was fewer than that of the control group.Besides,the concentrations of ethanol at 4%and above seriously affected the development and growth of nematodes.Therefore,1%ethanol was chosen for subsequent experiments to explore the mechanism of ethanol in anti-Alzheimer's disease.Since ethanol enters the organism,there will be a metabolic reaction to produce two main metabolites:acetaldehyde and acetic acid.It was confirmed that neither acetaldehyde nor acetic acid could prolong the average paralysis time of nematodes,indicating that the effects of ethanol on delaying the toxicity of A?relied on the role of ethanol itself rather than its metabolites.Afterwards,by q PCR and western blot,we found that ethanol did not affect the m RNA expression level of A?in nematodes,but reduced the expression of A?oligomers.In order to explore whether the alleviation of A?toxicity by ethanol depends on the proteasome or lysosomal pathway,the proteasome activity of nematodes was determined and results showed that ethanol had no effect on proteasome activity.However,lysosome staining experiments suggested ethanol could significantly increase the lysosome mass of nematodes.Besides,after treated with chloroquine,a kind of lysosomal inhibitor,ethanol could no longer effectively reduce the expression of A?oligomers and the number of A?deposition in nematodes.Meanwhile,the effects of ethanol on delaying nematode paralysis were significantly weakened.The above results indicated that the effects of ethanol on alleviating A?toxicity in nematodes were regulated by the lysosomal pathway.On the other hand,we found that the alleviation of A?toxicity by ethanol also depended on the stress response pathway.DAF-16 is an important transcription factor involved in regulating stress response in nematodes.After RNA interference of daf-16in nematodes,ethanol could no longer delay the paralysis of nematodes,suggesting that the effects of ethanol on delaying the paralysis of nematodes were regulated by DAF-16.Ethanol could also increase the nuclear translocation of DAF-16,and upregulate the expression of nematode stress-related genes such as daf-16,hsf-1,and shsps in nematodes.Besides,ethanol could significantly increase the expression of SOD-3 and HSP-16.2 proteins,reduce ROS content and increase the SOD activity.Overall,the above results indicated that low-concentration ethanol,as a stressor,activated the stress response in nematodes,thereby alleviating the toxicity of A?.Furthermore,the mechanism of ethanol alleviating A?toxicity was explored by a cell model.By CCK-8and ELISA assays,0.1%,0.5%,and 1%ethanol were proved to effectively alleviate the toxicity of human neuroblastoma cell SH-SY5Y induced by A?25-35 and reduce the secretion of A?1-42.DCFH-DA staining experiments confirmed that ethanol could reduce the ROS content in SH-SY5Y cells induced by A?25-35.Besides,by immunofluorescence staining experiments,chloroquine was found to block the reduction of A?protein in SH-SY5Y cells caused by A?25-35 induced by ethanol.In brief,the mechanism of the alleviation of A?toxicity mediated by ethanol in cells is similar to that of nematodes,which was regulated by oxidative stress and lysosomal pathways.On the basis of the above results,30 traditional Chinese medicines with anti-stress effects were selected to screen natural products with the ability of alleviating A?toxicity using a nematode model.Among them,three natural products with a stable effect were screened out,i.e.,the water extracts of Linderaaggregata(Sims)Kosterm,Radix Paeoniae Rubra,and Platycladus orientalis(L.)franco.Specifically,the optimal concentration of Linderaaggregata(Sims)Kosterm water extract for relieving A?toxicity is 2.5 mg/m L.The water extract of Radix Paeoniae Rubra at 10 mg/m L had the best effect on alleviating A?toxicity,while the water extract of Platycladus orientalis(L.)franco was able to alleviate A?toxicity at a lower concentration of 0.2 mg/m L.In summary,this study showed that low concentrations of ethanol could regulate the lysosomal pathway to increase A?degradation and activate the stress response pathway to promote A?disaggregation.Under the synergistic effects of these two pathways,ethanol could alleviate A?toxicity in both nematode and neural cell models.The benefits of low-dose ethanol in nematodes and cells provided a rationale for moderate alcohol consumption.Besides,this study also provided a reference basis for drug research and development of anti-Alzheimer's disease.3 natural products were also proved to alleviate the toxicity of A?in C.elegans,which provided new ideas for the development of AD therapeutic drugs. |
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