| Alzheimer’s disease(AD)is a class of neurodegenerative diseases,with progressive deterioration of cognitive and memory functions as the main clinical manifestations,involving behavioral disorders such as language,memory,understanding,attention,judgment and reasoning disorders.As the disease progresses,the patient’s executive ability decreases and eventually loses his daily living ability.The AD pathogenesis Aβ hypothesis holds that under normal circumstances,Aβ concentration balance in the brain is mainly regulated by its own production and clearance.If this balance is destroyed,Aβ will accumulate and deposit in the brain,leading to neuronal degeneration and necrosis.Therefore,the Aβ hypothesis is considered one of the current mainstream hypotheses.Current drugs used to treat AD can only improve Alzheimer’s symptoms,which cannot cure or reverse the occurrence of AD,and the treatment cannot achieve the desired effect.Therefore,AD has become one of the diseases that seriously detriment human health in the21 st century.Therefore,research on the pathogenesis of AD and the development of drugs for the effective treatment of AD have become a hotspot of clinical medical research.In this study,CL4176 of the AD model Caenorhabditis elegans(C.elegans)was used to explore the effect of cannabidiol(CBD)on Aβ protein aggregation in the AD model C.elegans,study the mechanism of CBD on Aβ protein aggregation,and provide a theoretical reference and clinical basis for the application of cannabinoids in the treatment of AD.1.Effect of CBD on the behavioral phenotype in C.elegansDifferent concentration gradients of CBD(50,100,and 400 μmol/L)were used on Aβtransgenic C.elegans CL4176 for the statistical analysis of its paralysis rate.The experimental results show that different concentrations of CBD can all alleviate the nematode paralysis phenotype,with the most effective concentration for reducing the paralysis rate of C.elegans being 100 μmol/L.Based on this,we chose 100 μmol/L CBD on the CL4176 nematodes for further statistical analysis of their longevity,number of swallowing,and number of body bending.The experimental results showed that a CBD of100 μmol/L had no effect on longevity,but it could increase the number of swallowing and body bending times of CL4176.2.Study of the antagonistic effects of CBD on Aβ protein aggregationFirst applying 100 μmol/L CBD on Aβ 1-42 peptide,and then using circular dichroism,CD to detect the antagonism of CBD on Aβ 1-42 peptide aggregation.Detection of the Aβ1-42 peptide spectrum at 190-250 nm by CD confirmed that CBD does not have the ability to directly antagonize Aβ1-42 peptide aggregation in vitro.Then,100 μmol/L CBD CL4176 was selected for use on Aβ transgenic C.elegans CL4176,and thioflavin S(Th S)was used to stain the Aβ protein.The experimental results showed that CBD can significantly reduce the aggregation of Aβ protein in C.elegans.Under the same experimental conditions,the Aβ protein in CL4176 nematode was detected by Western blot,and CBD treatment could reduce the expression of Aβ1-42 protein in nematode CL4176.The experiment further confirmed that CBD can alleviate AD symptoms caused by Aβ protein toxicity.3.Study of the mechanism of action of cannabidiol on Aβ protein aggregationPlant-derived polyphenols are often antioxidants,The antioxidant capacity of CBD and cannabidiol derivatives(CBD derivatives containing a phenolic hydroxyl group and a phenolic hydroxyl-methylated)and CBD-M2(CBD derivatives containing two phenolic hydroxymethylated groups)was measured in vitro by using DPPH and ABTS.It showed that as the number of phenolic hydroxyl substitutions increased,its antioxidant capacity also weakened,preliminarily confirming that the antioxidant capacity of CBD is determined by its own phenolic hydroxyl structure.CBD was applied to N2 in wild-type nematodes while using DCFH-DA as a fluorescent probe to detect ROS content.The experimental results showed that the ROS content in wild-type nematode N2 was reduced when treated with 100 μmol/L CBD,indicating that CBD has antioxidant capacity in wild-type nematode N2.The m RNA expression levels of catalase-related genes(ctl-1,ctl-2,and ctl-3)in wild-type nematode N2,superoxide dismutase-related genes(sod-1,sod-2,sod-3,and sod-4)and glutathione-s-transferase were detected by quantitative real-time PCR(Q-PCR).It was found that CBD could not affect the abovementioned gene expression.Both DAF-16 and SKN-1 are major transcription factors involved in the prevention of oxidative stress.To investigate the antioxidant pathway of CBD in vivo,C.elegans mutants daf-16(CF1038)and skn-1(EU1),which are closely related to oxidative stress,were used.Taking the normal wild-type nematode N2 as a control,CBD was found to improve the survival of the C.elegans mutants daf-16 and skn-1 and the wild-type nematode N2 after the oxidative stress induced by walnut quinone,thus indicating that the antioxidant activity of CBD is not mediated by daf-16 or skn-1 in vivo.Then,100 μmol/L CBD and its derivatives CBD-M1 and CBD-M2 were applied to Aβ transgenic C.elegans CL4176 for Th S staining experiments.A significant reduction in head region Aβ deposition in CBD-treated CL4176 cells was observed by Th S staining.However,there was a tendency toward decreased head region Aβ deposition in both CBD-M1 and CBD-M2 treated CL4176,but not as pronounced as CBD reduction,showing that as the number of phenolic hydroxyl substitutions gradually increases,its ability to alleviate Aβ deposition gradually decreases.When paralysis experiments of the Aβ transgenic nematode CL4176 were performed under the same conditions,CBD treatment significantly delayed the paralysis rate of CL4176 in Aβ transgenic C.elegans(P<0.01).As the number of substituted phenolic hydroxyl groups increased,the ability of CBD-M1 and CBD-M2 to delay the rate of paralysis in C.elegans gradually decreased.In conclusion,the phenolic hydroxy group of CBD is essential for scavenging ROS in vitro,mitigating Aβ aggregation in nematodes,and improving the neurotoxicity associated with Aβ in C.elegans and in vivo. |
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