A Multi-omics Investigation Of The Potential Mechanism Of Long-term Atorvastatin To Improve Cognitive Decline In Naturally Aging Rats

Objective:With the continuous improvement of China’s social development and medical level,people’s life expectancy is gradually increasing,and China is about to enter an aging society.The decline in cognitive function is one of the most obvious features of aging,and preventing and improving the decline in cognitive function during aging is a great challenge for an aging society.As early as the third decade of life,core human cognitive abilities,including processing speed,reasoning,situational memory,and spatial visualization,have begun to decline.Multiple growth curve models suggest that cognitive decline is not a sharp decline in old age,but a small but sustained decline throughout the life span.Therefore early and long-term interventions are important to prevent and improve the cognitive decline caused by aging.The current view is that neuropathological changes occur 20 years or more before the onset of dementia symptoms and that such pathological changes are difficult to reverse once they appear.Therefore,we need to find an intervention with high clinical prescription,early intervention,and long treatment duration as the target of the study that is more clinically relevant and realistic.Based on current evidence,a large number of people have been taking statins for a long period of time since middle age,with the primary goal of lipid-lowering or plaque stabilization,when they have shown little to no signs of cognitive decline.At the same time,a large number of studies in recent years have found the use of statins to be effective in reducing cognitive decline and neurodegenerative disease.Many clinical studies have looked at the effects of statins on patients almost exclusively over several years,while the long-term effects of long-term statin use on overall body function,including cognitive function,are not yet clear.Therefore,the choice of statins as an intervention in this study is most consistent with the current clinical use and pathogenesis.In conclusion,the present study used atorvastatin for early and long-term intervention in naturally aging rats to explore potential targets for the treatment of cognitive decompensation.Protein post-translational modifications play an important role in various biological pathways,regulating numerous important cellular processes,including enzyme activation,protein localization,and protein degradation.Lysine crotonylation is a newly discovered post-translational modification of histones in recent years that can be involved in cellular metabolism,cell cycle,and cellular reorganization processes.Meanwhile,some studies have revealed that crotonylation plays a regulatory epigenetic role in Alzheimer’s pathology.Because of this,this study conducted proteomic and crotonylation modification omic studies in the first two parts and investigated the mechanism of the potential therapeutic targets identified.The microbial-gut-brain axis plays an important role in the regulation of the nervous system.Several recent studies have confirmed that gut flora,brain inflammation,and cognitive impairment are closely related.Atorvastatin has been found to reduce the damage of neuroinflammation and improve cognitive function in AD mice.However,whether atorvastatin reduces neuroinflammation via the microbial-gut-brain axis remains unclear.Therefore,the present study explored the mechanism of the microbial-gut-brain axis involved in this effect in the third part.In summary,the present study proposes to use different doses of atorvastatin for long-term intervention in a rat model of natural aging.The potential targets of long-term atorvastatin effects on cognitive function were explored by proteomics,proteomic modification,and metagenomic microbial taxonomic sequencing approaches,and the mechanisms of their occurrence were investigated.Methods:This study is divided into three parts:the first part explores the potential targets of statin to improve cognitive function through cognitive-behavioral and proteomic studies;the second part validates the mechanism through cytological experiments;the third part explores the mechanism of statin to improve cognitive function and microbial-gut-brain axis through gut microbiome and immunological experiments.Part I:A total of 30 adult male SD rats（9 months old）were used for this study in three parts.The rats were randomly divided into a control group（saline 0.5 ml/d,10 rats）,a low-dose group（atorvastatin 2.1 mg/kg-d-1,10 rats）,and a high-dose group（atorvastatin 8.4mg/kg-d-1,10 rats）.We evaluated the cognitive behavior of rats by three methods:Y-maze experiment,novelty recognition experiment,and Morris water-maze experiment;screened the differential proteins by GO analysis and other methods based on proteomics and proteomic modification techniques;validated the screened proteins by western blot,and detected the relevant blood indexes by single molecular array for the detection of relevant blood indicators;and localization analysis by immunofluorescence double-staining method.Part II:We isolated and cultured rat hippocampal neuronal cells and investigated the regulation of multiple modifications of neurofilament light chain（NFL）and related loci by Co-IP,cell transfection,western blot,and PCR reactions.Part III:We analyzed the rat intestinal flora by 16s RNA microbiome sequencing and microbial community analysis;based on the results of the flora analysis,we further investigated the improvement of cognitive function by statins through microbial-gut-brain axis by cell isolation assay,flow cytometry,liquid chromatography-mass spectrometry,western blot,PCR,immunofluorescence staining,etc.The mechanism of cognitive improvement through the microbial-gut-brain axis was investigated.Results:Behavioral and proteomic results:The results of the Y-maze and novelty recognition experiments showed that the short-term（3 months）intervention did not significantly affect the cognitive function of rats,while the long-term（9 months）intervention showed that the cognitive function of the high-dose atorvastatin intervention group was significantly higher than that of the control group.the results of the Morris water maze experiment showed that the spatial learning ability and spatial memory ability of the high-dose atorvastatin intervention group were significantly higher than that of the control group.Proteomics results revealed that high-dose atorvastatin intervention significantly down-regulated the protein expression of NFL;crotonylation modification histology results showed that high-dose atorvastatin intervention significantly down-regulated the level of crotonylation modification of NFL at the K272 locus.Combining the above results,NFL was screened as the target protein in this study and the histological results were validated by protein blotting.We also measured NFL in blood using single-molecule array technology,and the results showed that NFL in serum was significantly decreased in the high-dose statin intervention group compared with the control group.To verify the relationship between NFL and cognitive decline,we performed co-localization of NFL and neurofibrillary tangles（NFT）by immunofluorescence double-staining,and the results showed that the co-localization of NFL and NFT was extremely high and increased with age,and decreased after atorvastatin intervention,especially in the high-dose group.There was a dose-dependent trend.2.Cytology experiments to verify the results:The presence of ubiquitination modifications at the K272 site of NFL was found by database analysis,and the long-term statin intervention could down-regulate the crotonylation level of NFL and up-regulate the ubiquitination level of NFL by immunoprecipitation method.The immunoprecipitation coupled with mass spectrometry screening identified silent information regulator 2（SIRT2）as a possible protein interactor with NFL,and long-term statin was found to enhance the interaction between SIRT2 and NFL and increase the enzymatic activity of SIRT2 by immunoprecipitation experiments.Treatment of neuronal cells with AGK2,a specific inhibitor of SIRT2,resulted in increased protein expression of NFL,increased levels of crotonylation,and decreased levels of ubiquitination.After SIRT2 overexpression in neuronal cells,the level of crotonylation of NFL decreased and the level of ubiquitination increased.After mutation of the K272 locus of NFL,the overall ubiquitination level of NFL did not change significantly,and the overall crotonylation level decreased.3.Results of gut microbiome and immunology studies:The results of liquid chromatography-mass spectrometry（LC-MS）showed that the expression levels of retinoic acid（RA）,retinol（ROH）,and retinol dehydrogenase 7（Rdh7）were significantly higher in the high-dose intervention group than in the control group,while retinyl esters（RE）was significantly reduced.Flow cytometric analysis showed that regulatory cells（Treg）in the high-dose intervention group were significantly higher than those in the control group,while IL-17+γδT was significantly lower than that in the control group.Interleukin 17（IL-17）in hippocampal tissue was significantly lower in the high-dose intervention group than in the control group.The results of theα-diversity analysis in the rat fecal 16s RNA microbiome sequencing and microbial community analysis showed that the ACE index,observed species index,and PD whole tree index was significantly higher in the high-dose intervention group than in the control group.The results of theβ-diversity analysis revealed significant differences between the flora of the high-dose intervention group and the rest of the groups.The significantly different bacteria in the rat intestinal flora at baseline were Clostridium and Ruminococcaceae,while the significantly different bacteria after the long-term high-dose intervention were Lactobacillale and d Bacill.Conclusion:1.long-term high-dose atorvastatin intervention significantly improved cognitive decline in naturally aged rats.2.long-term atorvastatin intervention improved cognitive function by increasing the expression of SIRT2 down-regulating the level of crotonylation modification at the NFL K272 locus,which in turn up-regulated the level of ubiquitination modification resulting in enhanced degradation of the NFL and reduced NFT.3.long-term atorvastatin intervention increased intestinal flora abundance and increase the abundance of probiotic flora.4.Long-term atorvastatin intervention can alter Rdh7expression in the intestine to affect RA metabolism,which in turn leads to proliferation of intestinal T_reg cells and suppression of IL-17⁺γδT cell function,ultimately reducing IL-17expression in the hippocampus to improve cognitive function.