| Studies have shown that emotional and psychological stress have many similarities in terms of concept,pathological conditions and clinical categories.Emotional pathogenic theory has a natural consistency with psychological stress response.With an increasing incidence of Type 2 Diabetes Mellitus(T2DM)in the country and the world,the correlation between psychological stress and diabetes has attracted more and more attention.Psychological stress can increase the risk of developing diabetes in patients with impaired glucose tolerance.Diabetes-Associated Cognitive Decline(DACD)is a brain-cerebral cognitive dysfunction disease caused by diabetes.The main manifestations are mild to moderate cognitive impairment,decreased learning and memory,and severe patients can develop into Alzheimer's disease(AD).The characteristic neuropathological changes of DACD have not been found yet,and several studies have shown that its pathogenesis and pathological changes are similar to those of AD.Studies have found that protein ubiquitination occurs in both AD patients and animals.Relying on E3 ligase,chronic psychological stress can cause cognitive impairment by increasing ubiquitination and degradation.It is suggested that DACD caused by emotion may be related to protein ubiquitination in the brain.In addition,the study also found that chronic psychological stress can increase the number of pathogenic bacteria and reduce the number of beneficial bacteria in the intestine.Dysregulation of the intestinal flora is an important factor leading to the development of cognitive dysfunction in diabetes and its complications.Proteomics can comprehensively and one-time expose specific disease protein molecules through high-throughput and high-resolution protein separation and identification technology.It is a large-scale study of all proteins expressed in the genome on an overall level,which can accelerate the recognition of pathological changes of diseases.The current proteomics research on cognitive impairment of diabetes is still very shallow,and there is less analysis of its ubiquitination proteomics.Multiple studies of AD have demonstrated that ubiquitinated proteins play a key role in neurodegenerative diseases such as AD.Therefore,studying the ubiquitination proteomics in the brain of DACD is of great significance for understanding the pathogenesis of DACD.The balanced gut flora not only plays an important role in the metabolism of the host metabolism,but also forms a natural barrier against the invasion of foreign pathogens.Once the balance of the intestinal flora is broken,the host is susceptible to disease.Many human diseases such as T2DM are related to the imbalance of intestinal flora.The intestinal flora plays an important role in the occurrence and prognosis of T2DM.In recent years,a large number of studies have also found a clear correlation between changes in intestinal flora and cognitive behavior.Changes in the intestinal flora can lead to changes in brain function,which in turn affects host behavior,such as anxiety,depression,and cognitive impairment.There are currently no valid and effective measures for DACD.The traditional chinese medicine considers that DACD belongs to "thirst",and "forgetfulness" or"dementia".Based on years of clinical experience,professor LiBin Zhan made ZiBu PiYin Recipe(ZBPYR),which was derived from "Bujvji"of Wu Cheng,Qing Dynasty.ZBPYR can not only improve the symptoms of cognitive impairment such as amnesia and dementia in clinical patients,but also improve the cognitive function of dementia rats with Spleen-Yin deficiency,aged rats,ZDF rats,DM rats induced by high-fat feeding combined with STZ injection and db/db mice.In summary,in this study,besides observing the behavioral performance of learning and memory in ZDF rats and whether emotional stress promotes the development of DACD,based on Label-free combined with Liquid chromatography-tandem mass spectrometry,we also found and screened out ubiquitinated proteins and biological pathways in the brain which are closely related to the pathogenesis of emotional-induced DACD and the mechanism of ZBPYR,in order to provide new clues for the study of the pathogenesis of emotional-induced DACD and the therapeutic mechanism of ZBPYR.In addition,based on 16S rRNA technology,we dynamically observed changes of intestinal flora during the development of emotional-induced DACD and ZBPYR intervention,and elucidated the dynamic regulation of ZBPYR on intestinal flora.1 Theoretical ResearchThis chapter systematically discusses the relationship between emotion and DACD,summarizes the current research progress in the understanding and treatment of DACD in traditional Chinese medicine and modern medicine,at the same time,it discusses "the theory of spleen" ZBPYR treating DACD and the establishment of animal model of DACD caused by emotion from many aspects,multi-level and multi-direction,in order to provide the basis and support for the theoretical connotation and basic research of "Spleen In Storing Idea and In Charge of Thought","Thinking-Impairing-Spleen",and causes DACD.2 Experimental ResearchObjective:(1)To observe the effect of ZBPYR on emotion-induced DACD rats'learning and memory ability;(2)To identify ubiquitin proteomics and screen out differentially expressed proteins in whole brain tissue of DACD rats induced by emotion;(3)Screening out differentially expressed proteins for bioinformatics analysis to understand the biological pathways and protein interaction networks closely related to the pathogenesis of DACD,and to analyze the possible targets of ZBPYR;(4)Defining the reliability of ubiquitinated proteomics results Sexuality,and preliminary analysis of the relationship between differential expression protein abundance changes and mRNA levels;(5)Dynamic observation of the effect of ZBPYR on the changes of intestinal flora in DACD rats induced by emotion.Methods:LZ rats were a lean control group,spontaneous type 2 diabetes model ZDF rats were randomly divided into diabetes model group(ZDF),diabetes model+emotional group(ZDF_P),diabetes model + emotional + ZBPYR treatment group(P_Z),a total of 4 groups.(1)By monitoring random blood glucose,fasting serum insulin,oral glucose tolerance test and insulin tolerance test,we observed the diabetic status of rats in each group and the intervention effect of ZBPYR.Corticosterone was detected to observe whether the HPA axis was activated in each group.(2)Through the open field experiment,dark-avoidance test and water maze test,the learning and memory ability of each group and the intervention effect of ZBPYR were observed.(3)Ubiquitous proteomics analysis based on Label-free and LC-MS/MS technology was carried out to find differentially expressed proteins.(4)Bioinformatics methods,including GO annotation,KEGG pathway and protein-protein interaction network,were used to analyze differentially expressed proteins.(5)Western blotting was used to validate the reliability of ubiquitinated proteomics results at protein level.qPCR was used to preliminarily analyze the relationship between the changes of differential ubiquitinated protein abundance and the changes of mRNA level.(6)Based on 16S rRNA technology,the intestinal flora analysis was carried out to analyze the abundance and diversity of intestinal flora species,and to find the target of intestinal flora of ZBPYR.Results:(1)The RBG of ZDF group increased significantly every week.ZDF rats had impaired glucose function,peripheral insulin resistance and abnormal glucose tolerance.Psychological stress aggravated the impairment of glucose function and peripheral insulin resistance in rats.After ZBPYR treatment,the glucose tolerance of rats was improved and peripheral insulin resistance was reduced.Psychological stress significantly increased corticosterone levels in rats,and ZBPYR treatment significantly reduced corticosterone levels.(2)In the open field experiment,ZBPYR treatment significantly increased the median residence time,central compartment shuttle and erecting times,and the total exercise distance increased.In the dark-avoidance test,ZBPYR treatment has a tendency to prolong the latency of rats.In the Morris water maze place navigation experiment,after ZBPYR treatment,the latency of rats in P_Z group was significantly shortened on the 5th day of training.In the Morris water maze space exploration experiment,ZBPYR treatment has a tendency to increase the time when the rat is looking for the original platform,the time in the quadrant of the original platform,and the number of times crossing the original platform.In Morris water maze visualization platform experiment,there was no significant difference in latency and swimming distance among the four groups.(3)A total of 1680 ubiquitinated sites,1620 ubiquitinated peptide segments and 842 ubiquitinated proteins were identified.Compared with LZ group and ZDF group,there were 200 differentially expressed proteins,of which 56 were up-regulated in ZDF group and 144 were down-regulated in ZDF group.Compared with ZDF group and ZDF_P group,127 proteins had significant quantitative changes,of which 69 were up-regulated in ZDF P group and 58 were down-regulated in ZDF_P group.A total of 176 proteins in ZDF_P group and P_Z group had significant quantitative changes.52 proteins were up-regulated in P_Z group and 124 proteins were down-regulated in P_Z group.Eventually 16 proteins were found in both comparisons showed significant differences.(4)Bioinformatics methods:Differentially expressed ubiquitinated proteins were mainly located in cells,organelles and membranes;the main molecular functions were binding,catalytic activity and transporting activity;the main biological processes were single-organism process,biological regulation,multicellular organismal process,cellular process and metabolic process.There are 10 KEGG pathways involved in differentially expressed proteins in LZ group and ZDF group,including insulin secretion,pancreatic secretion,cAMP signaling pathway,etc.There are 11 KEGG pathways involved in differentially expressed proteins in ZDF group and ZDF P group,including cAMP signaling pathways,Adrenergic signaling in cardiomyocytes,insulin secretion,etc.There are 21 KEGG pathways involved in differentially expressed proteins in ZDF_P group and P_Z group,including endocytosis,dopaminergic synapses,cAMP signaling pathway,etc.By using the IntACT database,it was found that there were 79,51,59 interacting differentially expressed proteins in the LZ group and the ZDF group,the ZDF group and the ZDF P group,the ZDF P group and the P_Z group,respectively.(5)Camk2?,Atp1b1,Gnao1,Tpi1 were selected for Western blotting verification,and the results were consistent with those of ubiquitinated proteomics analysis.The results of q-PCR showed that the expression of Camk2a mRNA in ZDF group was significantly lower compared with LZ group.The expression of Camk2a mRNA in P_Z group was significantly higher than that in ZDF P group.The expression changes of Atp1b1 mRNA,Gnao1 mRNA and Tpi1 mRNA were consistent with the change trend of protein level,but did not reach statistical difference.(6)Intestinal flora analysis:The results of microbial community alpha diversity index showed that ZBPYR could decrease Chao1 and ACE index dynamically.At the phylum level,all samples were dominated by Firmicutes and Bacteroides.Firmicules,Bacteroides,Tenericutes and Verrucomicrobia changed with time.At the genera level,the dominant bacteria in each group were Lactobacillus.The abundance of Bifidobacterium,Rohia,Streptococcus,Sutterella,Prevotella,Lactobacillus,etc changed with time.Principal component analysis,principal coordinate analysis and UniFrac distance analysis showed that samples in ZDF group and ZDF_P group were well separated from LZ samples,while samples in ZBPYR group were distributed between ZDF and ZDF_P group,indicating that ZBPYR could regulate the intestinal flora structure of DACD rats.Conclusion:(1)Spontaneous diabetic obese ZDF rats are consistent with the behavioral changes of DACD,so they can become a better animal model of DACD.Psychological stress promotes and aggravates the occurrence and development of DACD.ZBPYR can not only improve RBG level,glucose tolerance and peripheral insulin resistance in DACD ZDF rats,but also improve learning and memory ability.Psychological stress activates the HPA axis in ZDF rats,and ZBPYR can inhibit the activation of the HPA axis.(2)The ubiquitinated protein maps of LZ rats and ZDF rats were constructed,which laid a foundation for further study of the correlation between ubiquitinated proteins and DACD.We successfully screened out 200 differentially expressed ubiquitin proteins related to DACD,127 differentially expressed ubiquitin proteins related to psychological stress,and 16 differentially expressed proteins that may be the targets of ZBPYR.(3)Bioinformatics analysis revealed the GO classification,KEGG pathway and protein network interaction of differentially expressed ubiquitinated proteins in rats brain tissue,which laid a foundation for the future basic research on the pathogenesis of DACD and the therapeutic mechanism of ZBPYR.(4)The reliability of ubiquitinated proteomics techniques in this experiment was confirmed by Western blotting.The results of qPCR suggested that the change of protein expression might not be entirely due to the change of mRNA.(5)The results of intestinal flora analysis showed that ZBPYR could significantly improve the imbalance of intestinal flora diversity and dynamically reduce the diversity indices of Chao1 and ACE.The composition of the phylum was the same between human and rat.From the dynamic development process,ZBPYR can increase the abundance of Lactobacillus,Akkermansia and Anaerostipes,and decrease the abundance of Allobaculum,Alistipes,Proteus and Coprococcus.ZBPYR can significantly regulate probiotics and pathogenic bacteria disorders in DACD caused by psychological stress. |
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