To Explore The Microbiota-gut-brain Axis Dialogue Mechanism Of ZiBuPiYin Recipe For Preventing And Treating Diabetes-associated Cognitive Decline In Based On The "Prevention Of Disease"

Diabetes-associated cognitive decline(DACD)is considered to be one of the common complications of the central nervous system of type 2 diabetes mellitus(T2DM).Therefore,it is of great significance to carry out early and reasonable intervention and reduce risk factors to delay the occurrence,development and transformation of DACD.Gut microbiota is a key determinant of human health and disease,and scholars from all over the world has focused on exploring the relationship between T2DM and its cognitive dysfunction and gut microbiota.Targeted regulation of gut microbiota is considered as a new strategy to prevent and treat DACD.Traditional Chinese medicine believes that the pathogenesis of thirst and dementia is related to spleen deficiency.The balance and imbalance of the gut microbiota directly reflect the functional status of the spleen.The imbalance of the gut microbiota is the main physiological feature of the spleen governing the movement and the dysfunction of the gut microbiota is an important pathological factor of the spleen.Taking the spleen as the entry point for early intervention and discussing the prevention and treatment of DACD from the change and regulation of gut microbiota is a concrete manifestation of TCM’s "prevention of disease" idea.ZiBu PiYin recipe(ZBPYR)is derived from Zicheng Decoction,and has been widely used for the treatment of T2DM and the prevention of DACD.Here,we transplanted the gut microbiota of healthy or cognitive decline donor rats into ZDF or LZ rats,and integrated the method of 16S rRNA and targeted metabolomics to evaluate the directional effect of the gut microbiota on the recipient rats.In addition,we investigate whether the gut microbiota plays a key role in ZBPYR-mediated prevention of DACD and treatment of T2DM via incorporating microbiomics and metabolomics,and investigate the links between the microbiota-gut-brain axis interaction and the efficacy of ZBPYR in ZDF rats.Chapter One Theoretical ResearchThis chapter systematically discusses and analyzes the theory of "prevention of disease",the basis of "prevention of disease" from the theory of "spleen",and the basis of diabetes-associated cognitive decline based on the theory of "prevention of disease".Meanwhile,Through the analysis and summarize of ZBPYR’s biological activity and mechanism,it is concluded that ZBPYR and its active ingredients can effectively improve the learning and memory ability of diabetes-associated cognitive decline mainly through β-amyloid,synaptic plasticity,oxidative stress and mitochondrial dysfunction,endoplasmic reticulum stress and autophagy,leptin and insulin resistance,gut microbiota,etc.,,which could provide reference for clinical prevention and treatment of diabetes-associated cognitive decline,and provide new ideas for the development of brain health and intelligence new drugs.Chapter Two Experimental ResearchSection 1 The effect of changes in gut microbiota on diabetes-associated cognitive decline of ZDF hostObjective:Transplant the gut microbiota of healthy or cognitive decline donor rats into recipient LZ or ZDF rats,and integrated the method of 16S rRNA and targeted metabolomics to evaluate the directional effect of the gut microbiota on the T2DM phenotype and cognitive function in recipient rats.Method:1.Determine the basic metabolism and cognitive function of 15-week-old donor rats.The intestinal contents of the rats were collected for 16S rRNA analysis,and the gut microbiota supernatant of the donor rats were prepared for GMT.2.9-week-old recipient rats were divided into 6 groups:L-P group(LZ rats administrated with PBS),L-Lg group(LZ rats administrated with LZ donor gut microbiota supernatant),L-Zg group(LZ rats administrated with ZDF donor gut microbiota supernatant),Z-P group(ZDF rats administrated with PBS),Z-Lg group(ZDF rats administered with LZ donor gut microbiota supernatant),Z-Zg group(ZDF rats administrated with ZDF donor gut microbiota supernatant).Before gut microbiota transplantation,1 mL of a broad-spectrum antibiotic cocktail was administered once a day for 10 consecutive days to eliminate the gut microbiota,as the pseudo-sterile rat models were establish.3.For gut microbiota transplantation,750 μL of donor gut microbiota supernatant was given to each rat once a day for 28 consecutive days.Basic biochemical indicators were detected to assess the T2DM phenotype of rats;novel object recognition test and Morris water maze test were measured to assess the cognitive dysfunction of rats;Congo red staining were measurement to assess the amyloid load levels in hippocampus and cortex of rats;ELISA analysis were used to determine the contents of Aβ42 and Aβ40 in hippocampus and cortex of rats;western blotting were used to determine the expression levels of IRS2-Akt-FOXO1 insulin and JAK2-SOCS3-STAT3 leptin signaling pathway related protein in hippocampus and cortex of rats;16S rRNA sequencing analysis was used to determine the gut microbiota composition in the V3-V4 region of rats fecal and intestinal contents;targeted metabolomics analysis was used to determine the structure of the metabolites of the rats intestinal contents;spearman correlation analysis was used to explore the correlation among gut microbiota-metabolites-DACD host phenotype.Results:1.Donor ZDF rats showed T2DM phenotype and cognitive dysfunction at 15-week-old,and their gut microbiota composition were separated from the healthy LZ rats,ensure the difference in the composition of the transplanted gut microbiota.2.After administering a broad-spectrum antibiotic cocktail for intestinal preparation,the activity of the gut microbiota of recipient rats was eliminated,but the basic phenotype was not affected,indicating that the establishment of pseudo-sterile rats was successful.3.After gut microbiota transplantation,it was found that among the recipient ZDF rats,the T2DM phenotypic indicators were changed in Z-Lg group and Z-Zg group,which was manifested in that Z-Lg group was lower than Z-P group,while Z-Zg group was higher than Z-P group;among the recipient LZ groups,there was no significant difference in the T2DM phenotype.4.Novel object recognition showed that after training for 24 hours,Z-Lg group showed higher discrimination index than Z-P group,while Z-Zg group showed lower discrimination index than Z-P group,and the difference between Z-Zg group and Z-Lg group was significant.In the orientation navigation test of the Morris water maze test,Z-Lg group showed a lower escape latency than Z-P group,while Z-Zg group was the opposite;in addition,in the spatial exploration test,Z-Lg group showed improved spatial memory performance.There was no difference among the recipient LZ groups in the two behavioral tests.5.Among the recipient ZDF rats,Z-Lg group showed decreased amyloid plaque deposition in the hippocampus and cortex,compared with Z-P group;the number of plaques in Z-Zg group was closer to that of Z-P group.This result could be reproduced by the levels of insoluble and soluble Aβ in the hippocampus and cortex.In addition,the expression of p-IRS2,FOXO1,p-JAK2,SOCS3,and p-STAT3 increased in Z-P group,and the expression of p-AKT decreased,compared with L-P group;Z-Lg group showed the opposite trend,compared with Z-P group.But in the recipient LZ groups,there was no statistical difference among the groups.6.16S rRNA sequencing analysis showed that the gut microbiota composition of the Z-Lg group transplanted with healthy gut microbiota is closer to that of the donor L group,and the gut microbiota composition of the Z-Zg group is closer to the donor Z group,and gut micribiota transplantation did not change the gut microbiota composition of the recipient L-Lg group and L-Zg group;in addition,although the total number of OTU initially remained at a consistent level,the community diversity of the Z-Lg group has been significantly improved and the community structure of the species tends to be normal,while the community structure of the Z-Zg group tends to be more toward the Z-P group;after gut microbiota transplantation,the abundance level of 10 genera changes,which may be the key bacterial genus that contributes to the phenotypic differences of the transplanted individuals.7.A total of 136 metabolites were identified in the intestinal contents of rats,and amino acids,fatty acids and organic acids were the main types of metabolites;PCA and PLS-DA models showed clear separation among the groups;OPLS-DA model showed that gut microbiota transplantation changed the concentration of 20 metabolites.8.Spearman correlation analysis shows that 10 species of genera,such as Bacteroides,Parabacteroides,Prevotella,Blautia,Lactobacillus,can regulate 11 kinds of metabolites,such as ornithine,propanoic acid,acetic acid,citramalic acid,were significant negative correlated with host phenotype of DACD.Conclusion:After gut microbiota transplantation,the basal metabolic phenotype and cognitive dysfunction of ZDF rats have changed significantly.One possible mechanism is that the change of microbiota and metabolites activate the brain insulin and leptin signaling pathways,and regulate the deposition of Aβ in the brain.It is worth noting that 10 species of genera can regulate 20 kinds of metabolites,and having a significant improvement on the cognitive behavior of ZDF rats.In addition,through the evaluation of correlation analysis,a strong connection was observed among 10 species of genera,11 kinds of metabolites and DACD host phenotypes affected by gut microbiota transplantation.In summary,our study indicates that altering the microbiota-gut-brain axis by re-shaping the composition of gut microbiota is a viable strategy that has great potential for improving cognitive function and combatting DACD.Section 2 Based on microbiomics and metabolomics to explore the prevention and treatment of ZiBuPiyin Recipe on diabetes-associated cognitive decline in ZDF ratsObjective:Investigate whether the gut microbiota plays a key role in ZBPYR-mediated prevention of DACD and treatment of T2DM via incorporating microbiomics and metabolomics,investigate the links between the microbiota-gut-brain axis interaction and the efficacy of ZBPYR in ZDF rats,to interpret the mechanism of action of ZBPYR as therapy for DACD,to provide the theoretical basis for the clinical evaluation and diagnosis of DACD,and to provide a basis for elucidating the theory of "preventing disease" from the"spleen" theory.Method:1.5-week-old T2DM model ZDF rats and the lean control LZ rats were divided into 10 groups:9w-L group(LZ control rats treated with distilled water for 4 weeks),9w-Z group(ZDF model rats treated with distilled water for 4 weeks),9w-HZ group(ZDF model treated with high-dose ZBPYR for 4 weeks),9w-MZ(ZDF model treated with medium-dose ZBPYR for 4 weeks),9w-LZ group(ZDF model treated with low-dose ZBPYR for 4 weeks),15w-L group(LZ control rats treated with distilled water for 10 weeks),15w-Z group(ZDF model rats treated with distilled water for 10 weeks),15w-HZ group(ZDF model treated with high-dose ZBPYR for 10 weeks),15w-MZ(ZDF model treated with medium-dose ZBPYR for 10 weeks),15w-LZ group(ZDF model treated with low-dose ZBPYR for 10 weeks).The high/medium/low dose of ZBPYR were administered by oral gavage daily at dosage of 34.6 g/kg,17.3 g/kg and 8.7 g/kg,respectively,and the remaining rats were orally administered with same amount of distilled water,for 4 or 10 consecutive weeks.2.After 4 weeks of the experiment,the basic biochemical indicators of 9-week-old rats were detected to assess the T2DM phenotype of rats;after 10 weeks of the experiment,in addition to the above indicators,the novel object recognition test and Morris water maze test were measured to assess the cognitive dysfunction of 15-week-old rats;Congo red staining were measurement to assess the amyloid load levels in the hippocampus and cortex of the rats;ELISA analysis were used to determine the contents of Aβ42 and Aβ40 in hippocampus and cortex of rats;western blotting were used to determine the expression levels of IRS2-Akt-FOXO1 insulin and JAK2-SOCS3-STAT3 leptin signaling pathway related protein in hippocampus and cortex of rats;16S rRNA sequencing analysis was used to determine the gut microbiota composition in the V3-V4 region of rats intestinal contents;targeted metabolomics analysis was used to determine the structure of the metabolites of the rats intestinal contents and hippocampus;spearman correlation analysis was used to investigate the links between the microbiota-gut-brain axis interaction and the efficacy of ZBPYR.Results:1.After 4 and 10 weeks of the experiment,ZBPYR reduced body weight and abdominal circumference,and improved the disorder of glycolipid metabolism in ZDF rats,among which the high-dose treatment was significant.2.After 10 weeks of the experiment,ZBPYR improved the cognitive dysfunction of ZDF rats,among which the high-dose treatment was significant.3.16S rRNA sequencing analysis showed that the samples were clearly separated into different clusters at 4 and 10 weeks in the PC1 dimension;after 10 weeks of experiment,ZBPYR treatment transformed the gut micribiota community structure to healthy direction;in addition,after 4 or 10 weeks of experiment,the intervention of ZBPYR changes the abundance of phylum and genus level;6 species of bacteria had undergone dynamic changes during the development of DACD from T2DM;PICRUSt predictive analysis showed that Alzheimer’s disease and insulin signal pathway were mainly enriched in 15-week-old ZDF rats.4.After 10 weeks of the experiment,ZBPYR treatment reduced the amyloid plaque deposition in the hippocampus and cortex of ZDF rats,and reduced the levels of insoluble and soluble Aβ.In addition,ZBPYR treatment improved brain insulin resistance in ZDF rats,manifested by decreased levels of p-IRS2 and FOXO1,and increased levels of p-AKT;ZBPYR treatment improved brain leptin resistance in ZDF rats,manifested by decreased levels of p-JAK2,SOCS3 and p-STAT3.5.A total of 103 metabolites were detected in the intestinal contents of rats,and amino acids,fatty acids and organic acids were the main types of metabolites;PCA and PLS-DA models showed that ZBPYR treatment could significantly reverse the intestinal content metabolic profiles in ZDF rats over time;OPLS-DA model showed that 9 intestinal metabolites and 8 hippocampus metabolites were found to be dynamically changed during the development of DACD from T2DM.6.Spearman correlation analysis showed that Lactobacillus and Bacteroides among bacterial genera,L-glutamic acid,butyric acid,valeric acid,acetic acid,and propionic acid in intestinal metabolites;and 2-hydroxy-3-methylbutyric acid,linoleic acid,docosapentaenoic acid 22n-6,and 8,11,14-eicosatrienoic acid in hippocampus metabolites were significantly negatively correlated with DACD-related phenotypes.7.The related metabolite biomarkers of DACD disrupted by ZBPYR mainly involved in the following pathways:alanine,aspartic acid and glutamic acid metabolism;branched chain amino acid metabolism;short-chain fatty acid metabolism,and linoleic acid/unsaturated fatty acid metabolism.Conclusion:ZBPYR treatment produced lasting changes in gut microbiota community and metabolites,and remotely affected hippocampus metabolic changes,thereby improving memory deficits and reversing β-amyloid deposition and insulin/leptin resistance in the brain of ZDF rats from T2DM to DACD.This may be related to a series of metabolic changes affected by gut microbiota,including alanine,aspartic acid and glutamic acid metabolism pathway;branched-chain amino acid metabolism pathway;short-chain fatty acid metabolism pathway;and linoleic acid/unsaturated fatty acid metabolism pathway.Among them,the therapeutic prescription from the spleen-ZBPYR preventive effect of preventing DACD fdepends on the regulation of gut microbiota,and the regulation of intestinal metabolism and the relationship between intestinal and brain dialogue are ZBPYR is an important prevention mechanism.In short,this study is based on the "spleen" theory of "preventing disease",through ZBPYR regulating the change of gut microbiota to prevent the occurrence and development of DACD,exploring the cascade of microbiota-gut-brain axis after ZBOYR intervention,and constructing the"gut microbiota-metabolite-disease phenotype"association network,clarified the new biological mechanism of ZBPYR prevention and treatment of DACD,provided a theoretical basis for clinical development of a more suitable traditional Chinese medicine prevention and treatment plan for DACD,and enriched and perfected the theoretical connotation of the spleen in traditional Chinese medicine.