Predicting And Exploring The Synergistic Mechanisms Of Radix Astragali And Mulberry Leaf（morus Alba L.） In Treatment Of Diabetes Mellitus Based On Multi-omics

Diabetes mellitus（DM）and its complications,as the chronic and non-communicable disease that seriously affects human health and quality of life,have gradually developed into a major worldwide health problem.Traditional Chinese Medicine（TCM）has a long history in the treatment of DM.Radix Astragali and mulberry leaf（Morus alba L.）are commonly used in the treatment of DM,and the combination of them usually occurs in anti-diabetic Chinese prescriptions.However,the synergistic mechanism of Astragalus and mulberry leaf in the treatment of diabetes is still unclear.In order to determine the active components and potential molecular mechanisms of Astragalus and Mulberry Leaves in the intervention of diabetes,LC-MS non-targeted metabolomics was used to identify the chemical components.A diabetic mouse model induced by streptozotocin was established and then treated the diabetic mice with Mulberry Leaves and Astragalus.Meanwhile,the regulatory changes of genes in the livers of different groups of mice were analyzed by transcriptomics and proteomics and some significantly expressed differential genes were verified by q RT-PCR and Western blot.Finally,network pharmacology was used to predict and explore the synergistic mechanisms of Astragalus and Mulberry leaves in the treatment of DM.The main conclusions of this study are as follows:（1）Chemical constituents of Mulberry Leaves and Astragalus were identified by LC-MS.A total of 321 compounds were identified in Astragalus,76 of which had good pharmacological activity.These active ingredients mainly concentrated in flavonoids and isoflavones,such as astragalus,quercetin,formononein,genistine and so on.A total of 290 chemical substances were identified in Mulberry Leaves,and 68 compounds showed good pharmacological and pharmacodynamic activity,including flavonoids and isoflavones,such as quercetin,Robinoflavone,citrus flavone,amino acids such as histidine,lysine,arginine,and 1-deoxymonomycin,fructose,2-hydroxymethyl-5-hydroxy-γ-pyranone and other substances.（2）Effects of Astragalus and Mulberry Leaves on Hypoglycemia in Diabetic Mice: After Astragalus and Mulberry Leaves combined intragastric administration,the weight loss of diabetic mice was alleviated,and the symptoms of polydipsia and polyphagia were alleviated.The fasting blood glucose and serum insulin values of the mice were measured,and it was found that the blood glucose values decreased significantly compared with those before intragastric administration,and the serum insulin level gradually recovered to the same as that of normal mice.The results showed that the combination of Astragalus and Mulberry leaves could effectively control blood glucose in diabetic mice.（3）Results of Transcriptome Analysis: A total of 699 differential genes were screened in the diabetic model group（DC）compared with the combined treatment group of Astragalus and Mulberry Leaves（DD）,among which 306 genes were up-regulated and 393 genes were down-regulated.The GO and KEGG enrichment analysis of these differential genes showed that they were mainly involved in the regulation of steroid metabolism,long-chain fatty acid metabolism,lipid biosynthesis process,cofactor binding,monooxygenase activity,oxidoreductase activity,active transmembrane transporter activity,etc.The metabolic pathways involved include antigen processing and presentation,cytochrome P450 metabolic pathway,PPAR signaling pathway,glycolysis and gluconeogenesis.（4）Proteomic Analysis Results: The analysis of differential proteins of mice in the diabetic group and the Astragalus and Mulberry leaves combined treatment group showed that,compared with the diabetic model group,a total of 1041 proteins with significantly different expressions were screened in the Astragalus and Mulberry Leaves combined treatment group,among which 463 proteins were up-regulated and578 proteins were down-regulated.These differential proteins mainly played roles through retinol metabolism,steroid hormone biosynthesis,cysteine and methionine metabolism,PPAR signaling pathway,insulin signaling pathway.（5）Analysis of Common Differential Genes Between Transcriptome And Proteome and Verification Results of q RT-PCR: Compared with the diabetic model group,the 699 differentially expressed genes in the transcriptome and 169 differentially expressed proteins in the proteome in the Astragalus and Mulberry Leaves combination group had 35 specific genes in common.These common genes mainly played roles through retinol metabolism,steroid hormone biosynthesis,arachidonic acid metabolism,C5 isoprenoid biosynthesis,linoleic acid metabolism,drug metabolism-cytochrome P450 metabolism pathways.Then,the selected 28diabetes-related genes were verified by real-time quantitative PCR and Western blot,and most of the results were consistent with the results of transcriptome and proteome（6）Network Pharmacological Analysis of the Hypoglycemic Mechanism of Astragalus and Mulberry Leaves: The 39 active components,such as quercetin,genistine,calycin and formononetin,were mainly involved in 25 KEGG signaling pathways related to the treatment and prevention of diabetes,including AGE-RAGE signaling pathway,insulin resistance,HIF-1 signaling pathway and AMPK signaling pathway,through 47 corresponding anti-diabetes-related targets.In conclusion,it was showed that the potential hypoglycemia mechanism of Astragalus and Mulberry Leaves could regulate the protein expressions of Tap1,Ncoa4 and Alas2 through the main active components such as quercetin,genistein,calycin and formononetin.By down-regulating Fabp,Hmox1,up-regulating Hmgcr,Cyp7a1 and other genes,it promoted the metabolism and synthesis of glycolipids,and played an antioxidant role by participating in the retinol metabolism pathway and cytochrome P450 metabolism pathway,bile secretion,mitigates iron death,and with these,it finally maintained the glucose homeostasis of the body.