Study On The Mechanism Of Kaempferol-3-O-Glucuronide And Baicalin In Regulating Glucose Metabolism

As one of the chronic diseases,diabetes is marked by hyperglycemia,which induces multiple diseases and damages to health.Western medicine has a single target for the treatment of diabetes,the treatment effect is remarkable,but long-term medication will bring certain side effects.Traditional Chinese medicine(TCM)has a long history of treatment for diabetes,and it plays a unique role in the treatment of chronic diseases including diabetes with its characteristics of multi-target and multipathway.Although the research on the regulation of glycolipid metabolism by TCM currently is a hot topic,and there are reports on the role of TCM in the hypoglycemic effect.However,the specific mechanism of the regulation of glycolipid metabolism has not been thoroughly studied,and its targets are not clear.In this paper,the TCM and their active ingredients for the treatment of diabetes in Chinese medicine are systematically summarized,and the representative components and structure types of these commonly used TCMs are analyzed.The results show that the anti-diabetic drugs are mainly composed of bitter and sweet drugs,and their structure is polysaccharide,saponins,flavonoids,etc..Taking the flavonoid active ingredient as an example,the potential targets and pathways were analyzed by network pharmacology.The analysis results show that the PI3K-Akt signaling pathway,Ras signaling pathway,fluid shear stress and atherosclerosis represented the most core pathways,while the target proteins such as AKT and HRAS are the key nodes involved in those multiple signaling pathways.It promotes glycogen synthesis,regulates cell cycle,regulates glucose metabolism,resists oxidative stress,relieves inflammation,and balances lipid oxidation.From the combing results of the Xiaoke prescription system,it is found that for different medicinal parts of Paeonia lactiflora Pall.,the medication frequency of the flowers is higher than that of the roots,suggesting that flowers may contain unique components that are the material basis for hypoglycemic effect.In order to prove the hypothesis,UPLC-Q/TOF-MS technology combined with PCA method were used to analyze the differences of chemical constituents of flowers of P.lactiflora Pall.,Paeoniae Radix Rubra and Paeoniae Radix Alba.It was confirmed kaempferol derivative was the flavonoid chemical composition that is unique to the flowers of P.lactiflora Pall.contributes to the difference analysis.Further combined with in vivo drug metabolite analysis and literature search,kaempferol-3-O-glucuronide(K-3-G)was one of the important metabolites,and its' effect of improving glucose consumption was confirmed in the insulin resistance model of HepG2 cells.In order to reveal the mechanism of hypoglycemic effect of K-3-G,this paper demonstrates that the target may be AKT by network pharmacology prediction combined with enzyme activity assay,and speculated that K-3-G may activate AKT by acting on the PH domain of Lys14 and Asn53 amino acids using molecular docking.Meanwhile,the AKT PH domain transient transfected cell line carrying the green fluorescence(AKT PH-GFP)and control cell line AKT PH(R25C)-GFP which mutation the active site were used to investigate its effect on AKT membrane transport.The results jointly prove that K-3-G can directly activate AKT through binding to the AKT PH domain.Western Blot results showed that K-3-G can promote the phosphorylation of AKT(Ser 473)and improve the phosphorylation level of GSK3? in its downstream,thus improving the utilization of glucose by cells.Baicalin is a representative flavonoid in Scutellaria baicalensis Georgi,which has a variety of biological activities including regulation of glycolipid metabolism.In order to elucidate the mechanism of chemical biology,this paper prepared a probe for baicalin by alkyne modification firstly,next modified a alkynyl baicalin functionalized magnetic microsphere(baicalin-modified functionalized MMs)by click chemistry,then captured the target protein of baicalin from liver cells.AKT1 and HRAS may be potential targets of the captured protein by the analysis of networked pharmacological,and AKT is one of the main target proteins of baicalin after confirmed by SDS-PAGE and Western Blot.Furthermore,the distribution of baicalin and AKT in the cells was investigated by co-localization technique and the baicalin probe.It was found that the positions of the two were partially overlapped,which further confirmed the possibility of interaction between baicalin and AKT.Using AKT competition experiments of baicalin-modified functionalized MMs,it was found that the inhibitors and agonists of AKT PH domain can effectively antagonize the binding of baicalin to AKT,speculated that baicalin may bind to the PH domain of AKT.Further membrane transport of AKT PH domain confirmed that baicalin inhibited membrane transport by binding to AKT PH domain.Molecular docking indicated that baicalin may act on the Arg25 and Asn53 positions of the AKT PH domain,thereby activating the phosphorylation of AKT(Ser473)and causing an increase in the downstream of GSK3? phosphorylation.And has no significant effect on the PDK1 protein which is upstream of AKT.Based on the combing of traditional Chinese medicine hypoglycemic prescriptions and pharmacodynamic components,this paper focused on the flavonoids as a demonstration,and discover the pharmacodynamic basis of the drug through UPLC-Q/TOF-MS and PCA cluster analysis.Network pharmacology predicts the potential targets and pathways of glycolipid metabolism;Under the guidance of chemobiology,molecular probes are used to perform magnetic capture,cell localization and membrane transport functional analysis of target proteins.Finally,it was found that glucuronide,an in vivo metabolite of flavonoids,may be a molecular entity that exerts hypoglycemic effect.It directly activates AKT by acting on AKT PH domain and exerts hypoglycemic through AKT-GSK3? pathway.And provides new ideas of the mechanism of hypoglycemic of flavonoids.