Study On The Mechanism Of Sijunzi Decoction In Regulating Glucose And Lipid Metabolism Disorders In Type2 Diabetes Based On PI3K/AKT Pathway

ObjectiveTo investigate the pharmacodynamic changes of Sijunzi Decoction in regulating glucose and lipid metabolism disorders in type 2 diabetes mellitus(T2DM)mice and rats,and to screen their sites for reducing glucose and lipid,and to screen the possible role of Sijunzi Decoction in regulating T2 DM glucose and lipid metabolism disorders through network pharmacology The mechanism and experiments verify whether Sijunzi Decoction can protect the liver of T2 DM rats by regulating the PI3 K / AKT signal pathway(obtained by network pharmacology screening),thereby regulating glucose metabolism disorders.Methods:1 Sijunzi decoction preparation and preparation control: D-glucose was used as a reference substance,and phenol-concentrated sulfuric acid colorimetric method was used to determine the content of total polysaccharides in Sijunzi decoction;rutin was used as a reference substance,and sodium nitrite-aluminum nitrate was used.-The sodium hydroxide colorimetric method was used to determine the total flavonoid content in Sijunzi Decoction;the glycyrrhizic acid was used as a reference substance,and the 1% vanillin acetic acid-perchloric acid colorimetric method was used to determine the total saponin content in Sijunzi Decoction.Taking the comprehensive score of total polysaccharides,total flavones and total saponins as indicators,the optimal extraction process of Sijunzi Decoction was optimized by single factor experiment and orthogonal test method;the optimal purification process of Sijunzi Decoction was optimized by single factor experiment and response surface method..2 To investigate the regulatory effect of Sijunzi Decoction on the disorder of glucose and lipid metabolism in T2 DM mice: a high-sugar and high-fat diet combined with a small intraperitoneal injection of streptozotocin(STZ)was used to establish a mouse T2 DM insulin resistance model.The experiment set blank group,model group,positive group,Sijunzi Decoction high dose(SJZT-H),Sijunzi Decoction medium dose(SJZT-M),and Sijunzi Decoction low dose(SJZT-L)groups to investigate the body weight of mice(W),Liver index,oral glucose tolerance(OGTT),fasting blood-glucose(FBG)content,fasting insulin(FINS)content,insulin resistance index(HOMA-IR),total cholesterol(T-CHO)content,low Effects of LDL-C,HDL-C,TG and Liver Glycogen(LG)contents.3 Screening the active sites of Sijunzi Decoction to regulate glucose and lipid metabolism disorders in T2 DM mice: the experimental set was blank group,model group,positive drug group,Sijunzi Decoction water extract(SJZT)group,Sijunzi Decoction water extract and alcohol sediment(SJZT-D)group and Sijunzi decoction water extraction and alcohol precipitation(SJZT-U)group.The mice were examined for W,OGTT,FBG content,FINS content,HOMA-IR,T-CHO content,LDL-C content,HDL-C content,TG content,and LG content.4 Sijunzi Decoction’s pharmacodynamic research and active site screening of T2 DM rats’ glucose metabolism disorders: the experimental group consists of blank group,model group,positive drug group,SJZT group,SJZT-D group and SJZT-U group.Investigate rat W,liver index,OGTT,FBG content,FINS content,HOMA-IR,LG content,NO content,SOD activity,liver tissue morphology and pathological sections.5 Screening possible action mechanisms of Sijunzi Decoction for regulating T2 DM glucose metabolism disorders through network pharmacology: Screen the active ingredients of Sijunzi Decoction based on the TCMSP database,PubChem database,and SwissTargetPrediction database,and predict their target targets;according to the TTD database,DRUGBANK database,and DisGeNET database Screen T2 DM action targets;map the component targets to disease targets with Cytoscape software to build a Sijunzi soup component-target network,select core active components-core targets based on degrees of freedom,and perform GO analysis of core target genes through the DAVID database And KEGG analysis;finally,molecular docking was performed on the top 3proteins of the correlation and the top 5 components of the correlation.6 Validate the mechanism of network pharmacological screening through experiments:the experimental group is set to blank group,model group,positive drug group,SJZT group,real-time fluorescence quantitative PCR(qRT-PCR)method to detect INSR,IRS-1,PI3 K,AKT,FOXO1,GSK-3β expression of rat liver.Results:1 Preparation and control of Sijunzi Decoction liquid: Methodological investigation of total polysaccharides,total flavones,and total saponins of Sijunzi Decoction showed that the total polysaccharides,total flavones,and total saponins were between 6.54 ~ 43.6 ug/mL and 4.36 ~ 109 ug/mL,respectively.It exhibits good linearity in the range of 10.9 ~272.7ug/ml.The RSD of precision,stability and repeatability tests are less than 3.00%,and the sample recovery rates are 104.28%,102.235%,100.18%,and RSD <3.00%;preferably The optimal water extraction process conditions for Sijunzi Decoction are 10 times the amount of water added,the cooking time is 90 minutes,and the decoction is performed 3times.The optimal alcohol precipitation process conditions for Sijunzi Decoction are 85%alcohol precipitation concentration,and the relative density of the concentrated liquid is1.25 g / mL,let stand for 14 h.2 To investigate the regulatory effect of Sijunzi Decoction on glucose and lipid metabolism disorders in T2 DM mice: compared with the blank group,the W,AUC,FBG content,FINS content,HOMA-IR,T-CHO content,and liver index of the model group were extremely significant(P<0.001)increased,TG content increased significantly(P<0.01),LDL-C content increased significantly(P<0.05),LG content significantly decreased(P<0.001),and HDL-C content significantly(P< 0.05);compared with the model group,the W,AUC,FBG content,FINS content,HOMA-IR and liver index of the SJZT-H group were significantly reduced(P<0.001),and the T-CHO content and TG content were significantly(P<0.01)decreased,and the LG content was significantly increased(P<0.001);the AUC,FBG content,FINS content,and HOMA-IR of the SJZT-M group mice were significantly decreased(P<0.001),and the T-CHO content and TG The content was significantly reduced(P<0.05),and the LG content was significantly increased(P<0.001);HOMA-IR in the SJZT-L group was significantly reduced(P<0.001),and the AUC and FINS content were significantly reduced(P<0.01).,LG content increased significantly(P<0.05).3 Screening Sijunzi Decoction for regulating the active sites of glucose and lipid metabolism disorders in T2 DM mice: compared with the blank group,the W,AUC,FBG content,FINS content,HOMA-IR and T-CHO content in the model group were extremely significant(P<0.001)),The TG content increased significantly(P<0.01),the LG content decreased significantly(P<0.001),and the HDL-C content decreased significantly(P<0.05);compared with the model group,the AUC and FBG of the SJZT group mice Content,FINS content,and HOMA-IR content were significantly reduced(P<0.001),TG content,T-CHO content were significantly reduced(P<0.05),and LG content wasextremely significantly(P<0.001)increased;the SJZT-U group was smaller The AUC of rats was significantly reduced(P<0.01),and the FINS content and T-CHO content were significantly reduced(P<0.05).The AUC,FBG content,FINS content,and HOMA-IR of the SJZT-D group mice were significantly reduced(P<0.001),LG content increased significantly(P<0.001).4 Sijunzi Decoction Pharmacodynamics and Active Site Selection of T2 DM Rats’ Glucose Metabolism Disorders: Compared with the blank group,the AUC,FBG content,and HOMA-IR in the model group were significantly increased(P<0.001),and the liver index was significantly increased.(P<0.01)increased,FINS content increased significantly(P<0.05),W and LG content decreased significantly(P<0.001),SOD activity decreased significantly(P<0.05),and liver color of rats became yellow and qualitatively changed.Brittle,severe hepatocyte steatosis;compared with the model group,the AUC of the SJZT group rats was significantly reduced(P<0.001),the FBG was significantly reduced(P<0.05),and the LG content was significantly increased(P<0.001);SJZT-D group AUC,FBG,and NO contents were significantly reduced(P<0.05),the levels of LG were significantly increased(P<0.001),and the SOD activity was significantly increased(P<0.05);the contents of LG in the SJZT-U group were significantly increased Significantly increased(P<0.05);the color,texture,and liver cell degeneration of the rats in each administration group were improved to varying degrees.5 Screening possible mechanisms of Sijunzi Decoction regulating T2 DM glucose metabolism disorders through network pharmacology: 113 chemical components were screened from Sijunzi Decoction,which involved 47 targets for the treatment of diabetes;cores were selected based on node degrees of freedom ≥ average degree of freedom 22 components and 27 core targets;GO analysis results indicate that it involves 7 biological processes such as blood glucose homeostasis,positive regulation of adipose tissue development,4 molecular functions including steroid hormone activation,drug binding,including plasma membrane The nuclear chromatin is composed of 2 cells;KEGG analysis results indicate that it may be related to the treatment of diabetes through 7 signal pathways such as AMPK signaling pathway,PPAR signaling pathway,and insulin resistance.60% of the molecular docking has a strong binding activity,and 40% has a good binding activity.6 Validate the mechanism of network pharmacological screening through experiments:Compared with the blank group,the relative expression levels of INSR,IRS-1,PI3 K,AKT2,and mRNA in the model group were significantly reduced(P<0.001),and FOXO1 and GSK-3β mRNA were significantly reduced.The relative expression was extremely significant(P<0.001).Compared with the model group,the relative expression of PI3 KmRNA in the SJZT group was significantly increased(P<0.001),and the relative expressions of IRS-1 and AKT2 mRNA were significant(P<0.05)),The relative expression of INSR mRNA increased,and the relative expression of FOXO1 and GSK-3βmRNA decreased significantly(P<0.001).ConclusionUltraviolet spectrophotometry was used to determine the content of total polysaccharides,total flavonoids,and total saponins in Sijunzi Decoction,which was simple,accurate,and reliable.The optimal extraction and purification process is stable and feasible,which provides a scientific basis for the quality control of Sijunzi Decoction.The middle and high dose groups of Sijunzi Decoction can significantly improve insulin resistance in type 2 diabetic mice,promote insulin function recovery,and regulate glucose and lipid metabolism disorders.Screening results in rats and mice showed that the effective part of hypoglycemic and lipid-lowering was decoction.Network pharmacology research found that Sijunzi Decoction treats type 2 diabetes through multiple targets and multiple pathways,and selected the PI3 K / AKT signaling pathway for verification.It was found that Sijunzi Decoction can improve the insulin resistance of type 2 diabetic rats by activating the PI3 K / AKT signaling pathway in the liver,promote the synthesis of liver glycogen and the oxidative breakdown of glucose,and achieve the effect of regulating glucose metabolism disorders.