| Objective: To explore the target and pathway of Gegen Qinlian Decoction(GQD)in regulating type 2 diabetes mellitus.Methods:1.A systemic pharmacology study of GQD regulation in type 2 diabetes.(1)TCMSP and BATMAN-TCM databases and literature searching was integrated to find chemical constituents of Puerariae Lobatae Radix,Scutellariae Radix,Coptidis Rhizoma and Glycyrrhizae Radix et Rhizoma Praeparata cum Melle;(2)Retrieved from Swiss Target Prediction,SEA,Pub Chem,Pharmmapper and TCMSP databases,drug target prediction based on the pharmacophore model of chemical components were conducted,and target database was established;(3)Disease targets based on Gene Card and OMIM databases were collected,drug targets and disease targets wereintersected,thus targets related to GQD acting on diabetes was able to obtained;(4)Based on the Cell Marker database,the GQD and disease-related targets were subjected to cell enrichment analysis to obtain the target organs;(5)Pathway enrichment analysis was performed based on the KEGG database,and the pathways and targets related to diabetes were obtained;(6)The relevant targets obtained from pathway enrichment were analyzed by PPI and key targets were obtained.2.Study on GQD targeting liver cells for improving anti-hyperglycemic effects.(1)Quality control of GQD.Puerarin,baicalein,berberine and glycyrrhizin were used as traditional Chinese medicine markers of Gegen,Huangqin,Huanglian and Gancao in GQD,and the quality standard of GQD was constructed by HPLC spectrum of traditional Chinese medicine;(2)The insulin resistance model of Hep G2 cells was established by glucose,and the optimal concentration of IR-Hep G2 cell model was obtained;The cytotoxicity experiment was carried out with GQD to detect the effect of GQD on the proliferation of Hep G2 cells;Finally,the optimal concentration of GQD to improve IR Hep G2 cells was found in a certain range;(3)Q-PCR and Western Blot were used to detect the effects of GQD on genes and proteins related to cellular insulin resistance and cellular inflammation from gene and protein levels,respectively.3.Study on the anti-hyperglycemic effect of GQD acting on multiple organs in diabetic mice.(1)The diabetic mouse model was established by high-fat diet combined with multiple low-dose intraperitoneal injection of STZ;(2)After the model was established,the mice were randomly divided into model group,metformin group,GQD-L,GQD-M and GQD-H groups according to fasting blood glucose.(3)Continue to feed with the previous diet,the control and model group were given 10 m L/kg/d normal saline,and the GQD group was given with 1.14 g/kg/d,3.43g/kg/d,10.28 g/kg/d for seven weeks.(4)The OGTT and ITT values of mice were measured in the last week.(5)The serum,liver,skeletal muscle,epididymal fat,kidney and pancreas of mice in each group were obtained,and the physiological indexes were measured and stained with HE.Result:1.Systems pharmacological analysis of Gegen Qinlian Decoction.(1)A total of 642 related compounds and 2183 targets were obtained from Gegen Qinlian Decoction,of which 759 targets were associated with T2 DM.(2)Through the enrichment of target organs,the main target organs of Gegen Qinlian Decoction were liver,skeletal muscle,adipose tissue,kidney and pancreas,each containing 91,15,24,33 and 35 targets;(3)The KEGG enrichment analysis of these genes by cluster Profiler package showed that there were 28,32,34,20 and 22 pathways related to T2 DM in liver,skeletal muscle,adipose tissue,kidney and pancreas respectively.(4)The main pathways of liver included MAPK signaling pathway and PI3K-Akt signaling pathway;The main pathways of skeletal muscle were PI3K-Akt and focal adhesion signaling pathway;The main pathways of adipose tissue were PI3K-Akt and cell adhesion molecules;The main pathways of pancreas were pancreatic secret,insulin resistance and insulin secret signaling pathway;The main pathways of kidney were NF-kappa B,TNF and lipid and atherosclerosis signaling pathway.It indicated that GQD could treat T2 DM through multiple organs,multiple pathways and multiple targets.2.Study of the hypoglycemic effect of IR-Hep G2 cells improved by GQD.(1)Quality control of GQD.Taking puerarin,baicalein,berberine and glycyrrhizin as the markers of traditional Chinese medicine,the chromatograms of GQD were analyzed by high performance liquid chromatography(HPLC).The retention times were 9.37,16.93,32.91 and 36.74 min respectively,and the maximum absorption wavelengths were 248.63,215.6,200.3 and228.56 nm respectively.(2)The establishment of IR-Hep G2 cell model optimized the culture medium,and the optimum glucose content was 35 mmol/L;Cytotoxicity test showed that GQD had relatively little damage to cells at the concentration of 0 mg/m L ~10 mg/m L;The pharmacodynamic experiment of GQD on IR-Hep G2 cells showed that when the content of GQD was 5mmol/L,it not only did less damage to Hep G2 cells but also had a better therapeutic effect on cell insulin resistance.(3)The results of transcription level showed that compared with the control group,the expression of JNK and c-fos in the model group increased(p < 0.05,p < 0.001),and the expression of JNK and c-fos in the GQD treated group was inhibited(p < 0.01,p < 0.001);Meanwhile,there was no significant change in the expression of AKT2 in the model group,but GQD administration group promoted the expression of AKT2(p < 0.05).(4)Protein level results showed that GQD could affect PI3K-Akt pathway.Compared with the control group,the expression of PI3K/PI3 K in the model decreased,but the trend was not obvious.The expression of p-Akt/Akt decreased(p < 0.01).GQD administration group promoted the expression of p-PI3K/PI3 K and p-Akt/Akt.3.GQD affects the multiple organ hypoglycemic effect in diabetes mice.(1)The diabetic mouse model was established by high fat diet and intraperitoneal injection of STZ,GQD effectively reduced blood glucose and improve glucose tolerance and insulin tolerance in model group.(2)Compared with the control group,the content of TC,TG and LDL-C in serum of the "four items of blood lipids" in the model group increased,while the content of HDL-C decreased(p < 0.001).TC content decreased significantly in metformin group and GQD-M group(p< 0.001,p < 0.05);TG content decreased significantly in metformin group,GQD-M and GQD-H groups(p < 0.001 or p <0.01);LDL-C content increased significantly in metformin group and GQD-M administration group(p < 0.001,p < 0.05);HDL-C content increased slightly in metformin group and all GQD treated groups(p < 0.05),adjusting for abnormal lipid metabolism caused by T2 DM.(3)GQD reduced the liver damage caused by T2 DM.Compared with the control group,the contents of ALT and AST in the serum of the model group increased(p <0.001).Compared with the model group,the ALT content of metformin group and GQD-M,GQD-H administration groups decreased(p < 0.001);AST content decreased in metformin group and GQD-M,GQD-H groups(p < 0.05 or p < 0.01);GQD alleviated the renal tissue damage caused by T2 DM.Compared with the control group,the contents of BUN,Cr and UA in the serum of the model group increased(p < 0.01,p < 0.05,p <0.001).Compared with the model group,the bun content of metformin group and GQD-L,GQD-H administration groups decreased significantly(p < 0.01 or p < 0.001);Cr content decreased in metformin group and gqd-m group(p < 0.001 or p <0.05);UA content decreased significantly in all treatment groups(p < 0.001).(4)HE staining showed that GQD could reduce the vacuolization of liver and islets caused by T2DM;Inflammatory cell infiltration in liver,kidney,skeletal muscle,pancreas and adipose tissue.Conclusion:GQD improve hyperglycemic effects by regulating related target genes and pathways through multiple organs of liver,skeletal muscle,adipose tissue,pancreas and kidney.(1)GQD synergistically treat T2 DM by acting on MAPK and PI3K-Akt signaling pathways in liver and PI3K-Akt and focal adhesion signaling pathways in skeletal muscle;PI3K-Akt and cell adhesion molecules signaling pathways of adipose tissue;Pancreatic secret,insulin resistance and Insulin secretion signaling pathways of pancreas;NF-kappa B,TNF and lipid and atherosclerosis signaling pathways in kidney;(2)GQD regulates PI3K-Akt pathway and MAPK pathway of liver,regulates glycolipid homeostasis,reduces inflammatory response and enhances hypoglycemic effect.(3)GQD reduce blood glucose and reduce the physiological and morphological damage and inflammatory response of multiple organs caused by T2 DM. |
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