Based On The Network Pharmacology And Intestinal Germ Sequencing To Analyze The Possible Hypoglycemic Mechanisms Of Gegen Yam Capsule

Objective: At present,China's sub-health population has reached about 70%.Diabetes,as a high-incidence disease of sub-health population,has become another major health killer after cancer and cardiovascular disease.Once diagnosed with diabetes,it can only be controlled and accompanied,.which brings a certain economic burden to patients.Diabetes can also lead to diabetic foot,diabetic eye disease,diabetic nephropathy and other serious cardiovascular complications,which is also the main cause of death of diabetic patients.In view of the serious consequences of diabetes mellitus and its complications and the current status of sub-health population,if we can take some health food to supplement or prevent,it is undoubtedly an effective means to prevent the disease.Tongrentang Brand Gegen Yam Capsule(GYC)is a traditional Chinese medicine compound health food which has been on the market and has good hypoglycemic function.The whole prescription is composed of Radix Puerariae,Rhizoma Dioscoreae,Rehmannia glutinosa and Hedysarum M?ltijugum Maxim.The compatibility of four herbs is mild,qingrerunzao,yangyinshengjin,which is suitable for those with high blood sugar,but the mechanism of lowering blood sugar is still lacking.Traditional Chinese medicine(TCM)has many components and complex mechanisms.It is difficult to study TCM systematically with single component and single target.In recent years,network pharmacology has gradually attracted people's attention to the research methods of multi-component,multi-target and multi-pathway of traditional Chinese medicine.Many traditional Chinese medicines have also analyzed their mechanism of action through network pharmacology.The relationship between intestinal flora and metabolic diseases,especially diabetes mellitus,has become a research hotspot.It has been reported that some products produced by intestinal flora metabolism can affect blood sugar level.Taking Gegen Yam Capsule as an example,basing on the network pharmacology and intestinal germ sequencing to analyze its possible hypoglycemic mechanisms.Methods: 1.Testing the Auxiliary hypoglycemic function of Gegen Yam Capsule:The hyperglycemic rat model of islet injury was established.The rats were divided into control group,model group and GYC group.The blood glucose values of 0,0.5 and 2hours were measured.The insulin content in serum was measured and the insulin resistance index was calculated.2.Predicting the Possible Mechanism of Gegen Yam Capsule in Assisting Hypoglycemia by Network Pharmacology:Firstly,the potential active ingredients of Gegen Yam Capsule were screened by online database TCMSP,and then the components with hypoglycemic and lipid-lowering effects which were not included in the screening conditions were supplemented by text mining..Using the TCMSP and Drug Bank to screen out the target corresponding to or related to the selected constituent monomers.Cytoscape 3.2.1 software is used to construct network diagrams between components and targets,to display network relationships between components and targets,and the degree values..The screened potential targets were uploaded to Davida online database for gene ontology enrichment analysis(GO-BP)and KEGG pathway enrichment analysis.Molecular level verification of network pharmacological prediction results: Western Blot method was used to determine the relative changes of representative target proteins PI3 K,GSK-3beta,PTGS2,TNFalpha,NF-kappa B and TLR4 in network pharmacological prediction.Real-time PCR method was used to further verify the gene expression level of target proteins.3.Study on the correlation between the hypoglycemic effect of Gegen Yam Capsule and intestinal flora: The intestinal flora of experimental animals in each group were sequenced by 16 Sr DNA,the composition and relative abundance of intestinal flora were observed,and the content of LPS in serum of each group was determined.Pearson correlation analysis was carried out between the fasting blood glucose values of each group and the bacterial flora whose relative abundance changed more prominently to investigate the correlation between animal blood glucose and bacterial flora abundance.Results:1.GYC has hypoglycemic effect on hyperglycemia model rats: compared with the control group,the fasting blood glucose,0.5h,2h blood glucose and the area under the curve of blood glucose change in model group increased significantly(P <0.01);compared with model group,the fasting blood glucose,0.5h,2h blood glucose and the area under the curve of blood glucose change in GYC group decreased significantly(P < 0.01),and the 0.5h blood glucose value of each group was the highest(P < 0.01).Compared with the Control group,the insulin resistance index in Model group increased significantly(P < 0.01),while that in GYC group decreased significantly(P < 0.01).2.Based on the results of GYC network pharmacological analysis,75 active ingredients were screened out,corresponding to 286 targets.The main active ingredients with higher degree value were puerarin,allantoin,miscanthus,quercetin and kaempferol.The representative target proteins are PI3 K,Akt,GSK-3beta,PTGS2,PTGS1,TNFalpha and so on.There are 320 biological processes involved in glycolipid metabolism,immune inflammation,nervous system and other four categories.KEGG is enriched to 106 signaling pathways,involving glucose and lipid metabolism related pathways,immune inflammation related pathways,pathogenic microorganisms related pathways,nervous system related pathways,cancer and other six categories.Among them,14% are glycolipid metabolic signaling pathway and 8%are immune inflammation related signaling pathway.3.Validation of the target of glycolipid metabolism corresponding to the active components of GYC: Compared with Control group,the relative expression levels of PI3 K,Akt and protein in Model group decreased significantly(P<0.01),and the relative expression levels of GSK-3 beta protein increased significantly(P<0.01);compared with Model group,the relative expression levels of PI3 K,Akt and protein in GYC group increased significantly(P<0.01),GSK-3 beta protein increased significantly(P<0.01).Relative expression level of beta protein decreased significantly(P < 0.01),and the results of RNA level detection were consistent with the changes of relative expression level of the above proteins.4.Validation of targets related to inflammation of GYC active ingredients:Compared with Control group,the relative expression of PTGS2,TNFalpha,NFkappa B and TLR4 protein in Model group increased significantly(P < 0.01),while the relative expression of these proteins in GYC group decreased significantly(P < 0.01)compared with Model group.5.Improvement of intestinal flora in diabetic rats by GYC: At the generic level,the top 10 genera were selected.The changes in each group were mainly manifested in Mycoplasma,Lactobacillus,Streptococcus and Escherichia coli-Shigella.Compared with the Control group,the relative abundance of Mycoplasma,Streptococcus and Escherichia coli showed an upward trend,while that of Lactobacillus decreased.GYC could decrease the relative abundance of mycoplasma,Streptococcus and Shigella coli in intestinal tract of model rats,and increase the relative abundance of lactobacillus.Meanwhile,the serum LPS level in Model group was significantly higher than that in Control group(P < 0.01),while that in GYC group was significantly lower than that in Model group(P < 0.01).6.The hypoglycemic effect of GYC was correlated with the relative abundance of different intestinal flora bacteria.The correlation analysis showed that the relative abundance of Lactobacillus was negatively correlated with the fasting blood sugar(P< 0.01).The higher the fasting blood sugar,the lower the relative abundance of lactobacillus.The relative abundance of mycoplasma,Streptococcus and Shigella coli was positively correlated with fasting blood glucose(P < 0.01).The higher the fasting blood glucose,the higher the relative abundance of the three bacteria.Conclusion: 1.The results of network pharmacological analysis showed that increasing the relative expression levels of PI3 K and Akt,protein and m RNA and decreasing the relative expression levels of GSK-3 beta protein and m RNA might be one of the hypoglycemic mechanisms of Gegen Yam Capsule,and decreasing the relative expression levels of TLR4,NFk B,PTGS2 and TNFalpha might be one of the hypoglycemic mechanisms of Gegen Yam Capsule.2.Sequencing analysis of intestinal flora showed that increasing the relative abundance of lactic acid bacteria,decreasing the relative abundance of mycoplasma,Streptococcus and Shigella coli,and lowering the level of LPS in serum might be one of the mechanisms of hypoglycemic effect of Gegen Yam Capsule.