| Diabetes mellitus(DM)is a chronic non communicable disease caused by genetic and environmental factors,which has become a major health problem threatening global human health.However,the total number of diabetic diseases in China is the highest in the world,and the vast majority are type 2 diabetes mellitus(T2DM).At present,the specific pathogenesis of T2 DM has not been studied clearly.The core pathological mechanism has been confirmed is insulin resistance(IR)and impaired islet cell function.Although there are many types of hypoglycemic drugs on the market,in addition to controlling blood sugar,drugs also have the function of preventing complications.The adverse reactions of Western medicine and the fluctuation of blood glucose can not be ignored.Intestinal flora is an important part of human flora,which affects the host's nutrition,immunity and energy metabolism.Once the flora is out of balance,it will lead to dysfunction of some body functions and induce various diseases.A large number of studies have confirmed that there is a close relationship between T2 DM and intestinal flora disorder,which is an important factor in the pathogenesis of T2 DM.Therefore,it is an effective way to control T2 DM by regulating the flora and improving the metabolism of internal environment.In recent years,a large number of studies about the effect of Chinese herbal medicine on intestinal flora have been carried out.It is expected that Chinese herbal medicine can intervene in intestinal microecosystem to treat T2 DM.A traditional Chinese medicine prescription called Shenqi compound,which was founded by my tutor Professor Xie in many years' efforts to prevent and treat diabetes and its chronic complications.Shenqi compound has the effect of Tonifying Qi and spleen,nourishing yin and activating blood circulation.In the past,the treatment of diabetes and the reduction of various complications have a very good effect.Previous studies show that Shenqi compound can reduce blood glucose and increase insulin sensitivity on the one hand,and participate in the regulation of intestinal flora on the other hand.However,we can not know how Shenqi compound regulates intestinal flora,and is intestinal flora the potential mechanism of Shenqi compound in reducing blood glucose? More experiments are needed to confirm these questions.Therefore,we intend to carry out this study,this study is mainly to observe the effect of Shenqi compound on intestinal flora and intestinal metabolites of diabetic GK rats.We try to find a new mechanism of Shenqi compound in the treatment of T2 DM from the point of view of flora,so as to provide microbiological and molecular theoretical basis for the wide promotion and use of Shenqi compound in clinic.ObjectivesTo study the effect of Shenqi compound on intestinal flora and endogenous metabolites in diabetic GK rats.From the perspective of intestinal flora and metabolic disorder,we use macrogenomic technology and metabolomics to explore the potential molecular mechanism of Shenqi compound in intervention of T2 DM.The purpose of this study is to provide microbiological and metabonomic evidence for clinical treatment.MethodsThis study was conducted in diabetic GK rats.After one week of adaptive feeding,the GK rats with two times of random blood glucose greater than or equal to11.1mmol/l were included in the observation.Then we randomly divided GK rats into three groups: model group,sitagliptin group and Shenqi compound group.The above three groups were fed with high-fat diet.The normal group was composed of Wistar rats,which mainly ate general diet.There were 8 rats in each group,and the whole observation period was 8 weeks.We used normal saline to gavage GK rats in the model group and Wistar rats in the normal group,used the solution made by sitagliptin to gavage GK rats in the sitagliptin group,and used Shenqi compound decoction to gavage rats in the Shenqi compound group.During the experiment,the general state,weight and blood glucose of rats in each group were recorded by the staff.The rats were weighed on the Sunday of each week.The blood glucose was randomly determined for one day a week.After 8 weeks of intervention,The pathological specimens of colon were stained with HE and observed under microscope.The inflammatory index lipolysacharide(LPS)and intestinal secreted immunoglobulin A(SIgA)were measured by ELISA technique.At the same time,2-4feces of the lower third of colon were collected aseptically,and the fecal samples of 3rats in each group were randomly selected to detect the community structure and differential distribution of intestinal microflora by metagenomics technology,and Kyoto Encyclopedia of Genes and Genomes(KEGG)was used to detect the community structure and differential distribution of intestinal microflora Genes and genomes database was used to analyze the key genes of the differences.Secondly,the fecal samples of 32 rats were analyzed by Ultra High Performance Liquid Chromatography-Q Exactive Orbitrap-Mass Spectrometry(UHPLC-QE-MS)technology to find the differential metabolites,and the pathway analysis was carried out by using the differential metabolites to clarify the molecular mechanism and biological significance of Shenqi compound on T2 DM.Results1.After 8 weeks of intervention,Shenqi compound can improve the mental state,behavioral activity,skin color and other general conditions of diabetic GK rats.With the extension of drug intervention time,the weight of GK rats in this three groups,namely model group,Shenqi compound group and sitagliptin group,were lower than Wistar rats in normal group(P < 0.01).In addition,we also found that Shenqi compound did not show any effect on the weight of rats with the extension of treatment time.With the extension of intervention time,Shenqi compound and sitagliptin could reduce the random blood glucose of GK rats(P < 0.05).2.Shenqi compound can significantly reduce the content of serum inflammatory factor LPS in diabetic GK rats(P < 0.05),increase the SIgA level in intestinal tissue(P < 0.05),can also increase the protein content of ZO-1 in colon tissue(P < 0.05);HE pathological results showed that there were some pathological manifestations in the intestinal tract of diabetic GK rats,such as destruction of glandular structure,decrease of goblet cells,necrosis and abscission of intestinal villi,and infiltration of a large number of inflammatory cells.After treatment with Shenqi compound and sitagliptin,only inflammatory cells infiltrated into GK rats,which could improve the pathological state of intestinal tract to a certain extent.3.The results of metagenomics showed that the number and diversity of bacterial flora in diabetic GK rats were more abundant than that in normal rats,which indicated that GK rats had dysbacteriosis.Shenqi compound could regulate the abundance of flora at all levels of phylum,class,order,family,genus and species,and significantly increase the abundance of fusobacteria at phylum level(P < 0.01).At the family level,it could significantly increase the abundance of mariniflaceae(P < 0.05).KEGG gene function analysis showed that a large proportion of intestinal microorganisms performed metabolism related functions,such as carbohydrate metabolism,amino acid metabolism,coenzyme and vitamin metabolism,nucleotide metabolism,energy metabolism,etc.Shenqi compound can treat T2 DM by influencing lipid metabolism pathway,secondary metabolite synthesis,degradation of heterobiotin and metabolism pathway,up regulating the expression of k00045,k00625,k00560,k00641,k00929 and k00764 genes,and inhibiting the expression of k00196,k00261,k00936 and k00275 genes.4.Metabonomics analysis showed that the intestinal metabolites of diabetic GK rats were significantly different from those of normal rats,indicating that GK rats had metabolic disorder.Shenqi compound can improve 54 kinds of intestinal metabolites,including glycerophospholipids,l-sulfoalanine,isoleucine tyrosine,L-?-amino-1h-pyrrole-1-hexanoic acid,?-guanidine propionic acid,2-ethyl-2-hydroxybutyric acid,prostaglandin H2,prostaglandin E3,etc.The 54 different metabolites were mainly enriched in 12 metabolic pathways related to diabetes metabolism,including biosynthesis of phenylalanine,tryptophan and tyrosine,glycine,serine and threonine metabolism,phenylalanine metabolism,?-alanine metabolism,tyrosine metabolism,cysteine and methionine metabolism,etc.,as well as the synthesis and downregulation of ketones There are many pathways,such as glycolysis,pyruvate metabolism,dicarboxylic acid and glyoxylic acid metabolism,butyric acid metabolism,glycolysis/ gluconeogenesis.These results indicate that Shenqi compound can affect the process of glucose metabolism by regulating the above metabolic pathways and metabolites.Conclusions1.Compared with normal rats,GK rats have intestinal flora structural disorder and metabolic disorder.2.Shenqi compound can improve the general condition of diabetic GK rats and reduce blood glucose.3.Shenqi compound can repair intestinal mucosal barrier and inhibit the growth of intestinal mucosa LPS leakage,increase the contents of SIgA and ZO-1 in intestine and reduce colitis injury;can increase the abundance of fusobacteria and marinifilaceae,affect the lipid metabolism pathway,the synthesis of secondary metabolites,the degradation of heterobiotin and metabolism pathway,up regulate k00045,k00625,k00560,k00641,k00929,k00764 genes,down regulate k00196,k00261,k00936,k00275 genes to treat T2 DM.4.Shenqi compound can regulate 54 kinds of intestinal metabolites,such as glycerophospholipids,l-sulfoalanine,isoleucine tyrosine,L-?-amino-1h-pyrrole-1-hexanoic acid,?-guanidine propionic acid,2-ethyl-2-hydroxybutyric acid,prosta glandin H2,prostaglandin E3,etc.these 54 kinds of metabolites can affect glucose metabolism through multiple pathways,so as to achieve the effect of treating T2 DM. |
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