| Objectives:Type 2 diabetes mellitus(T2DM),characterized by insulin resistance,impaired insulin secretion or both,is a chronic metabolic disease.Chronic inflammatory,oxidative stress,glucotoxicity,lipotoxicity and intestinal flora are all involved in the occurrence and development of T2 DM and its complications.Antidiabetic drugs are important means to treat T2 DM.Although there are a variety of effective antidiabetic drugs available for clinicians to choose,they still cannot satisfy the large diabetic population and reduce the overall compliance rate of blood glucose.At present,a number of scholars at home and abroad are dedicated to searching for active compounds from natural products that can assist in treatment of diabetes and clarifying the mechanism from various aspects,hoping to bring more choices to patients with diabetes.As a scientific and technological resource,traditional Chinese medicine have therapeutic and health care functions and are widely used in the research of diabetic treatment.Ixeris chinensis(Thunb.)Nakai is widely distributed in China,which is consumed as wild vegetable in folk and used as herbal medicine to treat bronchitis,pneumonia,pharyngitis,dysentery,enteritis and other diseases.Modern pharmacological studies show that Ixeris chinensis is rich in multiple active ingredients,which possesses many pharmacological activities including antibacterial,immune regulation,anti-inflammatory,antioxidant,hepatoprotective effect,as well as alleviating nonalcoholic fatty liver disease,but the study on T2 DM is relatively scarce.In this study,we invested the effects of different Ixeris chinensis extracts on alleviating T2 DM and their mechanisms by intestinal flora analysis and metabonomic analysis.Methods:1.We formulated extraction scheme,prepared Ixeris chinensis ethanol extract and aqueous extract.The Ixeris chinensis aqueous extracts were separated by ultrafiltration using cellulose ultrafiltration membrane.Chemical methods and HPLC-QTrap MS/MS were used to identify the chemical components of the extracts,and the antioxidant activities of the extracts in vitro were compared.Finally,Ixeris chinensis ethanol extract(ICEE),Ixeris chinensis aqueous extract <1k Da(ICAE<1k Da)and Ixeris chinensis aqueous extract >10k Da(ICAE>10k Da)were selected for follow-up study.2.T2 DM mouse model was induced by high-fat diet combined with STZ.The three extracts were all divided into low and high dosage groups,metformin was used as a positive control.The blood glucose,body weight and food intake of mice were monitored every week.OGTT,serum insulin,HOMA-IR,glycogen,GLP-1,blood lipid and liver function were compared.The serum antioxidant enzyme activity and malondialdehyde level were measured by assay kits.The levels of inflammatory factors in liver and pancreas tissues were assessed by RT-q PCR.The histopathological changes were observed by H&E staining,and the distributions of islet cells were observed by immunofluorescence staining.RT-q PCR,western blot and immunohistochemical staining were used to detect the expression levels of PI3K/AKT pathway-associated proteins,glycogen synthesis key enzymes,gluconeogenesis key enzymes and related transcription factors,as well as lipid synthesis key enzymes in liver.3.RT-q PCR was used to detect the expression of intestinal barrier related proteins and inflammatory factors in colon tissues,and the serum LPS level was measured by ELISA assays.The concentration of short-chain fatty acids(SCFAs)of cecum contents was determined by HPLC.The composition of the intestinal microbiota in the caecum of mice was analyzed based on high-throughput 16 S r DNA sequencing.4.LC-MS-based non-targeted metabolomics technology was used to identify the composition of urine metabolites of mice.Results:1.The results of the chemical method on the active components of the extracts showed that ICAE>10k Da was mainly composed of macromolecular polysaccharides and uronic acid.The gel permeation chromatography(GPC)showed that its molecular weight was 118.889 k Da.ICAE<1k Da mainly consisted of flavonoids and triterpenes,while the effective components measured were less in ICEE.The small molecular substances were detected by HPLC-QTrap-MS/MS.The results indicated that the three extracts were rich in amino acid and their derivatives,flavonoids,carbohydrate and their derivatives,organic acid and their derivatives,terpenoids,nucleotide and their derivatives,polyphenols,alkaloid and their derivatives,but the relative abundance of various compounds in the three extracts were different.The three extracts all showed antioxidant effects in vitro,but their scavenging capacities to different free radicals were different.2.The three extracts lowed fasting and post-load blood glucose levels in diabetic mice,improved insulin resistance,recovered β cell damage and decreased the ratio of glucagon positive cells to insulin positive cells.ICAE>10k Da showed the best effects among the three extracts,followed by ICAE<1k Da and ICEE.The levels of serum lipid and liver lipid deposition decreased after the three extracts supplementation,and expressions of the key enzymes associated with fatty acid synthesis(Fasn and Acc)and cholesterol synthesis(Hmgcr)were inhibited.The three extracts enhanced the activity of antioxidant enzymes,reduced the level of malondialdehyde and also down-regulated the expression of inflammatory factors in pancreas and liver.The three extracts activated PI3K/AKT signal pathway,up-regulated the expression of Glut2 in liver,inhibited the expression of Gsk3β and promoted Gys2 expression,thereby increasing liver glycogen content.The key enzymes of gluconeogenesis(Pepck and G-6-pase)and related regulatory factors(Creb and Pgc-1α)were also down-regulated.3.The three extracts up-regulated the expression of intestinal barrier related protein,down-regulated the expression of inflammatory factors,reduced the pathological damage of colon tissue and the serum LPS level.The SCFAs in cecum contents of diabetic mice were reduced,and two kinds of Ixeris chinensis aqueous extracts promoted the production of acetic acid and butyric acid.Compared with normal mice,the intestinal flora of diabetic mice altered,the relative abundance of some harmful bacteria,such as Candidatus＿Stoquefichus,Collinsella,etc.increased,while the relative abundance of the SCFAs-producing bacteria Faecalibaculum and Anaerostipes decreased.The three extracts regulated the intestinal flora structure of diabetic mice.ICEE altered the gut microbiota slightly,whereas ICAE>10k Da reshaped the gut microbiota most significantly,and the effects of the three extracts on the intestinal flora were dose-dependent,which were more significant in the high-dose groups.High-dose of ICEE reduced the relative abundance of Candidatus＿Stoquefichus,Eubacterium＿coprostanoligenes＿group,etc.and increased the relative abundance of Megasphaera and Frisingicoccus.High-dose of ICAE<1k Da reduced the relative abundance of Candidatus＿Stoquefichus and Collinsella,raised the relative abundance of the SCFAs-producing bacterium Butyricicocus,and also enriched Actatifactor and Ruminococus＿torques＿Group,etc.High-dose of ICAE>10k Da also lowed the relative abundance of Candidatus＿Stoquefichus and Collinsella,enriched beneficial bacteria Prevotella,Muribaculateae,Roseburia and Christensenellacea＿R-7＿Group,etc.The correlation analysis between the differential bacteria and the physiological indexes of mice indicated that two kinds of Ixeris chinensis aqueous extracts-enriched bacteria were beneficial to improve glucose and lipid metabolism,oxidative stress,colon barrier function and inflammation.In addition,Christensenellacea＿R-7＿Group enriched in high-dose of ICAE>10k Da group was positively correlated with butyric acid,and Roseburia was positively correlated with propionic acid.4.The urine metabolic profile in T2 DM mice changed compared with that in normal control mice.The pathways enrichment of differential metabolites mainly related to energy metabolism.ICEE impacted the metabolic profile least in diabetic mice compared with the other two extracts.It was found that high-dose of ICEE could reduce L-isoleucine by screening differential metabolites related to diabetes.The significant pathway enrichment of differential metabolites involved purine metabolism only.The high-dose of ICAE<1k Da also altered the composition of urinary metabolites in diabetic mice significantly,down-regulating D-galactose,taurocholic acid,Lphenylalanine and 5-Hydroxy-N-formyl kynurenine,while up-regulating cholic acid,5-hydroxylysine and traumatic acid.Pathways enrichment of differential metabolites included amino acid metabolism and bile acid metabolism.ICAE>10k Da also demonstrated the best effect on regulating the metabolism of diabetic mice.More abnormal metabolites had obvious callback after high-dose of ICAE>10k Da supplementation in diabetic mice,including down-regulating D-tagatose,D-galactose,L-phenylalanine,5-Hydroxy-N-formyl kynurenine,taurocholic acid and Diglyceride.Besides,ICAE>10k Da up-regulated phosphatidylcholine and phosphatidylethanolamine,which might be beneficial,meanwhile increased cholic acid.Pathways enrichment of differential metabolites consisted of carbohydrate metabolism,lipid metabolism,amino acid metabolism and bile acid metabolism.Conclusions:1.The three Ixeris chinensis extracts alleviate T2 DM,reduce hyperglycemia and hyperlipidemia,improve insulin resistance and decrease islets β Cell damage in T2 DM mice.ICAE>10k Da,which is rich in polysaccharides,shows the best effects on alleviating T2 DM and can be the first choice.2.The three Ixeris chinensis extracts show anti-inflammatory and antioxidant activity,activate the PI3K/AKT signal pathway,increase glucose uptake in liver,promote liver glycogen synthesis and inhibit hepatic gluconeogenesis.3.The three Ixeris chinensis extracts can improve intestinal barrier function,regulate intestinal microbiota,change composition of metabolites and regulate metabolic pathways.ICAE>10k Da can enrich more beneficial bacteria in diabetes mice and callback more abnormal metabolites. |
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