Effect Of A Neutral Polysaccharide From Acanthopanax Trifoliatus(L.)Merr.on Intestinal Disaccharidase And Liver Glucose Metabolism In Diabetic Mice

Acanthopanax trifoliatus（L.）Merr.is a shrub from Acanthopananx of the family Araliaceae.It is widely distributed in central and southern China,especially in the Lingnan regiones.A hypoglycemic effect of neutral polysaccharide from Acanthopanax trifoliatus（L.）Merr.（ATP1-1）on type 1 diabetes mellitus was proved in previous experiments.On the basis of the research,the purpose of this article is to investigate the hypoglycemic effect of ATP1-1 on type 2 diabetic mice,and to explore its hypoglycemic mechanism based on intestinal disaccharidase,glycogen synthesis,glycogenolysis,and gluconeogenesis pathway.The main research contents,results and conclusions of the study are as follows:1.Type 2 diabetic mice model was induced by high-fat diet and high-dose streptozotocin,and all diabetic mice were divided into 4 groups:model group,metformin group（185 mg/kg/d）,ATP1-1 high-dose（80mg/kg/d）and ATP1-1 low-dose group（40 mg/kg/d）.Normal group was also set separately.During intragastric administration for 8 weeks,body weight,diet,water intake,fasting blood glucose（FBG）,oral glucose tolerance test（OGTT）and organ indexes of liver and epididymal fat were determined.,as well as the blood lipids,glycosylated hemoglobin（GHb）,were measured by biochemical analyzer,the pathological morphology of liver and pancreas were explored by HE staining.The results showed that compared with the model group,ATP1-1 treatment could significantly increase the body weight（P<0.05）,reduce the levels of food intake,water intake and FBG（P<0.01）,and improve the glucose tolerance and the organ index of of diabetic mice（P<0.01）.At the mean time,it could also repair islet cell injury.In addition,the expressions of TG,TCHO,and LDL-C in the serum of diabetic mice were significantly lower than those in the model group,and the content of HDL-C was significantly increased（P<0.01）.The results showed that ATP1-1 can regulate hyperglycemia and repair islet cell damage in type 2 diabetic mice,and can improve dyslipidemia in type2 diabetic mice.2.Sucrose tolerance test（OSTT）and maltose tolerance test（OMTT）were determined in the last week of the experiment,and mouse intestine was collected for in vivo and in vitro disaccharidase activity inhibition test.The content and m RNA expression of glucagon-like peptide-1（GLP-1）and sucrase-isomaltase（SI）in serum and ileum were detected by enzyme-linked immunosorbent assay（ELISA）and real-time quantitative PCR（q RT-PCR）.The results indicated that ATP1-1 could improve the sucrose and maltose tolerance of diabetic mice（P<0.01）.According to the in vivo experimental results,the sucrase inhibition rates of high-dose ATP1-1 on the duodenum,jejunum and ileum were 48.29%,75.09%and 31.41%,respectively,and the maltase inhibition rates were 26.23%,18.34%,and34.18%,respectively.In vitro experimental results showed that the IC₅₀ of ATP1-1 on sucrase and maltase in duodenum were 3381.00μg/m L and226.50μg/m L,respectively.Meanwhile,ATP1-1 could up-regulate GLP-1 m RNA and down-regulate SI m RNA expression（P<0.01）.In conclusion,ATP1-1 can inhibit the activity and expression of intestinal disaccharidase,and promote the expression of GLP-1 to improve the hyperglycemia in type2 diabetic mice.3.Periodic acid schiff stain（PAS）and dnthrone sulfate colorimetric method were used to observe glycogen distribution and contents.The content of insulin,glucagon in serum were determined by ELISA,and the m RNA and protein expression of factors involved in AMPK,PI3K/AKT/GSK3βand c AMP/PKA signaling pathways were determined by q RT-PCR and Western Blot.The results of PAS staining and glycogen content determination showed that with ATP1-1 treatment,the liver and muscle glycogen of diabetic mice were significantly decreased,and the distribution of liver glycogen was more uniform（P<0.01）.ELISA results showed that ATP1-1 could increase serum insulin level and decrease glucagon content（P<0.01）.q RT-PCR results showed that ATP1-1treatment could up-regulate the m RNA expressions of FOXO1,IRS1,PI3K,GSK3β,Akt and PKA,and down-regulate the m RNA expressions of G6Pase,GPa and Glucagon（P<0.01,P<0.05）.Western Blot results showed that ATP1-1 treatment could promote the phosphorylation of AMPK,FOXO1,and PKA,and inhibit the phosphorylation of PK,thereby inhibiting the protein expression of PGC1-α,PEPCK,and GPa.At the same time,it could also down-regulate p-Akt protein expression and promote GS phosphorylation to inhibit its overexpression.In conclusion,ATP1-1 can play a hypoglycemic effect and improve insulin resistance by promoting insulin secretion and inhibiting glucagon secretion,and its mechanism is related to activation of AMPK pathway,inhibition of c AMP/PKA pathway and PI3K/Akt/GSK3 pathway.Thereby inhibition of gluconeogenesis is associated with glycogenolysis and reduction of glycogen oversynthesis in hyperglycemic states,reducing hepatic glycogen accumulation.