Study On The Anti-T2DM Mechanism Of Oral Lycium Barbarum Polysaccharide Based On The "Incretin Effect" Of GLP-1

Objective:Based on the"Incretin Effect"of GLP-1,the hypoglycemic mechanism of Lycium barbarum polysaccharide（LBP）was investigated at three levels,namely,gene transcription level,GLP-1 secretion level and post-secretion activity maintenance,respectively,in order to provide a new theoretical basis for LBP the aim is to provide a new theoretical basis for the mechanism of action of LBP in improving blood glucose in T2DM patients.Methods:1 Characterization of LBP samples:（1）Phenol-sulfuric acid method was used to determine the percentage of polysaccharide in LBP Sample.（2）Bicinchoninic Acid method was used to detect protein concentration in LBP samples.（3）High performance liquid mass spectrometry（HPLC）was used to determine the composition of monosaccharides in the sample.（4）It is also be anylzed by ultraviolet and infrared spectroscopy.（5）SDS-PAGE gel electrophoresis was used for detection of protein molecular weight in LBP.2 The effect of LBP on GLP-1 secretion by cell experiment:（1）The expression ofβ-catenin in the cytoplasm and nucleus,and the cellular expression ofβ-catenin,cAMP,Epac,and SGLT1 were detected by Western Blot.（2）In addition,immunofluorescence（IF）was used to locatedβ-catenin and GLP-1 in cells.（3）Co-immunoprecipitation（COIP）was used to identify the interaction between proteinβ-catenin and TCF7L2 or FOXO4.（4）RT-PCR was used to detect GCG gene expression in cells.（5）Calcium ion concentration and GLP-1 in the cell supernatant was detected by ELISA.3 The effect of LBP on GLP-1 secretion and the activity maintenance after secretion through animal experiments:（1）KK^Ay mouse special diet was used to construct a type 2 diabetes model.After the model is formed,the model group is randomly divided into three groups,namely the model group,the LBP group,and the Acarbose group.During treatment,the corresponding drugs were given by gavage（the model group was given normal saline）,and C57BL/6 mice were used as the control group,and the normal saline was given by gavage.（2）Blood glucose and body weight of mice were measured once a week during modeling and treatment intervention.（3）The kit detects the activity ofα-glucosidase in the intestinal content of mice.（4）PT-PCR was used to detect the expression of GCG gene in mice intestinal（5）Immunohistochemistry for localization of GLP-1 in mouse intestinal terminal tissue.ELISA for detection of GLP-1 and DPP-4 concentrations in mouse serum.Note:Black C57BL/6J mice are often used as control groups due to their gene homology with KKAy mice.Results1 Characterization of LBP samples:（1）The polysaccharide content of LBP is70.3%.（2）Samples of LBP contain trace amounts of protein,which is less than 5.06%and weights about 55k D.（3）LBP is composed of six monosaccharides,D-galacturonic acid,D-mannose,L-rhamnose,D-glucose,D-galactose,and L-arabinose,which ratio is0.47:2.37:1.28:11.9:0.77:1.2 Effect of LBP on GLP-1 in STC-1 cells via Wnt/β-catenin cAMP/Epac and SGLT1 pathway:（1）Compared with control group,50μg/m L and 100μg/m L LBP can notably increase the concentration ofβ-catenin in the cytoplasm and nucleus.25μg/m L,50μg/m L,100μg/m L LBP significantly increased the expression ofβ-catenin protein,cAMP and Epac protein in the cell（P<0.05）.（2）After immunofluorescence detection ofβ-catenin protein in cells,β-catenin is obviously transferred from the cytoplasm to the nucleus（3）The COIP results suggested that compared with the control,LBP could distinctly increase the dimer of TCF7L2/β-catenin（P<0.05）.On the contrary,the binding of FOXO4/β-catenin complex was significantly reduced（P>0.05）.（4）According to RT-PCR,with different concentrations of LBP treatment,50μg/m L and100μg/m L LBP treatment increased the expression of GCG gene significantly in cells（P<0.05）.（5）GLP-1 was significantly higher in the 50μg/m L and 100μg/m L LBP treatment groups than in the control group.（P<0.05）.3 The regulatory effect of LBP on GLP-1 secretion:（1）In a low-glucose environment,SGLT1 protein is secreted to a certain extent.High sugar significantly increased SGLT1 protein expressed compared to low sugar（P<0.05）.SGLT1expression was significantly decreased in the LBP group compared with the group without LBP addition（P<0.05）.（2）Compared to the low-sugar group,green fluorescence is more obvious in high-glucose culture conditions.At the same time,compared with the group without LBP,the intensity of the green fluorescent representing GLP-1 increased after the addition of LBP.（3）Compared with the low-glucose group,the concentration of GLP-1 in the low-glucose+LBP,high-glucose,and high-glucose+LBP groups all increased significantly（P<0.05）.（4）Ca²⁺levels were increased markedly in the high sugar group compared to the low sugar group（P<0.05）.and the Ca²⁺concentration in the low-glucose+LBP and high-glucose+LBP groups decreased significantly（P<0.05）.（5）After successful modeling,the weight and blood glucose of the three groups of T2DM mice were significantly greater than those of the control group（P<0.05）.After the intervention,there were no obvious effects on body weight and blood glucose among the three groups of mice with T2DM.LBP group and acarbose group（P>0.05）.But the weight and blood glucose of these three groups were clearly higher than control（P<0.05）.（6）Theα-glucosidase enzyme activity in the LBP group was significantly inferior to that in the model group（P<0.05）at 30 min and120 min.Theα-glucosidase activity was significantly smaller in the acarbose group than in the model group（P<0.05）at 30 min,90 min and 120 min.At 60 min,There was no significant change between the four groups（P>0.05）.（7）At 60 min,The level of GCG gene was found to be remarkably high in the acarbose group than in the model group（P<0.05）.The LBP group and acarbose groups at 90 min was considerably more than the model control group（P<0.05）.（8）At 30 minutes,The prevalence of GLP-1positive cells in the LBP and acarbose groups was significantly above that in the model control group（P<0.05）.At the other three time points,there will be no apparent difference between the three groups of diabetic model mice（P>0.05）.4 LBP maintains its activity after GLP-1 secretion:In the LBP group,DPP-4production gradually increased with time,but there was no statistically difference between the four time points（P>0.05）.The LBP group was significantly more than the model group at 30 minutes（P<0.05）.60 min post-gavage,GLP-1 serum collection in mice in LBP and acarbose groups was significantly higher than that in the model group and other time points（P<0.05）.At 30 min,90 min,and 120 min,there was no significant difference between the model control group,LBP group and acarbose group（P>0.05）.Conclusion:At the level of GCG gene transcription,LBP can activate the downstream target gene GCG gene by activating the Wnt/β-catenin signaling pathway and cAMP/Epac pathway,thereby regulating the secretion of GLP-1.At the level of LBP’s regulation of GLP-1 secretion,LBP inhibits the expression of SGLT1 protein to reduce the concentration of Ca2+in the cell,thereby increasing the expression of GLP-1.In addition,by inhibiting the activity ofα-glucosidase,the concentration of GLP-1can be increased in a short period of time,thereby achieving the effect of reducing blood sugar.At the level of activity maintenance after GLP-1 secretion,LBP does not increase the concentration of GLP-1 by inhibiting the activity of DPP-4 and avoiding the degradation of GLP-1.