枸杞多糖促进小肠内分泌细胞分泌肠促胰液素

The Effect of Lycium Barbarum Polysaccharide in Stimulating Secretin Secretion in Small Intestinal Endocrine Cells

目的探讨枸杞多糖(LBP)通过Wnt/β-catenin信号通路对小鼠肠道内分泌细胞(STC-1)中GCG基因的影响以及调节胰高血糖素样肽-1(GLP-1)分泌的作用机制。方法(1)将STC-1细胞分为4组:空白对照组、25μg·mL^(-1) LBP干预组、50μg·mL^(-1) LBP干预组、100μg·mL^(-1) LBP干预组,用Western blot法分别检测4组中细胞质、细胞核以及全蛋白中β-catenin的表达;采用免疫荧光法检测每组细胞中β-catenin的原位表达。Western blot检测4组细胞全蛋白中cAMP、Epac蛋白的表达水平。(2)将STC-1细胞分为4组:空白对照组、重组蛋白Wnt3a组(加入50 ng·mL^(-1)重组蛋白Wnt3a)、LBP组(加入100μg·mL^(-1) LBP)、LBP联合重组蛋白Wnt3a组(50 ng·mL^(-1)重组Wnt3a蛋白与100μg·mL^(-1) LBP联合),通过免疫共沉淀法检测转录因子FOXO4和TCF7L2分别与β-catenin结合。(3)将STC-1细胞分为4组:空白对照、25μg·mL^(-1) LBP干预组、50μg·mL^(-1) LBP干预组、100μg·mL^(-1) LBP干预组,ELISA试剂盒检测4组GLP-1浓度;从每组细胞中提取总RNA,反转录后利用RT-PCR法检测细胞中GCG基因的表达水平。结果50μg·mL^(-1)和100μg·mL^(-1) LBP干预可上调细胞核和细胞质中β-catenin的表达。随着LBP干预浓度的增加,核转位愈发明显。在LBP刺激下,转录因子TCF7L2与β-catenin的结合能力增强。随着LBP干预浓度的增加,GCG基因和GLP-1的表达增加。结论LBP可通过激活调节Wnt/β-catenin信号通路促进GCG基因的表达,从而上调GLP-1的浓度。

Objective To investigate the mechanism of lycium barbarum polysaccharide(LBP)on stimulating secretion of GLP-1 by STC-1 cells.Methods STC-1 cells were divided into four groups:control,25μg·mL^(-1) LBP,50μg·mL^(-1) LBP,100μg·mL^(-1) LBP.The expression ofβ-catenin in cytoplasm,nucleus and total protein was detected by Western blot.Immunofluorescence method was used to detect the in situ expression ofβ-catenin in each group of cells.In addition,Western blot was used to detect the expression level of cAMP and Epac protein in the total protein of the above four groups of cells.STC-1 cells were divided into control group,positive control group(adding 50 ng·mL^(-1) recombinant protein Wnt3a),therapy group(adding 100μg·mL^(-1) LBP),LBP+Wnt3a combined group(50 ng·mL^(-1) recombinant Wnt3a protein combined with 100μg·mL^(-1) LBP),the binding of transcription factors FOXO4 and TCF7L2 toβ-catenin was detected by immunoprecipitation.STC-1 cells were divided into four groups:control,25μg·mL^(-1) LBP group,50μg·mL^(-1) LBP group and 100μg·mL^(-1) LBP group.The concentration of GLP-1 in the four groups was detected by ELISA kit.Total RNA was extracted from each group of cells,and the expression level of GCG gene in cells was detected by RT-PCR after reverse transcription.Results 50μg·mL^(-1) and 100μg·mL^(-1) LBP could up-regulate the expression ofβ-catenin in nucleus and cytoplasm.As the concentration of LBP intervention increased,nuclear translocation became more obvious.Under the stimulation of LBP,the binding ability of transcription factor TCF7L2 toβ-catenin increased in STC-1 cells.As the concentration of group with intervention by LBP,the expression significantly increased in GCG gene and the concentration of GLP-1.Conclusion LBP may promote the expression of GCG gene by regulating the Wnt/β-catenin signal pathway,thus up-regulating the concentration of GLP-1.