从AMPK/PKC信号通路探讨盐酸药根碱促进NCI-H716细胞分泌GLP-1的作用机制

Study on the Mechanism of Jatrorrhizine Hydrochloride Promote NCI-H716 Cell Secretion GLP-1 Through AMPK/PKC Signal Pathway

目的研究盐酸药根碱(JAT)对NCI-H716细胞分泌GLP-1的促进作用,并初步阐明JAT恢复内源性GLP-1分泌作用与AMPK/PKC信号通路相关。方法采用CCK-8法检测不同浓度JAT(6、30、60、90、150、300μmol/L)对NCI-H716细胞活性的影响,筛选150μmol/L JAT作为本实验基本浓度。分别选择JAT低、中、高浓度(100、150、200μmol/L)、150μmol/L JAT+不同浓度抑制剂Dorsomorphin(2、10、50μmol/L)、150μmol/L JAT+不同浓度抑制剂Chelerythrine(2、10μmol/L)对NCI-H716细胞进行干预,采用CCK-8方法检测细胞活性,酶联免疫吸附法(ELISA)测定GLP-1含量,RT-PCR法检测GLP-1 mRNA水平,Western blot法检测GLP-1蛋白表达。结果JAT可促进NCI-H716细胞分泌GLP-1(P<0.05);AMPK通路抑制剂Dorsomorphin和PKC通路抑制剂Chelerythrine均可抑制JAT的作用(P<0.05),且抑制剂Dorsomorphin的作用具有浓度依赖性。结论JAT通过AMPK/PKC信号通路促进NCI-H716细胞分泌GLP-1,具有恢复内源性GLP-1分泌作用。

Objective To investigate the promotion effect of jatrorrhizine hydrochloride(JAT)on the secretion of GLP-1 by NCI-H716 cells,and to preliminarily clarify the mechanism of the recovery of endogenous GLP-1 secretion is related to the AMPK/PKC signaling pathway.Methods NCI-H716 cells were treated with different concentrations of JAT(6,30,60,90,150,300μmol/L),cell viability was determined by CCK-8 method,and the optimal JAT concentration was selected,150μmol/L JAT was screened as the basic concentration in this experiment.JAT low,medium and high concentrations(100,150,200μmol/L),150μmol/L JAT combined with different concentrations of inhibitor Dorsomorphin(2,10,50μmol/L),150μmol/L JAT combined with different concentrations of inhibitor Chelerythrine(2,10μmol/L)were selected to intervene in the NCI-H716 cells.The cell activity was detected by CCK-8 method,the GLP-1 content was determined by enzyme-linked immunosorbent assay(ELISA),the GLP-1 mRNA level was detected by RT-PCR method,and the protein expression was detected by Western blot method.Results JAT significantly promoted the secretion of GLP-1 by NCI-H716 cells,while Dorsomorphin,an inhibitor of PKC pathway,and Chelerythrine,a PKC pathway inhibitor,inhibited the JAT activity in a dose-dependent manner.Conclusion JAT promotes NCI-H716 cell secretion GLP-1 through AMPK/PKC signaling pathway,has the function of restoring endogenous secretion.