他克莫司联合人胰高血糖素样肽-1对肝细胞中胰岛素信号通道的活化作用

Activation effect of tacrolimus combined with human glucagon like peptide-1 on the insulin signalling pathway in hepatocytes

目的研究人胰高血糖素样肽-1(GLP-1)联合他克莫司对肝中胰岛素信号通路的影响及作用机制。方法培养人HL7702细胞系长到对数生长期,并将细胞分为4组:空白对照组(空白培养基)、他克莫司组(5 mg·L-1他克莫司)、GLP-1组(100 nmol·L-1GLP-1)和联合组(以5 mg·L-1他克莫司+100nmol·L-1GLP-1),均处理细胞90 min。用免疫印迹法检检测Thr 308位点的磷酸化蛋白激酶B (p-AktThr389)、Ser 473位点的磷酸化蛋白激酶B(p-AktSer473)和Thr 389位点的磷酸化核糖体蛋白S6激酶(p-S6K1Thr389)的相对表达水平。结果空白对照组、他克莫司组、GLP-1组和联合组中p-AktThr389的相对表达量分别为1. 00±0,1. 60±0. 24,1. 81±0. 28和1. 95±0. 44;上述4组的p-S6K1Thr389的相对表达量分别为1. 00±0,1. 86±0. 41,1. 61±0. 22和1. 50±0. 18;他克莫司组、GLP-1组和联合组与空白对照组相比,上述指标的差异均有统计意义(均P <0. 05)。空白对照组、他克莫司组、GLP-1组和联合组中p-AktSer473的相对表达量分别为1. 00±0,1. 30±0. 08,1. 21±0. 23和1. 13±0. 09,他克莫司组、GLP-1组和联合组与空白对照组相比,差异均无统计意义(均P> 0. 05)。结论他克莫司联合GLP-1能够激活胰岛素信号通路中的Akt-m TOC-S6K1通路,提示GLP-1改善他克莫司对肝脂肪紊乱可能与Akt-m TOC-S6K1信号通路无关。

Objective To investigate the impact and mechanisms of the human glucagon like peptide-1(GLP-1)plus tacrolimus on insulin signalling pathway in hepatocytes.Methods HL7702 cells at the logarithmic growth phase were divided into four groups:blank control group(blank medium),tacrolimus group(5 mg·L-1 tacrolimus),GLP-1 group(100 nmol·L-1 GLP-1),combination group(5 mg·L-1 tacrolimus+100 nmol·L-1 GLP-1),with fresh media for 90 min respectively.The expression levels of phosph protein kinase B in Thr389(p-AktThr389),phosph protein kinase B in Ser473(p-AktSer473),phosph ribosomal protein S6 kinase in Thr389(p-S6 K1 Thr389)were detected by Western blot analysis.Results The relative expression of p-AktThr389 in blank control group,tacrolimus group,GLP-1 group,and combination group were 1.00±0,1.60±0.24,1.81±0.28,1.95±0.44;the relative expression of p-S6 K1 Thr389 in the above 4 groups were 1.00±0,1.86±0.41,1.61±0.22,1.50±0.18.Comparing between tacrolimus group,GLP-1 group,and combination group with blank control group,the differences of the factors were significant(all P<0.05).The relative expression of p-AktSer473 in the above 4 groups were 1.00±0,1.30±0.08,1.21±0.23,1.13±0.09,Comparing between tacrolimus group,GLP-1 group,and combination group with blank control group,the difference of the factor was not significant(all P>0.05).Conclusion Akt-m TOC-S6 K1 pathway of insulin signaling pathway in liver were activated by tacrolimus combined with GLP-1,which suggested that the improvement of tacrolimus-induced liver fat disorder by GLP-1 may be independent on Akt-GSK3-GS signaling pathway.