FK506对人肝HL-7702细胞中Akt表达的影响

Effects of tacrolimus on the expression of Akt in human hepatocytes line HL-7702

目的观察他克莫司(FK506)对人肝HL-7702细胞系胰岛素信号通路关键位点Akt表达的影响,以探究FK506诱导血糖升高的分子机制。方法选用处于对数生长期的人肝HL-7702细胞系,分为3个处理组和1个对照组。处理组分别用不同浓度的FK506(0.1、1、5 mg/L)处理24 h,对照组用等量培养基培养24 h。采用蛋白质免疫印迹法(western blot)检测各组Akt蛋白的表达量及其磷酸化水平,同时采用蛋白磷酸酶2A(protein phosphatase 2A,PP2A)活性测定试剂盒测定PP2A活性。结果各组Akt的表达水平及磷酸化位点p-Akt(Ser473)水平无明显变化,另一磷酸化位点p-Akt(Thr308)水平(F=5.657,P=0.022)差异有统计学意义;当FK506浓度为5 mg/L时与对照组相比显著升高,差异有统计学意义(P=0.037),随着FK506浓度的升高,PP2A活性有一定程度升高,但差异无统计学意义(F=0.857,P=0.501)。结论 FK506引起HL-7702细胞中Akt磷酸化水平升高,这一变化可能与PP2A无关。

Objective The study aimed to investigate the effects of tacrolimus (FK506) on the expression of Akt, a key site of insulin signalling pathway in human hepatocytes line (HL-7702), thereby exploring the molecular mechanisms of hyperglycemia induced by FK506. Methods HL-7702 cells in logarithmic growth phase were selected and divided into three treatment groups and one control group. The three treatment groups were respectively treated with different concentrations of FK506 (0.1, 1, 5 mg/L) for 24 hours, the control group was cultured for 24 hours with equal amount of medium. The expression of Akt protein and its phosphorylation level were detected by western blot, and the activity of protein phosphatase 2A (PP2A) was measured by means of PP2A kit. Results The expression of Akt and the level of phosphorylation of Akt (Ser473) had no significant change in each group, the difference of the other phosphotylation of Akt (T308) was statistically significant (F=5.657, P=0.022) among all the groups; The treatment group with the concentration of tacrolimus 5 mg/L was significantly higher as compared to the control group (P=0.037); With the increase of FK506 concentration, the PP2A activity was increased to a certain degree, but the difference was not statistically significant (F=0.857, P=0.501). Conclusions FK506 can increase the phosphorylation level of Akt in HL-7702, which may not be related to PP2A.