武警总医院科研项目(编号:WZ20130403)。
目的 观察环孢素A(cyclosporin A,CsA)对肝细胞磷酸化AKT (phosphorylated AKT,P-AKT)表达的影响,探讨CsA导致胰岛素抵抗的机制。方法 本研究分两部分:(1)体外研究:用不同浓度CsA(0.1 μmol/L、1 μmol/L、5 μmol/L)作用于人肝细胞(HL7702细胞)48 h,用Western blot方法检测P-AKT蛋白的表达水平。(2)体内研究(动物实验):建立环孢素A诱发的大鼠糖尿病模型,用免疫组化方法分析P-AKT蛋白在糖尿病大鼠肝细胞中的表达水平。结果 (1)各浓度(0.1 μmol/L、1 μmol/L、5 μmol/L)CsA组与对照组相比,HL7702肝细胞中的P-AKT蛋白的表达呈现逐渐下降趋势,差异有统计学意义(P<0.05、P<0.01、P<0.01)。(2)环孢素A引起大鼠血糖升高,在服用环孢素A的第5个月,大鼠平均血糖为9.28 mmol/L,说明环孢素A诱导的糖尿病大鼠模型建立成功,糖尿病大鼠肝细胞中P-AKT蛋白的表达明显低于服药前和正常对照组(P<0.05)。结论 服用治疗剂量的环孢素A导致大鼠血糖升高,环孢素A抑制大鼠肝细胞中P-AKT的表达,环孢素A抑制人肝细胞HL7702中P-AKT的表达,提示影响PI3K/AKT信号通路可能是环孢素A导致胰岛素抵抗的机制之一。
Objective To observe the effects of cyclosporin A on the expression of phosphorylated AKT in hepatocytes, and to investigate the mechanism of insulin resistance caused by cyclosporin A. Methods This study included two parts. 1. In vitro experiment: Human hepatocyte HL77022 cell line was cultured at different concentrations of cyclosporin A (0.1 μmol/L, 1 μmol/L, 5 μmol/L) for 48 hours. The expressions of phosphorylated AKT (P-AKT) in HL77022 cells were measured by Western blot assay. 2. Rat in vivo experiment: SD rats were randomly divided into 2 groups. The rats in the control group were administrated with distilled water 1 mL/Kg/d. The rats in the cyclosporine group were administrated with cyclosporine 25 mg/Kg/d. The total experiment time was 5 months. The levels of fasting blood glucose and insulin were tested at the end of the experiment. The insulin resistance index and insulin sensitivity index were calculated. The expression of P-AKT in the rat hepatocytes was measured by immunohistochemistry. Results 1. Each group of the HL7702 cells treated by CsA (0.1 μmol/L, 1 μmol/L, 5 μmol/L) showed a significantly decreased expression of P-AKT (P<0.05, P<0.01, and P<0.01). 2. After 5 months of therapy, the fasting blood glucose level of rats in the cyclosporine group was 9.28 mmol/L, indicating that cyclosporine-induced diabetic rat models were established. The insulin sensitivity index in the cyclosporine group was lower than that in the control group (P<0.05). The expression of P-AKT in liver in the cyclosporine group was significantly lower than that in the control group (P<0.05). Conclusions Therapeutic dose of cyclosporine has hyperglycemic effects on rats. Cyclosporine can reduce the expression of phosphorylated AKT in hepatic tissue in rats and also decrease the expression of P-AKT in human hepatocyte HL77022 cells, which indicate that cyclosporine may cause insulin resistance by interfering PI3K/AKT signal transduction pathway.
杨柳,刘晓军,王宏宇,曹永,马永华,徐春.环孢素A对肝细胞中磷酸化AKT蛋白表达的影响[J].中国比较医学杂志,2016,26(6):18~22.
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