沉默信息调节因子6对慢加急性肝衰竭糖异生的影响和机制探讨

Effect and mechanism of silent information regulator 6 on gluconeogenesis in chronic-on-acute liver failure

目的探讨慢加急(亚急)性肝衰竭(ACLF)患者以及氧化应激损伤大鼠肝细胞沉默信息调节因子6(SIRT6)和糖异生限速酶表达的变化以及可能机制。方法选取2016年8月至2018年5月首都医科大学附属北京佑安医院10例ACLF患者纳入ACLF组,10例正常肝移植供者纳入正常对照组。收集入选者空腹血糖、总胆红素、白蛋白、丙氨酸氨基转移酶(ALT)等资料。分离Sprague Dawley大鼠肝细胞,分为对照组(无任何干预)、模型组(H2O2干预6 h)、哺乳动物雷帕霉素靶蛋白(mTOR)激活组(模型组基础上加入mTOR激活剂)、mTOR抑制组(模型组基础上加入mTOR抑制剂)。蛋白质电泳和聚合酶链反应检测葡萄糖-6-磷酸酶(G6P)、磷酸烯醇式丙酮酸羧激酶(PEPCK)、SIRT6以及mTOR的相对表达。结果ACLF组ALT、总胆红素高于正常对照组,差异均有统计学意义(均P<0.05)。ACLF组SIRT6含量(0.15±0.07)μg/L、空腹血糖(3.19±0.59)mmol/L,明显低于正常对照组(0.46±0.15)μg/L、(7.07±2.07)mmol/L,差异均有统计学意义(均P<0.05)。ACLF组肝组织PEPCK、G6P蛋白相对表达明显低于正常对照组。模型组SIRT6、PEPCK、G6P mRNA相对表达低于对照组,差异均有统计学意义(均P<0.05)。激活mTOR后PEPCK、G6P、SIRT6相对表达高于模型组,抑制mTOR后PEPCK、G6P、SIRT6相对表达低于模型组。结论ACLF时SIRT6可能与mTOR信号途径相互作用,抑制糖异生,并与低血糖的发生相关,降低SIRT6水平,可能起到延缓ACLF进展的作用。

Objective To investigate the effect and mechanism of silent information regulator 6(SIRT6)and gluconeogenesis-dependent rate-limiting enzymes in hepatocytes in oxidative stress injury rats and chronic-on-acute(sub-acute)liver failure(ACLF)patients.Methods From August 2016 to May 2018,10 patients with ACLF from Beijing Youan Hospital Affiliated to Capital Medical University were included in the ACLF group,and 10 normal donors were included in the normal control group.Level of fasting blood glucose,total bilirubin,albumin,and alanine aminotransferase(ALT)were studied.Sprague Dawley rat hepatocytes were isolated and divided into control group(without any intervention),model group(H2O2 intervention for 6 h),mammalian rapamycin target protein(mTOR)activation group(mTOR activation was added to the model group),mTOR inhibition group(mTOR inhibitor was added on the basis of the model group).Protein electrophoresis and polymerase chain reaction was used to detect the relative expression of glucose-6-phosphatase(G6P),phosphoenolpyruvate(PEPCK),SIRT6,and mTOR.Results The ALT and total bilirubin level in ACLF group were significantly higher than those in the normal control group,and the differences were statistically significant(all P<0.05).In ACLF group,level of SIRT6(0.15±0.07)μg/L and fasting blood glucose(3.19±0.59)mmol/L were significantly lower than those in the normal control group(0.46±0.15)μg/L and(7.07±2.07)mmol/L,the difference was statistically significant(all P<0.05).The relative expression of PEPCK and G6P protein in liver tissue of ACLF group was significantly lower than that of normal control group.The relative expression of SIRT6,PEPCK,and G6P in the model group were lower than those in the control group,and the differences were statistically significant(all P<0.05).When mTOR is activated,the relative expression of PEPCK,G6P,and SIRT6 was higher than those in the model group,and after mTOR inhibition,the relative expression of PEPCK,G6P,and SIRT6 was lower than in the model group.Conclusion ACLF,SIRT6 may inhibit gluconeogenesis,and increased the occurrence of hypoglycemia through activating mTOR signaling pathway.Blocking of SIRT6 levels may slow down the progress of ACLF.