抑制高硒致肝细胞内糖代谢重塑的体外干预研究

Intervention study to inhibit the glucose metabolic remodeling in hepatocytes induced by high-selenium in vitro

目的观察外源性丝氨酸或甘氨酸对体外高硒培养的肝细胞内硒蛋白和内源性丝氨酸合成与代谢酶表达的影响及其剂量-反应关系。方法以L02细胞为干预对象,实验分为抑制实验与剂量-反应实验。抑制实验中设置空白对照组、高硒(SeMet)对照组、丝氨酸对照组和高硒+丝氨酸干预组。其中,SeMet和丝氨酸的浓度均为0.05μmol/L,空白对照组给予等量生理盐水。剂量-反应实验中,SeMet的浓度为0.05μmol/L,丝氨酸或甘氨酸的干预浓度梯度均为0、0.05、0.1、0.5、1、5、10、50、100和500μmol/L。采用免疫印迹法检测裂解液中磷酸甘油酸脱氢酶(phosphoglycerate dehydrogenase,PHGDH)、丝氨酸羟甲基转移酶1(serine hydroxymethyltransferase 1,SHMT1)、亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase,MTHFR)以及硒蛋白P(selenoprotein P,SELENOP)和谷胱甘肽过氧化物酶1(glutathione peroxidase 1,GPX1)的表达水平。结果(1)抑制实验中,与空白对照组比较,其他3组的L02细胞内硒蛋白(GPX1和SELENOP)表达均显著增加(P<0.05);而与高硒对照组L02细胞内PHGDH高表达相比,高硒+丝氨酸干预组的PHGDH、SHMT1和MTHFR表达均显著降低(P<0.05)。(2)剂量-反应实验中,L02细胞内PHGDH表达均随着丝氨酸和甘氨酸浓度的增加而逐步降低,呈现明显的剂量-反应关系。而其他代谢酶(SHMT1和MTHFR)蛋白表达均未呈现类似趋势。结论外源性补充丝氨酸,可以直接反馈抑制高硒培养的肝细胞中糖酵解旁路——丝氨酸从头合成通路之关键酶的过表达,减少内源性丝氨酸合成;外源性添加的甘氨酸则可以通过在肝细胞内直接转化为内源性丝氨酸,达到类似效果。

OBJECTIVE To observe the effect of exogenous serine or glycine on the synthesis of selenoprotein and endogenous serine and the expression of metabolic enzymes in hepatocytes cultured with high-selenium in vitro and its dose-response relationship.METHODS The experiment was divided into two parts,namely a inhibition experiment and a dose-response experiment,using L02 cells as the intervention target.In the inhibition experiment,the blank control group,high-Se(SeMet)group,serine intervention group and high-Se+serine intervention group were set up.Both SeMet and serine were given at a level of 0.05μmol/L,and the blank control group was given the same volumes of saline.In the dose-response experiment,the concentration of SeMet was 0.05μmol/L,and the intervention concentration gradients of serine or glycine were 0,0.05,0.1,0.5,1,5,10,50,100 and 500μmol/L.The expression of phosphoglycerate dehydrogenase(PHGDH)、serine hydroxymethyltransferase 1(SHMT1)、methylenetetrahydrofolate reductase(MTHFR)、selenoprotein P(SELENOP)and glutathione peroxidase 1(GPX1)was detected by Western Blot(WB).RESULTS(1)In the inhibition experiment,compared with the blank control group,the expression of selenium proteins(GPX1 and SELENOP)in L02 cells of the other three groups were significantly increased(P<0.05).Compared with the high expression of PHGDH in L02 cells of high-Se group,the expressions of PHGDH,SHMT1 and MTHFR in high-Se+serine group were significantly decreased(P<0.05).(2)In the dose-response experiment,the expression of PHGDH enzyme in L02 cells gradually decreased with the increase of the concentration of exogenous serine or glycine,showing an obvious dose-dependent effect.In contrast,none of the other metabolic enzymes(SHMT1 and MTHFR)showed similar trends in protein expression.CONCLUSION The upregulated expression of PHGDH,the key enzyme in the de novo synthesis pathway of serine in hepatocytes cultured with high-selenium can be inhibited feedback by exogenous serine or endogenous serine transformed from exogenous glycine directly.