脂多糖预处理导致的糖原合成酶激酶-3抑制对肝糖原的影响和机制

Effect of endotoxin pretreatment-induced glycogen synthase kinase-3 inhibition on glycogen metabolism in rat liver and the mechanism

目的探讨脂多糖预处理时糖原合成酶激酶3(glycogen synthase kinase-3,GSK-3)功能活性的变化及其对肝组织糖原代谢的影响和机制。方法雄性SD大鼠随机分为正常对照、脂多糖预处理和GSK-3抑制剂氯化锂预处理组,分别进行相应处理后再接受大剂量脂多糖(10 mg/kg)攻击以建立脂多糖诱导的急性肝损伤模型;采用PAS染色法观察肝组织糖原聚集,用试剂盒法定量检测肝组织糖原含量,以Western Blot法半定量分析GSK-3的蛋白表达和抑制性磷酸化水平,采用考马斯亮兰比色法测定肝组织钙依赖蛋白酶的活性。结果尽管大剂量脂多糖攻击后各组动物肝组织糖原含量组间比较均无显著差异(P>0.05),但均较攻击前有显著降低(P<0.05),且脂多糖和氯化锂预处理均可导致肝组织糖原含量增加(P<0.05);尽管诱导脂多糖预处理并未改变GSK-3的蛋白表达水平(P>0.05),但导致GSK-3β抑制性磷酸化(P<0.05)和GSK-3α不完全裂解;大剂量脂多糖攻击后肝组织钙依赖蛋白酶活性较前显著升高(P<0.05),但组间比较无显著差异(P>0.05)。结论脂多糖预处理导致GSK-3β抑制性磷酸化和GSK-3α不完全裂解,促进肝组织糖原合成和聚集,但不影响钙依赖蛋白酶活性,有利于增加肝组织糖原储备并可能在遭受大剂量脂多糖攻击时提供能量需求。

Objective To investigate the changes in the functional activity of glycogen synthase kinase-3 （GSK-3） in the hepatic tissue after endotoxin （lipopolysaccharide, LPS） tolerance and explore the effects of LPS-induced GSK-3 inhibition on glycogen metabolism in the liver. Methods Male SD rats were randomly divided into normal control, endotoxin pretreatment and GSK-3 inhibitor （lithium chloride） groups with corresponding pretreatments prior to a large dose of LPS challenge （10 mg/kg） to induce liver injury. Glycogen deposition and content in the hepatic tissue was detected using periodic acid-Schiff （PAS） staining and a glycogen quantification kit, respectively. Western blotting was performed for semi-quantitative analysis of protein level and inhibitory phosphorylation of GSK-3, and a Coomassie brilliant blue G-250-based colorimetric assay was used to detect calpain activity in the liver. Results Glycogen content in the liver decreased significantly after LPS challenge in all the 3 groups （P＆lt;0.05） but showed no significant difference among the groups （P＆gt;0.05）. Both LPS and lithium chloride pretreatments caused a significant increase of liver glycogen content （P＆lt;0.05）. LPS pretreatment induced inhibitory phosphorylation of GSK-3β （P＆lt;0.05） and partial cleavage of GSK-3α but did not affect the expression of GSK-3 protein （P＆gt;0.05）. Large-dose LPS challenge significantly increased the activity of calpain in the liver tissue （P＆lt;0.05） to a comparable level in the 3 groups （P＆gt;0.05）. Conclusion Endotoxin pretreatment induces inhibitory phosphorylation of GSK-3β and partial cleavage of GSK-3α and promotes the deposition of liver glycogen but does not affect the activity of calpain, which may contribute to an increased glycogen reserve for energy supply in the event of large-dose LPS challenge.