抑制 GSK-3β 通过 GSK-3β/β-连环蛋白信号通路减轻小鼠肝缺血再灌注损伤。
Inhibition of GSK-3beta ameliorates hepatic ischemia-reperfusion injury through GSK-3beta/beta-catenin signaling pathway in mice.
机构信息
Liver Transplantation Center, Nanjing Medical University, Nanjing, China.
出版信息
Hepatobiliary Pancreat Dis Int. 2012 Jun;11(3):278-84. doi: 10.1016/s1499-3872(12)60161-1.
BACKGROUND
Glycogen synthase kinase (GSK)-3beta/beta-catenin signaling regulates ischemia-reperfusion (I/R)-induced apoptosis and proliferation, and inhibition of GSK-3beta has beneficial effects on I/R injury in the heart and the central nervous system. However, the role of this signaling in hepatic I/R injury remains unclear. The present study aimed to investigate the effects and mechanism of GSK-3beta/beta-catenin signaling in hepatic I/R injury.
METHODS
Male C57BL/6 mice (weighing 22-25 g) were pretreated with either SB216763, an inhibitor of GSK-3beta, or vehicle. These mice were subjected to partial hepatic I/R. Blood was collected for test of alanine aminotransferase (ALT), and liver specimen for assays of phosphorylation at the Ser9 residue of GSK-3beta, GSK-3beta activity, axin 2 and the anti-apoptotic factors Bcl-2 and survivin, as well as the proliferative factors cyclin D1 and proliferating cell nuclear antigen, and apoptotic index (TUNEL). Real-time PCR, Western blotting and immunohistochemical staining were used.
RESULTS
SB216763 increased phospho-GSK-3beta levels and suppressed GSK-3beta activity (1880+/-229 vs 3280+/-272 cpm, P<0.01). ALT peaked at 6 hours after reperfusion. Compared with control, SB216763 decreased ALT after 6 hours of reperfusion (4451+/-424 vs 7868+/-845 IU/L, P<0.01), and alleviated hepatocyte necrosis and vacuolization. GSK-3beta inhibition led to the accumulation of beta-catenin in the cytosol (0.40+/-0.05 vs 1.31+/-0.11, P<0.05) and nucleus (0.62+/-0.14 vs 1.73+/-0.12, P<0.05), beta-catenin further upregulated the expression of axin 2. Upregulation of GSK-3beta/beta-catenin signaling increased Bcl-2, survivin and cyclin D1. Serological and histological analyses showed that SB216763 alleviated hepatic I/R-induced injury by reducing apoptosis (1.4+/-0.2% vs 3.6+/-0.4%, P<0.05) and enhanced liver proliferation (56+/-8% vs 19+/-4%, P<0.05).
CONCLUSION
Inhibition of GSK-3beta ameliorates hepatic I/R injury through the GSK-3beta/beta-catenin signaling pathway.
背景
糖原合成酶激酶（GSK）-3β/β-catenin 信号通路可调节缺血再灌注（I/R）诱导的细胞凋亡和增殖，而抑制 GSK-3β 对心脏和中枢神经系统的 I/R 损伤具有有益作用。然而，该信号通路在肝 I/R 损伤中的作用尚不清楚。本研究旨在探讨 GSK-3β/β-catenin 信号通路在肝 I/R 损伤中的作用及机制。
方法
雄性 C57BL/6 小鼠（体重 22-25g）用 GSK-3β 抑制剂 SB216763 或载体预处理，然后进行部分肝 I/R。采集血液用于检测丙氨酸氨基转移酶（ALT），采集肝脏标本用于检测 GSK-3β 丝氨酸 9 位磷酸化、GSK-3β 活性、轴蛋白 2 和抗凋亡因子 Bcl-2 和 survivin 以及增殖因子 cyclin D1 和增殖细胞核抗原的表达，以及凋亡指数（TUNEL）。采用实时 PCR、Western blot 和免疫组织化学染色法进行检测。
结果
SB216763 增加了磷酸化 GSK-3β 水平并抑制了 GSK-3β 活性（1880±229 对 3280±272cpm，P<0.01）。ALT 在再灌注后 6 小时达到峰值。与对照组相比，SB216763 降低了再灌注 6 小时后的 ALT（4451±424 对 7868±845IU/L，P<0.01），并减轻了肝细胞坏死和空泡化。GSK-3β 抑制导致β-catenin 在细胞质（0.40±0.05 对 1.31±0.11，P<0.05）和核内（0.62±0.14 对 1.73±0.12，P<0.05）积聚，β-catenin 进一步上调了轴蛋白 2 的表达。GSK-3β/β-catenin 信号通路的上调增加了 Bcl-2、survivin 和 cyclin D1。血清学和组织学分析表明，SB216763 通过减少细胞凋亡（1.4±0.2% 对 3.6±0.4%，P<0.05）和增强肝增殖（56±8% 对 19±4%，P<0.05）来减轻肝 I/R 诱导的损伤。
结论
抑制 GSK-3β 通过 GSK-3β/β-catenin 信号通路改善肝 I/R 损伤。
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