Modulation of intracellular signal pathway for preventing liver damage after extended hepatectomy

肝脏缺血再灌注（I/R）损伤是扩大肝切除术或肝移植术后的主要问题之一。Nrf 2是一种转录因子，通过激活保护性抗氧化酶和解毒酶来保护细胞和组织免受氧化应激的影响。术前激活肝脏组成细胞中的Nrf 2可能导致肝脏I/R损伤的预防。在本研究中，我们评估了Nrf 2激活是否赋予肝脏I/R损伤的耐受性，使用了奥替普拉、15-脱氧-Δ 12，14-藜芦定J2（PGJ 2）和二烯丙基硫醚（DAS）来确认这些药物是否激活了肝脏组成细胞中的Nrf 2。从大鼠和小鼠肝脏中分离并培养肝上皮细胞和肝星状细胞。免疫荧光检测显示，Nrf-2在这两种类型的细胞中被这些药物刺激后立即易位到细胞核中。PGJ 2还能诱导HO-1的表达，且呈剂量依赖性，有报道称，肝星状细胞的激活在肝I/R过程中具有不利影响。为了评价PGJ 2对星状细胞活化的作用，通过真实的时间PCR检测α平滑肌肌动蛋白和I型胶原的mRNA表达。PGJ 2可显著抑制这两个基因的表达，提示PGJ 2可有效抑制星状细胞的活化。最后，研究了PGJ 2对I/R损伤的影响。使用C57/B16小鼠。采用70%缺血60 min模型。缺血前3 h经尾静脉给予PGJ 2，可明显抑制再灌注后3、6 h AST/ALT的升高。此外，TUNEL法检测发现，PGJ 2激活Nrf 2后，I/R后的细胞凋亡也受到抑制，提示PGJ 2激活Nrf 2可能在肝脏外科领域发挥有益作用。

Ischemia-reperfusion (I/R) injury of the liver is one of major problems after extended hepatectomy or liver transplantation. To perform operation much safer, some strategies to prevent I/R injury has been required.Nrf2 is a transcription factor that protects cell and tissues from oxidative stress by activating protective antioxidant and detoxifying enzymes. It is likely that preoperative activation of Nrf2 in liver-consisting cells results in prevention of I/R injury of the liver. In this protocol, we evaluated whether Nrf2 activation confer the tolerance for I/R injury of the liver.Oltipraz, 15-deoxy-Δ 12, 14-prostagrandine J2 (PGJ2), and diallyl sulfide (DAS) were used to confirm whether these drugs activate Nrf2 in liver-consisting cells. Liver epithelial cells and liver stellate cells were isolated from rat and mouse liver and cultured. Immunofluorescent assay revealed that Nrf-2 translocated into the nucleus immediately after stimulating by these drugs in both types of cells. The expression of HO-1 was also induced by PGJ2 in a dose dependent manner.It has been reported that activation of stellate cells has detrimental effect during hepatic I/R. To evaluate the effect of PGJ2 on stellate cell activation, the mRNA expression of alpha smooth muscle actin and type I collagen was examined by real time PCR. PGJ2 significantly inhibited the expression of both genes, suggesting that PGJ2 effectively suppressed the activation of stellate cells.Finally, the effect of PGJ2 on I/R injury was examined. C57/B16 mouse was used. We applied 70% ischemia model (60 min). PGJ2 was administrated intravenously via tail vein 3 hours before ischemia.The increasing of AST/ALT level 3 and 6 hours after reperfusion was significantly suppressed by PGJ2. In addition, TUNEL assay revealed that apoptosis after I/R was also suppressed.These data suggested that Nrf2 activation by PGJ2 may bring the beneficial effect in the field of liver surgery.