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

肝缺血再灌注（I/R）损伤是肝切除术或肝移植术后的主要问题之一。为了使手术更加安全，需要采取一些策略来防止I/R损伤。Nrf2是一种转录因子，通过激活保护性抗氧化和解毒酶来保护细胞和组织免受氧化应激。可能是术前肝组成细胞中Nrf2的激活导致了肝脏I/R损伤的预防。在这个方案中，我们评估了Nrf2激活是否赋予肝脏对I/R损伤的耐受性。使用Oltipraz， 15-deoxy-Δ 12,14 -前列腺素J2 （PGJ2）和二烯丙基硫醚（DAS）来确认这些药物是否激活肝构成细胞中的Nrf2。从大鼠和小鼠肝脏中分离肝上皮细胞和肝星状细胞进行培养。免疫荧光分析显示，在两种类型的细胞中，Nrf-2在这些药物刺激后立即易位到细胞核中。PGJ2也能诱导HO-1的表达，并呈剂量依赖性。据报道，在肝I/R过程中，星状细胞的激活具有不利影响。为评价PGJ2对星状细胞活化的影响，采用real - time PCR检测α -平滑肌肌动蛋白和I型胶原mRNA的表达。PGJ2显著抑制了这两个基因的表达，表明PGJ2有效抑制了星状细胞的活化。最后观察PGJ2对I/R损伤的影响。采用C57/B16小鼠。采用70%缺血模型（60 min）。缺血前3小时经尾静脉给予PGJ2。再灌注后3、6 h， PGJ2明显抑制AST/ALT水平升高。此外，TUNEL实验显示，I/R后细胞凋亡也受到抑制。这些数据提示PGJ2激活Nrf2可能在肝脏手术领域带来有益的效果。

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.