缺血预处理减轻大鼠减体积肝移植损伤并促进肝再生

doi:10.3969/j.issn.1673-8225.2010.53.042

摘要/Abstract

摘要：

背景：近年来，肝移植技术迅速发展，如何预防缺血再灌注损伤并有效保护肝再生成为研究的热点。缺血预处理是保护肝缺血损伤的有效方法，但其确切机制尚存争议。
目的：研究缺血预处理在大鼠减体积肝移植肝损伤和肝再生中的作用及机制。
方法：动物随机分为3组，肝移植组建立大鼠减体积肝移植模型。缺血预处理+肝移植组在供肝灌注前阻断第1肝门行缺血预处理10 min，再灌注15 min。假手术组在开腹后游离肝周韧带，然后关腹。分别于术后0.5，2，6，24 h取材。通过血清谷丙转氨酶水平和移植肝组织病理检查评估肝损伤。半定量免疫组织化学和western blot法测定氧化还原蛋白1表达水平，检测移植肝细胞增殖细胞核抗原评估肝再生情况。
结果与结论：与肝移植组相比，缺血预处理+肝移植组术后6，24 h受体血清谷丙转氨酶明显降低(P < 0.05；P < 0.01)。病理学分析显示肝移植组术后24 h可见到门脉周围大量炎细胞浸润，肝窦扩张明显，肝组织损伤较重；而缺血预处理+肝移植组则损伤较轻。半定量免疫组织化学显示缺血预处理+肝移植组移植肝中Ref-1蛋白表达明显增加，这一结果同样在westernblot检测中得到验证：缺血预处理+肝移植组移植肝术后24 h Ref-1蛋白表达较肝移植组明显增强 (P < 0.05)。同时，术后2，6和24 h 缺血预处理+肝移植组增殖细胞核抗原阳性细胞数较肝移植组明显增加(P < 0.05)。结果提示缺血预处理可减轻大鼠减体积肝移植术后早期移植物肝损伤并促进肝再生，这与Ref-1蛋白高表达密切相关。

关键词: 缺血预处理, 肝再生, 损伤, 肝移植, 大鼠

Abstract:

BACKGROUND: Recently, liver transplantation technique has been developed rapidly, and prevention of ischemia/reperfusion injury and protection of liver regeneration have become a research focus. Ischemic preconditioning (IPC) is an effective method for protecting liver ischemic injury. However, the mechanism remains controversial.
OBJECTIVE: To investigate the mechanism of IPC on hepatic injury and regeneration after reduced-size rat liver transplantation.
METHODS: Animals were randomly divided into 3 groups. Rat reduced-size liver transplantation model was established in liver transplantation group. IPC +liver transplantation group underwent first porta hepatis blocking for 10 minutes before liver graft reperfusion, followed by reperfusion for 15 minutes. The ligament around the liver was dissociated in the sham-surgery group. The samples were collected 0.5, 2, 6 and 24 hours post-operation. The hepatic injury was examined by the serum alanine aminotransferase (ALT) and hepatic tissue histopathology analysis of grafts. Semi-quantitative immunohistochemistry and westernblotting were used to examine the redox factor-1 (Ref-1) protein expression. The hepatic regeneration of the grafts was examined by the expression of proliferating cell nuclear antigen (PCNA) in hepatic cells.
RESULTS AND CONCLUSION: Compared with liver transplantation group, the ALT values at 6 and 24 hours after operation in IPC group decreased significantly (P < 0.05; P < 0.01). Pathological analysis indicated that there were lots of inflammation cells around the portal veins, the serious sinus hepaticus dilation and damage of hepatic tissue in liver transplantation group. However, the tissue injury observed in IPC group was comparatively slight. Semi-quantitative immunohistochemistry revealed that Ref-1 protein was more abundant in IPC grafts tissue compared to liver transplantation group. These observations were supported by westernblotting studies where Ref-1 protein was shown to be over-expressed in IPC specimens at 24 hours after reduced-size liver transplantation (P < 0.05). In addition, the number of PCNA-positive cells in IPC group was more than liver transplantation group at 2, 6 and 24 hours after operation (P < 0.05). IPC improves hepatic regeneration and relieves grafts injury in earlier period after reduced-size rat liver transplantation, which is associated with the over-expression of Ref-1 protein.

中图分类号:

R617

刘现忠，姚爱华，王轩，仲跻巍，李相成. 缺血预处理减轻大鼠减体积肝移植损伤并促进肝再生[J]. 中国组织工程研究, 2010, 14(53): 10053-10057.

Liu Xian-zhong, Yao Ai-hua, Wang Xuan, Zhong Ji-wei, Li Xiang-cheng. Ischemic preconditioning improves hepatic regeneration with reduced injury following reduced-size rat liver transplantation[J]. Chinese Journal of Tissue Engineering Research, 2010, 14(53): 10053-10057.

参考文献

引言

材料方法

讨论

During liver transplantation, there is an unavoidable ischemic stage, which results in grafts injury after reperfusion. Living-related liver transplantation is developed to alleviate the mortality resulting from the scarcity of suitable cadaveric grafts[12-13]. The main problem in using living-related liver transplantation for adults is graft size disparity[14-15]. In addition, I/R, which is inevitable in liver transplantation, reduces liver regeneration after hepatectomy[15].
As one strategy against I/R injury, recent studies have paid attention to IPC[16-18], a phenomenon in which brief periods of ischemia followed by reperfusion render tissues resistant to subsequent prolonged ischemia. IPC was originally characterized in dog hearts[19] and has since been recognized in a wide spectrum of animals and humans[20-21]. Studies have shown that IPC protected livers against cold I/R injury and improved recipient survival after liver transplantation[22-23].
The main mechanism of IPC protecting against I/R injury is to release active oxygen in reperfusion phase[24-25], and the burst of active oxygen is the triggering factor of Ref-1 protein expression[26], which imply IPC could activate the expression of Ref-1 protein by releasing active oxygen. At present, the studies with respect to the role of Ref-1 in IPC procedure are few. In the present study, we used Lewis rats to attempt to minimize the grafts injury from reject reaction. Model of 50% partial rat liver transplantation was established according to reduced liver grafts in clinical adult living donor liver transplantation.
The injury in liver transplantation is complicated. Oxidative stress is recognized as a critical cause of injury in reperfused tissue and remains a major concern in liver transplantation. Increasing studies suggest that intracellular as well as extracellular oxidative stress may play an important role in inducing postischemic tissue injury. At a very early phase of reperfusion, production of reactive oxygen species associated with Rac1 has recently been reported to mediate intracellular oxidative stress[8], which consequently activates redox-dependent pathologic signals including NF-κB. However, Ref-1 can defend the injury results from oxidative stress in cell[8,27] and protect hepatic I/R injury.
Ref-1, a 37-kDa protein, is initially described as a key endonuclease involved in base excision repair pathways of a variety of DNA lesions. In addition to this function, Ref-1 is known to act as a redox-dependent regulator of various transcription factors, such as AP-1 and NF-κB, thereby affecting transcriptional regulation of their target genes. Ref-1 protein contains two distinct domains. The N-terminal domain contains the nuclear localization sequence which is essential for redox activity while the endonuclease activity resides in the C-terminal region[26]. It is reported that adenoviral overexpression of Ref-1 in hepatic tissue results in significant suppression of reperfusion-induced oxidative stress, NF-κB activation, apoptosis, and acute hepatic injury[9]. Recently, it has been reported that constitutive activation of Stat3 protects against Fas-induced liver injury partly by up-regulation of Ref-1 in a redox-dependent manner[28-29].
In the present study, the expression of Ref-1 protein was increased in both IPC group and PLT group compared with the sham-surgery group. Significantly, the expression of Ref-1 protein in IPC group was higher than the PLT group in early period after reduced-size liver transplantation (P < 0.05), suggesting that the IPC promotes the expression of Ref-1 protein which relieves grafts injury.
Ape1/Ref-1 is located in the cytoplasm of highly metabolically active or proliferative cells, such as spermatocytes, hippocampal cells, and hepatocytes. Since highly metabolically active cells experience an increase in oxidative stress, the presence of Ape1/Ref-1 in the cytoplasm may reflect an involvement in cellular responses to oxygen tension. Usually, the factor that spurs Ref-1 expression will facilitate its intracellular activity. Stimulating factor changes the subcellular localization of Ref-1[30], almost shift from cytoplasm to nucleus. Results from the present study reveal that hepatic nucleus expression of Ref-1 in IPC group was less at 6 hours and increased at 24 hours after reperfusion, which may relate with the endonuclease function of Ref-1. However, the regulatory mechanism of Ref-1 distribution in cells remains unclear.
Results from the present study provide the evidence that Ref-1 participates in the protecting mechanism of IPC for graft I/R injury and regeneration after partial liver transplantation. Compared with PLT group, the serum ALT values at 6 and 24 hours after reperfusion were degraded obviously in IPC group (P < 0.05), and the ALT value at 5 days dropped to preoperative level, but in the PLT group its level remained high. These results reveal IPC relieve the grafts injury in early period after reduced-for-size liver transplantation. In order to investigate the effect of IPC on liver regeneration after transplantation, PCNA staining of hepatic tissue was performed. The expression of PCNA in hepatic tissue in IPC group was more obvious than that in PLT group (P < 0.05), which demonstrates IPC promotes the expression of PCNA and improves liver regeneration.
In summary, results from the present study show that IPC relieved grafts I/R injury and improved liver regeneration in early period after reduced-for-size liver transplantation of rats. Moreover, IPC promoted Ref-1 protein activity and protected grafts injury by relieving cellular oxidative stress. The precise mechanism by which IPC promotes Ref-1 expression and the principal role of Ref-1 defending postischemic injury or/and promoting regeneration require further investigation.

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