Zhang Li, Zhang Sheng-Ning, Li Li, Zhang Xi-Bing, Wu Rui-Chao, Liu Jun-Han, Huang Zhao-Yu, Li Wang, Ran Jiang-Hua
Department of Hepatopancreatobiliary Surgery, The Affiliated Calmette Hospital of Kunming Medical University, The First People's Hospital of Kunming, Calmette Hospital Kunming, Yunnan Province, China.
Department of Hepatopancreatobiliary, The First People's Hospital of Yunnan Province, The Affiliated Hospital of Kunming University of Science and Technology Kunming, Yunnan Province, China.
Int J Clin Exp Pathol. 2019 Jan 1;12(1):217-228. eCollection 2019.
This study was conducted to investigate the effect of warm ischemia duration on hepatocyte mitochondrial damage after liver transplantation, and confirm the role of CaMKIIγ in this process. Rat donation after cardiac death (DCD) liver transplantation model was established by exposing donor liver to 0 (W group), 15 (W group), and 30 (W group) min warm ischemia. Some rats in W group were transfected with CaMKIIγ and CaMKIIγ-shRNA lentivirus. On day 1, 3, and 7 post-transplantation, a series of experiments, including HE staining, TEM observation, ALT and AST measurement, flow cytometry analysis, qRT-PCR, and Western blotting were performed to evaluate the extent of hepatic and mitochondria damage. Within 7 days post-transplantation, prolonged ischemia led to an obvious deterioration of hepatic and mitochondria damage, presenting with a marked increase of apoptotic hepatocytes, ALT and AST levels, cells with low MMP, and AIF and Cyt C expression. CaMKIIγ overexpression caused the significant ultrastructural damage of hepatic cells, increase of cells with low MMP, enhancement of AIF and Cyt C expression, and augmented Ca/CaM/CaMKIIγ, while blocking CaMKIIγ showed an opposite result. In conclusion, ischemia duration is proportional to the extent of hepatic mitochondria damage, and CaMKIIγ plays a negative regulatory role in this process by regulating the Ca/CaM/CaMKII signaling pathway.
本研究旨在探讨热缺血时间对肝移植后肝细胞线粒体损伤的影响，并证实CaMKIIγ在此过程中的作用。通过将供体肝脏暴露于0（W组）、15（W组）和30（W组）分钟的热缺血来建立大鼠心脏死亡后供体（DCD）肝移植模型。W组的一些大鼠用CaMKIIγ和CaMKIIγ-shRNA慢病毒进行转染。在移植后第1、3和7天，进行了一系列实验，包括HE染色、透射电镜观察、谷丙转氨酶（ALT）和谷草转氨酶（AST）测定、流式细胞术分析、qRT-PCR和蛋白质免疫印迹，以评估肝脏和线粒体损伤的程度。在移植后7天内，缺血时间延长导致肝脏和线粒体损伤明显恶化，表现为凋亡肝细胞、ALT和AST水平显著升高，线粒体膜电位（MMP）降低的细胞以及凋亡诱导因子（AIF）和细胞色素C（Cyt C）表达增加。CaMKIIγ过表达导致肝细胞超微结构显著损伤，MMP降低的细胞增加，AIF和Cyt C表达增强，以及Ca/CaM/CaMKIIγ增加，而阻断CaMKIIγ则产生相反的结果。总之，缺血时间与肝脏线粒体损伤程度成正比，CaMKIIγ通过调节Ca/CaM/CaMKII信号通路在此过程中发挥负性调节作用。
