Hernandez-Alejandro Roberto, Zhang Xusheng, Croome Kris P, Zheng Xiufen, Parfitt Jeremy, Chen Dong, Jevnikar Anthony, Wall William, Min Wei-Ping, Quan Douglas
Multi-Organ Transplant Program, London Health Sciences Centre, The University of Western Ontario, London, Canada.
J Surg Res. 2012 Aug;176(2):614-20. doi: 10.1016/j.jss.2011.10.004. Epub 2011 Nov 1.
Tumor necrosis factor-alpha (TNF-α) is a central mediator in the hepatic response to ischemia/reperfusion. Short hairpin RNA (shRNA) has been proven to be an effective means of harnessing the RNA interference pathway in mammalian cells. In the current study, we investigated whether silencing TNF-α gene with shRNA can prevent liver ischemic reperfusion injury (IRI).
Male BalB/c mice were randomized to TNF-α shRNA, scramble shRNA, or sham operation groups. TNF-α shRNA and scramble shRNA groups were injected 48 h before inducing IRI. IRI was induced via microaneurysm clamps applied to the left hepatic artery and portal vein. Six hours after reperfusion, IRI injury was examined by serum level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver histopathology, MPO, and MDA level, as well as by relative quantities of TNF-α mRNA.
TNF-α expression induced by ischemia reperfusion in the liver was significantly suppressed after treatment with TNF-α shRNA compared with the group treated with scramble shRNA (P < 0.001). Mice treated with TNF-α shRNA showed lower peak values of AST and ALT than scramble shRNA treated mice (P < 0.001). On histopathologic slides, mice treated with TNF-α shRNA had significantly less ischemia/reperfusion injury based on Suzuki score than the scramble shRNA group, 3.57 ± 2.30 and 8.83 ± 0.98 respectively (P < 0.001), while the sham group was not significantly different from the TNF-alpha shRNA group, 0 ± 0 and 3.57 ± 2.30, respectively (P = 0.075). Liver tissue MDA levels were significantly lower in mice treated with TNF-α shRNA as compared with the group treated with scramble shRNA (P < 0.01). Immunohistochemical staining for MPO was significantly lower in mice treated with TNF-α shRNA compared with the group treated with shRNA (compared with treated with scramble shRNA group.)
Liver IRI can be minimized through gene silencing of TNF-α. This may represent a novel therapy in the setting of transplantation and in other conditions associated with IRI of the liver.
肿瘤坏死因子-α（TNF-α）是肝脏对缺血/再灌注反应的核心介质。短发夹RNA（shRNA）已被证明是在哺乳动物细胞中利用RNA干扰途径的有效手段。在本研究中，我们调查了用shRNA沉默TNF-α基因是否能预防肝脏缺血再灌注损伤（IRI）。
将雄性BalB/c小鼠随机分为TNF-α shRNA组、乱序shRNA组或假手术组。在诱导IRI前48小时注射TNF-α shRNA和乱序shRNA。通过应用于左肝动脉和门静脉的微动脉瘤夹诱导IRI。再灌注6小时后，通过血清丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）水平、肝脏组织病理学、MPO和MDA水平以及TNF-α mRNA的相对量来检查IRI损伤。
与乱序shRNA处理组相比，TNF-α shRNA处理后肝脏缺血再灌注诱导的TNF-α表达明显受到抑制（P < 0.001）。TNF-α shRNA处理的小鼠AST和ALT的峰值低于乱序shRNA处理的小鼠（P < 0.001）。在组织病理学切片上，根据铃木评分，TNF-α shRNA处理的小鼠缺血/再灌注损伤明显少于乱序shRNA组，分别为3.57±2.30和8.83±0.98（P < 0.001），而假手术组与TNF-α shRNA组无显著差异，分别为0±0和3.57±2.30（P = 0.075）。与乱序shRNA处理组相比，TNF-α shRNA处理的小鼠肝脏组织MDA水平明显较低（P < 0.01）。与乱序shRNA处理组相比，TNF-α shRNA处理的小鼠MPO免疫组化染色明显较低（与乱序shRNA处理组相比）。
通过TNF-α基因沉默可使肝脏IRI最小化。这可能代表了一种在移植及其他与肝脏IRI相关疾病中的新型治疗方法。
