Pieper Galen M, Nilakantan Vani, Nguyen Thanh K, Hilton Gail, Roza Allan M, Johnson Christopher P
Division of Transplant Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Antioxid Redox Signal. 2008 Jun;10(6):1031-40. doi: 10.1089/ars.2007.1867.
Apoptosis is a significant factor in cardiac dysfunction and graft failure in cardiac rejection. In this study, we examined potential signaling molecules responsible for caspase 3 activation in a model of acute cardiac allograft rejection. The roles of reactive oxygen species (ROS) and nitric oxide (NO) were determined in untreated allografts and allograft recipients treated with either cyclosporine (CsA), alpha-phenyl-t-butylnitrone (PBN, a spin-trapping agent), vitamin C (VitC), Mn(III)tetrakis (1-methyl-4-pyridyl)porphyrin); MnTmPyP, a superoxide dismutase (SOD) mimetic), or L-(1-iminoethyl)lysine) (L-NIL), an inhibitor of inducible NO synthase (iNOS) enzyme activity. Graft tissue was taken for measuring superoxide radical production, Western blotting, and direct measurement of caspase 3 activity. Activation of caspase 3 in untreated allografts was revealed by the appearance of cleaved caspase 3 from pro-caspase 3 by Western blotting and functional caspase 3 catalytic activity. CsA or PBN inhibited iNOS expression and caspase 3 activity. VitC and MnTmPyP did not alter iNOS expression or decrease NO levels but did inhibit caspase 3 activity. In contrast, L-NIL completely inhibited the increase in NO production without altering iNOS expression and inhibited caspase 3 activity. The prevention of TUNEL staining by MnTmPyP and L-NIL confirmed downstream effects of superoxide and NO on apoptosis. These studies indicate that both superoxide and NO (precursors of peroxynitrite formation) play a significant role in caspase 3 activation in cardiac allograft rejection.
细胞凋亡是心脏功能障碍和心脏移植排斥反应中移植物衰竭的一个重要因素。在本研究中，我们在急性心脏移植排斥反应模型中检测了负责半胱天冬酶3激活的潜在信号分子。在未处理的同种异体移植物以及用环孢素（CsA）、α-苯基叔丁基硝酮（PBN，一种自旋捕获剂）、维生素C（VitC）、四（1-甲基-4-吡啶基）锰卟啉（MnTmPyP，一种超氧化物歧化酶（SOD）模拟物）或L-（1-亚氨基乙基）赖氨酸（L-NIL，一种诱导型一氧化氮合酶（iNOS）酶活性抑制剂）处理的同种异体移植物受体中，确定了活性氧（ROS）和一氧化氮（NO）的作用。采集移植物组织用于测量超氧自由基产生、蛋白质印迹法以及直接测量半胱天冬酶3活性。通过蛋白质印迹法检测到前体半胱天冬酶3裂解为半胱天冬酶3以及半胱天冬酶3的功能催化活性，揭示了未处理的同种异体移植物中半胱天冬酶3的激活。CsA或PBN抑制iNOS表达和半胱天冬酶3活性。VitC和MnTmPyP没有改变iNOS表达或降低NO水平，但确实抑制了半胱天冬酶3活性。相反，L-NIL完全抑制了NO产生的增加，而不改变iNOS表达，并抑制了半胱天冬酶3活性。MnTmPyP和L-NIL对TUNEL染色的抑制证实了超氧化物和NO对细胞凋亡的下游作用。这些研究表明，超氧化物和NO（过氧亚硝酸盐形成的前体）在心脏移植排斥反应中半胱天冬酶3激活中起重要作用。
