TNFα1型受体敲除对小鼠急性后肢缺血的作用

目的研究肿瘤坏死因子α1型受体(tumornecrosisfactorαreceptor1,TNFαR1)信号通路在小鼠急性后肢缺血中的作用,并探讨其作用机制。方法通过结扎TNFαR1/和野生型小鼠股动静脉及其主要分支建立后肢急性缺血模型,激光多普勒测手术前后后肢血液灌流,TUNEL染色观察细胞凋亡,凝胶电泳观察DNA凋亡梯带,免疫蛋白印迹法测定Caspase3、Bax蛋白的表达。结果术后1天TNFαR1/组缺血侧后肢血液灌流显著高于野生型组,腓肠肌TUNEL阳性细胞显著低于野生型组。TNFαR1/组缺血评分则显著低于野生型组,两组的自发截肢率分别为50%、0%。TNFαR1/组DNA凋亡梯带、Caspase3和Bax蛋白表达减弱。结论TNFαR1敲除可抑制TNFα信号通路下游死亡相关蛋白的激活,减少细胞死亡和调亡,对急性后肢缺血有保护作用。

The protective effects of tumor necrosis factor receptor 1 deletion on hindlimb ischemia

Objective Signals in the tumor necrosis factor α TNFα pathway, including TNFα and tumor necrosis factor α receptor 1TNFR1, are upregulated after acute hindlimh ischemia. It is unclear, however, whether this upregulation is apathophysiological consequence or a compensatory response. In the present study, the effect of TNFR1 deletion in a mouse model of hindlimb ischemia was investigated.Methods Hindlimb ischemia was produced through ligation of the proximal and distal left femoral artery and its branches in TNFR1 knockout TNFR1-/- and wild type WT mice. Laser Doppler blood flow measurement showed that limb perfusion was significantly higher in TNFR1-/- mice than that in the WT mice at 1 day after operation.Results The calculated ischemic scores were 3.67 ± 0.52 in WT group and 1.83 ± 0.41 in TNFR1-/- group P ＜0.05. The rate of amputation was 50% in WT mice versus 0% in TNFR1-/- mice. There were less TUNEL positive cells in the calf muscle of TNFR1-/- mice than that of WT mice. Furthermore, DNA fragmentation was more obvious in WT mice.Western blot showed less expression of Bax and cleaved caspase 3 in TNFR1-/- mice 1 day after ischemia, suggesting a reduced apoptotic process in the absence of TNFR1.Conclusion In mice with hindlimb ischemia, knockout of TNFR1 prevents the activation of death-related proteins down streaming to TNF α and attenuated cell death including apoptosis. Systemic block of the TNFR1 might be a new interventional methods-to preserve the limbs from acute ischemic attack in patients with peripheral arterial obstructive disease.