程序性坏死在自体原位肝移植术大鼠肺损伤中的作用

Role of necroptosis in lung injury in a rat model of autologous orthotopic liver transplantation

目的评价程序性坏死在自体原位肝移植术大鼠肺损伤中的作用。方法清洁级健康成年雄性SD大鼠24只,体重250~280 g,10~12周龄,采用随机数字表法分为3组(n=8):假手术组(S组)、自体原位肝移植术组(T组)和程序性坏死抑制剂Necrostatin-1组(N组)。S组仅单纯进行开腹,游离相应的血管和韧带,最后关腹;T组和N组均制备自体原位肝移植术大鼠肺损伤模型,N组术前30 min腹腔注射Necrostatin-1 1 mg/kg。于再灌注6 h时处死大鼠取肺组织,计算肺组织湿/干重(W/D)比值,采用ELISA法测定TNF-α和IL-6含量,采用免疫组化法测定混合系列蛋白酶样结构域(MLKL)表达,采用Western blot法检测受体相互作用蛋白激酶1(RIPK1)和受体相互作用蛋白激酶3(RIPK3)的表达。结果与S组比较,T组和N组肺组织W/D比值、TNF-α和IL-6含量升高;RIPK1、RIPK3和MLKL表达上调(P<0.05);与T组比较,N组肺组织W/D比值、TNF-α和IL-6含量降低,肺组织RIPK1、RIPK3和MLKL表达下调(P<0.05)。S组肺组织形态未见明显异常,N组肺组织病理学损伤较T组明显减轻。结论程序性坏死参与了自体原位肝移植术大鼠肺损伤的过程。

Objective To evaluate the role of necroptosis in lung injury in a rat model of autologous orthotopic liver transplantation.Methods Twenty-four clean-grade healthy adult male Sprague-Dawley rats,weighing 250-280 g,aged 10-12 weeks,were divided into 3 groups(n=8 each)by a random number table method:sham operation group(S group),autologous orthotopic liver transplantation group(T group)and necroptosis inhibitor necrostatin-1 group(N group).In S group,the abdomen was opened,the corresponding blood vessels and ligaments were isolated,and the abdomen was finally closed.The lung injury models were established after autologous orthotopic liver transplantation in T and N groups,and necrostatin-11 mg/kg was intraperitoneally injected at 30 min before surgery in N group.Rats were sacrificed at 6 h of reperfusion,and lung tissues were removed for determination of the wet/dry weight ratio(W/D ratio),contents of tumor necrosis factor-alpha(TNF-α)and interleukin-6(IL-6)(by enzyme-linked immunosorbent assay),expression of mixed lineage kinase domain-like protein(MLKL)(by immunohistochemistry),and expression of receptor-interacting protein kinase 1(RIPK1)and RIPK3(by Western blot).Results Compared with S group,the W/D ratio and contents of TNF-αand IL-6 were significantly increased,and the expression of RIPK1,RIPK3 and MLKL was up-regulated in T and N groups(P<0.05).Compared with T group,the W/D ratio and contents of TNF-αand IL-6 were significantly decreased,and the expression of RIPK1,RIPK3 and MLKL was down-regulated in N group(P<0.05).No obvious abnormality was found in the morphology of lung tissues in S group,and the pathological changes were significantly attenuated in N group when compared with T group.Conclusion Necroptosis is involved in the process of lung injury in a rat model of autologous orthotopic liver transplantation.