背景:心脏移植(HTX)是终末期心力衰竭的标准治疗方法。然而,缺血期后再灌注可导致心肌损伤。中性粒细胞浸润,随着组织降解中性粒细胞弹性蛋白酶(NE)相关的丝氨酸蛋白酶和氧衍生自由基的释放,与不良移植物结局相关。已显示组织蛋白酶C(CatC)的抑制阻断NE相关蛋白酶活化。我们假设CatC抑制剂BI-9740改善HTX后的移植物功能。
方法:在HTX大鼠模型中,受体Lewis大鼠口服安慰剂(n=12)或BI-9740(n=11,20mg/kg),每日一次,共12天.未治疗的刘易斯大鼠的供体心脏被移出,保存在心脏停搏液中,随后异位植入。再灌注1小时后评估体内左心室(LV)移植物功能。在来自BI-9740处理和对照大鼠的骨髓裂解物中测定嗜中性粒细胞丝氨酸蛋白酶的蛋白水解活性。此外,检查心肌形态变化,和心脏样本进行了免疫组织化学和蛋白质印迹分析。
结果:与安慰剂组相比,BI-9740治疗的大鼠骨髓细胞裂解物中NE相关的蛋白水解活性明显降低。组织病理学病变,升高的CatC和髓过氧化物酶阳性细胞浸润,与安慰剂组相比,在接受BI-9740治疗的动物心脏中,与聚(ADP-核糖)聚合酶(PARP)-1阳性细胞数量增加的硝基酪氨酸免疫反应性降低。关于植入移植物的功能参数,两种收缩功能均有改善(LV收缩压110±6vs74±6mmHg;dP/dtmax2782±149vs2076±167mmHg/s,低压发展压力,在200微升的脑室内容积下,p<0.05)和BI-9740治疗的动物与接受唯一安慰剂的动物相比,心脏的舒张功能。此外,与安慰剂组相比,给予BI-9740导致移植物再搏动时间更短.然而,这项研究没有提供DNA片段的证据,超氧阴离子和过氧化氢的产生,与缺乏与细胞凋亡相关的蛋白质改变有关,如HTX后移植物中的蛋白质印迹所证明。
结论:我们提供了实验证据,表明CatC的药理抑制作用可以改善大鼠HTX后的移植物功能。
Heart transplantation (HTX) is the standard treatment for end-stage heart failure. However, reperfusion following an ischemic period can contribute to myocardial injury. Neutrophil infiltration, along with the subsequent release of tissue-degrading neutrophil elastase (NE)-related serine proteases and oxygen-derived radicals, is associated with adverse graft outcomes. The inhibition of cathepsin C (CatC) has been shown to block NE-related protease activation. We hypothesized that the CatC inhibitor BI-9740 improves graft function after HTX.
In a rat model of HTX, the recipient Lewis rats were orally administered with either a placebo (n = 12) or BI-9740 (n = 11, 20 mg/kg) once daily for 12 days. Donor hearts from untreated Lewis rats were explanted, preserved in a cardioplegic solution, and subsequently heterotopically implanted. In vivo left-ventricular (LV) graft function was assessed after 1 h of reperfusion. The proteolytic activity of neutrophil serine proteases was determined in bone marrow lysates from BI-9740-treated and control rats. Additionally, myocardial morphological changes were examined, and heart samples underwent immunohistochemistry and western blot analysis.
The NE-related proteolytic activity in bone marrow cell lysates was markedly decreased in the BI-9740-treated rats compared to those of the placebo group. Histopathological lesions, elevated CatC and myeloperoxidase-positive cell infiltration, and nitrotyrosine immunoreactivity with an increased number of poly(ADP-ribose) polymerase (PARP)-1-positive cells were lowered in the hearts of animals treated with BI-9740 compared to placebo groups. Regarding the functional parameters of the implanted graft, improvements were observed in both systolic function (LV systolic pressure 110 ± 6 vs 74 ± 6 mmHg; dP/dtmax 2782 ± 149 vs 2076 ± 167 mmHg/s, LV developed pressure, at an intraventricular volume of 200 µl, p < 0.05) and diastolic function in the hearts of BI-9740 treated animals compared with those receiving the only placebo. Furthermore, the administration of BI-9740 resulted in a shorter graft re-beating time compared to the placebo group. However, this study did not provide evidence of DNA fragmentation, the generation of both superoxide anions and hydrogen peroxide, correlating with the absence of protein alterations related to apoptosis, as evidenced by western blot in grafts after HTX.
We provided experimental evidence that pharmacological inhibition of CatC improves graft function following HTX in rats.