Department of Surgery, University of Connecticut School of Medicine, Farmington, CT 06032, USA.
Molecular Cardiology and Angiogenesis Laboratory, University of Connecticut School of Medicine, Farmington, CT 06032, USA.
Cells. 2023 Jun 1;12(11):1527. doi: 10.3390/cells12111527.
Intra-abdominal sepsis is commonly diagnosed in the surgical population and remains the second most common cause of sepsis overall. Sepsis-related mortality remains a significant burden in the intensive care unit despite advances in critical care. Nearly a quarter of the deaths in people with heart failure are caused by sepsis. We have observed that overexpression of mammalian Pellino-1 (Peli1), an E3 ubiquitin ligase, causes inhibition of apoptosis, oxidative stress, and preservation of cardiac function in a myocardial infarction model. Given these manifold applications, we investigated the role of Peli1 in sepsis using transgenic and knockout mouse models specific to this protein. Therefore, we aimed to explore further the myocardial dysfunction seen in sepsis through its relation to the Peli 1 protein by using the loss of function and gain-of-function strategy.
A series of genetic animals were created to understand the role of Peli1 in sepsis and the preservation of heart function. Wild-type, global Peli1 knock out (Peli1), cardiomyocyte-specific Peli1 deletion (CP1KO), and cardiomyocyte-specific Peli1 overexpressing (alpha MHC (αMHC) Peli1; AMPEL1) animals were divided into sham and cecal ligation and puncture (CLP) surgical procedure groups. Cardiac function was determined by two-dimensional echocardiography pre-surgery and at 6- and 24-h post-surgery. Serum IL-6 and TNF-alpha levels (ELISA) (6 h), cardiac apoptosis (TUNEL assay), and Bax expression (24 h) post-surgery were measured. Results are expressed as mean ± S.E.M.
AMPEL1 prevents sepsis-induced cardiac dysfunction assessed by echocardiographic analysis, whereas global and cardiomyocyte-specific deletion of Peli1 shows significant deterioration of cardiac functions. Cardiac function was similar across the sham groups in all three genetically modified mice. ELISA assay displayed how Peli 1 overexpression decreased cardo-suppressive circulating inflammatory cytokines (TNF-alpha, IL-6) compared to both the knockout groups. The proportion of TUNEL-positive cells varied according to Peli1 expression, with overexpression (AMPEL1) leading to a significant reduction and Peli1 gene knockout (Peli1 and CP1KO) leading to a significant increase in their presence. A similar trend was also observed with Bax protein expression. The improved cellular survival associated with Peli1 overexpression was again shown with the reduction of oxidative stress marker 4-Hydroxy-2-Nonenal (4-HNE).
Our results indicate that overexpression of Peli1 is a novel approach that not only preserved cardiac function but reduced inflammatory markers and apoptosis following severe sepsis in a murine genetic model.
腹腔脓毒症在外科人群中通常被诊断出来，仍然是总体脓毒症的第二大常见原因。尽管重症监护取得了进展，但与脓毒症相关的死亡率仍然是重症监护病房的一个重大负担。心力衰竭患者近四分之一的死亡是由脓毒症引起的。我们观察到，哺乳动物 Pellino-1（Peli1）的过表达，一种 E3 泛素连接酶，在心肌梗死模型中导致细胞凋亡、氧化应激和心脏功能的抑制。鉴于这些多方面的应用，我们使用特定于该蛋白的转基因和敲除小鼠模型来研究 Peli1 在脓毒症中的作用。因此，我们旨在通过使用功能丧失和功能获得策略，进一步探索脓毒症中观察到的心肌功能障碍与其 Peli1 蛋白的关系。
创建了一系列遗传动物来了解 Peli1 在脓毒症和心脏功能保护中的作用。野生型、全局 Peli1 敲除（Peli1）、心肌细胞特异性 Peli1 缺失（CP1KO）和心肌细胞特异性 Peli1 过表达（α MHC（αMHC）Peli1；AMPEL1）动物分为假手术和盲肠结扎穿孔（CLP）手术组。术前和术后 6-24 小时通过二维超声心动图确定心脏功能。术后 6 小时（ELISA）测量血清白细胞介素 6（IL-6）和肿瘤坏死因子-α（TNF-α）水平，术后 24 小时测量心脏细胞凋亡（TUNEL 检测）和 Bax 表达。结果表示为平均值±标准误差。
AMPEL1 可预防脓毒症诱导的心脏功能障碍，通过超声心动图分析评估，而全局和心肌细胞特异性 Peli1 缺失导致心脏功能显著恶化。在所有三种基因修饰小鼠中，假手术组的心脏功能相似。ELISA 检测显示，与两个敲除组相比，Peli1 过表达如何降低了心脏抑制性循环炎症细胞因子（TNF-α、IL-6）。TUNEL 阳性细胞的比例根据 Peli1 的表达而变化，过表达（AMPEL1）导致其数量显著减少，而基因敲除（Peli1 和 CP1KO）则导致其数量显著增加。Bax 蛋白表达也出现了类似的趋势。与 Peli1 过表达相关的细胞存活改善再次通过减少氧化应激标志物 4-羟基-2-壬烯醛（4-HNE）得到证明。
我们的结果表明，Peli1 的过表达不仅在严重脓毒症的小鼠遗传模型中保留了心脏功能，而且降低了炎症标志物和细胞凋亡，是一种新的方法。
