Müllebner Andrea, Herminghaus Anna, Miller Ingrid, Kames Martina, Luís Andreia, Picker Olaf, Bauer Inge, Kozlov Andrey V, Duvigneau Johanna Catharina
Ludwig Boltzmann Institute for Traumatology, The Research Center in Cooperation With AUVA, Vienna, Austria.
Department of Biomedical Sciences, Institute for Medical Biochemistry, University of Veterinary Medicine Vienna, Vienna, Austria.
Front Med (Lausanne). 2022 Mar 16;9:785285. doi: 10.3389/fmed.2022.785285. eCollection 2022.
Abdominal surgery is an efficient treatment of intra-abdominal sepsis. Surgical trauma and peritoneal infection lead to the activation of multiple pathological pathways. The liver is particularly susceptible to injury under septic conditions. Liver function is impaired when pathological conditions induce endoplasmic reticulum (ER) stress. ER stress triggers the unfolded protein response (UPR), aiming at restoring ER homeostasis, or inducing cell death. In order to translate basic knowledge on ER function into the clinical setting, we aimed at dissecting the effect of surgery and peritoneal infection on the progression of ER stress/UPR and inflammatory markers in the liver in a clinically relevant experimental animal model.
Wistar rats underwent laparotomy followed by colon ascendens stent peritonitis (CASP) or surgery (sham) only. Liver damage (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and De Ritis values), inflammatory and UPR markers were assessed in livers at 24, 48, 72, and 96 h postsurgery. Levels of inflammatory (IL-6, TNF-α, iNOS, and HO-1), UPR (XBP1, GRP78, CHOP), and apoptosis (BAX/Bcl-XL) mRNA were determined by qPCR. Splicing of XBP1 (XBP1s) was analyzed by gel electrophoresis, p-eIF2α and GRP78 protein levels using the western blots.
Aspartate aminotransferase levels were elevated 24 h after surgery and thereafter declined with different kinetics in sham and CASP groups. Compared with sham De Ritis ratios were significantly higher in the CASP group, at 48 and 96 h. CASP induced an inflammatory response after 48 h, evidenced by elevated levels of IL-6, TNF-α, iNOS, and HO-1. In contrast, UPR markers XBP1s, p-eIF2α, GRP78, XBP1, and CHOP did not increase in response to infection but paralleled the kinetics of AST and De Ritis ratios. We found that inflammatory markers were predominantly associated with CASP, while UPR markers were associated with surgery. However, in the CASP group, we found a stronger correlation between XBP1s, XBP1 and GRP78 with damage markers, suggesting a synergistic influence of inflammation on UPR in our model.
Our results indicate that independent mechanisms induce ER stress/UPR and the inflammatory response in the liver. While peritoneal infection predominantly triggers inflammatory responses, the conditions associated with organ damage are predominant triggers of the hepatic UPR.
腹部手术是治疗腹腔内脓毒症的有效方法。手术创伤和腹膜感染会激活多种病理途径。在脓毒症状态下，肝脏特别容易受到损伤。当病理状况诱导内质网（ER）应激时，肝功能会受损。ER应激触发未折叠蛋白反应（UPR），旨在恢复ER稳态或诱导细胞死亡。为了将关于ER功能的基础知识转化为临床应用，我们旨在剖析手术和腹膜感染对临床相关实验动物模型中肝脏ER应激/UPR进展及炎症标志物的影响。
Wistar大鼠接受剖腹手术，随后进行升结肠支架性腹膜炎（CASP）或仅进行手术（假手术）。在术后24、48、72和96小时评估肝脏损伤（天冬氨酸转氨酶（AST）、丙氨酸转氨酶（ALT）和德瑞蒂斯值）、炎症和UPR标志物。通过qPCR测定炎症（IL-6、TNF-α、诱导型一氧化氮合酶（iNOS）和血红素加氧酶-1（HO-1））、UPR（X盒结合蛋白1（XBP1）、葡萄糖调节蛋白78（GRP78）、C/EBP同源蛋白（CHOP））和凋亡（BAX/Bcl-XL）mRNA水平。通过凝胶电泳分析XBP1的剪接（XBP1s），使用蛋白质印迹法检测磷酸化真核起始因子2α（p-eIF2α）和GRP78蛋白水平。
术后24小时天冬氨酸转氨酶水平升高，此后假手术组和CASP组以不同动力学下降。与假手术组相比，CASP组在48和96小时的德瑞蒂斯比值显著更高。CASP在48小时后诱导炎症反应，表现为IL-6、TNF-α、iNOS和HO-1水平升高。相比之下，UPR标志物XBP1s、p-eIF2α、GRP78、XBP1和CHOP并未因感染而增加，而是与AST和德瑞蒂斯比值的动力学平行。我们发现炎症标志物主要与CASP相关，而UPR标志物与手术相关。然而，在CASP组中，我们发现XBP1s、XBP1和GRP78与损伤标志物之间的相关性更强，表明在我们的模型中炎症对UPR有协同影响。
我们的结果表明，独立的机制诱导肝脏中的ER应激/UPR和炎症反应。虽然腹膜感染主要触发炎症反应，但与器官损伤相关的状况是肝脏UPR的主要触发因素。
