Yu Qing, Zou Liying, Yuan Xiu, Fang Fang, Xu Feng
Department of Intensive Care Unit, Children's Hospital of Chongqing Medical University, Chongqing, China.
Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children's Hospital of Chongqing Medical University, Chongqing, China.
Front Pharmacol. 2021 Apr 26;12:658677. doi: 10.3389/fphar.2021.658677. eCollection 2021.
Liver injury is one of the serious complications of sepsis. Previous studies suggested that dexmedetomidine (DEX) could alleviate cecal ligation and puncture (CLP)-induced liver injury. However, it is unclear whether the protective effect of DEX on sepsis-induced liver injury is related to autophagy. Mice ( = 105) were randomly divided into the following groups: (i) CON group (Sham); (ii) CLP group (CLP-induced liver injury + saline); (iii) CLP + DEX group (CLP-induced liver injury + DEX). Mouse models of sepsis-induced liver injury were established using CLP. DEX or normal saline was administered by intraperitoneal injection at 0, 2, and 4 h after CLP surgery. The mortality rate within 120 h was calculated. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and inflammatory cytokines were measured at 6, 12, and 24 h in each group. Hematoxylin and eosin staining assay was carried out to detect the morphological changes of mouse liver cells in each group. The levels of autophagy-associated proteins LC3II, Beclin-1, p62, and LAMP-2 were detected in three groups of mice using western blotting. The expression of LC3II was detected using immunofluorescence. Transmission electron microscopy (TEM) of liver tissue was used to observe autophagosomes and autophagosome-lysosomes. Lastly, the effect of DEX on the AMPK/SIRT1 pathway-associated protein levels were detected using western blotting. Meanwhile, we used L0-2 cells infected with mRFP-GFP-LC3 adenovirus to further analyze the role of SIRT1 in DEX-induced autophagy in liver injury model . DEX significantly improved the survival rate of septic mice at the early stage and ameliorated the pathology of sepsis-induced liver injury. The level of autophagy-associated proteins, phosphorylated ()-AMPK/AMPK, and SIRT1 in the liver of CLP-induced sepsis mice peaked at 12 h post-CLP and decreased significantly at 24 h. In the CLP + DEX group, the levels of autophagy-associated proteins, -AMPK/AMPK, and SIRT1 increased, whereas inflammatory cytokines decreased at 24 h. The autophagosome structure was clearly observed at different time points in the CLP + DEX group. In the hepatocyte injury model, the SIRT1 inhibitor significantly increased intracellular ROS levels and reversed the effect of DEX on autophagy flux. We demonstrated a novel mechanism in which DEX protects against CLP-induced liver injury. DEX enhances autophagy, which alleviates the inflammatory responses in CLP-induced liver injury by regulating the SIRT1/AMPK pathway.
肝损伤是脓毒症的严重并发症之一。先前的研究表明，右美托咪定（DEX）可减轻盲肠结扎穿孔（CLP）诱导的肝损伤。然而，DEX对脓毒症诱导的肝损伤的保护作用是否与自噬有关尚不清楚。将105只小鼠随机分为以下几组：（i）CON组（假手术组）；（ii）CLP组（CLP诱导的肝损伤+生理盐水）；（iii）CLP + DEX组（CLP诱导的肝损伤+ DEX）。采用CLP建立脓毒症诱导的肝损伤小鼠模型。在CLP手术后0、2和4小时通过腹腔注射给予DEX或生理盐水。计算120小时内的死亡率。在每组的6、12和24小时测量丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）和炎性细胞因子水平。进行苏木精-伊红染色检测每组小鼠肝细胞的形态变化。采用蛋白质免疫印迹法检测三组小鼠中自噬相关蛋白LC3II、Beclin-1、p62和LAMP-2的水平。采用免疫荧光法检测LC3II的表达。用肝脏组织的透射电子显微镜（TEM）观察自噬体和自噬体-溶酶体。最后，采用蛋白质免疫印迹法检测DEX对AMPK/SIRT1通路相关蛋白水平的影响。同时，我们使用感染了mRFP-GFP-LC3腺病毒的L0-2细胞进一步分析SIRT1在DEX诱导的肝损伤模型自噬中的作用。DEX显著提高了脓毒症小鼠的早期存活率，并改善了脓毒症诱导的肝损伤病理。CLP诱导的脓毒症小鼠肝脏中自噬相关蛋白、磷酸化（p）-AMPK/AMPK和SIRT1的水平在CLP后12小时达到峰值，并在24小时显著下降。在CLP + DEX组中，自噬相关蛋白、p-AMPK/AMPK和SIRT1的水平在24小时升高，而炎性细胞因子水平下降。在CLP + DEX组的不同时间点均清晰观察到自噬体结构。在肝细胞损伤模型中，SIRT1抑制剂显著提高细胞内活性氧水平，并逆转了DEX对自噬流的影响。我们证明了DEX保护免受CLP诱导的肝损伤的新机制。DEX增强自噬，通过调节SIRT1/AMPK通路减轻CLP诱导的肝损伤中的炎症反应。
