目的 从线粒体自噬角度探讨羟基红花黄色素A（hydroxysafflor yellow A,HSYA）减轻小鼠脓毒症肝损伤的可能作用机制。方法 50只C57BL/6小鼠随机分为假手术组（10只）、CLP组（10只）、HSYA组（20只）。采用盲肠结扎穿刺（cecal ligation and puncture,CLP）构建脓毒症模型。HSYA组造模前24 h、2 h及造模后2 h皮下注射羟基红黄色素A（120 mg·kg-1）。通过小鼠脓毒症评分系统评估各组动物的生存状态;使用HE染色法和透射电镜分别观察肝脏组织病理学改变和线粒体形态改变;通过ELISA法检测小鼠血清中谷丙转氨酶、谷草转氨酶水平以及炎症因子白细胞介素-6、肿瘤坏死因子-α、白细胞介素-1β水平。使用Western blot法检测肝脏组织线粒体自噬相关蛋白PTEN诱导假定激酶1（PTEN induced putative kinase 1,PINK1）、E3泛素蛋白连接酶（E3 ubiquitin ligase,Parkin）、LC3结合蛋白p62（the ubiquitin-and LC3-binding protein p62,p62）、微管轻链蛋白3B-II（microtubule-associated protein 1 light chain 3 beta-II,LC3B-II）的表达。结果 HSYA有效改善CLP小鼠的精神状态、反应速度、活动、呼吸质量;与CLP组小鼠相比,HSYA组肝损伤好转,线粒体自噬小体增加,HSYA组血清ALT、AST水平、IL-6、TNF-α、IL-1β水平及IL-6、TNF-α、IL-1β mRNA水平均降低;与CLP组相比,HSYA组小鼠肝脏组织中PINK1、Parkin、LC3B-II蛋白表达水平升高,P62蛋白表达水平降低。结论 HSYA可抑制脓毒症小鼠炎症因子释放,减轻肝损伤,其机制可能与调控线粒体自噬以及PINK1/Parkin通路相关。
Abstract
Objective To explore the possible mechanism of hydroxysafflor yellow A (HSYA) in alleviating liver injury in septic mice from the perspective of mitochondrial autophagy. Methods Fifty C57BL/6 mice were randomly divided into sham operation group (10 mice), sepsis group (10 mice), and HSYA group (20 mice). The sepsis model was established by cecal ligation and puncture (CLP). HSYA (120 mg·kg-1) was subcutaneously injected 24 hours, 2 hours before and 2 hours after the model establishment in the HSYA group. The survival status of each group was evaluated by the mouse sepsis scoring system. The pathological changes of liver tissue and the morphological changes of mitochondria were observed by HE staining and transmission electron microscopy, respectively. The levels of alanine aminotransferase, aspartate aminotransferase, and inflammatory factors interleukin-6, tumor necrosis factor-α, and interleukin-1β in mouse serum were detected by ELISA. The expression levels of mitochondrial autophagy-related proteins PTEN-induced putative kinase 1(PINK1), E3 ubiquitin ligase (Parkin), ubiquitin-and LC3-binding protein p62(p62), and Microtubule-Associated Protein 1 Light Chain 3 Beta-II (LC3B-II) in liver tissue were detected by Western blot. Results HSYA effectively improved the mental state, reaction speed, activity, and respiratory quality of CLP mice. Compared with the CLP group, liver injury in the HSYA group was improved, and the number of mitochondrial autophagosomes increased. The levels of ALT, AST, IL-6, TNF-α, IL-1β in serum and the mRNA levels of IL-6, TNF-α, and IL-1β in the HSYA group were all decreased. Compared with the CLP group, the expression levels of PINK1, Parkin, and LC3B-II proteins in the liver tissue of the HSYA group were increased, and the expression level of p62 protein was decreased. Conclusion HSYA can inhibit the release of inflammatory factors in septic mice and alleviate liver injury. The mechanism may be related to the regulation of mitochondrial autophagy and the PINK1/Parkin pathway.
关键词
脓毒症 /
肝损伤 /
羟基红花黄色素A /
线粒体自噬 /
PINK1/Parkin通路
Key words
sepsis /
liver injury /
hydroxysafflor yellow A /
mitochondrial autophagy /
PINK1/Parkin pathway
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基金
湖南省自然科学基金项目“羟基红花黄素A通过抑制ROS-NLRP3通路对小鼠脓毒症肝损伤的保护作用及机制研究”（2023JJ60102）; 长沙市自然科学基金“基于mRNA-代谢网络探讨电针刺激足三里配伍肝俞对大鼠脓毒症肝损伤的作用机制”（kq2208124）; 湖南省教育厅优秀青年基金“电针刺激足三里对大鼠脓毒症肝损伤的保护作用及代谢组学研究”（22B0049）
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