The Protective Effect of Esculentoside A on Experimental Acute Liver Injury in Mice

Inflammatory response and oxidative stress are considered to play an important role in the development of acute liver injury induced by carbon tetrachloride (CCl4) and galactosamine (GalN)/lipopolysaccharides (LPS). Esculentoside A (EsA), isolated from the Chinese herb phytolacca esculenta, has the effect of modulating immune response, cell proliferation and apoptosis as well as anti-inflammatory effects. The present study is to evaluate the protective effect of EsA on CCl4 and GalN/LPS-induced acute liver injury. In vitro, CCK-8 assays showed that EsA had no cytotoxicity, while it significantly reduced levels of TNF-α and cell death rate challenged by CCl4. Moreover, EsA treatment up-regulated PPAR-γ expression of LO2 cells and reduced levels of reactive oxygen species (ROS) challenged by CCl4. In vivo, EsA prevented mice from CCl4-induced liver histopathological damage. In addition, levels of AST and ALT were significantly decreased by EsA treatment. Furthermore, the mice treated with EsA had a lower level of TNF-α, Interleukin (IL)-1β and IL-6 in mRNA expression. EsA prevented MDA release and increased GSH-Px activity in liver tissues. Immunohistochemical staining showed that over-expression of F4/80 and CD11b were markedly inhibited by EsA. The western bolt results showed that EsA significantly inhibited CCl4-induced phosphonated IkBalpha (P-IκB) and ERK. Furthermore, EsA treatment also alleviated GalN/LPS-induced acute liver injury on liver enzyme and histopathological damage. Unfortunately, our results exhibited that EsA had no effects on CCl4-induced hepatocyte apoptosis which were showed by TUNEL staining and Bax, Caspase-3 and cleaved Caspase-3 expression. Our results proved that EsA treatment attenuated CCl4 and GalN/LPS-induced acute liver injury in mice and its protective effects might be involved in inhibiting inflammatory response and oxidative stress, but not apoptosis with its underlying mechanism associated with PPAR-γ, NF-κB and ERK signal pathways.炎症反应与氧化应激被认为在四氯化碳（carbon tetrachloride, CCl4）、半乳糖胺（galactosamine, GalN）/脂多糖（lipopolysaccharides, LPS）诱导的急性肝损伤的发生发展中发挥关键作用。从中药垂序商陆（phytolacca esculenta）中分离得到的商陆皂苷A（Esculentoside A, EsA），具有调节免疫反应、调控细胞增殖与凋亡以及抗炎的药理活性。本研究旨在评估EsA对CCl4及GalN/LPS诱导的急性肝损伤的保护作用。体外实验中，CCK-8检测结果显示，EsA无细胞毒性，且可显著降低CCl4刺激下的肿瘤坏死因子-α（TNF-α）水平与细胞死亡率。此外，EsA处理可上调LO2细胞过氧化物酶体增殖物激活受体γ（PPAR-γ）的表达，并降低CCl4刺激下的活性氧（ROS）水平。体内实验表明，EsA可拮抗CCl4诱导的小鼠肝脏组织病理学损伤；同时，EsA处理可显著降低血清中天冬氨酸氨基转移酶（AST）与丙氨酸氨基转移酶（ALT）的水平。进一步研究发现，EsA处理组小鼠的TNF-α、白细胞介素（IL）-1β及IL-6的mRNA表达水平均显著降低；EsA可抑制肝脏组织中丙二醛（MDA）的释放，并提升谷胱甘肽过氧化物酶（GSH-Px）的活性。免疫组织化学染色结果显示，EsA可显著抑制巨噬细胞表面标志物F4/80与CD11b的过表达。蛋白质印迹（Western blot）实验结果显示，EsA可显著抑制CCl4诱导的磷酸化IκBα（P-IκBα）与细胞外调节蛋白激酶（ERK）的激活。此外，EsA处理同样可减轻GalN/LPS诱导的急性肝损伤，表现为肝功能酶学指标异常及肝脏组织病理学损伤均得到改善。遗憾的是，本研究结果显示，EsA对CCl4诱导的肝细胞凋亡无显著影响，该结论通过TUNEL染色及Bcl-2相关X蛋白（Bax）、半胱氨酸天冬氨酸蛋白酶3（Caspase-3）与活化半胱氨酸天冬氨酸蛋白酶3（cleaved Caspase-3）的表达水平得以验证。本研究结果证实，EsA处理可减轻小鼠CCl4及GalN/LPS诱导的急性肝损伤，其保护作用可能通过抑制炎症反应与氧化应激实现，而非调控细胞凋亡，具体机制可能与PPAR-γ、核因子κB（NF-κB）及ERK信号通路相关。