Yang Yi, Han Chenyang, Sheng Yongjia, Wang Jin, Zhou Xiaohong, Li Wenyan, Guo Li, Ruan Shuiliang
Department of Pharmacy, The Second Affiliated Hospital of Jiaxing University, Jiaxing, China.
Department of Center Laboratory, The Second Affiliated Hospital of Jiaxing University, Jiaxing, China.
Front Pharmacol. 2020 Oct 28;11:570776. doi: 10.3389/fphar.2020.570776. eCollection 2020.
In this study, we mainly explored the mechanism and target of the anti-inflammatory effects of Aureusidin (Aur) in acute liver injury.
Lipopolysaccharide (LPS) was used to induce inflammatory injury in Kupffer cells (KCs) . After Aur treatment with gradient concentration, flow cytometry, propidium iodide (PI) staining, and Hoechst 33342 staining were used to detect the apoptotic level of KCs, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of inflammatory factors, including Interleukin-1β (IL-1β), Interleukin-18 (IL-18), and tumor necrosis factor alpha (TNF-α). Western blot was used to detect the expression of toll-like receptor 4 (TLR4), myeloid differentiation protein-2 (MD2), MyD88, and p-P65. Aur was labeled with biotin, followed by a pull-down assay to detect its binding with MD2. Moreover, D-GalN/LPS was used to induce acute liver injury in mice , followed by Aur treatment by gavage. H&E staining was used to detect the pathological changes of liver tissue, an IF assay was used to detect the expression of MD2, Western blot was used to detect the expression of relevant proteins.
Aur pretreatment could significantly inhibit LPS-induced KC injury, downregulate the apoptotic level, inhibit the expression of inflammatory factors, decrease the level of MDA, and downregulate the expression of MD2 in cells. Aur could inhibit the activation level of TLR4/MD2-NF-κB in a dose-dependent pattern, a high dose of Aur had a superior effect compared to low-dose Aur. In the case of MD2 deletion, the effects of Aur were suppressed. Additionally, pull-down and co-immunoprecipitation assays show that Aur can bind with the MD2 protein to inhibit the activation of TLR4/MD2-NF-κB. Results of mice experiments also showed that Aur could relieve liver injury, decrease the levels of ALT and AST, and simultaneously downregulate the levels of inflammatory factors in tissues and peripheral blood.
We found that Aur exerted an anti-inflammatory effect by directly targeting the MD2 protein, further inhibiting the expression of TLR4/MD2-NF-κB, thereby relieving acute liver injury. Therefore, Aur might be a potential inhibitor for MD2.
在本研究中，我们主要探讨了金黄菌素（Aur）在急性肝损伤中抗炎作用的机制和靶点。
采用脂多糖（LPS）诱导库普弗细胞（KCs）发生炎性损伤。用不同梯度浓度的Aur处理后，采用流式细胞术、碘化丙啶（PI）染色和Hoechst 33342染色检测KCs的凋亡水平，并用酶联免疫吸附测定（ELISA）检测炎性因子白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）和肿瘤坏死因子-α（TNF-α）的表达水平。采用蛋白质免疫印迹法检测Toll样受体4（TLR4）、髓样分化蛋白-2（MD2）、髓样分化因子88（MyD88）和磷酸化P65（p-P65）的表达。用生物素标记Aur，然后通过下拉实验检测其与MD2的结合。此外，用D-氨基半乳糖/脂多糖（D-GalN/LPS）诱导小鼠急性肝损伤，然后通过灌胃给予Aur。采用苏木精-伊红（H&E）染色检测肝组织的病理变化，采用免疫荧光（IF）实验检测MD2的表达，采用蛋白质免疫印迹法检测相关蛋白的表达。
Aur预处理可显著抑制LPS诱导的KC损伤，下调凋亡水平，抑制炎性因子的表达，降低丙二醛（MDA）水平，并下调细胞中MD2的表达。Aur可呈剂量依赖性抑制TLR4/MD2-核因子κB（NF-κB）的激活水平，高剂量Aur的作用优于低剂量Aur。在MD2缺失的情况下，Aur的作用受到抑制。此外，下拉实验和免疫共沉淀实验表明，Aur可与MD2蛋白结合，抑制TLR4/MD2-NF-κB的激活。小鼠实验结果还表明，Aur可减轻肝损伤，降低谷丙转氨酶（ALT）和谷草转氨酶（AST）水平，同时下调组织和外周血中炎性因子的水平。
我们发现Aur通过直接靶向MD2蛋白发挥抗炎作用，进一步抑制TLR4/MD2-NF-κB的表达，从而减轻急性肝损伤。因此，Aur可能是一种潜在的MD2抑制剂。
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