Objectives: To explore the role of the cGAS-STING signaling pathway in the therapeutic mechanism of Liangxue Jiedu Huayu Formula (LXJDHYF) for acute-on-chronic liver failure (ACLF) in mice.
Methods: Thirty C57BL/6 mice were randomly divided into blank control group, model group, low- and high-dose LXJDHYF groups, and H151 (a specific cGAS-STING pathway inhibitor) group (n=6). In all but the control group, the mice were treated with CCl4 to induce liver cirrhosis followed by intraperitoneal injections of lipopolysaccharide and D-amino galactose to establish mouse models of ACLF. After the treatments, the mouse livers were collected for HE and TUNEL staining, and serum levels of ALT, AST and TBil were determined. In bone marrow-derived macrophages (BMDMs) and liver tissues of ACLF mice, the expressions of cGAS-STING signaling pathway-related mRNAs including IFN‑β, ISG15, IL-6 and TNF-α were determined with RT-qPCR, and the phosphorylation levels of IRF3 and STING proteins were investigated using Western blotting.
Results: Compared with the mice in the model group, the LXJDHYF-treated mice exhibited milder hepatocyte necrosis and inflammatory cell infiltration in the liver with significantly reduced hepatocyte apoptosis. LXJDHYF treatment also significantly lowered serum levels of ALT, AST, TBil, IL-6 and TNF-α in ACLF mice and effectively suppressed the expressions of cGAS-STING signaling pathway-related mRNA in both the BMDMs and the liver tissues and the phosphorylation of IRF3 and STING proteins in the BMDMs.
Conclusions: LXJDHYF can significantly improve liver function and attenuate inflammation in ACLF mice possibly by inhibiting excessive activation of the cGAS-STING signaling pathway.
目的: 基于cGAS-STING 信号通路，研究中药凉血解毒化瘀方（LXJDHYF）治疗小鼠慢加急性肝衰竭（ACLF）的可能作用机制。方法: 30只C57BL/6小鼠随机分为空白组、模型组、凉血解毒化瘀方高剂量组、凉血解毒化瘀方低剂量组及cGAS-STING 信号通路特异性抑制剂H151组，6只/组。除空白组外，其余各组采用CCl4诱导形成肝硬化，后予以脂多糖联合D-氨基半乳糖腹腔注射急性攻击形成ACLF小鼠模型。取小鼠肝组织进行HE以及TUNEL染色，生化法测定血清ALT、AST及TBil水平以检测凉血解毒化瘀方对小鼠肝功能的影响。同时采用RT-qPCR测定凉血解毒化瘀方对小鼠骨髓巨噬细胞（BMDMs）β干扰素（IFN-β）、干扰素刺激基因15（ISG15）、白细胞介素6（IL-6）及肿瘤坏死因子-α（TNF-α）mRNA表达水平的影响；Western blotting分析凉血解毒化瘀方对BMDMs干扰素调节因子3（IRF3）及干扰素基因刺激因子（STING）蛋白磷酸化水平的影响；结合RT-qPCR检测ACLF小鼠肝组织中上述mRNA表达水平，ELISA法检测ACLF小鼠血清 IL-6及TNF-α水平以观察凉血解毒化瘀方对cGAS-STING 信号通路的作用。结果: HE染色显示，凉血解毒化瘀方组肝细胞坏死及炎症浸润程度与模型组相比较轻，TUNEL染色提示凉血解毒化瘀方组凋亡阳性细胞比例低于模型组（P<0.001）。凉血解毒化瘀方组血清ALT、AST及TBil水平均低于模型组（P<0.001）。RT-qPCR结果表明，凉血解毒化瘀方能抑制BMDMs及小鼠肝组织IFN-β、ISG15、IL-6及TNF-α的mRNA表达（P<0.05）。Western blotting结果显示，随着凉血解毒化瘀方给药剂量增加，BMDMs细胞中IRF3及STING蛋白磷酸化水平降低（P<0.05），凉血解毒化瘀方组血清IL-6及TNF-α水平均低于模型组（P<0.05）。结论: 凉血解毒化瘀方能够改善ACLF小鼠肝功能，降低小鼠血清促炎细胞因子水平，其机制可能与抑制cGAS-STING 通路过度激活有关。.
Keywords: Liangxue Jiedu Huayu Formula; acute-on-chronic liver failure; cGAS-STING signaling pathway; interferon stimulating gene 15; interferon‑β.