Data Sheet 1_Paeoniflorin attenuates sepsis-induced liver injury by reprogramming macrophage polarization via the TLR4/NF-κB pathway.pdf

BackgroundSepsis-associated liver injury (SALI) increases mortality in critically ill patients but lacks targeted treatments. Although the natural compound Paeoniflorin shows anti-inflammatory and immunomodulatory potential, its specific function and mechanism in SALI remain unclear.
MethodsA murine model of polymicrobial sepsis was established using cecal ligation and puncture (CLP). Male C57BL/6 mice were randomly allocated to Sham, CLP, CLP+Paeoniflorin (30, 60, 120 mg/kg), CLP+Paeoniflorin+TLR4 agonist (RS09 TFA), and Paeoniflorin-only control groups. Liver injury was assessed through serum ALT/AST measurements, histopathological evaluation, and TUNEL apoptosis assay. Hepatic inflammatory cytokine expression was quantified by qPCR. Macrophage polarization was analyzed via immunohistochemistry for F4/80, CD86 (M1), and CD206 (M2) markers. TLR4/NF-κB pathway activity was examined using Western blotting and immunohistochemistry. Transcriptomic profiling was performed through RNA sequencing and KEGG pathway analysis.
ResultsPaeoniflorin administration significantly attenuated CLP-induced elevations in serum ALT and AST levels in a dose-dependent manner, ameliorated histopathological liver damage, and reduced hepatocyte apoptosis. Treatment with Paeoniflorin substantially downregulated hepatic mRNA expression of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β). Immunohistochemical analysis revealed that Paeoniflorin treatment was associated with a shift in macrophage marker expression, characterized by a reduction in cells co-staining for F4/80 and the classic M1 marker CD86, and an increase in cells co-staining for F4/80 and the classic M2 marker CD206. This suggests a potential modulation of macrophage polarization balance towards an anti-inflammatory phenotype. Both transcriptomic and protein analyses confirmed that Paeoniflorin suppressed activation of the TLR4/NF-κB signaling pathway. The protective effects of Paeoniflorin were completely abolished by co-administration of the TLR4 agonist RS09 TFA.
ConclusionPaeoniflorin confers protection against sepsis-induced liver injury by modulating macrophage polarization from the pro-inflammatory M1 phenotype toward the anti-inflammatory M2 phenotype through inhibition of the TLR4/NF-κB signaling pathway. These findings identify Paeoniflorin as a promising candidate for further development as an immunomodulatory therapy for SALI.

背景 脓毒症相关性肝损伤（Sepsis-associated liver injury, SALI）会升高重症患者的死亡率，但目前尚无针对性治疗方案。尽管天然化合物芍药苷（Paeoniflorin）具备抗炎与免疫调节潜力，但其在SALI中的具体功能及作用机制仍未明确。

方法 本研究采用盲肠结扎穿刺术（cecal ligation and puncture, CLP）构建多微生物脓毒症小鼠模型。将雄性C57BL/6小鼠随机分为假手术组、CLP模型组、CLP+芍药苷（30、60、120 mg/kg）组、CLP+芍药苷+Toll样受体4（Toll-like receptor 4, TLR4）激动剂（RS09 TFA）组以及单纯芍药苷对照组。通过血清谷丙转氨酶（ALT）/谷草转氨酶（AST）检测、组织病理学评估及脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记（TUNEL）凋亡实验评估肝损伤程度。采用实时定量聚合酶链反应（qPCR）定量检测肝脏炎症细胞因子的mRNA表达水平。通过免疫组化检测F4/80、CD86（M1型巨噬细胞标志物）与CD206（M2型巨噬细胞标志物）的表达，分析巨噬细胞极化状态。采用蛋白质印迹法与免疫组化检测TLR4/核因子κB（Nuclear Factor-kappa B, NF-κB）通路的活化水平。通过RNA测序及京都基因与基因组百科全书（KEGG）通路分析开展转录组谱分析。

结果 给予芍药苷可呈剂量依赖性显著缓解CLP诱导的血清ALT、AST水平升高，改善肝脏组织病理学损伤，并减少肝细胞凋亡。芍药苷治疗可显著下调肝脏促炎细胞因子（IL-6、TNF-α、IL-1β）的mRNA表达。免疫组化分析显示，芍药苷处理可改变巨噬细胞标志物的表达模式：共表达F4/80与经典M1标志物CD86的细胞数量减少，而共表达F4/80与经典M2标志物CD206的细胞数量增加，提示芍药苷可将巨噬细胞极化平衡向抗炎表型调控。转录组学与蛋白质分析均证实，芍药苷可抑制TLR4/NF-κB信号通路的活化。联合给予TLR4激动剂RS09 TFA可完全抵消芍药苷的肝脏保护作用。

结论 芍药苷可通过抑制TLR4/NF-κB信号通路，调控巨噬细胞从促炎M1表型向抗炎M2表型极化，从而发挥对抗脓毒症相关性肝损伤的保护作用。本研究结果表明，芍药苷有望成为治疗SALI的免疫调节类候选药物，具备进一步开发价值。