Abstract: Background　The incidence of obstructive cholestatic liver injury（OCLI） has been increasing year by year，accompanied by a high mortality rate. Current technical means mainly focus on relieving obstruction，but their efficacy in treating liver injury is limited. The occurrence and progression of OCLI are closely related to the NF-κB pathway and NLRP3-induced pyroptosis. Dachaihu Decoction has been proven effective in improving liver function in clinical practice and is closely associated with the NF-κB pathway and NLRP3，providing a new approach for the integrated traditional Chinese and Western medicine treatment of OCLI. Objective　To explore the therapeutic effects and mechanisms of Dachaihu Decoction-containing serum on hepatocyte injury induced by mixed bile acids based on the NF-κB/NLRP3 signaling pathway. Methods　Between June 2024 and December 2024，Human normal L02 hepatocytes were randomly divided into five groups：blank control group，mixed bile salt group，drug-containing serum group，NF-κB inhibitor group，and drug-containing serum group +NF-κB inhibitor group. After 24 hours of drug intervention，the expression of IL-1β，IL-6，and IL-18 was detected by ELISA；the protein expression of NF-κB p65，NLRP3，Caspase-1，and other molecules was detected by Western Blot；and the mRNA levels of NF-κB，NLRP3，and Caspase-1 were detected by RT-PCR. Results　Compared with the blank group，the mixed bile salt group showed increased levels of IL-1β，IL-6，IL-18，NF-κB p65，NLRP3，Caspase-1 protein and mRNA expression（P<0.05）；Compared with the mixed bile salt group，the levels of IL-1β，IL-6，IL-18，NF-κB p65，NLRP3，Caspase-1 protein and mRNA expression were all reduced in the drug containing serum group，NF-κB inhibitor group，and the drug containing serum group+NF-κB inhibitor group（P<0.05）；Compared with the mixed bile salt group，the levels of IL-1β，IL-6，and IL-18，as well as the protein and mRNA levels of NF-κB p65，NLRP3，and Caspase-1，were all reduced in the drug containing serum group（P<0.05）；Compared with the drug containing serum group，the drug containing serum group+NF-κB inhibitor group showed increased levels of IL-1β，IL-6，and IL-18，as well as protein expression levels of NLRP3，and RNA expression levels of NF-κB p65（P<0.05）. Conclusion　Dachaihu Decoction-containing serum exerts hepatoprotective effects by inhibiting the NF-κB/NLRP3 signaling pathway，reducing the activation of Caspase-1 and inflammatory factors，alleviating pyroptosis，and regulating hepatocyte function.

Key words: Hepatocyte injury, Dachaihu decoction, NF-κB, NLRP3

摘要： 背景　梗阻性胆汁淤积性肝损伤（OCLI）发病率逐年升高，伴有病死率，现有技术手段以解除梗阻，但对于肝损伤的疗效有限，OCLI 的发生与进展与核因子 κB（NF-κB）通路及核苷酸结合寡聚化结构 3（NLRP3）引发的细胞焦亡密切相关，大柴胡汤临床改善肝功能疗效确切，且与 NF-κB 通路及 NLRP3 关系密切，为 OCLI 的中西医结合治疗提供新的思路。目的　基于 NF-κB/NLRP3 信号通路，探讨大柴胡汤含药血清对混合胆汁酸诱导的肝细胞损伤的疗效与机制。方法　2024 年 6—12 月，将 20 只雄性 SPF 级 SD 大鼠随机分为空白对照组和含药血清组，每组各 10 只。空白对照组持续 7 d 灌胃等体积 0.9% 氯化钠溶液，用于制备空白血清；含药血清组连续灌胃大柴胡汤，7 d 后采集血清，制备大柴胡汤含药血清。CCK-8 法筛选含药血清最佳干预浓度为 15%。将人血浆中含量最高的 6 种胆汁酸：胆酸、鹅脱氧胆酸、脱氧胆酸、石胆酸、熊脱氧胆酸及甘胺鹅脱氧胆酸在生理浓度范围内配制复合胆酸盐溶液，CCK-8 法筛选诱导 L02 细胞损伤的最佳胆酸盐剂量为 100×。将人正常 L02 肝细胞随机分为空白组、混合胆酸盐组、含药血清组、NF-κB 抑制剂组及含药血清组 +NF-κB 抑制剂组，药物干预 24 h 后，酶联免疫吸附法（ELISA）检测白介素（IL）-1β、IL-6 和 IL-18 水平；Western Blotting 法检测 NF-κB p65、NLRP3、Caspase-1 蛋白表达水平；实时定量 PCR 法（RT-PCR）检测 NF-κB p65、NLRP3、Caspase-1 的 mRNA 表达水平。结果　与空白组比较，混合胆酸盐组 IL-1β、IL-6、IL-18 水平，NF-κB p65、NLRP3、Caspase-1 蛋白及 mRNA 表达水平均升高（P<0.05）；与混合胆酸盐组比较，含药血清组、NF-κB 抑制剂组、含药血清组 +NF-κB 抑制剂组 IL-1β、IL-6、IL-18 水平，NF-κB p65、NLRP3、Caspase-1 蛋白及 mRNA 表达水平均降低（P<0.05）；与混合胆酸盐组相比，含药血清组中 IL-1β、IL-6 和 IL-18 水平，NF-κB p65、NLRP3、Caspase-1 蛋白及 mRNA 水平均降低（P<0.05）；与含药血清组相比，含药血清组 +NF-κB 抑制剂组中 IL-1β、IL-6 和 IL-18 水平，NLRP3 蛋白表达水平，NF-κB p65 mRNA 表达水平有所升高（P<0.05）。结论　大柴胡汤含药血清通过抑制 NF-κB/NLRP3 信号通路，降低 Caspase-1 及炎症因子活化，改善细胞焦亡，调节肝细胞功能，改善从而发挥保护肝细胞的作用。

关键词: 肝细胞损伤, 大柴胡汤, 核因子 κB, 核苷酸结合寡聚化结构3