Tan Zhangkui, Chen Lifeng, Ye Zhiqin, Lu Qiping
Department of Rheumatology and Immunology, General Hospital of Central Theater Command of the People's Liberation Army, Wuhan 430070, China.
Department of Rheumatology, Hubei Provincial Hospital of Traditional Chinese Medicine, affiliated with Hubei University of Chinese Medicine, Wuhan 430061, China.
Chin J Nat Med. 2025 Apr;23(4):457-470. doi: 10.1016/S1875-5364(25)60854-5.
Xiaohuang Qudan decoction (XHQDD) is a classical traditional Chinese medicine (TCM) formula widely used in the treatment of cholestatic liver injury. Despite its widespread use, the protective mechanism of XHQDD against cholestatic liver injury remains incompletely understood. The aim of this study was to investigate whether XHQDD mediates its beneficial effects by inhibiting the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway and regulating TH17/Treg balance. To this end, the researchers used Sprague-Dawley (SD) rats and established a cholestatic liver injury model by oral administration of alpha-naphthylisothiocyanate (ANIT). The experimental group was divided into six groups: Control (CON), ANIT, ursodeoxycholic acid (UDCA), XHQDD-low dose (XHQDD-L) group, XHQDD-medium dose (XHQDD-M) group, and XHQDD-high dose (XHQDD-H) groups. Then, after 7 d of treatment, various tests were performed to verify the results. Firstly, XHQDD and its drug-containing serum were analyzed by ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UPLC-MS/MS), and 14 blood-entry components were identified. Then, bile flow was monitored and found to be significantly reduced in the model group, which was significantly reversed in the UDCA and XHQDD groups. To further assess ANIT-induced liver injury, hematoxylin and eosin (H&E) and Sirius red staining, alongside transmission electron microscopy (TEM), were employed to observe liver tissues, revealing hepatocellular injury, cholestasis, and hepatic fibrotic changes. Serum inflammatory factors and liver injury indicators were assessed using enzyme-linked immunosorbent assay (ELISA), indicating an inflammatory state in ANIT-induced liver injury rats. The expression levels of JAK2/STAT3-related genes and proteins in liver and intestinal tissues were measured via quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, immunofluorescence (IF) staining, and Western blottting (WB) assays. These studies revealed that the inflammatory state of liver-injured rats was inextricably linked to the inflammatory cascade associated with the JAK2/STAT3 pathway and that XHQDD may exert anti-inflammatory efficacy by inhibiting the JAK2/STAT3 pathway. Flow cytometry was used to determine the percentage of T helper 17 (Th17)/regulatory T (Treg) cells in serum and hepatocytes, and it was further found that XHQDD was able to regulate Th17/Treg immune homeostasis in liver-injured rats. The findings suggest that XHQDD markedly alleviates inflammation in ANIT rats, potentially treating cholestasis and liver injury through JAK2/STAT3 inhibition and Th17/Treg balance regulation.
小黄祛疸汤（XHQDD）是一种广泛用于治疗胆汁淤积性肝损伤的经典中药方剂。尽管其应用广泛，但XHQDD对胆汁淤积性肝损伤的保护机制仍未完全明确。本研究旨在探讨XHQDD是否通过抑制Janus激酶2（JAK2）/信号转导和转录激活因子3（STAT3）通路并调节TH17/Treg平衡来发挥其有益作用。为此，研究人员使用Sprague-Dawley（SD）大鼠，通过口服α-萘异硫氰酸酯（ANIT）建立胆汁淤积性肝损伤模型。实验组分为六组：对照组（CON）、ANIT组、熊去氧胆酸（UDCA）组、XHQDD低剂量（XHQDD-L）组、XHQDD中剂量（XHQDD-M）组和XHQDD高剂量（XHQDD-H）组。然后，在治疗7天后，进行各种测试以验证结果。首先，通过超高效液相色谱-质谱/质谱（UPLC-MS/MS）分析XHQDD及其含药血清，鉴定出14种入血成分。接着监测胆汁流量，发现模型组胆汁流量显著降低，而UDCA组和XHQDD组胆汁流量显著逆转。为进一步评估ANIT诱导的肝损伤，采用苏木精-伊红（H&E）和天狼星红染色以及透射电子显微镜（TEM）观察肝组织，发现肝细胞损伤、胆汁淤积和肝纤维化改变。使用酶联免疫吸附测定（ELISA）评估血清炎症因子和肝损伤指标，表明ANIT诱导的肝损伤大鼠处于炎症状态。通过定量逆转录聚合酶链反应（qRT-PCR）、免疫组织化学、免疫荧光（IF）染色和蛋白质免疫印迹（WB）分析测定肝和肠组织中JAK2/STAT3相关基因和蛋白的表达水平。这些研究表明，肝损伤大鼠的炎症状态与JAK2/STAT3通路相关的炎症级联反应密切相关，且XHQDD可能通过抑制JAK2/STAT3通路发挥抗炎作用。使用流式细胞术测定血清和肝细胞中辅助性T细胞17（Th17）/调节性T（Treg）细胞的百分比，进一步发现XHQDD能够调节肝损伤大鼠的Th17/Treg免疫稳态。研究结果表明，XHQDD可显著减轻ANIT大鼠的炎症，可能通过抑制JAK2/STAT3和调节Th17/Treg平衡来治疗胆汁淤积和肝损伤。
