Xiaohuang Qudan decoction alleviates ANIT-induced cholestatic liver injury by inhibiting the JAK2/STAT3 pathway and regulating TH17/Treg

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.