Yinhuang oral liquid (YOL) protects against APAP-driven hepatotoxicity by enhancing autophagy and inhibiting NLRP3 inflammasome via the activation of Nrf2-Keap1 pathway

This study aims to investigate the mechanism of YOL alleviated drug-induced liver injury (DILI). Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS) was used to screen potential bioactive ingredients of YOL, while molecular docking was performed to identify target proteins. An in vitro model of DILI was established by exposing BRL3A to APAP to elucidate the protective effects of YOL. Thirteen major compounds with peak areas exceeding 1% were identified in YOL, mainly including flavonoids (26.78%), phenylpropanoids (16.80%), carbohydrates and glycosides (11.61%), terpenes (10.25%), amino acids, peptides and their derivatives (7.10%), and others types. Molecular docking suggested that Baicalin, Wogonoside, Oroxylin A-7-O-glucuronide, and Cryptochlorogenic acid showed extremely high affinity to Nrf2. In APAP-treated BRL3A cells, YOL improved the cell viability and morphology, alleviated oxidative damage by reducing malondialdehyde (MDA) and reactive oxygen species (ROS) levels, and restored mitochondrial membrane potential (MMP). YOL also rescued impaired autophagy by elevating the LC3II/I ratio, p-mTOR, Beclin-1, Atg7 and Atg4B expression, while reducing mTOR level. In addition, YOL mitigated inflammatory damage by suppressing NLRP3 inflammasome activation, evidenced by decreased levels of NLRP3 and cysteinyl aspartate specific proteinase-1 (caspase-1) proteins, as well as decreased mRNA levels of NLRP3, IL-1β and IL-6. Furthermore, YOL activated the Nrf2-Keap1 signaling pathway by promoting Nrf2 nuclear translocation and HO-1 expression, alongside downregulating of p62 and Keap1 protein levels. YOL also enhanced autophagy, mitigated oxidative damage and NLRP3 inflammasome activation in BRL3A Nrf2-KD cells. YOL alleviates APAP-induced hepatotoxicity by enhancing autophagy and suppressing NLRP3 inflammasome activation through the upregulation of the Nrf2-Keap1 pathway. These findings provide mechanistic evidence supporting the therapeutic potential of YOL for the management of DILI.