Under Chronic, High-Dose Administration of Wenjing Decoction, the Activation of the Hepatic Nrf2/HO-1 Pathway Mediates Increased Bilirubin Levels and Associated Reversible Neurologic Dysfunction in Mice.

Background: Hyperbilirubinemia is a prevalent manifestation of drug-induced liver injury, which can result in neurological complications in severe cases. Despite the extensive clinical application of Wenjing Decoction (WJD) over a millennium, there is a significant lack of systematic nonclinical safety evaluation data. Furthermore, the toxicity characteristics and mechanisms associated with long-term high-dose exposure remain unclear. Aim: This study seeks to conduct a systematic assessment of the toxicological characteristics of WJD via a 28-day repeated-dose toxicity investigation. Specifically, our focus lies in examining its impacts on hepatic and neurological functions, as well as elucidating its underlying molecular mechanisms. Methods: A bioinformatics approach was initially utilized to predict the potential molecular targets of WJD associated with hyperbilirubinemia. Subsequently, Kunming mice were randomized into either a control group or a high-dose WJD treatment group (18.75 g/kg, equivalent to 20 times the standard human daily dose). Animals received intragastric administration of the respective treatments daily for 28 consecutive days. A subset of animals was assigned to a recovery phase to monitor the reversibility of any observed effects. Serum biochemical parameters, including total bilirubin (TBIL) and unconjugated bilirubin (UCB), hepatic oxidative stress markers such as glutathione disulfide (GSSG) and malondialdehyde (MDA), and neurobehavioral performance (evaluated via pole climbing and shuttle box tests) were systematically measured. Furthermore, Western blot, quantitative real-time polymerase chain reaction (qRT-PCR), and immunohistochemistry were employed to analyze the expression levels of the Nrf2/Heme oxygenase-1 (HO-1) signaling pathway and related inflammatory cytokines. Finally, comprehensive histopathological examinations were conducted on both liver and brain tissues. Results: Bioinformatics predictions indicate that HO-1 is a key target. Animal experiments demonstrate that the administration of WJD results in a significant increase in serum TBIL and UCB in mice, exhibiting characteristics of nonhemolytic hyperbilirubinemia. Mild oxidative stress, characterized by increased GSSG and MDA levels, along with elevated ALT activity, occurs in the liver. This is accompanied by the activation of the Nrf2/HO-1 pathway and the upregulation of inflammatory factors such as IL-6, IL-1β, and TNF-α; however, no significant histopathological damage is observed. Regarding the nervous system, mice in the administration group show a decrease in anal temperature, impaired motor coordination, and abnormal avoidance behavior. Although the expression of HO-1 in brain tissue is downregulated, no organic lesions are detected in the brain. All of the aforementioned abnormal indicators can be reversed during the recovery period following drug withdrawal. Conclusion: Long-term and high-dose exposure to WJD can induce hyperbilirubinemia in mice by activating the hepatic Nrf2/HO-1 signaling pathway, as well as causing mild hepatic oxidative damage and neurobehavioral abnormalities. This toxic reaction is reversible, indicating that when WJD is used clinically for extended periods or at high doses, careful monitoring of bilirubin metabolism and related functional indicators is essential.