Acute liver injury (ALI) is a clinical syndrome characterized by jaundice, ascites, coagulopathy, or hepatic encephalopathy [1]. It results from abrupt impairment of liver function within a short timeframe and can stem from various causes such as chemical exposure, alcohol consumption, pathogens, parasites, and autoimmune factors [2]. A subset of ALI cases progresses to the advanced stage known as acute liver failure (ALF), which carries a high mortality rate [3]. ALI exhibits a diverse range of etiologies, including alcoholic liver injury, drug-induced liver injury, and viral hepatitis [4]. Globally, drug intoxication stands as the predominant cause of ALI. In the United States and the United Kingdom, approximately 57% and 51% of ALF cases, respectively, are attributed to drug overdoses, with acetaminophen (APAP) overdose being the primary culprit [5]. Currently, N-acetylcysteine (NAC) serves as the primary clinical intervention for APAP-induced ALI. While NAC, as a precursor of l-cysteine, can enhance glutathione biosynthesis and act as a potent free radical scavenger, effectively mitigating liver damage, its prolonged use may hinder hepatocyte regeneration and impede subsequent recovery of liver function [6]. Furthermore, in cases where patients do not respond favorably to NAC therapy, liver transplantation remains the sole recourse [7]. Therefore, the quest for safer and more universally applicable strategies for the prevention and treatment of ALI holds immense clinical significance.

Traditional Chinese medicine encompasses several active ingredients known for their hepatoprotective properties and ability to mitigate liver tissue damage [8]. These compounds include flavonoids, terpenoids, organic acids, and alkaloids. Commonly used in TCM for targeted treatment are diammonium glycyrrhizinate [9], ursodeoxycholic acid [10], and silymarin [11]. However, their administration often comes with adverse effects like gastrointestinal reactions and secretion dysfunction [12]. Thus, there is an urgent imperative to explore safer and more efficacious drugs for liver injury inhibition. Citri Reticulatae Pericarpium (CRP), derived from the dried and mature peel of Citrus reticulata ‘Chachi,’ is a traditional authentic medicinal herb native to the southern Lingnan region of China [13]. CRP is recognized for its therapeutic functions, including qi regulation, spleen invigoration, alleviation of stomach vomiting, and liver soothing [14]. It is frequently employed in clinical practice to address symptoms such as vomiting, diarrhea, excessive cough, and phlegm due to dietary insufficiency [15]. Studies have demonstrated the effectiveness of CRP as the primary ingredient in liver damage treatment [16]. Research has spotlighted "Chaihu Shugan Powder” a classic Chinese medicine formula for liver disease treatment that features CRP as a key ingredient [17]. CRP contains various active constituents, including volatile oils, alkaloids and flavonoids. Among these, flavonoids like Nobiletin (NOB), Tangeretin (TANG), Naringenin (NIN), and Naringin (NRG) are prominent [18]. However, to date, the precise role of CRP's active ingredients in preventing and treating ALI, along with the specific molecular mechanisms involved, remains insufficiently elucidated, warranting further investigation.

In this study, we employed a network pharmacology approach to analyze and forecast the active constituents in CRP associated with hepatoprotective effects and to uncover potential molecular mechanisms for treating ALI. We also analyzed key targets. Our primary focus was on investigating the hepatoprotective properties of NIN, a bioactive compound found in the traditional Chinese medicine CRP, using the acetaminophen APAP-induced drug-induced liver injury model as our principal research subject. Our investigation spanned both in vivo and in vitro settings, with the aim of shedding light on the hepatoprotective effects and relevant molecular pathways of NIN. These findings provide essential preliminary experimental data for a deeper understanding of the molecular mechanisms governing CRP's active ingredients in regulating ALI.