Imidazole Alkaloids Epiisopilosine and Epiisopiloturine Attenuate Acetaminophen‐Induced Liver Toxicity in Mice via Autophagy Modulation and Anti‐Inflammatory Effects

There is increasing interest in natural metabolites, such as alkaloids, due to their potential in treating liver diseases, including acetaminophen (APAP)-induced hepatotoxicity. Alkaloids are known to modulate autophagy, a mechanism associated with liver protection. Epiisopilosine (EPIIS) and epiisopiloturine (EPI), imidazole alkaloids derived from Pilocarpus microphyllus (Jaborandi), exhibit anti-inflammatory and hepatic immunomodulatory effects. Therefore, this study aimed to compare the hepatoprotective and autophagy-modulating effects of EPI and EPIIS in a murine model of APAP-induced hepatotoxicity. In the experimental design, male BALB/c mice received APAP (750 mg/kg, i.p.) to induce hepatotoxicity, followed by phosphate-buffered saline (PBS), N-acetylcysteine (NAC-318 mg/kg, i.p.), or alkaloids (0.3, 1, or 3 mg/kg, i.p.) 30 min later. To assess the involvement of autophagy, hydroxychloroquine (HCQ; 80 mg/kg, i.p.), an autophagy inhibitor, was administered 2 h before APAP. Treatment with EPI, EPIIS, or NAC significantly reduced liver toxicity (p < 0.05). APAP-treated mice exhibited marked centrilobular necrosis, which was markedly reduced following the treatment with Jaborandi alkaloids. Furthermore, EPIIS and EPI significantly reduced the inflammatory and oxidative markers. Administration of HCQ 2 h before APAP abolished these effects. Western blotting analysis revealed increased LC3B expression, a marker of autophagy, in the hepatic tissues of the treated mice, indicating that EPIIS and EPI modulate autophagy. Molecular docking analysis suggested potential interactions between the alkaloids and CXCL10, a chemokine linked to inflammation and autophagy inhibition. These findings demonstrate that EPIIS and EPI protect against APAP-induced hepatotoxicity in mice, potentially by modulating autophagy and reducing inflammation.