PORIMIN: The key to (+)-Usnic acid-induced liver toxicity and oncotic cell death in normal human L02 liver cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Usnic acid (UA) is one of the well-known lichen metabolites that induces liver injury. It is mainly extracted from Usnea longissima and U. diffracta in China or from other lichens in other countries. U. longissima has been used as traditional Chinese medicine for treatment of cough, pain, indigestion, wound healing and infection. More than 20 incidences with hepatitis and liver failure have been reported by the US Food and Drug Administration since 2000. UA is an uncoupler of oxidative phosphorylation causing glutathione and ATP depletion. Previous histological studies observed extensive cell and organelle swellings accompanied with hydrotropic vacuolization of hepatocytes. AIM OF THE STUDY: This study was to investigate the mechanism of UA-induced liver toxicity in normal human L02 liver cells and ICR mice using various techniques, such as immunoblotting and siRNA transfection. MATERIALS AND METHODS: Assays were performed to evaluate the oxidative stress and levels of GSH, MDA and SOD. Double flouresencence staining was used for the detection of apoptotic cell death. The protein expressions, such as glutathione S transferase, glutathione reductase, glutathione peroxidase 4, catalase, c-Jun N-terminal protein kinase, caspases, gastamin-D and porimin were detected by Western blotting. Comparisons between transfected and non-transfected cells were applied for the elucidation of the role of porimin in UA-induced hepatotoxicity. Histopathological examination of mice liver tissue, serum total bilirubin and hepatic enzymes of alanine aminotransferase and aspatate aminotransferase were also studied. RESULTS: The protein expressions of glutathione reductase, glutathione S transferase and glutathione peroxidase-4 were increased significantly in normal human L02 liver cells. Catalase expression was diminished in dose-dependent manner. Moreover, (+)-UA did not induce the activation of caspase-3, caspase-1 or gasdermin-D. No evidence showed the occurrence of pyroptosis. However, the porimin expressions were increased significantly. In addition, (+)-UA caused no cytotoxicity in the porimin silencing L02 cells. CONCLUSIONS: In conclusion, (+)-UA induces oncotic L02 cell death via increasing protein porimin and the formation of irreversible membrane pores. This may be the potential research area for future investigation in different aspects especially bioactivity and toxicology.

Review: Usnic acid-induced hepatotoxicity and cell death.

Increasing prevalence of herbal and dietary supplement-induced hepatotoxicity has been reported worldwide. Usnic acid (UA) is a well-known hepatotoxin derived from lichens. Since 2000, more than 20 incident reports have been received by the US Food and Drug Administration after intake of UA containing dietary supplement resulting in severe complications. Scientists and clinicians have been studying the cause, prevention and treatment of UA-induced hepatotoxicity. It is now known that UA decouples oxidative phosphorylation, induces adenosine triphosphate (ATP) depletion, decreases glutathione (GSH), and induces oxidative stress markedly leading to lipid peroxidation and organelle stress. In addition, experimental rat liver tissues have shown massive vacuolization associated with cellular swellings. Additionally, various signaling pathways, such as c-JNK N-terminal kinase (JNK), store-operated calcium entry, nuclear erythroid 2-related factor 2 (Nrf2), and protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathways are stimulated by UA causing beneficial or harmful effects. Nevertheless, there are controversial issues, such as UA-induced inflammatory or anti-inflammatory responses, cytochrome P450 detoxifying UA into non-toxic or transforming UA into reactive metabolites, and unknown mechanism of the formation of vacuolization and membrane pore. This article focused on the previous and latest comprehensive putative mechanistic findings of UA-induced hepatotoxicity and cell death. New insights on controversial issues and future perspectives are also discussed and summarized.

Identification of photodegraded derivatives of usnic acid with improved toxicity profile and UVA/UVB protection in normal human L02 hepatocytes and epidermal melanocytes.

This study focused on the investigation of photodegradation of usnic acid (UA) which is a natural UV absorbing agent in lichens. Despite years of historical use in food supplement, traditional medicine or cosmetic products in many countries, liver toxicity has been found to be one of the severe and life threatening adverse effects in early 2000's. Such severe side effect has limited UA or its synthesized derivatives for further use clinically or commercially. In this study, extracted UA from Usnea longissima in methanol was exposed to natural sunlight for 21 days. Five photodegraded derivatives (1 to 5) with two new and three previously explored compounds were isolated and purified by column chromatography and preparative liquid chromatography. The structures of these derivatives were identified based on the data of nuclear magnetic resonance spectrum, mass spectrum, optical rotation, infrared spectrum, X-ray crystallography and/or electronic circulation dichroism. The cytotoxicity of (+)-UA and 2 to 5 in liver L02 cells and melanocytes were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Experimental results indicated that IC50 of (+)-UA in liver L02 cells and melanocytes were 24.4 and 6.9 µM respectively, while compound 2 to 5 have lower cytotoxicity with IC50 of 326.7, 1085.0, 62.7 and 152.4 µM in L02 cells and 87.7, 297.7, 60.2 and 85.0 µM in melanocytes respectively. Besides, (+)-UA and these derivatives were exposed to fix dosed of UVA or UVB. The anti-UVA/UVB activity was determined via Hoechst33342/propidium iodide double staining method, and quantified by computer linked fluorescence microscope equipped with CellsSense Dimension system. Based on analysis, Compound 2 to 5 captured prominent UVA/UVB protection capacity in both hepatocytes and melanocytes (p < .001). In addition, the effects of chemicals on tyrosinase were evaluated via Western Blot analysis. In terms of tyrosinase expression, only 2 showed significant stimulating effect (p < .05). However, the safe use of these derivatives cutaneously should be further studied. In conclusion, the photodegraded derivatives (2 to 5) of extracted UA have lower hepatotoxicity than (+)-UA and captured significant UV protection activities.