Zanthoxylum armatum DC. extract induces liver injury via autophagy suppression and oxidative damage by activation of mTOR/ULK1 pathway.

Zanthoxylum armatum DC. (ZADC) has anti-inflammatory, antioxidative, and antibacterial effects. The cytotoxicity of methanol extract of Zanthoxylum armatum DC. (MZADC) has been reported for BRL 3 A cell lines. However, whether MZADC can induce liver damage in vivo remains unclear. Therefore, it is essential to explore whether ZADC causes liver injury and, if the results confirm hepatotoxicity, to further study the potential mechanisms for the in-vitro cytotoxicity of the BRL 3 A cell lines. In vivo, different doses (0.346, 0.519, and 1.038 g/kg/day) of MZADC treatment were given by intragastric administration among male Sprague Dawley rats for 28 days. Levels of serum alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) in the high dose group increased. Steatosis and focal necrosis were found in liver cells in rats in the high dose group. In vitro, BRL 3 A cells were cultivated with MZADC at different concentrations (30, 50, and 70 µg/mL) for 24 h. The cell viability, the number of autophagosomes, and the expression levels of LC3 and Beclin-1 were on a decreasing trend. Besides, proportions of p-mTOR/mTOR and p-ULK1/ULK1 increased. Meanwhile, reactive oxygen species (ROS) accumulation and the content of malondialdehyde (MDA) were on the rise while the activity of superoxide dismutase (SOD) and the content of glutathione (GSH) was on the decline. This research suggests that MZADC may cause rats liver injury and inhibit autophagy in BRL 3 A cells by the mTOR/ULK1 pathway, and further induce intracellular oxidative damage.

Activation of ATM/Chk2 by Zanthoxylum armatum DC extract induces DNA damage and G1/S phase arrest in BRL 3A cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum armatum DC is a traditional medicinal plant. It is widely used in clinical treatment and disease prevention in China, India and other regions. Modern studies have reported the phytotoxicity, cytotoxicity and the animal toxicity of Zanthoxylum armatum DC, and the damage of genetic material has been observed in plants, but the detailed mechanism has not been explored. Besides, the toxicity of normal mammalian cells has not been evaluated. AIM OF THE STUDY: To evaluate the effects and underlying mechanism of genetic material damage in BRL 3A cells induced by Zanthoxylum armatum DC. MATERIALS AND METHODS: Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry was used for identification of compounds in methanol extract of Zanthoxylum armatum DC. BRL 3A cells were incubated with different concentrations of methanol extract of Zanthoxylum armatum DC (24 h). The cytotoxicity of extract was assessed with cell viability, LDH release rate, and ROS production. The damage of genetic material was assessed with OTM value of comet cells, cell cycle and the expression levels of p-ATM, p- Chk2, Cdc25A, and CDK2. RESULTS: Ultra-High Performance Liquid Chromatography and Orbitrap High-Resolution Mass Spectrometry investigation revealed the presence of compounds belonging to flavonoid, fatty acid and alkaloid groups. The viability of BRL 3A cells was reduced in a time-dose dependent manner treated by methanol extract of Zanthoxylum armatum DC. It increased LDH release rate and ROS production, activated the DNA double strand damage marker of γH2AX and produced comet cells. In addition, methanol extract of Zanthoxylum armatum DC caused ATM-mediated DNA damage, further phosphorylated Chk2, inhibited cell cycle related proteins, and arrested the G1/S cycle. CONCLUSIONS: Methanol extract of Zanthoxylum armatum DC induces DNA damage and further leads G1/S cell cycle arrest by triggering oxidative stress in the BRL 3A cells. This study provides some useful evidences for its development as an antitumor drug via activation of ATM/Chk2.

Aconitine induces autophagy via activating oxidative DNA damage-mediated AMPK/ULK1 signaling pathway in H9c2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Aconitum species, with a medicinal history of 2000 years, was traditionally used in the treatment of rheumatism, arthritis, bruises, and pains. However, many studies have reported that Aconitum species can cause arrhythmia in experimental animals, resulting in myocardial fibrosis and cardiomyocyte damage. Cardiotoxicity is the main toxic effect of aconitine, but the detailed mechanism remains unclear. AIM OF THE STUDY: This study aimed to explore the effects and underlying mechanism of autophagy in H9c2 cardiomyocytes induced by aconitine. MATERIALS AND METHODS: H9c2 cells were incubated with different concentrations of aconitine for 24 h, and the intervention sections were pretreated with various inhibitors for 1 h. The effects of aconitine on the oxidative DNA damage, autophagy and viability of H9c2 cells were evaluated by flow cytometry, confocal microscopy, enzyme-linked immunosorbent assay and Western blot. RESULTS: In H9c2 cells, the cell viability declined, LDH release rate, the number of autophagosomes, protein expression levels of LC3 and Beclin-1 increased significantly after 24 h of aconitine incubation. The pretreatment of autophagy inhibitor 3-MA decreased markedly autophagosomes and protein expression levels of LC3 and Beclin-1, which suggested that aconitine could induce cell autophagy. The significant increase of ROS and 8-OHdG showed that aconitine could cause oxidative DNA damage through ROS accumulation. Meanwhile, treatment of aconitine dramatically increased AMPKThr172 and ULK1Ser317 phosphorylation, and Compound C inhibited AMPKThr172 and ULK1Ser317 phosphorylation, which proved that aconitine induced autophagy via AMPK activation mediated ULK1 phosphorylation. Antioxidant NAC significantly reduced LDH, ROS and 8-OHdG, inhibited the phosphorylation of AMPKThr172 and ULK1Ser317, and down-regulated autophagosomes and proteins expression levels of LC3 and Beclin-1. Consequently, the inhibition of oxidative DNA damage and AMPK/ULK1 signaling pathway alleviated the aconitine-induced autophagic death of H9c2 cells. CONCLUSIONS: These results showed that aconitine induces autophagy of H9c2 cardiomyocytes by activating AMPK/ULK1 signaling pathway mediated by oxidative DNA damage. The autophagy induced by aconitine in cardiomyocytes is dependent on the activation of the AMPK pathway, which may provide novel insights into the prevention of aconitine-related toxicity.