Realgar-Induced Neurotoxicity: Crosstalk Between the Autophagic Flux and the p62-NRF2 Feedback Loop Mediates p62 Accumulation to Promote Apoptosis.

Realgar is a traditional Chinese medicine that contains arsenic. It has been reported that the abuse of medicine-containing realgar has potential central nervous system (CNS) toxicity, but the toxicity mechanism has not been elucidated. In this study, we established an in vivo realgar exposure model and selected the end product of realgar metabolism, DMA, to treat SH-SY5Y cells in vitro. Many assays, including behavioral, analytical chemistry, and molecular biology, were used to elucidate the roles of the autophagic flux and the p62-NRF2 feedback loop in realgar-induced neurotoxicity. The results showed that arsenic could accumulate in the brain, causing cognitive impairment and anxiety-like behavior. Realgar impairs the ultrastructure of neurons, promotes apoptosis, perturbs autophagic flux homeostasis, amplifies the p62-NRF2 feedback loop, and leads to p62 accumulation. Further analysis showed that realgar promotes the formation of the Beclin1-Vps34 complex by activating JNK/c-Jun to induce autophagy and recruit p62. Meanwhile, realgar inhibits the activities of CTSB and CTSD and changes the acidity of lysosomes, leading to the inhibition of p62 degradation and p62 accumulation. Moreover, the amplified p62-NRF2 feedback loop is involved in the accumulation of p62. Its accumulation promotes neuronal apoptosis by upregulating the expression levels of Bax and cleaved caspase-9, resulting in neurotoxicity. Taken together, these data suggest that realgar can perturb the crosstalk between the autophagic flux and the p62-NRF2 feedback loop to mediate p62 accumulation, promote apoptosis, and induce neurotoxicity. Realgar promotes p62 accumulation to produce neurotoxicity by perturbing the autophagic flux and p62-NRF2 feedback loop crosstalk.

Crosstalk between autophagy and the Keap1-Nrf2-ARE pathway regulates realgar-induced neurotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar, the main component of which is As2S2 or As4S4 (≥90%), is a traditional Chinese natural medicine that has been used to treat carbuncles, furuncles, snake and insect bites, abdominal pain caused by parasitic worms, and epilepsy in China for many years. Because realgar contains arsenic, chronic or excessive use of single-flavor realgar and realgar-containing Chinese patent medicine can lead to drug-induced arsenic poisoning, but the exact mechanism underlying its toxicity to the central nervous system is unclear. AIM OF THE STUDY: The aim of this study was to clarify the mechanism of realgar-induced neurotoxicity and to investigate the effects of realgar on autophagy and the Keap1-Nrf2-ARE pathway. MATERIALS AND METHODS: We used rats treated with the autophagy inhibitor 3-methyladenine (3-MA) or adeno-associated virus (AAV2/9-r-shRNA-Sqstm1, sh-p62) to investigate realgar-induced neurotoxicity and explore the specific relationship between autophagy and the Keap1-Nrf2-ARE pathway (the Nrf2 pathway) in the cerebral cortex. Molecular docking analysis was used to assess the interactions among the Nrf2, p62 and Keap1 proteins. RESULTS: Our results showed that arsenic from realgar accumulated in the brain and blood to cause neuronal and synaptic damage, decrease exploratory behavior and spontaneous movement, and impair memory ability in rats. The mechanism may have involved realgar-mediated autophagy impairment and continuous activation of the Nrf2 pathway via the LC3-p62-Keap1-Nrf2 axis. However, because this activation of the Nrf2 pathway was not sufficient to counteract oxidative damage, apoptosis was aggravated in the cerebral cortex. CONCLUSIONS: This study revealed that autophagy, the Nrf2 pathway, and apoptosis are involved in realgar-induced central nervous system toxicity and identified p62 as the hub of the LC3-p62-Keap1-Nrf2 axis in the regulation of autophagy, the Nrf2 pathway, and apoptosis.