Niuhuang jiedu prescription alleviates realgar-induced dopaminergic and GABAergic neurotoxicity in Caenorhabditis elegans

Ethnopharmacological relevance

Niuhuang Jiedu (NHJD) is a Chinese medicine prescription containing realgar (As₂S₂), which is neurotoxic, and seven other traditional Chinese medicines (TCMs). However, whether the multiple TCMs contained in NHJD can mitigate the neurotoxicity of realgar is yet to be elucidated.

Aim of the study

This study aimed to investigate the effect of NHJD on realgar-induced neurotoxicity and elucidate its underlying mechanisms.

Material and methods

Caenorhabditis elegans was treated with realgar suspension (1–8 mg/mL) for 48 h. Transgenic nematode strains labeled with green fluorescent protein were used to evaluate neuronal structural damage. Locomotion and perception behaviors were assessed by measuring head thrashes, body bends, and chemotaxis. Oxidative stress was determined by detecting reactive oxygen species (ROS), lipofuscin, and glutathione-S-transferase-4 (GST-4) levels. Subsequently, a quantitative reverse transcription–polymerase chain reaction was employed to analyze gene expression associated with oxidative stress, whereas the role of the p38 MAPK pathway was investigated using KU25 nematodes. Finally, arsenic species in the nematodes were estimated using high-performance liquid chromatography–atomic fluorescence spectrometry. Data analysis was performed using analysis of variance or rank tests with SPSS 26.0.

Results

Treatment of nematodes with 8 mg/mL realgar resulted in significant damage to dopaminergic and GABAergic neurons, impaired locomotor and perceptual behavior (at 4–8 mg/mL), and induced oxidative damage (at 2–8 mg/mL). A neuron-defective model was established using 8 mg/mL realgar to gauge the effects of NHJD on realgar-induced damage. The findings indicated that NHJD, containing the same dose of realgar, significantly alleviated neuronal damage and neurobehavioral impairment and increased ROS, lipofuscin, and GST-4 levels. In addition, NHJD reduced the expressions of oxidative stress-related genes (gst-4, skn-1, gcs-1, gss-1, ctl-2, ctl-3, and sod-1) compared with realgar alone. Nonetheless, significant differences in skn-1 expression, neurobehavior, ROS, or lipofuscin levels were not observed between the realgar and NHJD groups in KU25 nematodes. Moreover, arsenic methylation metabolites were not identified in the nematodes.

Conclusions

The multiple TCMs contained in NHJD effectively mitigated realgar-induced dopaminergic and GABAergic neurotoxicity in nematodes, and pmk-1 may play a crucial role in NHJD's alleviating realgar-induced neurotoxicity via the p38 MAPK signaling pathway.