Protective role of cinnabar and realgar in Hua-Feng-Dan against LPS plus rotenone-induced neurotoxicity and disturbance of gut microbiota in rats

Hua-Feng-Dan (HFD) is a traditional Chinese medicine used for neurological disorders. HFD contains cinnabar (HgS) and realgar (As4S4). The ethnopharmacological basis of cinnabar and realgar in HFD is not known. Aim of the study: To address the role of cinnabar and realgar in HFD-produced neuroprotection against neurodegenerative diseases and disturbance of gut microbiota. Lipopolysaccharide (LPS) plus rotenone (ROT)-elicited rat dopaminergic (DA) neuronal damage loss was performed as a Parkinson's disease animal model. Rats were given a single injection of LPS. Four months later, rats were challenged with the threshold dose of ROT. The clinical dose of HFD was administered via feed, starting from ROT administration for 46 days. Behavioral dysfunction was detected by rotarod and Y-maze tests. DA neuron loss and microglial activation were assessed via immunohistochemical staining and western bolt analysis. The colon content was collected to extract bacterial DNA followed by real-time PCR analysis with 16S rRNA primers. LPS plus ROT induced neurotoxicity, as evidenced by DA neuron loss in substantia nigra, impaired behavioral functions and increased microglial activation. HFD-original (containing 10% cinnabar and 10% realgar) rescued loss of DA neurons, improved behavioral dysfunction and attenuated microglial activation. Compared with HFD-original, HFD-reduced (3% cinnabar and 3% realgar) was also effective, but to be a less extent, while HFD-removed (without cinnabar and realgar) was ineffective. In analysis of gut microbiome, the increased Verrucomicrobiaceae and Lactobacteriaceae, and the decreased Enterobacteeriaceae by LPS plus ROT were ameliorated by HFD-original, and to be the less extent by HFD-reduced. Cinnabar and realgar are active ingredients in HFD to exert beneficial effects in a neurodegenerative model and gut microbiota. [Display omitted] •Hua-Feng-Dan (HFD) protected rats from LPS plus Rotenone-induced dopamine neuronal loss and behavioral dysfunction.•HFD ameliorated LPS plus Rotenone-induced microglial activation.•HFD attenuated LPS plus Rotenone-induced disruption of gut microbiome.•Cinnabar and realgar were the active ingredients in HFD to exert beneficial effects.