Sarin is a potent organophosphorus nerve agent that causes cognitive dysfunction, but its underlying molecular mechanisms are poorly understood. In this study, a rat model of repeated low-level sarin exposure was established using the subcutaneous injection of 0.4 × LD50 for 21 consecutive days. Sarin-exposed rats showed persistent learning and memory impairment and reduced hippocampal dendritic spine density. A whole-transcriptome analysis was applied to study the mechanism of sarin-induced cognitive impairment, and a total of 1035 differentially expressed mRNA (DEmRNA), including 44 DEmiRNA, 305 DElncRNA, and 412 DEcircRNA, were found in the hippocampus of sarin-treated rats. According to Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and Protein–Protein Interaction (PPI) analysis, these DERNAs were mainly involved in neuronal synaptic plasticity and were related to the pathogenesis of neurodegenerative diseases. The circRNA/lncRNA–miRNA–mRNA ceRNA network was constructed, in which Circ_Fmn1, miR-741-3p, miR-764-3p, miR-871-3p, KIF1A, PTPN11, SYN1, and MT-CO3 formed one circuit, and Circ_Cacna1c, miR-10b-5p, miR-18a-5p, CACNA1C, PRKCD, and RASGRP1 constituted another circuit. The balance between the two circuits was crucial for maintaining synaptic plasticity and may be the regulatory mechanism by which sarin causes cognitive impairment. Our study reveals the ceRNA regulation mechanism of sarin exposure for the first time and provides new insights into the molecular mechanisms of other organophosphorus toxicants.
Background: The incidence of sleep disorders is more than 27% in the worldwide, and the development of novel sleep drugs that target GABAA receptors is of great interest. Traditional drug screening methods restrict the discovery of lead compounds, the high-throughput screening system is a powerful means for the lead compounds discovery of sleep drug. Methods: The GABAA1-CHO cell line stably expressing α1β2γ2L was constituted by co-transfection of α1, β2 and γ2L subunits into CHO-T-Rex cells. The high-throughput screening method of membrane potential targeting GABAAR was established and optimized. The optimized method was used to screen the compound library, and the compounds with high activity were obtained. The active compounds were confirmed in vitro by electrophysiological detection technique, and the sleep effects of compounds in vivo were detected by pentobarbital sodium sleep model in mice. Results: A stable cell line expressing human GABAA1 receptor in CHO-T-Rex cells was generated and used to establish a functional high-throughput screening assay based on the measurement of membrane potential changes in living cells by fluorometric imaging plate reader (FLIPR). The assay was further used to detect the dose-effect relationships of tool compounds, the EC50 valus of agonist GABA (137.42±26.31 nM), positive allosteric modulator diazepam (3.22±0.73 μM), and antagonist gabazine (0.16±0.04 μM), blocking agents bicuculine (0.47±0.06 μM), PTX (6.39±1.17 μM). In the meanwhile, the compounds were screened from a compound library (10000) by the membrane potential dye assay. Selected 4 active compounds were further identified for their EC50 values in vitro by electrophysiological method, the EC50 values of 4 compounds were further determined as 1.37±0.43 μM, 0.69±0.17 μM, 0.77±0.16 μM, and 1.62±0.29 μM, respectively. Furthermore, the pentobarbital sleep rate and the sleep time of mice pretreated with 4 active compounds by oral administration were significantly increased compared with mice pretreated with negative control in vivo experiment. Conclusion: We successfully generated a stable CHO cell line expressing human GABAA1 by induced expression strategy which decreased cytotoxicity. Then developed an efficient membrane potential detection method for high-throughput screening, the assay based on the stable cell line could distinguish different types of GABAA1 modulators, which would be an effective in vitro system to screen the GABAAR-targeted compounds. Compared with the patch clamp electrophysiological detection method, the membrane potential detection method has higher detection flux for compounds and higher detection sensitivity for active compounds.
This work aimed to assess whether mitochondrial damage in the liver induced by subacute soman exposure is caused by peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) and whether PGC-1α regulates mitochondrial respiratory chain damage. Toxicity mechanism research may provide theoretical support for developing anti-toxic drugs in the future. First, a soman animal model was established in male Sprague-Dawley (SD) rats by subcutaneous soman injection. Then, liver damage was biochemically evaluated, and acetylcholinesterase (AChE) activity was also determined. Transmission electron microscopy (TEM) was performed to examine liver mitochondrial damage, and high-resolution respirometry was carried out for assessing mitochondrial respiration function. In addition, complex I-IV levels were quantitatively evaluated in isolated liver mitochondria by enzyme-linked immunosorbent assay (ELISA). PGC-1α levels were detected with a Jess capillary-based immunoassay device. Finally, oxidative stress was analyzed by quantifying superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG), and reactive oxygen species (ROS) levels. Repeated low-level soman exposure did not alter AChE activity, while increasing morphological damage of liver mitochondria and liver enzyme levels in rat homogenates. Complex I, II and I II activities were 2.33, 4.95, and 5.22 times lower after treatment compared with the control group, respectively. Among complexes I-IV, I-III decreased significantly (p < 0.05), and PGC-1α levels were 1.82 times lower after soman exposure than in the control group. Subacute soman exposure significantly increased mitochondrial ROS production, which may cause oxidate stress. These findings indicated dysregulated mitochondrial energy metabolism involves PGC-1α protein expression imbalance, revealing non-cholinergic mechanisms for soman toxicity.
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