Plasma exosomes proteome profiling discovers protein markers associated with the therapeutic effect of Chaihu-Longgu-Muli decoction on temporal lobe epilepsy

Traditional Chinese medicine (TCM) uses Chaihu-Longgu-Muli decoction (CLMD) to alleviate disease, clear away heat, calm the mind, and temper excitation. It has been widely used for the therapy of neuropsychiatric disorders including epilepsy, dementia, anxiety, insomnia, and depression for several centuries in China. This study aims to analyze differentially expressed proteins (DEPs) in the plasma exosomes of patients with temporal lobe epilepsy (TLE) and after the Chaihu-Longgu-Muli Decoction (CLMD) therapy and to explore the biomarkers of TLE and the potential targets of CLMD in treating TLE. The plasma exosomes of normal people and patients with TLE before the treatment of oxcarbazepine (OXC) and combined treatment of OXC and CLMD (OXC.CLMD) were harvested. The exosomes were separated from plasma through ultracentrifugation and then identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. The DEPs were analyzed by proteomics and then subjected to gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The protein level of key genes was detected using Western blot. A lithium chloride-pilocarpine-induced epilepsy rat model was established and treated with OXC alone, OXC. CLMD, and CLMD alone (low dose and high dose). Neuronal injury in the hippocampal dentate gyrus and ribosomal protein L6 (RPL6) expression in the brain tissues were detected using H&E staining, Nissl staining, and Western blot. The proteomic analysis showed several DEPs were present among plasma exosomes in the four groups; DEPs were enriched in epilepsy-related function and pathway. Four key proteins were screened, including RPL6, Nucleolin (NCL), Apolipoprotein A1 (APOA1), and Lactate Dehydrogenase A (LDHA). Among them, RPL6, NCL, and LDHA protein levels were downregulated and APOA1 protein level was upregulated in the plasma exosomes of TLE patients. After OXC and OXC. CLMD treatment, the protein level of RPL6, NCL, and LDHA was increased, and the APOA1 protein level was decreased. Moreover, the RPL6 protein level was further elevated after OXC. CLMD treatment than that after OXC treatment. In the TLE rat model, neuronal degeneration and necrosis in the hippocampal dentate gyrus increased and RPL6 expression level decreased. After the treatment with OXC, OXC. CLMD, and CLMD alone, the degeneration and necrosis of neurons decreased, and the RPL6 expression level was increased; RPL6 upregulation was remarkably obvious after CLMD treatment. RPL6, NCL, LDHA and APOA1 are the DEPs in the plasma exosomes of patients with TLE before and after therapy. RPL6 might be a potential biomarker of CLMD in treating TLE.