Chloride intracellular channel 4 participate in the protective effect of Ginkgolide B in MPP+ injured MN9D cells: insight from proteomic analysis

BACKGROUND: Ginkgolide B (GB), the extract of G. biloba leaves, has been shown to be protective against many neurological disorders, including Parkinson’s disease (PD). Efforts have been made to synthesized ginkgolides analogs and derivatives with more targeted and smaller molecular weight. In the present study, four GB derivatives (GBHC-1-GBHC-4) were synthesized, and their protective roles in N-methyl-4-phenylpyridinium (MPP +) injured MN9D dopaminergic neuronal cell line were evaluated. Also, cell response mechanisms upon these GB derivatives treatment were analyzed by iTRAQ proteomics. METHODS: MN9D cells were treated with MPP + to induce in vitro cell models of PD. Four GB derivatives (GBHC-1-GBHC-4) were synthesized, and their protective roles on cell viability and apoptosis in in vitro PD model cells were evaluated by CCK8 assay, fluorescence-activated cell sorting and DAPI staining, respectively. The proteomic profiles of MPP+ injured MN9D cells pretreated with or without GB and GB derivatives were detected using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique. RESULTS: Pretreatment with GBHC-1-GBHC-4 noticeably increased cell viability and attenuated cell apoptosis in MPP+ -injured MN9D cells. Using proteomic analysis, we identified differentially expressed proteins upon GB and GB derivatives treatment. Chloride intracellular channel 4 (CLIC4) and “protein processing in endoplasmic reticulum” pathways participated in the protective roles of GB and GBHC-4. GB and GBHC-4 pretreatment could significantly reverse MPP+ -induced CLIC4 expression and translocation from cytoplasm to nucleus of MN9D cells. CONCLUSIONS: Quantitative comparative proteomic analysis identified differentially expressed proteins associated with GB and GB derivatives. We further verified the expression of CLIC4 by western blotting and immunocytochemistry assay. This bio-information on the identified pathways and differentially expressed proteins such as CLIC4 provide more targeted directions for the synthesis of more effective and targeted GB derivatives for the treatment of neurological disorders.