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Chloride intracellular channel 4 participate in the protective effect of Ginkgolide B in MPP+ injured MN9D cells: insight from proteomic analysis.
Clinical Proteomics ( IF 2.8 ) Pub Date : 2020-09-05 , DOI: 10.1186/s12014-020-09295-6
Zili Feng 1 , Zhibin Zhu 1 , Wang Chen 1 , Yu Bai 1 , Daihua Hu 1 , Jia Cheng 1
Affiliation  

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. 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. 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. 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.

中文翻译:

氯化物细胞内通道 4 参与银杏内酯 B 在 MPP+ 损伤 MN9D 细胞中的保护作用:蛋白质组学分析的见解。

银杏内酯 B (GB) 是银杏叶的提取物,已被证明对许多神经系统疾病具有保护作用,包括帕金森病 (PD)。已努力合成靶向性更强、分子量更小的银杏内酯类似物和衍生物。在本研究中,合成了四种 GB 衍生物 (GBHC-1-GBHC-4),并评估了它们在 N-甲基-4-苯基吡啶 (MPP +) 损伤的 MN9D 多巴胺能神经元细胞系中的保护作用。此外,通过 iTRAQ 蛋白质组学分析了这些 GB 衍生物处理后的细胞反应机制。MN9D细胞用MPP + 处理以诱导PD的体外细胞模型。合成了四种GB衍生物(GBHC-1-GBHC-4),并通过CCK8法评估了它们对体外PD模型细胞的细胞活力和凋亡的保护作用,分别是荧光激活细胞分选和 DAPI 染色。使用等压标签进行相对和绝对定量 (iTRAQ) 标记技术检测用或不用 GB 和 GB 衍生物预处理的 MPP+ 损伤 MN9D 细胞的蛋白质组学特征。在 MPP+ 损伤的 MN9D 细胞中,用 GBHC-1-GBHC-4 预处理显着增加细胞活力并减弱细胞凋亡。使用蛋白质组学分析,我们鉴定了 GB 和 GB 衍生物处理后差异表达的蛋白质。氯化物细胞内通道 4 (CLIC4) 和“内质网中的蛋白质加工”途径参与了 GB 和 GBHC-4 的保护作用。GB和GBHC-4预处理可以显着逆转MPP+诱导的CLIC4表达和MN9D细胞从细胞质到细胞核的易位。定量比较蛋白质组学分析确定了与 GB 和 GB 衍生物相关的差异表达蛋白质。我们通过蛋白质印迹和免疫细胞化学测定进一步验证了 CLIC4 的表达。这种关于已识别途径和差异表达蛋白质(如 CLIC4)的生物信息为合成更有效和靶向 GB 衍生物以治疗神经系统疾病提供了更有针对性的方向。
更新日期:2020-09-07
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