Citrate Synthase (Cs) gene mutation (locus ahL4) has been found to play an important role in progressive hearing loss of A/J mice. HEI-OC1 cells have been widely used as an in vitro system to study cellular and molecular mechanisms related to hearing lose. We previously reported the increased apoptosis and the accumulation of reactive oxygen species in shRNACs-1429 cells, a Cs low-expressed cell model from HEI-OCI. The details of the mechanism of ROS production and apoptosis mediated by the abnormal expression of Cs needed to research furtherly. iTRAQ proteomics was utilized to detect the differentially expressed proteins (DEPs) caused by low expression of Cs. The GO and KEGG pathways analysis were performed for annotation of the differentially expressed proteins. Protein–protein interaction network was constructed by STRING online database. Immunoblotting was utilized to confirm the protein levels of the the differentially expressed proteins. The differentially expressed proteins were significantly enriched in various signaling pathways mainly related to mitochondrial dysfunction diseases including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, et al. Most noteworthy, the oxidative phosphorylation pathway was most significantly suppressed in the shRNACs-1429 cells,, in which a total of 10 differentially expressed proteins were enriched and were all downregulated by the abnormal expression of Cs. The downregulations of Ndufb5, Ndufv1 and Uqcrb were confirmed by immunoblotting. Meanwhile, the ATP levels of shRNACs-1429 cells were also reduced. These results suggest that low level expression of Cs induces the inhibition of oxidative phosphorylation pathway, which is responsible for the high level production of reactive oxygen species and low level of ATP, leading to the apoptosis of cochlear cells. This study may provide new theories for understanding and therapy of progressive hearing loss.

中文翻译：

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下调柠檬酸合酶对HEI-OC1细胞氧化磷酸化信号通路的影响

已发现柠檬酸合酶 (Cs) 基因突变（位点 ahL4）在 A/J 小鼠的进行性听力损失中起重要作用。HEI-OC1 细胞已被广泛用作体外系统来研究与听力损失相关的细胞和分子机制。我们之前报道了 shRNACs-1429 细胞中细胞凋亡增加和活性氧的积累，这是一种来自 HEI-OCI 的 Cs 低表达细胞模型。Cs异常表达介导的ROS产生和细胞凋亡的机制细节有待进一步研究。iTRAQ 蛋白质组学用于检测由 Cs 低表达引起的差异表达蛋白 (DEP)。进行 GO 和 KEGG 通路分析以注释差异表达的蛋白质。蛋白质-蛋白质相互作用网络由STRING在线数据库构建。免疫印迹用于确认差异表达蛋白质的蛋白质水平。差异表达的蛋白质在主要与线粒体功能障碍疾病相关的各种信号通路中显着富集，包括帕金森病、阿尔茨海默病、亨廷顿病等。最值得注意的是，在 shRNACs-1429 细胞中，氧化磷酸化途径受到最显着抑制，其中总共富集了 10 种差异表达的蛋白质，并且都因 Cs 的异常表达而下调。通过免疫印迹证实 Ndufb5、Ndufv1 和 Uqcrb 的下调。同时，shRNACs-1429细胞的ATP水平也降低。这些结果表明 Cs 的低水平表达诱导了氧化磷酸化途径的抑制，它负责高水平产生活性氧和低水平的ATP，导致耳蜗细胞凋亡。这项研究可能为理解和治疗进行性听力损失提供新的理论。