Department of Anesthesiology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230032, China.
Department of Biochemistry and Molecular Biology, Anhui Medical University, Hefei, 230022, China; The Key Laboratory of Zoonoses and Pathogen Biology Anhui, Hefei, China.
Brain Res Bull. 2019 Jul;149:129-136. doi: 10.1016/j.brainresbull.2019.04.010. Epub 2019 Apr 16.
BACKGROUND: Patients suffering from diabetes mellitus experience poor outcomes after ischemic stroke. RTN1-C, ER-associated proteins localized in endoplasmic reticulum (ER) membrane, plays an important role in ER stress-induced apoptosis and regulates cellular susceptibility to different apoptosis pathways. Overexpression of RTN1-C can aggravate cerebral ischemia/reperfusion injury (IRI). ER stress plays a crucial role in hyperglycemia-aggravated cerebral IRI. In this study, we aimed to investigate the role of RTN1-C in high glucose-aggravated OGD/R-induced cell damage. MATERIALS AND METHODS: N2a cells and primary neuronal cells were cultured in normal glucose or high glucose conditions. We used a model of oxygen-glucose deprivation followed by reoxygenation (OGD/R). RTN1-C shRNA was used to knock down RTN1-C. The chemical chaperone 4-phenylbutyric acid (4-PBA) is a low molecular weight fatty acid that has the ability to stabilize mutant proteins and facilitate their folding, was used to inhibited ER stress. Cell viability and apoptosis were measured by CCK-8 and flow cytometry assays. The contents of ER stress-associated proteins, such as GRP78, cleaved caspase-12, CHOP and cleaved caspase-3, were detected by western blot. RESULTS: High glucose significantly decreased cell viability and increased cell apoptosis in OGD/R-treated neuronal cells. The contents of GRP78, cleaved caspase-12, CHOP and cleaved caspase-3 under high glucose conditions were higher than those under normal glucose conditions after OGD/R. Importantly, inhibition of ER stress by 4-PBA alleviated the high glucose-aggravated OGD/R-induced cell damage. Here, we demonstrated that high glucose increases RTN1-C expression in OGD/R-treated cells. More importantly, knockdown of RTN1-C expression dramatically reversed the high glucose-aggravated change in cell viability and apoptosis and relieved ER stress in OGD/R-treated cells. CONCLUSIONS: High glucose significantly increases RTN1-C expression in OGD/R-treated cells. RTN1-C affects high glucose-treated OGD/R cells by exacerbating ER stress.
背景：患有糖尿病的患者在经历缺血性中风后会出现较差的预后。RTN1-C 是内质网（ER）膜中 ER 相关蛋白，在 ER 应激诱导的细胞凋亡中发挥重要作用，并调节细胞对不同凋亡途径的敏感性。RTN1-C 的过表达会加重脑缺血再灌注损伤（IRI）。内质网应激在高血糖加重的脑 IRI 中起着至关重要的作用。在本研究中，我们旨在探讨 RTN1-C 在高糖加重的 OGD/R 诱导的细胞损伤中的作用。
材料和方法：在正常葡萄糖或高葡萄糖条件下培养 N2a 细胞和原代神经元细胞。我们使用氧葡萄糖剥夺后再氧合（OGD/R）的模型。使用 RTN1-C shRNA 敲低 RTN1-C。化学伴侣 4-苯丁酸（4-PBA）是一种低分子量脂肪酸，具有稳定突变蛋白并促进其折叠的能力，用于抑制内质网应激。通过 CCK-8 和流式细胞术测定细胞活力和细胞凋亡。通过 Western blot 检测内质网应激相关蛋白，如 GRP78、裂解的 caspase-12、CHOP 和裂解的 caspase-3 的含量。
结果：高葡萄糖显著降低 OGD/R 处理的神经元细胞的细胞活力并增加细胞凋亡。在 OGD/R 后，高葡萄糖条件下的 GRP78、裂解的 caspase-12、CHOP 和裂解的 caspase-3 含量高于正常葡萄糖条件下的含量。重要的是，4-PBA 抑制内质网应激可减轻高葡萄糖加重的 OGD/R 诱导的细胞损伤。在这里，我们证明高葡萄糖增加 OGD/R 处理细胞中的 RTN1-C 表达。更重要的是，RTN1-C 表达的敲低显著逆转了高葡萄糖加重的细胞活力和凋亡变化，并缓解了 OGD/R 处理细胞的内质网应激。
结论：高葡萄糖显著增加 OGD/R 处理细胞中的 RTN1-C 表达。RTN1-C 通过加重内质网应激影响高葡萄糖处理的 OGD/R 细胞。
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