Department of Vascular Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, China.
Endoscopy Center, China-Japan Union Hospital of Jilin University, Changchun 130033, China.
Life Sci. 2019 Apr 15;223:88-94. doi: 10.1016/j.lfs.2019.03.015. Epub 2019 Mar 8.
Gluaredoxin1 (GRX1) is an important protein of the cellular antioxidant defense system, but its role in renal epithelial cell injury caused by ischemia remains unclear. In this study, we aimed to gain insight into the role of GRX1 in HK-2 cells with oxygen glucose deprivation (OGD) injury, which served as an in vitro cell model of renal epithelial cell ischemic injury. We investigated the underlying regulation of GRX1, DJ-1, and HSP70 as well as the role of the GRX1/DJ-1/HSP70 signaling pathway in this model.
The protein and mRNA expressions were measured by Western blot and qRT-PCR assays, respectively. GRX1 was overexpressed by transfection of pcDNA.3.1-GRX1 and DJ-1 was inhibited by transfection with DJ-1 siRNA. Cell apoptosis, caspase-3 activity, lactate dehydrogenase (LDH) leakage, or superoxide dismutase (SOD) content was tested by the related detection kit. Reactive oxygen species (ROS) level was detected via carboxy-HDCF-DA.
We found that GRX1 was distinctly down-regulated in HK-2 cells after incubation under the OGD condition. GRX1 overexpression markedly constrained cell apoptosis, caspase-3 activity, LDH leakage, and the ROS level, while SOD content was elevated. GRX1 up-regulation increased DJ-1 and HSP70 protein expression, while DJ-1 inhibition significantly offset the effect of GRX1 overexpression on HSP70, indicating that GRX1 could regulate HSP70 via control of DJ-1. Moreover, we observed that HSP70 inhibition removed the constraints imposed by GRX1 overexpression on ROS level, LDH leakage, and caspase-3 activity.
Overall, this study showed that GRX1 minimizes cell injury and apoptosis in HK-2 cells under OGD conditions via regulation of DJ-1 and HSP70 expression.
谷氧还蛋白 1（GRX1）是细胞抗氧化防御系统的重要蛋白，但它在缺血引起的肾上皮细胞损伤中的作用尚不清楚。在这项研究中，我们旨在深入了解 GRX1 在氧葡萄糖剥夺（OGD）损伤的 HK-2 细胞中的作用，该细胞作为肾上皮细胞缺血性损伤的体外细胞模型。我们研究了 GRX1、DJ-1 和 HSP70 的潜在调节作用，以及该模型中 GRX1/DJ-1/HSP70 信号通路的作用。
通过 Western blot 和 qRT-PCR 测定分别测量蛋白质和 mRNA 的表达。通过 pcDNA.3.1-GRX1 转染过表达 GRX1，通过 DJ-1 siRNA 转染抑制 DJ-1。通过相关检测试剂盒检测细胞凋亡、caspase-3 活性、乳酸脱氢酶（LDH）漏出或超氧化物歧化酶（SOD）含量。通过羧基-HDCF-DA 检测活性氧（ROS）水平。
我们发现，在 OGD 条件下孵育后，HK-2 细胞中的 GRX1 明显下调。GRX1 过表达显著抑制细胞凋亡、caspase-3 活性、LDH 漏出和 ROS 水平，而 SOD 含量升高。GRX1 上调增加了 DJ-1 和 HSP70 蛋白表达，而 DJ-1 抑制显著抵消了 GRX1 过表达对 HSP70 的影响，表明 GRX1 可以通过控制 DJ-1 来调节 HSP70。此外，我们观察到 HSP70 抑制消除了 GRX1 过表达对 ROS 水平、LDH 漏出和 caspase-3 活性的抑制作用。
总的来说，这项研究表明，GRX1 通过调节 DJ-1 和 HSP70 的表达，最大限度地减少了 OGD 条件下 HK-2 细胞的细胞损伤和凋亡。
