Hwang Hyun Sook, Park In Young, Kim Hyun Ah, Choi Soo Young
Cell Physiol Biochem. 2017;41(1):252-264. doi: 10.1159/000456090. Epub 2016 Jan 23.
The protein transduction domain (PTD) enables therapeutic proteins to directly penetrate the membranes of cells and tissues, and has been increasingly utilized. Glutaredoxin-1 (GRX-1) is an endogenous antioxidant enzyme involved in the cellular redox homeostasis system. In this study, we investigated whether PEP-1-GRX-1, a fusion protein of GRX-1 and PEP-1 peptide, a PTD, could suppress catabolic responses in primary human articular chondrocytes and a mouse carrageenan-induced paw edema model.
Human articular chondrocytes were isolated enzymatically from articular cartilage and cultured in a monolayer. The transduction efficiency of PEP-1-GRX-1 into articular chondrocytes was measured by western blot and immunohistochemistry. The effects of PEP-1-GRX-1 on matrix metalloproteinases (MMPs) and catabolic factor expression in interleukin (IL)-1β- and lipopolysaccharide (LPS)-treated chondrocytes were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and western blot. The effect of PEP-1-GRX1 on the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) signaling pathway were also analyzed by western blot. Finally, the inhibitory effect of PEP-1-GRX-1 on MMP-13 production was measured in vivo in a mouse carrageenan-induced paw edema model.
PEP-1-GRX-1 significantly penetrated into human chondrocytes and mouse cartilage, whereas GRX-1 did not. PEP-1-GRX-1 significantly suppressed MMP-13 expression and nitric oxide (NO) production in LPS-stimulated chondrocytes, and NO production in IL-1β-stimulated chondrocytes, compared with GRX-1. In addition, PEP-1-GRX-1 decreased IL-1β- and LPS-induced activation of MAPK and NF-κB. In the mouse model of carrageenan-induced paw edema, PEP-1-GRX-1 significantly suppressed carrageenan-induced MMP-13 production as well as paw edema.
These results demonstrate that PEP-1-GRX-1 can be transduced efficiently in vitro and in vivo into human chondrocytes and mouse cartilage tissue and downregulate catabolic responses in chondrocytes by inhibiting the MAPK and NF-κB pathway. PEP-1-GRX-1 thus has the potential to reduce catabolic responses in chondrocytes and cartilage.
蛋白质转导结构域（PTD）能使治疗性蛋白质直接穿透细胞和组织的膜，且其应用日益广泛。谷氧还蛋白-1（GRX-1）是一种参与细胞氧化还原稳态系统的内源性抗氧化酶。在本研究中，我们探究了GRX-1与一种PTD即PEP-1肽的融合蛋白PEP-1-GRX-1是否能抑制原代人关节软骨细胞中的分解代谢反应以及在小鼠角叉菜胶诱导的爪肿胀模型中发挥作用。
从关节软骨中酶解分离出人关节软骨细胞并进行单层培养。通过蛋白质印迹法和免疫组织化学法检测PEP-1-GRX-1转入关节软骨细胞的效率。通过实时定量逆转录-聚合酶链反应和蛋白质印迹法分析PEP-1-GRX-1对白细胞介素（IL）-1β和脂多糖（LPS）处理的软骨细胞中基质金属蛋白酶（MMPs）和分解代谢因子表达的影响。还通过蛋白质印迹法分析PEP-1-GRX1对丝裂原活化蛋白激酶（MAPK）和核因子κB（NF-κB）信号通路的影响。最后，在小鼠角叉菜胶诱导的爪肿胀模型中体内检测PEP-1-GRX-1对MMP-13产生的抑制作用。
PEP-1-GRX-1能显著穿透进入人软骨细胞和小鼠软骨，而GRX-1不能。与GRX-1相比，PEP-1-GRX-1能显著抑制LPS刺激的软骨细胞中MMP-13的表达和一氧化氮（NO）的产生，以及IL-1β刺激的软骨细胞中NO的产生。此外，PEP-1-GRX-1降低了IL-1β和LPS诱导的MAPK和NF-κB的激活。在角叉菜胶诱导的爪肿胀小鼠模型中，PEP-1-GRX-1显著抑制了角叉菜胶诱导的MMP-13产生以及爪肿胀。
这些结果表明，PEP-1-GRX-1在体外和体内均能有效地转入人软骨细胞和小鼠软骨组织，并通过抑制MAPK和NF-κB途径下调软骨细胞中的分解代谢反应。因此，PEP-1-GRX-1有潜力减少软骨细胞和软骨中的分解代谢反应。
