Division of Medical Biotechnology, Department of Medical Laboratory Sciences, School of Paramedical Sciences, Shiraz University of Medical Sciences, Meshkin-Fam St., Shiraz, Iran.
Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
Mol Biol Rep. 2022 Feb;49(2):1103-1111. doi: 10.1007/s11033-021-06935-4. Epub 2021 Nov 14.
The kynurenine pathway (KP) can be involved in the pathogenesis of neurodegenerative diseases and excessive neurotoxic metabolite production. This study aimed to evaluate the effects of overexpression of murine 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (Acmsd) gene in inflammatory conditions in RAW 264.7 cell line to present more information about the effect of this gene on inflammatory conditions and the KP cycle.
The coding sequence of the Acmsd gene was cloned into pCMV6-AC-IRES-GFP expression vector with a green fluorescent protein (GFP) marker. To simulate inflammatory conditions, RAW 264.7 macrophage cells were stimulated by Lipopolysaccharide (LPS) 24 h before transfection, and transfected by Polyethyleneimine (PEI) with constructed plasmids expressing the Acmsd gene. The effect of Acmsd gene expression level on murine Interferon-gamma (Ifn-γ) and murine Indoleamine 2,3-dioxygenase 1 (Ido1) gene expression level was investigated by Real-Time PCR. According to the results of this study, good transfection efficiency was observed 72 h after transfection, and Acmsd expression level increased 29-fold (P < 0.001) in transfected LPS-stimulated cells compared to the control group (LPS-stimulated cells that were not transfected). Additionally, increased Acmsd expression level significantly down-regulated Ifn-γ (P < 0.001) and Ido1 (P < 0.01) expression level in transfected LPS-stimulated cells compared to LPS-stimulated cells.
Acmsd gene overexpression in inflammatory conditions can reduce the expression levels of the Ido1 gene, and its regulator, Ifn-γ. Consequently, it may be considered as a novel regulatory factor in the KP balance.
犬尿氨酸途径（KP）可能参与神经退行性疾病的发病机制和过多神经毒性代谢产物的产生。本研究旨在评估在 RAW 264.7 细胞系的炎症条件下过表达鼠 2-氨基-3-羧基戊烯酸-6-半醛脱羧酶（Acmsd）基因的影响，以提供更多关于该基因对炎症条件和 KP 循环影响的信息。
Acmsd 基因的编码序列被克隆到 pCMV6-AC-IRES-GFP 表达载体中，带有绿色荧光蛋白（GFP）标记。为了模拟炎症条件，在转染前 24 小时用脂多糖（LPS）刺激 RAW 264.7 巨噬细胞，并用聚乙二烯亚胺（PEI）转染表达 Acmsd 基因的构建质粒。通过实时 PCR 研究 Acmsd 基因表达水平对鼠干扰素-γ（Ifn-γ）和鼠吲哚胺 2,3-双加氧酶 1（Ido1）基因表达水平的影响。根据本研究的结果，转染后 72 小时观察到良好的转染效率，与对照组（未转染的 LPS 刺激细胞）相比，转染的 LPS 刺激细胞中的 Acmsd 表达水平增加了 29 倍（P<0.001）。此外，与 LPS 刺激细胞相比，Acmsd 表达水平的增加显著下调了转染的 LPS 刺激细胞中的 Ifn-γ（P<0.001）和 Ido1（P<0.01）表达水平。
在炎症条件下过表达 Acmsd 基因可以降低 Ido1 基因及其调节剂 Ifn-γ的表达水平。因此，它可以被认为是 KP 平衡的一个新的调节因子。
