Miyoshi Ken-ichi, Okura Takafumi, Fukuoka Tomikazu, Higaki Jitsuo
Department of Integrated Medicine and Informatics, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan.
Clin Exp Nephrol. 2007 Mar;11(1):26-33. doi: 10.1007/s10157-006-0445-4. Epub 2007 Mar 28.
Interleukin (IL)-6, cyclooxygenase (COX)-2, and monocyte chemoattractant protein (MCP)-1 contribute to renal injury. The promoter regions of these genes contain CCAAT/enhancer-binding protein (C/EBP)-binding sites. In this study, we investigated the role of C/EBP-delta in mesangial cells (MCs).
In an in vivo study, anti-Thy 1.1 glomerulonephritis rats were generated and C/EBP-delta, IL-6, COX-2, and MCP-1 expressions were assessed by immunohistochemistry. In an in vitro study, cultured MCs were transfected with non-silencing (NS) short interfering RNA (siRNA) or C/EBP-delta siRNA. Subsequently, after stimulation with IL-1beta, C/EBP-delta, IL-6, COX-2, and MCP-1 mRNA expression levels were evaluated using real-time polymerase chain reaction (PCR). IL-6 concentration in the culture medium was determined by enzyme-linked immunosorbent assay. In addition, cell proliferative activity against IL-1beta or platelet-derived growth factor-BB was assessed by bromodeoxyuridine incorporation.
In the in vivo study, C/EBP-delta, IL-6, COX-2, and MCP-1 were expressed in the mesangial region of anti-Thy 1.1 glomerulonephritis rats on day 1. In the in vitro study, IL-1beta increased C/EBP-delta mRNA levels in NS siRNA-transfected MCs (7.3-fold), but no increase was evident in C/EBP-delta siRNA-transfected MCs. IL-6, COX-2, and MCP-1 mRNA levels in C/EBP-delta siRNA-transfected MCs were all lower than those in NS siRNA-transfected MCs (decreases of 57.7%, 85.7%, and 69.3%, respectively). The IL-6 concentration in the culture medium from C/EBP-delta siRNA transfected MCs (7.37 +/- 4.3 pg/ml) was also lower than that in the culture medium from NS siRNA-transfected MCs (25.2 +/- 3.4 pg/ml). Cell proliferative activity in C/EBP-delta siRNA-transfected MCs was lower than that in NS siRNA transfected MCs.
C/EBP-delta was induced in the mesangial region during the early stages of anti-Thy1.1 glomerulonephritis. C/EBP-delta contributes to inflammatory gene expression and MC proliferation.
白细胞介素（IL）-6、环氧化酶（COX）-2和单核细胞趋化蛋白（MCP）-1参与肾损伤。这些基因的启动子区域含有CCAAT/增强子结合蛋白（C/EBP）结合位点。在本研究中，我们调查了C/EBP-δ在系膜细胞（MCs）中的作用。
在体内研究中，制备抗Thy 1.1肾小球肾炎大鼠，通过免疫组织化学评估C/EBP-δ、IL-6、COX-2和MCP-1的表达。在体外研究中，用非沉默（NS）小干扰RNA（siRNA）或C/EBP-δ siRNA转染培养的MCs。随后，在用IL-1β刺激后，使用实时聚合酶链反应（PCR）评估C/EBP-δ、IL-6、COX-2和MCP-1 mRNA表达水平。通过酶联免疫吸附测定法测定培养基中的IL-6浓度。此外，通过溴脱氧尿苷掺入评估针对IL-1β或血小板衍生生长因子-BB的细胞增殖活性。
在体内研究中，抗Thy 1.1肾小球肾炎大鼠第1天系膜区表达C/EBP-δ、IL-6、COX-2和MCP-1。在体外研究中，IL-1β使NS siRNA转染的MCs中C/EBP-δ mRNA水平增加（7.3倍），但在C/EBP-δ siRNA转染的MCs中未见明显增加。C/EBP-δ siRNA转染的MCs中IL-6、COX-2和MCP-1 mRNA水平均低于NS siRNA转染的MCs（分别降低57.7%、85.7%和69.3%）。C/EBP-δ siRNA转染的MCs培养基中的IL-6浓度（7.37±4.3 pg/ml）也低于NS siRNA转染的MCs培养基中的浓度（25.2±3.4 pg/ml）。C/EBP-δ siRNA转染的MCs中的细胞增殖活性低于NS siRNA转染的MCs。
在抗Thy1.1肾小球肾炎早期，系膜区诱导表达C/EBP-δ。C/EBP-δ促进炎症基因表达和MC增殖。
