Yang Mengxue, Gan Hua, Shen Qing
Department of Nephrology, First Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2012 Jun;37(6):578-85. doi: 10.3969/j.issn.1672-7347.2012.06.007.
To characterize the expression of Toll-like receptor 4 (TLR4) in monocytes of diabetic nephropathy (DN) patients and the response of TLR4 to lipopolysaccharide (LPS), and, further, to explore the potential effects of inflammatory immune response in DN.
Thirty DN patients with uremia, ten early-type 2 DN patients, and twenty healthy volunteers were enrolled for the determination of TLR4 expression in monocytes by using peripheral blood flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated and subjected to 1 μg/mL LPS for 24 h. Monocytes were collected to assay NF-κB p65 and Notch1 expression by Western blot, with immuneofluorescence detection. Serum and supernatants were sampled for the determination of interleukin-6 (IL-6) concentration by using ELISA. Serum C-reactive protein (CRP) level was determined by using the immunoturbidimetry.
Compared with the normal control, type 2 DN uremic patients had a significantly higher TLR4 fluorescence-blot intensities (FI), and serum CRP and IL-6 levels [TLR4 FI: DN uremia patients 2.8±0.9; early type 2 DN patients 3.4 ±0.7; healthy subjects 1.6±0.7. IL-6 concentration: DN uremia patients (84.8±20.7) pg/mL; early type 2 DN patients (63.20±14.4) pg/mL; healthy subjects (11.0±2.0) pg/mL. CRP concentraton: DN uremia patients (5.4±2.8) mg/L; early type 2 DN patients (3.7±1.7) mg/L; healthy subjects (1.7±0.7) mg/L. P<0.01 for any DN-group vs control]. In early type 2 DN patients, following exposure to LPS, PBMCs showed a significant upregulation in TLR4 and NF-κB p65 expression and a remarked increase in serum IL-6 level (all P<0.05), and NF-κB p65 transfer to the nucleus is enhanced. Notch1 protein expression was not significantly altered in any group.
A disturbance in proinflammatory CD14(+)CD16(+) monocytes occurs in type 2 DN patients. Such immunological dysfunction may be related to activation in NF-κB/TLR4 signaling pathways, and have nothing to do with the Notch1 signaling pathway.
研究糖尿病肾病（DN）患者单核细胞中Toll样受体4（TLR4）的表达及TLR4对脂多糖（LPS）的反应，进一步探讨炎症免疫反应在DN中的潜在作用。
选取30例尿毒症期DN患者、10例早期2型DN患者和20名健康志愿者，采用外周血流式细胞术检测单核细胞中TLR4的表达。分离外周血单个核细胞（PBMCs），用1μg/mL LPS处理24小时。收集单核细胞，采用蛋白质免疫印迹法检测NF-κB p65和Notch1的表达，并进行免疫荧光检测。采集血清和上清液，采用酶联免疫吸附测定法（ELISA）检测白细胞介素-6（IL-6）浓度。采用免疫比浊法测定血清C反应蛋白（CRP）水平。
与正常对照组相比，2型DN尿毒症患者的TLR4荧光强度（FI）、血清CRP和IL-6水平显著升高[TLR4 FI：DN尿毒症患者2.8±0.9；早期2型DN患者3.4±0.7；健康受试者1.6±0.7。IL-6浓度：DN尿毒症患者（84.8±20.7）pg/mL；早期2型DN患者（63.20±14.4）pg/mL；健康受试者（11.0±2.0）pg/mL。CRP浓度：DN尿毒症患者（5.4±2.8）mg/L；早期2型DN患者（3.7±1.7）mg/L；健康受试者（1.7±0.7）mg/L。各DN组与对照组相比，P<0.01]。在早期2型DN患者中，PBMCs经LPS刺激后，TLR4和NF-κB p65表达显著上调，血清IL-6水平显著升高（均P<0.05），且NF-κB p65向细胞核的转移增强。各实验组Notch1蛋白表达均无明显变化。
2型DN患者促炎性CD14(+)CD16(+)单核细胞存在紊乱。这种免疫功能障碍可能与NF-κB/TLR4信号通路的激活有关，而与Notch1信号通路无关。
