Gao Li-jie, Li Chong-hui, Wang Jiang-huai, Huang Zhi-qiang
Hepatobiliary Surgery Institute, PLA General Hospital, Beijing 100853, China.
Zhongguo Wei Zhong Bing Ji Jiu Yi Xue. 2011 May;23(5):267-70.
To approach the nuclear factor-ΚB (NF-ΚB) nuclear translocation mechanism in bacterial lipoprotein (BLP) tolerance.
Human monocytic THP-1 cells were first pretreated with 10, 100, 1 000 ng/ml BLP for 20 hours to induce BLP tolerance. Then THP-1 cells without BLP pretreatment (control group) or with BLP pretreatment (tolerance group) were stimulated with 0, 10, 100, 1 000 ng/ml BLP again for 6 hours. The tumor necrosis factor-α (TNF-α) content in culture medium was measured by enzyme linked immunosorbent assay (ELISA) in order to determine the most suitable BLP pretreatment and stimulation concentration. Western blotting was used to detect the protein level, nuclear translocation and phosphorylation of NF-ΚB p50 and p65 in the cells of control and tolerance groups treated with respective conditions for 0, 0.5, 1, 2 and 6 hours.
In control group BLP stimulation (10, 100, 1 000 ng/ml) could induce THP-1 activation and TNF-α production (pg/ml: 184.86±32.51, 3 215.88±167.09, 6 042.96±245.37) in a dose-dependent manner. In tolerance group, 100 ng/ml BLP pretreatment resulted in almost complete inhibition of TNF-α production as induced by 101 000 ng/ml BLP stimulation. Therefore, 100 ng/ml BLP pretreatment and 1 000 ng/ml stimulation were selected for following cell treatment. Western blotting analysis showed that there was an increase of p50 protein level in BLP-tolerant cells comparing with control group (0 hour: 542.9±15.6 vs. 272.8±13.2, 0.5 hour: 558.0±16.9 vs. 236.4±11.8, 1 hour: 524.7±17.5 vs. 211.6±9.8, 2 hours: 584.9±15.6 vs. 222.4±12.3, all P<0.01), whereas the p65 protein level was similar between the two groups. BLP stimulation also induced the nuclear translocation of p50 and p65 in control group (1-hour p50: 344.2±13.6 vs. 79.0±5.2, p65: 78.4±4.5 vs. 0, both P<0.05), but not in tolerance group. In addition, the phosphorylation of p65 at serine 536 was induced after BLP stimulation in control THP-1 cells (0.5 hour: 0.67±0.08 vs. 0.04±0.01, 1 hour: 0.71±0.11 vs. 0.04±0.01, both P<0.05), but this change was not detected in BLP-tolerant cells.
It was found that in BLP-tolerant cells, the expression of inhibitory subunit p50 was increased and the nuclear translocation and phosphorylation of p65 with trans-activation ability was inhibited. These changes are likely responsible for the reduced gene expression of NF-ΚB dependent genes in BLP-tolerant cells.
探讨细菌脂蛋白（BLP）耐受中核因子-κB（NF-κB）核转位机制。
人单核细胞THP-1细胞先用10、100、1000 ng/ml BLP预处理20小时以诱导BLP耐受。然后，未用BLP预处理的THP-1细胞（对照组）或经BLP预处理的THP-1细胞（耐受组）再次分别用0、10、100、1000 ng/ml BLP刺激6小时。采用酶联免疫吸附测定（ELISA）法检测培养基中肿瘤坏死因子-α（TNF-α）含量，以确定最适宜的BLP预处理及刺激浓度。采用蛋白质印迹法检测对照组和耐受组细胞在各自条件下处理0、0.5、1、2和6小时后NF-κB p50和p65的蛋白水平、核转位及磷酸化情况。
在对照组中，BLP刺激（10、100、1000 ng/ml）可呈剂量依赖性诱导THP-1激活及TNF-α产生（pg/ml：184.86±32.51、3215.88±167.09、6042.96±245.37）。在耐受组中，100 ng/ml BLP预处理几乎完全抑制了1000 ng/ml BLP刺激诱导的TNF-α产生。因此，选择100 ng/ml BLP预处理及1000 ng/ml刺激用于后续细胞处理。蛋白质印迹分析显示，与对照组相比，BLP耐受细胞中p50蛋白水平升高（0小时：542.9±15.6 vs. 272.8±13.2，0.5小时：558.0±16.9 vs. 236.4±11.8，1小时：524.7±17.5 vs. 211.6±9.8，2小时：584.9±15.6 vs. 222.4±12.3，均P<0.01），而两组间p65蛋白水平相似。BLP刺激也可诱导对照组中p50和p65的核转位（1小时p50：344.2±13.6 vs. 79.0±5.2，p65：78.4±4.5 vs. 0，均P<0.05），但在耐受组中未诱导。此外，在对照THP-1细胞中，BLP刺激后可诱导p65丝氨酸536位点磷酸化（0.5小时：0.67±0.08 vs. 0.04±0.01，1小时：0.71±0.11 vs. 0.04±0.01，均P<0.05），但在BLP耐受细胞中未检测到这种变化。
发现在BLP耐受细胞中，抑制亚基p50的表达增加，具有反式激活能力的p65的核转位及磷酸化受到抑制。这些变化可能是BLP耐受细胞中NF-κB依赖性基因表达降低的原因。
