Yang Jing, Su Nan, Du Xiaolan, Chen Lin
State Key Laboratory of Trauma, Burns and Combined Injury, Center of Bone Metabolism and Repair, Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China.
Cell Mol Biol Lett. 2014 Dec;19(4):611-22. doi: 10.2478/s11658-014-0216-2. Epub 2014 Oct 29.
Bone displays suppressed osteogenesis in inflammatory diseases such as sepsis and rheumatoid arthritis. However, the underlying mechanisms have not yet been clearly explained. To identify the gene expression patterns in the bone, we performed Affymetrix Mouse Genome 430 2.0 Array with RNA isolated from mouse femurs 4 h after lipopolysaccharide (LPS) administration. The gene expressions were confirmed with real-time PCR. The serum concentration of the N-terminal propeptide of type I collagen (PINP), a bone-formation marker, was determined using ELISA. A total of 1003 transcripts were upregulated and 159 transcripts were downregulated (more than twofold upregulation or downregulation). Increased expression levels of the inflammation-related genes interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were confirmed from in the period 4 h to 72 h after LPS administration using real-time PCR. Gene ontogene analysis found four bone-related categories involved in four biological processes: system development, osteoclast differentiation, ossification and bone development. These processes involved 25 upregulated genes. In the KEGG database, we further analyzed the transforming growth factor β (TGF-β) pathway, which is strongly related to osteogenesis. The upregulated bone morphogenetic protein 2 (BMP2) and downregulated inhibitor of DNA binding 4 (Id4) expressions were further confirmed by real-time PCR after LPS stimulation. The osteoblast function was determined through examination of the expression levels of core binding factor 1 (Cbfa1) and osteocalcin (OC) in bone tissues and serum PINP from 4 h to 72 h after LPS administration. The expressions of OC and Cbfa1 decreased 6 h after administration (p < 0.05). Significantly suppressed PINP levels were observed in the later stage (from 8 h to 72 h, p < 0.05) but not in the early stage (4 h or 6 h, p > 0.05) of LPS stimulation. The results of this study suggest that LPS induces elevated expressions of skeletal system development- and osteoclast differentiation-related genes and inflammation genes at an early stage in the bone. The perturbed functions of these two groups of genes may lead to a faint change in osteogenesis at an early stage of LPS stimulation. Suppressed bone formation was found at later stages in response to LPS stimulation.
在脓毒症和类风湿性关节炎等炎症性疾病中，骨骼表现出成骨作用受到抑制。然而，其潜在机制尚未得到明确解释。为了确定骨骼中的基因表达模式，我们在给予脂多糖（LPS）4小时后，从小鼠股骨中分离RNA，进行了Affymetrix小鼠基因组430 2.0芯片检测。通过实时PCR对基因表达进行了确认。使用酶联免疫吸附测定法（ELISA）测定了骨形成标志物I型胶原N端前肽（PINP）的血清浓度。共有1003个转录本上调，159个转录本下调（上调或下调超过两倍）。使用实时PCR证实，在给予LPS后4小时至72小时期间，炎症相关基因白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）和肿瘤坏死因子α（TNF-α）的表达水平升高。基因本体分析发现四个与骨骼相关的类别涉及四个生物学过程：系统发育、破骨细胞分化、骨化和骨骼发育。这些过程涉及25个上调基因。在京都基因与基因组百科全书（KEGG）数据库中，我们进一步分析了与成骨密切相关的转化生长因子β（TGF-β）信号通路。LPS刺激后，通过实时PCR进一步证实了骨形态发生蛋白2（BMP2）的上调和DNA结合抑制因子4（Id4）的下调。通过检测给予LPS后4小时至72小时骨组织中核心结合因子1（Cbfa1）和骨钙素（OC）的表达水平以及血清PINP，确定成骨细胞功能。给予LPS 6小时后，OC和Cbfa1的表达下降（p<0.05）。在LPS刺激后期（8小时至72小时，p<0.05）观察到PINP水平显著降低，但在早期（4小时或6小时，p>0.05）未观察到。本研究结果表明，LPS在早期可诱导骨骼中骨骼系统发育和破骨细胞分化相关基因以及炎症基因表达升高。这两组基因功能的紊乱可能导致LPS刺激早期成骨作用出现轻微变化。在LPS刺激后期发现骨形成受到抑制。
