Department of Periodontology and Oral Biology, Boston University School of Dental Medicine, Boston, MA, USA.
J Bone Miner Res. 2010 Jul;25(7):1604-15. doi: 10.1002/jbmr.59.
To gain insight into the effect of diabetes on fracture healing, experiments were carried out focusing on chondrocyte apoptosis during the transition from cartilage to bone. Type 1 diabetes was induced in mice by multiple low-dose streptozotocin injections, and simple transverse fractures of the tibia or femur was carried out. Large-scale transcriptional profiling and gene set enrichment analysis were performed to examine apoptotic pathways on total RNA isolated from fracture calluses on days 12, 16, and 22, a period of endochondral bone formation when cartilage is resorbed and chondrocyte numbers decrease. Tumor necrosis factor alpha (TNF-alpha) protein levels were assessed by ELISA and caspase-3 by bioactivity assay. The role of TNF was examined by treating mice with the TNF-specific inhibitor pegsunercept. In vitro studies investigated the proapoptotic transcription factor FOXO1 in regulating TNF-induced apoptosis of chondrogenic ATDC5 and C3H10T1/2 cells as representative of differentiated chondrocytes, which are important during endochondral ossification. mRNA profiling revealed an upregulation of gene sets related to apoptosis in the diabetic group on day 16 when cartilage resorption is active but not day 12 or day 22. This coincided with elevated TNF-alpha protein levels, chondrocyte apoptosis, enhanced caspase-3 activity, and increased FOXO1 nuclear translocation (p < .05). Inhibition of TNF significantly reduced these parameters in the diabetic mice but not in normoglycemic control mice (p < .05). Silencing FOXO1 using siRNA in vitro significantly reduced TNF-induced apoptosis and caspase activity in differentiated chondrocytes. The mRNA levels of the proapoptotic genes caspase-3, caspase-8, caspase-9, and TRAIL were significantly reduced with silencing of FOXO1 in chondrocytic cells. Inhibiting caspase-8 and caspase-9 significantly reduced TNF-induced apoptosis in chondrogenic cells. These results suggest that diabetes causes an upregulation of proapoptotic genes during the transition from cartilage to bone in fracture healing. Diabetes increased chondrocyte apoptosis through a mechanism that involved enhanced production of TNF-alpha, which stimulates chondrocyte apoptosis and upregulates mRNA levels of apoptotic genes through FOXO1 activation.
为了深入了解糖尿病对骨折愈合的影响，实验专注于软骨向骨转化过程中的软骨细胞凋亡。通过多次小剂量链脲佐菌素注射诱导小鼠 1 型糖尿病，并对胫骨或股骨进行简单的横向骨折。在软骨吸收和软骨细胞数量减少的软骨内成骨时期（骨折痂形成的第 12、16 和 22 天），从分离的骨折痂总 RNA 上进行大规模转录谱和基因集富集分析，以检查凋亡途径。通过 ELISA 评估肿瘤坏死因子-α (TNF-α) 蛋白水平，通过生物活性测定法评估半胱氨酸天冬氨酸蛋白酶-3 (caspase-3)。通过用 TNF 特异性抑制剂 pegsunercept 处理小鼠来检查 TNF 的作用。体外研究探讨了促凋亡转录因子 FOXO1 在调节 TNF 诱导的软骨细胞 ATDC5 和 C3H10T1/2 细胞凋亡中的作用，这些细胞是软骨内骨化过程中的重要细胞。mRNA 谱分析显示，在软骨吸收活跃的第 16 天，糖尿病组中与凋亡相关的基因集上调，但在第 12 天或第 22 天则没有上调。这与 TNF-α 蛋白水平升高、软骨细胞凋亡、caspase-3 活性增强以及 FOXO1 核易位增加（p <.05）相一致。在糖尿病小鼠中抑制 TNF 可显著降低这些参数，但在血糖正常的对照小鼠中则没有（p <.05）。在体外使用 siRNA 沉默 FOXO1 可显著降低分化软骨细胞中 TNF 诱导的凋亡和 caspase 活性。在软骨细胞中沉默 FOXO1 可显著降低促凋亡基因 caspase-3、caspase-8、caspase-9 和 TRAIL 的 mRNA 水平。抑制 caspase-8 和 caspase-9 可显著降低软骨细胞中 TNF 诱导的凋亡。这些结果表明，在骨折愈合的软骨向骨转化过程中，糖尿病导致促凋亡基因的上调。糖尿病通过一种机制增加软骨细胞凋亡，该机制涉及 TNF-α 的产生增加，TNF-α 通过 FOXO1 激活刺激软骨细胞凋亡并上调凋亡基因的 mRNA 水平。
