糖尿病与长骨和颌骨骨折的风险增加相关。糖尿病也与延迟愈合或非愈合的发病率增加相关。我们之前的研究认为，一种主导机制是终板骨形成过程中软骨的过早消失与增加的破骨细胞活性有关。我们测试了FOXO1在糖尿病受损血管生成和软骨细胞凋亡中的关键作用的假设。我们通过特异性删除软骨细胞中的FOXO1基因，在小鼠中诱导股骨闭合性骨折。对照组包括具有FOXO1基因的小鼠。糖尿病组的小鼠通过多次链脲霉素注射使其发生糖尿病，而正常血糖组的小鼠接收了载体。第16天收集标本。样品经固定、脱钙和包埋于石蜡块中，然后进行免疫染色，利用抗cleaved caspase-3或CD31特异性抗体与匹配的对照IgG抗体进行比较，并通过TUNEL法检测凋亡。此外，ATDC5软骨细胞在体外通过RT-PCR、荧光素酶报告基因和染色质免疫共沉淀实验进行检测。糖尿病小鼠的血管数量比正常血糖小鼠少约50%。在糖尿病小鼠中，FOXO1的删除部分恢复了较少的血管数量（p < 0.05）。此外，糖尿病使caspase-3阳性和凋亡性软骨细胞增加了50%。在糖尿病动物中，FOXO1的删除阻止了两者增加到与正常血糖动物相当的水平（p < 0.05）。高葡萄糖（HG）和高晚期糖基化终产物（AGE）水平在体外刺激了FOXO1与caspase-3启动子的结合，并且过度表达FOXO1增加了荧光素酶报告基因实验中的caspase-3启动子活性。此外，我们对之前的机制研究进行了回顾，证明肿瘤坏死因子（TNF）抑制剂能够逆转骨折愈合过程中受损的血管生成和高水平软骨细胞凋亡的情况。本文提供了新的研究结果，结合最近的研究，全面阐述了糖尿病如何通过高血糖水平、AGE和增加的炎症影响血管生成，刺激软骨细胞凋亡，从而干扰愈合过程。糖尿病骨折中的FOXO1具有负面作用，通过减少新血管形成和增加软骨细胞死亡，这与其在正常骨折愈合中的作用不同。版权所有© 2023 Alharbi and Graves.

Diabetes mellitus is associated with higher risks of long bone and jaw fractures. It is also associated with a higher incidence of delayed union or non-union. Our previous investigations concluded that a dominant mechanism was the premature loss of cartilage during endochondral bone formation associated with increased osteoclastic activities. We tested the hypothesis that FOXO1 plays a key role in diabetes-impaired angiogenesis and chondrocyte apoptosis.Closed fractures of the femur were induced in mice with lineage-specific FOXO1 deletion in chondrocytes. The control group consisted of mice with the FOXO1 gene present. Mice in the diabetic group were rendered diabetic by multiple streptozotocin injections, while mice in the normoglycemic group received vehicle. Specimens were collected 16 days post fracture. The samples were fixed, decalcified, and embedded in paraffin blocks for immunostaining utilizing anti cleaved caspase-3 or CD31 specific antibodies compared with matched control IgG antibody, and apoptosis by the TUNEL assay. Additionally, ATDC5 chondrocytes were examined in vitro by RT-PCR, luciferase reporter and chromatin immunoprecipitation assays.Diabetic mice had ~ 50% fewer blood vessels compared to normoglycemic mice FOXO1 deletion in diabetic mice partially rescued the low number of blood vessels (p < 0.05). Additionally, diabetes increased caspase-3 positive and apoptotic chondrocytes by 50%. FOXO1 deletion in diabetic animals blocked the increase in both to levels comparable to normoglycemic animals (p < 0.05). High glucose (HG) and high advanced glycation end products (AGE) levels stimulated FOXO1 association with the caspase-3 promoter in vitro, and overexpression of FOXO1 increased caspase-3 promoter activity in luciferase reporter assays. Furthermore, we review previous mechanistic studies demonstrating that tumor necrosis factor (TNF) inhibition reverses impaired angiogenesis and reverses high levels of chondrocyte apoptosis that occur in fracture healing.New results presented here, in combination with recent studies, provide a comprehensive overview of how diabetes, through high glucose levels, AGEs, and increased inflammation, impair the healing process by interfering with angiogenesis and stimulating chondrocyte apoptosis. FOXO1 in diabetic fractures plays a negative role by reducing new blood vessel formation and increasing chondrocyte cell death which is distinct from its role in normal fracture healing.Copyright © 2023 Alharbi and Graves.