AhR-Notch信号通路对糖尿病状态巨噬细胞表型转化的调控及其在糖尿病牙槽骨缺损愈合中的作用

Impaired healing of alveolar bone defect in diabetes is one of great problems in oral disease treatment. Disorder of macrophage (Mφ) phenotype transition is the major cause of this disease, while the mechanism of this abnormal transition remains to be elucidated. In recent years, the aryl hydrocarbon receptor (AhR) has been found to play an important role in the phenotype transition of different immune cells. However, it remains unknown whether AhR regulates the Mφ phenotype transition in bone defect under diabetic conditions. Our previous studies have shown that Mφ in alveolar bone defects of diabetic rats had reduced AhR expression, and exhibited a pro-inflammatory phenotype. Thus, we hypothesize that increased AhR expression may regulate its downstream Notch signaling, which is essential in Mφ phenotype transition. And the regulation of Mφ phenotype transition by AhR-Notch pathway may improve alveolar bone healing in diabetes. In this study, we propose to enhance AhR expression in both in vitro and in vivo models of diabetes, and detect the changes of AhR-Notch pathway, Mφ phenotype transition, and bone healing using western blot analysis, immunocytochemical staining, etc. Additionally, we propose to examine the AhR target genes of Notch pathway members using chromatin immunoprecipitation. It is hoped to demonstrate the changes of Notch pathway, Mφ phenotype transition, and alveolar bone healing by regulation of AhR expression in the diabetic condition, and to provide new ideas for the treatment of impaired healing of alveolar bone defect in diabetes.

糖尿病牙槽骨缺损愈合障碍是口腔临床医疗难点之一，缺损部位巨噬细胞（Mφ）表型转化异常是该疾病的重要原因，但转化异常的机制尚不清楚。近年发现芳香烃受体（AhR）在多种免疫细胞表型转化中有重要调节作用，但是否调节糖尿病骨缺损部位Mφ表型转化尚未明确。本课题组前期研究发现，糖尿病大鼠牙槽骨缺损部位Mφ过度向促炎表型转化，且AhR表达减少。本研究推测增强AhR表达，可能调节下游对Mφ表型转化作用关键的Notch通路，改善Mφ转化异常及糖尿病牙槽骨缺损愈合。本研究拟在体外及动物糖尿病模型中增强AhR表达，以免疫印迹实验、免疫细胞化学染色等检测Mφ中AhR-Notch信号通路、Mφ表型转化与骨再生的变化，以染色质免疫共沉淀法检测AhR与Notch通路成员基因结合位点，探讨糖尿病状态增强AhR对Notch通路、Mφ表型转化的调节作用、及对牙槽骨缺损愈合的影响，为改善糖尿病牙槽骨缺损愈合障碍提供新思路。

糖尿病牙槽骨缺损愈合障碍是口腔临床医疗难点之一，缺损部位巨噬细胞（Mφ）表型转化异常是该疾病的重要原因，但转化异常机制尚不清楚。近年发现芳香烃受体（AhR）及其下游Notch信号通路在多种免疫细胞表型转化中有重要调节作用，但是否调节糖尿病骨缺损部位Mφ表型转化尚未明确。本研究目的是探讨糖尿病状态下，调节AhR-Notch信号通路对Mφ表型转化及牙槽骨缺损愈合的影响及机制。本研究在体外糖尿病状态下增强颌骨来源Mφ的AhR表达，以流式细胞术、免疫印迹实验、ELISA、碱性磷酸酶活性检测等技术检测Mφ表型转化、Mφ中AhR-Notch通路、炎症因子表达、共培养的骨髓间充质干细胞（BMSC）骨向分化的变化，发现在糖尿病状态下，增强颌骨来源Mφ的AhR表达，能抑制Mφ向促炎M1表型转化，促进向抑炎M2表型转化，使Mφ的Notch1信号通路激活减少，促炎因子肿瘤坏死因子α（TNFα）、白介素6（IL6）表达减少，抑炎因子白介素10（IL10）表达增多，并提高BMSC碱性磷酸酶活性；对糖尿病大鼠牙槽骨缺损部位组织增强AhR表达后，以HE染色检测骨缺损部位形态，以实时荧光PCR检测骨缺损部位炎症因子、Mφ表型标记物及AhR-Notch通路主要成员及成骨相关蛋白的基因表达，发现牙槽骨缺损愈合水平增高，缺损局部组织促炎因子TNFα、IL6表达减少，抑炎因子IL10表达增多，M1表型标记物表达减少，M2表型标记物表达增多，缺损局部组织中Notch1信号通路激活受到抑制，碱性磷酸酶基因表达增高。这些结果提示，在糖尿病状态下，增强Mφ的AhR表达，能改善Mφ表型转化，抑制炎症反应，并促进骨缺损愈合，这可能与抑制Notch1信号通路有关。该研究为改善糖尿病牙槽骨缺损愈合提供了新思路。