SIRT1激活线粒体自噬的干预机制对AGEs影响下PDLSCs成骨分化过程的调控作用

Insufficient osteogenic capacity of periodontal ligament stem cells (PDLSCs) is the key to periodontal bone defect caused by diabetes-associated periodontitis. Studies have shown that advanced glycation end products (AGEs) are important culprit in the occurrence and development of diabetes-associated periodontitis. In previous studies, we found that AGEs inhibited osteogenic differentiation of PDLSCs, decreased SIRT1 expression and affected mitophagy activation. However, the effect of SIRT1 in the activation of mitophagy in AGEs intervention of PDLSCs osteogenic differentiation has not been well illustrated. Therefore, in the present study, we focused on the following aspects. Firstly, we intend to determine the role of mitochondrial damage in the inhibition of osteogenic differentiation of PDLSCs by AGEs. Secondly, we will detect the role of PINK1/Parkin-mediated mitophagy on the osteogenic differentiation of PDLSCs after the stimulation of AGEs. Thirdly, we try to disclose the mechanism of osteogenic differentiation of PDLSCs affected by PINK1/Parkin-mediated mitophagy which might be regulated by SIRT1. Finally, animal model will be used to investigate the function of SIRT1 in the osteogenesis of PDLSCs in diabetes-associated periodontitis. This study attempts to reveal the molecular mechanism of AGEs-affected osteogenic differentiation of PDLSCs, and provide a theoretical basis for the precise treatment of diabetes-associated periodontitis.

牙周膜干细胞（PDLSCs）成骨能力不足是糖尿病相关的牙周炎导致牙周缺损的关键，研究表明晚期糖基化终末端产物（AGEs）是糖尿病相关的牙周炎发生和发展的重要元凶。我们前期研究发现AGEs抑制PDLSCs成骨分化，且能降低SIRT1表达并影响线粒体自噬激活，但SIRT1激活线粒体自噬在AGEs干预下PDLSCs成骨分化中的作用并不明确。据此，本课题拟首先在AGEs抑制PDLSCs成骨分化中，明确线粒体损伤的影响；其次探讨AGEs作用下，PINK1/Parkin介导的线粒体自噬对PDLSCs成骨分化作用；进而揭示SIRT1调控PINK1/Parkin介导的线粒体自噬影响PDLSCs成骨分化的机制；最后利用动物模型，研究SIRT1对糖尿病相关的牙周炎中PDLSCs成骨的促进作用。本课题力图揭示AGEs影响下PDLSCs成骨分化的分子机理，为糖尿病相关的牙周炎精准治疗提供一定的理论依据。

糖尿病加重牙周炎的发展，与单纯牙周炎患者（P）相比，糖尿病相关牙周炎（DP）患者牙槽骨吸收更严重，临床并无特殊治疗方式。促进糖尿病微环境下牙周组织再生是DP治疗目标，牙周膜干细胞（PDLSCs）被认为是最理想的细胞来源。在糖尿病微环境下，PDLSCs的Sirt1表达降低，抑制Pink1介导的线粒体自噬，成骨潜力受损。以往研究提示，激活Sirt1促进间充质干细胞（MSCs）成骨抑制骨丧失。本研究探究是否可以通过激活SIRT1/PINK1干预线粒体自噬促进糖尿病微环境下PDLSCs成骨分化，寻找DP治疗的有效靶点。本研究利用自发性Ⅱ型糖尿病大鼠 (GK）丝线结扎法建立DP动物模型，LPS、AGEs联合刺激PDLSCs建立细胞模型。micro-CT检测大鼠牙槽骨嵴顶至釉牙骨质界（CEJ-ABC）距离评估牙槽骨吸收和再生程度，免疫组化检测牙周组织OPG表达。生化检测成骨分化关键基因（ALP、RUNX-2、OCN）及成骨标志物（ALP）活性；透射电镜观察线粒体数量、融合和分裂等超微结构；免疫荧光检测线粒体Mitotracker的表达；DCFH-DA检测细胞ROS水平；JC-1荧光探针检测线粒体膜电位（MMP）；免疫荧光检测LC3和Pink1的表达。SRT1720和EX527上下调sirt1的表达，检测成骨分化和线粒体自噬的变化。RES灌胃DP大鼠，micro-CT检测CEJ-ABC距离，免疫组化检测Sirt1、Pink1蛋白表达，Masson染色检测新骨形成。研究结果提示DP组相较于P组CEJ-ABC距离大，成骨相关蛋白表达下降。LPS和AGEs联合刺激PDLSCs，钙化结节减少，ALP活性降低，成骨相关指标表达降低；ROS升高；线粒体内JC-1绿色荧光强度增强，红色荧光强度下降；线粒体自噬相关基因表达降低。DP组Sirt1、Pink1蛋白下降。上下调Sirt1，Sirt1、Pink1蛋白、钙化结节、ALP发生相应的变化。RES灌胃，大鼠CEJ-ABC距离减少，新骨形成增加，Sirt1、Pink1表达升高。本研究从关键线粒体自噬分子PINK1切入，探究糖尿病相关牙周炎微环境中，Sirt1调节线粒体自噬参与PDLSCs成骨分化的机制。目前的结果有助于阐述Sirt1/PINK1信号轴上调线粒体自噬促进PDLSCs的成骨分化，为糖尿病相关牙周炎的治疗提供了理论基础和依据。