Abstract：
OBJECTIVE: This study aimed to explore the effects of small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells (L-D-sEV) on periodontal ligament cells from periodontitis affected teeth (p-PDLCs) in vitro and experimental periodontitis in mice.
METHODS: In vitro, the biological function of p-PDLCs and the underlying molecular mechanism were investigated by flow cytometry, Western blot, and quantitative real-time PCR (qRT-PCR) analysis. Eighteen-eight-week-old male C57BL/6 mice were randomly divided into three groups: control (Con), periodontitis (Peri), and L-D-sEV groups. Mice periodontitis model was induced by placing the 5-0 silk thread (around the maxillary second molar) and P.gingivalis (1 ×107 CFUs per mouse). In vivo, the alveolar bone loss, osteoclast activity, and macrophage polarization were measured by micro-computed tomography and histological analysis.
RESULTS: In vitro, the RANKL/OPG ratio and phosphorylation of JNK and P38 protein levels of p-PDLCs were significantly decreased after L-D-sEV administration. Besides, flow cytometry and qRT-PCR analysis showed that L-D-sEV reduced apoptosis of p-PDLCs, down-regulated apoptosis-related genes Caspase-3 and BCL-2-Associated X expression, and up-regulated B-cell lymphoma-2 gene levels. In vivo, L-D-sEV administration significantly reduced alveolar bone loss, inhibited osteoclast activity, and induced M2 polarization. The histological analysis showed that iNOS/CD206, RANKL/OPG, p-JNK/JNK, and p-P38/P38 ratios were significantly lower in the L-D-sEV group than in the Peri group.
CONCLUSIONS: L-D-sEV administration alleviated alveolar bone loss by mediating RANKL/OPG-related osteoclast activity and M2 macrophage polarization, alleviating p-PDLCs apoptosis and proliferation via the JNK and P38 pathways.
METHODS: In vitro, the biological function of p-PDLCs and the underlying molecular mechanism were investigated by flow cytometry, Western blot, and quantitative real-time PCR (qRT-PCR) analysis. Eighteen-eight-week-old male C57BL/6 mice were randomly divided into three groups: control (Con), periodontitis (Peri), and L-D-sEV groups. Mice periodontitis model was induced by placing the 5-0 silk thread (around the maxillary second molar) and P.gingivalis (1 ×107 CFUs per mouse). In vivo, the alveolar bone loss, osteoclast activity, and macrophage polarization were measured by micro-computed tomography and histological analysis.
RESULTS: In vitro, the RANKL/OPG ratio and phosphorylation of JNK and P38 protein levels of p-PDLCs were significantly decreased after L-D-sEV administration. Besides, flow cytometry and qRT-PCR analysis showed that L-D-sEV reduced apoptosis of p-PDLCs, down-regulated apoptosis-related genes Caspase-3 and BCL-2-Associated X expression, and up-regulated B-cell lymphoma-2 gene levels. In vivo, L-D-sEV administration significantly reduced alveolar bone loss, inhibited osteoclast activity, and induced M2 polarization. The histological analysis showed that iNOS/CD206, RANKL/OPG, p-JNK/JNK, and p-P38/P38 ratios were significantly lower in the L-D-sEV group than in the Peri group.
CONCLUSIONS: L-D-sEV administration alleviated alveolar bone loss by mediating RANKL/OPG-related osteoclast activity and M2 macrophage polarization, alleviating p-PDLCs apoptosis and proliferation via the JNK and P38 pathways.
摘要：
目的：本研究旨在探讨脂多糖预处理的卵泡细胞（L-D-sEV）来源的小细胞外囊泡在体外和实验性牙周炎小鼠中对牙周炎患牙（p-PDLCs）牙周膜细胞的影响。
方法：体外，通过流式细胞术研究了p-PDLCs的生物学功能和潜在的分子机制，蛋白质印迹,和定量实时PCR(qRT-PCR)分析。将十八周龄雄性C57BL/6小鼠随机分为三组：对照组（Con），牙周炎（Peri），和L-D-sEV组。通过放置5-0丝线（上颌第二磨牙周围）和牙龈卟啉单胞菌（每只小鼠1×107CFU）来诱导小鼠牙周炎模型。在体内,牙槽骨丢失,破骨细胞活性，和巨噬细胞极化通过显微计算机断层扫描和组织学分析进行测量。
结果：体外，L-D-sEV给药后,p-PDLCs的RANKL/OPG比值以及JNK和P38蛋白磷酸化水平均显著降低.此外,流式细胞术和qRT-PCR分析显示L-D-sEV减少p-PDLCs的凋亡,凋亡相关基因Caspase-3和BCL-2相关X表达下调,和上调B细胞淋巴瘤-2基因水平。在体内,L-D-sEV给药显著减少牙槽骨丢失，抑制破骨细胞活性，并诱导M2极化。组织学分析显示iNOS/CD206、RANKL/OPG、p-JNK/JNK,和p-P38/P38比值在L-D-sEV组明显低于Peri组。
结论：L-D-sEV通过介导RANKL/OPG相关的破骨细胞活性和M2巨噬细胞极化减轻牙槽骨骨丢失，通过JNK和P38途径减轻p-PDLCs的凋亡和增殖。
方法：体外，通过流式细胞术研究了p-PDLCs的生物学功能和潜在的分子机制，蛋白质印迹,和定量实时PCR(qRT-PCR)分析。将十八周龄雄性C57BL/6小鼠随机分为三组：对照组（Con），牙周炎（Peri），和L-D-sEV组。通过放置5-0丝线（上颌第二磨牙周围）和牙龈卟啉单胞菌（每只小鼠1×107CFU）来诱导小鼠牙周炎模型。在体内,牙槽骨丢失,破骨细胞活性，和巨噬细胞极化通过显微计算机断层扫描和组织学分析进行测量。
结果：体外，L-D-sEV给药后,p-PDLCs的RANKL/OPG比值以及JNK和P38蛋白磷酸化水平均显著降低.此外,流式细胞术和qRT-PCR分析显示L-D-sEV减少p-PDLCs的凋亡,凋亡相关基因Caspase-3和BCL-2相关X表达下调,和上调B细胞淋巴瘤-2基因水平。在体内,L-D-sEV给药显著减少牙槽骨丢失，抑制破骨细胞活性，并诱导M2极化。组织学分析显示iNOS/CD206、RANKL/OPG、p-JNK/JNK,和p-P38/P38比值在L-D-sEV组明显低于Peri组。
结论：L-D-sEV通过介导RANKL/OPG相关的破骨细胞活性和M2巨噬细胞极化减轻牙槽骨骨丢失，通过JNK和P38途径减轻p-PDLCs的凋亡和增殖。
