| Background and purposeChronic periodontitis is an infectious disease of the periodontal support tissue caused by a variety of periodontal pathogens,mainly Porphyromonas gingivalis,and has become the main cause of adult dentition defects.However,the pathogenesis of periodontitis is still unclear.In recent years,studies have confirmed that periodontitis is related to high levels of reactive oxygen species(ROS).The excessively produced ROS in periodontitis cannot be balanced by the antioxidant defense system,which eventually leads to periodontal tissue damage.Periodontal ligament fibroblasts(PDLFs)are the most important cellular components in the periodontal ligament,and Porphyromonas gingivalis can damage the periodontal ligament by destroying the morphology and function of PDLFs.But the exact mechanism of PDLFs injury in periodontitis is still unclear.Periostin(POSTN)is a cell adhesion protein originally discovered in mouse osteoblasts,which is mainly expressed in collagen-rich fibrous connective tissues subjected to constant mechanical stress,such as the periodontal ligament,and periostin is mainly secreted by PDLFs in the periodontal ligament.Periostin is not only involved in the development and maturation of periodontal tissue,but also plays an important role in the homeostasis of the periodontal microenvironment.However,the specific expression patterns and regulatory mechanisms of periostin in periodontitis remain controversial,and targeting periostin or its related signaling pathways may contribute to the development of new diagnostic and therapeutic strategies for periodontitis.Therefore,this project intends to establish mouse periodontitis models and a PDLFs inflammation model to verify the expression pattern of periostin in periodontitis,to clarify whether periostin is involved in lipopolysaccharide(LPS)-induced oxidative stress injury,and to further explore the potential regulation mechanism of periostin in PDLFs injury.Materials and methods1.In the first part of this project,mouse periodontitis models were constructed.Micro computed tomography(Micro-CT)and hematoxylin-eosin staining(HE)were used to observe the destruction of periodontal ligament and alveolar bone resorption.Immunohistochemical staining(IHC)was used to detect the location and expression change of periostin.A PDLFs inflammatory model was constructed to explore the expression change of periostin in periodontitis.2.In the second part of this project,TdT-mediated dUTP nick end labeling(TUNEL),flow cytometry,real-time quantitative polymerase chain reaction(qRT-PCR)and western blot were used to observe the apoptosis of PDLFs after LPS treatment.We also established PDLFs overexpressing periostin by lentiviral vector infection.Then qRT-PCR and western blot were used to detect the expression change of apoptosis-related markers,and to investigate whether periostin was involved in regulating apoptosis of PDLFs in periodontitis.3.In the third part of this project,the ROS inhibitor N-acetylcysteine(NAC)was added to explore whether the expression change of periostin and the apoptosis of PDLFs were affected by oxidative stress.Subsequently,the nuclear factor erythroid 2-related factor 2(Nrf2)inhibitor ML385 was added to explore whether periostin regulated LPS-induced apoptosis of PDLFs through the Nrf2/hemeoxygenase-1(HO-1)signaling pathway.Results1.The results of Micro-CT and HE staining showed periodontal ligament damage and horizontal resorption of alveolar bone in the periodontitis group.IHC staining images revealed that periostin was mainly expressed in the periodontal ligament,and the expression of periostin decreased in the periodontal ligament of mice with periodontitis.Meanwhile,the expression of periostin decreased in PDLFs after LPS treatment in vitro.2.The results of TUNEL,flow cytometry,qRT-PCR and western blot showed that the apoptosis of PDLFs increased after LPS treatment.However,after PDLFs infected with lentivirus overexpressing periostin were treated with LPS,the expression of B-cell lymphoma-2(Bcl-2)increased,and the expressions of Bcl2-associated X protein(Bax)and cleaved cysteinyl aspartate specific proteinase 3(cleaved-caspase3)decreased,indicating that periostin inhibited LPS-induced apoptosis of PDLFs.3.Fluorescence images and flow cytometry results showed that LPS activated ROS in PDLFs,and NAC reversed the LPS-induced decrease of periostin expression and apoptosis of PDLFs.The results of immunofluorescence,qRT-PCR and western blot revealed that the expression of nuclear Nrf2 and total HO-1 decreased after LPS treatment,but the above phenomena were reversed by periostin.Furthermore,the inhibitor of Nrf2 ML385 attenuated the protective effect of periostin against LPS-induced apoptosis in PDLFs.ConclusionThe expression of periostin decreased in periodontal ligament of periodontitis mice and LPS-induced PDLFs,and periostin inhibits the apoptosis of PDLFs induced by oxidative stress in periodontitis via the Nrf2/HO-1 signaling pathway. |
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