Role Of Toll-like Receptor 4 In The Mechanism Of Bone Destruction In Periodontitis

Periodontitis is a chronic infectious disease which is often accompanied by inflammation of the gums,absorption of the alveolar bone and eventually causing the loosening of the teeth and severely affecting oral health.It is one of the most common diseases in the oral cavity and the main reason to the loss of teeth in adults.At the same time,periodontitis also affects the occurrence of various diseases in the body and endangers human health.In the process of periodontitis,most of the tissue damage is caused by the host’s response to infection.The host’s immune inflammatory response is an important cause of its occurrence and development,including innate immune response and acquired immune response.As the first defensive barrier of the immune system,innate immunity can identify periodontal pathogens through pattern-recognition receptors（PRRs）,which can lead to immune response disorders,leading to an imbalance of pathogenic bacterial populations and microecological imbalances.The formation of destructive inflammation,which damages periodontal tissue,becomes a destructive immune response.As one of the PRRs,toll-like receptor 4（TLR4）is thought to play an important role in the development of periodontitis.Objective:To investigate the role of TLR4 in the mechanism of bone destruction in periodontitis.Methods:In this study,C57BL/6 mice（WT）and TLR4^-/-mice(TLR4^-/-)were used as experimental subjects to establish a model of periodontitis by unilateral silk ligation and sacrificed after 1 week.Maxillary bones on both sides were dissected.Reconstruction of the maxillary three-dimensional model after micro CT scans and analysis of bone destruction related parameters;specimens were sectioned for hematoxylin-eosin（HE）staining,tartrate-resistant acid phosphatase（TRAP）staining and modified Masson trichrome staining.In vitro,we extracted bone marrow mononuclear cells from WT mice and TLR4^-/-mice to differentiate into osteoclasts,stained with TRAP kit and immunofluorescence,and counted under microscope.At the same time,RNA and protein were extracted for the detection of osteoclast related indicators using real-time RT-PCR and western-blot.Primary culture of cranial bones from WT mice and TLR4^-/-mice was used to isolate osteoblasts and extract RNA and protein.Real-time RT-PCR and western-blot detection were also used to detect bone related indicators.Results:The WT and TLR4^-/-mouse model of periodontitis was successfully constructed.No significant uptake was observed in the control jaws of the WT and TLR4^-/-mice by micro CT three-dimensional reconstruction,whereas the experimental side of WT and TLR4^-/-mice was not observed.The bone mass of maxillary second molar was reduced not only in the proximal and distal side but also in the buccal and tongue in the WT and TLR4^-/-mice,and the TLR4^-/-mice experimental side was less absorbed than the WT experimental bone tissue.CT-related data analysis revealed that bone resorption was significantly increased in the experimental side in WT and TLR4^-/-mice compared to the control side,and the decrease of bone mineral density（BMD）,bone volume fraction（BVF）,and number of trabecular bone（Tb.N）and trabecular resolution（Tb.Sp）is more pronounced in the WT experimental side than in TLR4^-/-experimental side.HE staining showed that there was no significant difference in the degree of soft tissue destruction between experimental TLR4^-/-mice and WT mice,but less alveolar bone resorption was showed in the experimental side of TLR4^-/-mice.TRAP staining further revealed that the WT experimental side showed more osteoclasts than the TLR4^-/-experimental side.At the same time,improved Masson trichrome staining showed that the reduction in bone mass was more pronounced on the WT side than on the TLR4^-/-experimental side,but the infiltration was more obvious in the TLR4^-/-experimental side.In vitro,we extracted bone marrow mononuclear cells from WT and TLR4^-/-mice to differentiate into osteoclasts.TRAP staining and immunofluorescence results showed that WT group had stronger osteoclast formation ability than TLR4^-/-group.Real time RT-PCR and western-blot results further indicated that the expression level of osteoclast related markers including NFATc1,CTSK,TRACP decreased after TLR4knockout.In the meantime,we used cranial parietal bone from WT mice and TLR4^-/-mice to culture primary osteoblasts.The expression of OCN and RUNX2 in TLR4^-/-was decreased by real time RT-PCR,and the expression of OPN was increased,but western-blot showed that the expression of OCN,OPN and RUNX2 in TLR4^-/-was all significantly increased,indicating that the osteogenic capacity of TLR4 knockout mice was stronger than that of WT mice.Conclusions:TLR4 promotes periodontal bone destruction in the progression of periodontitis.