MT01Inhibit The MRNA Expression Of Osteoclast-related Factors In RAW264.7Stimulated By Periodontitis Patient’s Own Tissue Nucleic Acid

Periodontitis is regarded as a kind of periodontal tissue destructiondue to unbalanced local immune disease, the disease progression andseverity is closely related to host their own immune inflammatoryresponse. The key factor in autoimmune inflammation of periodontaldisease process, periodontitis patient’s own tissue nucleic acid is not onlyinvolved in the development of periodontal disease, but there is likely tobe the mechanism of periodontitis play regulatory role. Periodontitispatient’s own tissue nucleic acid as part of the damage-associatedmolecular patterns,which is "endogenous danger signals". Researchconfirms that the nucleic acid class DAMPs can start to produce theinnate immune response with the characteristics of a large number ofinflammatory cytokines. Such innate immune response is a"double-edged sword", both fast and effective control of bacterialinfections, but also in the condition of excessive activation ofinflammation caused by a sexually transmitted disease damage. Thisinflammation disease reason damage in periodontal disease mainly forinflammatory alveolar bone resorption. Considering these factors as you can see, the periodontitis patient’s own tissue nucleic acid may be used asa class DAMPs excessive activation of innate immune responses andtrigger an inflammatory alveolar bone loss in the development ofperiodontal disease.To confirm this inference, First of all,we use the periodontitispatient’s own tissue nucleic acid stimulate murine macrophageRAW264.7. Then,determine the mRNA expression of osteoclast-relatedfactors. We found that the periodontitis patient’s own tissue nucleic acidcan promote osteoclast activation, indicating that the periodontitispatient’s own tissue nucleic acid involved in the innate immune responsesin periodontal disease and lead an inflammatory alveolar bone loss byincreasing the osteoclast-related factors gene expression level.Secondly, we use MT01,an oligodeoxynucleotide designed based onthe sequence of human mitochondrial DNA, to intervene the stimulationof murine macrophage RAW264.7by the periodontitis patient’s owntissue nucleic acid.It was found that MT01can inhibit the activation of the periodontitispatient’s own tissue nucleic acid promote osteoclast activation. Furtherconfirmed that the periodontitis patient’s own tissue nucleic acid may beused as a class DAMPs excessive activation of innate immune responsesand trigger an inflammatory alveolar bone loss in the development ofperiodontal disease. Methods Inflammatory periodontal tissue samples of chronicperiodontitis patients were taken in periodontal flap surgery, and healthygingival tissue samples were taken from orthodontic patients during toothextractions. Total RNA from periodintal tissue was extracted and reversetranscribed into cDNA, cryopreserved for further use. Firstly, Specificsequence oligodeoxynucleotide MT01at concentration of1μg/ml wasadded in murine macrophage RAW264.7and the cells were incubated foranother3hours. Cells with PBS were used as negative controls. Secondly,the inflammatory periodontal tissue cDNA and healthy periodontal tissuecDNA (1μg/ml) was added subsequently. There were four experimentalgroups: healthy periodontal tissue cDNA+RAW264.7, inflammatoryperiodontal tissue cDNA+RAW264.7, MT01+healthy periodontal tissuecDNA+RAW264.7, MT01+inflammatory periodontal tissuecDNA+RAW264.7. Real-time PCR was used to detect the mRNAexpression of osteoclast-related factors IL-6, IL-12p35, IL-12p40,MMP-9, NFATc1, RANK and TNF-α mRNA after3,6,12and24hours.Result The mRNA levels of osteoclast-related factors NFATc1,MMP-9, TNF-α, IL-6, IL-12p40, IL-12p35and RANK in RAW264.7were markedly unregulated with the treatment of periodontitis patient’sown tissue nucleic acid. However the mRNA expression ofosteoclast-related factors was inhibited by use of immunosuppressant. Conclusion Periodontitis patient’s own tissue nucleic acid to theactivation of osteoclast can be as the basis of periodontal DAMPs in localtissue. MT01inhibit the mRNA expression of osteoclast-related factors inRAW264.7stimulated by periodontitis patient’s own tissue nucleic acidmay prompt that periodontitis patient’s own tissue nucleic acid may beinvolved in periodontal local immune responses.