The Role Of DNA Damage And CGAS In Oral Inflammatory Diseases

PartⅠ:DNA damage and c GAS expression in rat model of periodontitis and LPS-induced h PDLCsAim:To investigate the existence of DNA damage response（DDR）and expression of c GAS in rat model of periodontitis and LPS-induced h PDLCs.Methodology:Experimental periodontitis model of SD rats was established by ligation of silk thread.Samples of mandibular bone were taken for analysis after 4weeks of induction.After assessing levels of periodontitis,immunohistochemical staining of control and inflammation periodontal tissue was performed to detect the expression ofγ-H2A.X（a biomarker for DNA double-strand break）and c GAS.Primary human periodontal ligament cells（h PDLCs）were classified and identified,and then were respectively stimulated by LPS with concentration gradient and time gradient.The expression changes ofγ-H2A.X and c GAS were detected by Western blotting,RT-q PCR,and immunofluorescence.Results:It was showed in immunohistochemical staining that the number ofγ-H2A.X and c GAS positive cells obviously increased in periodontal tissue in the successfully constructed rats periodontitis（P<0.05）.In LPS-stimulated periodontal ligament cells,cell immunofluorescence results showed a significantly increased expression ofγ-H2A.X and c GAS compared with the NC group（P<0.01）.Meanwhile,results of western blotting and RT-q PCR analysis confirmed that the expression of c GAS was enhanced in a concentration-dependent and duration-dependent manner in a certain range of LPS stimulation.Conclusions:Significant DNA damage and c GAS expression were detected in the periodontitis of rats.At the same time,LPS can induce DNA double-strand breaks（DSBS）in human periodontal ligament cells,and the expression of c GAS is positively correlated with LPS stimulation in a certain range in a concentration-dependent and duration-dependent manner.PartⅡ:Identify the Role of LPS-induced DNA Damage Response and c GAS in periodontitisObjective:To explore the potential role of LPS-induced DNA damage and c GAS in periodontitis,and to further understand the pathogenesis and regulatory mechanism of periodontitis.Methodology:c GAS-knockdown models of h PDLCs were constructed with si RNA（Small interfering RNA）technique.Subsequently,RT-q PCR analysis was performd to detect the expression levels of related inflammatory cytokines,such as IL-1,IL-8 and TNF-αin c GAS knockdown h PDLCs.At the same time,4 groups were set:（1）Negative Control（NC）respectively;（2）si-c GAS;（3）NC+LPS;（4）si-c GAS+LPS.The effect of c GAS on NF-κB signaling pathway in LPS-induced h PDLCs were detected by cellular immunofluorescence and Western blotting.Caffeine（10μmol/L）and Parthenolide（25μmol/L）were respectively used as inhibitor for DNA damage and NF-κB signaling pathway to pretreat h PDLCs for 6h prior to the LPS stimulus.Subsequently,the expression ofγ-H2A.X,c GAS and p65 nuclear translocation were analysised by Western blotting to further elucidate the effect of DNA damage on the c GAS and NF-κB signaling pathways in periodontitis.Results:Results of RT-q PCR analysis showed a significantly reduced expression levels of related inflammatory cytokines,such as IL-1,IL-8 and TNF-αin c GAS knockdown h PDLCs compared to the control group（P<0.05）.Meanwhile,immunofluorescence and Western blotting detection also confirmed that the activation of NF-κB signaling pathway was significantly inhibited in LPS-induced h PDLCs transfected with si-c GAS（P<0.05）.After the DNA damage was supressed by caffeine,the up-regulation ofγ-H2A.X,c GAS and p65 nuclear translocation induced by LPS were abrogated（P<0.05）.While no obvious change of the expression levels ofγ-H2A.X and c GAS were found in h PDLCs treated with NF-κB signaling pathway inhibitors.PartⅡI:Exploration on the Prevention and Treatment of Yunnan Baiyao Combined with Kangfuxin Liquid on Radioactive Oral Mucosal InflammationObjective:To preliminary explore the prevention and treatment effects of Yunnan Baiyao combined with Kangfuxin liquid in radioactive oral mucosal inflammation provides clinical evidence for the follow-up study of the role of DNA damage in oral inflammatory diseases.Methodology:According to the inclusion and exclusion criteria,90 patients with nasopharyngeal carcinoma were selected for analysis.The patients were randomly divided into study group and control group（45 cases in each group）.All patients with nasopharyngeal carcinoma received inhalation therapy of normal saline combined with dexamethasone,gentamicin,and vitamin B₁₂one day after radiotherapy.Among them,45 patients were treated with Yunnan Baiyao 0.5g/time/day plus Kangfuxin solution 10m L morning and evening gargling 2min treatment as the research group.The oral mucosal symptoms of patients after radiotherapy were scored,including pain after radiotherapy,mucosal hyperemia and edema,mucosal ulcers and dry mouth.At the same time,the sera of patients before and after treatment were collected,the expression changes of related inflammatory factors were detected by ELISA experiment,and the changes of T lymphocytes were detected by flow cytometry.Results:The oral mucosal symptoms of patients before and after treatment were scored.The results showed that in the experimental group treated with Yunnan Baiyao combined with Kangfuxin liquid,the symptoms of pain,ulcers,redness and swelling related to the oral mucosa and inflammation were significantly improved compared with the control group（P<0.05）.).The results of enzyme-linked immunosorbent assay showed that compared with the control group,after the treatment of Yunnan Baiyao combined with Kangfuxin liquid,the serum levels of related inflammatory cytokines such as CRP,TNF-α,IL-1 and MMP-9 in the experimental group The expression level was significantly reduced（P<0.05）.At the same time,the expression levels of tumor-related markers VEGF and HIF-1a in the experimental group were also suppressed.After measuring the T cell types in the peripheral blood,it was found that in the experimental group,CD4+cells,which play an important role in innate immunity,were maintained at a stable level compared to the control group.Conclusion:Radiation injury can induce inflammatory response in the oral mucosa,and the treatment of Yunnan Baiyao combined with Kangfuxin Liquid can reduce the expression of inflammatory factors to a certain extent and reduce the inflammatory response damage.The mutual mechanism between this inflammation inhibitory effect and DNA damage still needs further study.