A Preliminary Study On The Relationship Between Radiation Induced Oral Mucositis, Immune Level And Oral Flora

The relationship between oral flora and radiation induced oral mucositis(RIOM)Background and ObjectionThe incidence of malignant tumors in the head and neck is on the rise.Oral cavity is one of the parts with the highest density and most species of microorganism in the whole body.There is a potential interaction between oral flora and oral mucositis.RIOM is the most common early oral toxicity during or after radiotherapy in patients with head and neck cancer(HNC),and it is also an early oral toxicity that limits the dose of radiotherapy.Current research is devoted to the use of interventions to change the existence or composition of oral flora in an effort to reduce the incidence and severity of oral mucositis.However,no guidelines have been established to guide clinical practice.The purpose of this study was to investigate the effect of head and neck radiotherapy on oral flora in mice and humans,and the effect of oral microbial transplantation(OMT)on RIOM in normal mice.Methods1.Effect of head and neck radiotherapy on oral flora in mice and effect of oral flora transplantation on radiation induced oral mucositis in normal mice.Male C57BL/6J mice were irradiated with a single dose of 8Gy gamma ray for 3 days to establish a model of RIOM in normal mice.Healthy male C57BL/6J mice were anesthetized and then wiped with aseptic oral swabs(including upper jaw,cheeks and tongue)to prepare oral microorganisms of normal mice.The mice were divided into three groups according to body weight:blank control group,simple irradiation group(THI)and irradiation after OMT group.The mice in the three groups were dissected,and their tongues were stained with hematoxylin and eosin(H&E)and immunohistochemical staining.The plasma and tongue tissues of mice were taken for enzyme-linked immunosorbent assay(Elisa)for interleukin-1(IL-1),IL-6,IL-10,tumor necrosis factor-α(TNF-α)and tumor growth factor β(TGF-β).Fresh mouse oral samples were collected and DNA was extracted for 16S ribosomal RNA(16S rRNA)gene analysis to evaluate bacterial diversity.The statistical method was student t test.2.Effect of radical Intensity Modulated Radiation Therapy(IMRT)on oral flora in patients with HNC.49 cases from Nanfang Hospital,Southern Medical University,with an average age of 50.35 years.Every HNC patient was confirmed by pathology and every patient’s posterior wall of oral pharynx was taken before radiotherapy,during radiotherapy(between the 18th and 22nd times of radiotherapy)and after radiotherapy.All patients underwent a comprehensive oral and dental assessment to assess the potential risk factors for oral complications prior to radiotherapy.All patients received IMRT.Fresh samples of oral flora were collected to extract DNA,and then 16S rRNA gene analysis was carried out to evaluate bacterial diversity.Results1.Effect of head and neck radiotherapy on oral flora in mice and effect of oral flora transplantation on radiation induced oral mucositis in normal mice.Local head irradiation significantly increased the levels of inflammatory related factors IL-1,IL-6,IL-10,TNF-α and TGF-β in peripheral blood and tongue tissue of mice and the OMT could significantly decrease the levels of them.After irradiation,the tongue tissue structure of mice was destroyed,and the OMT could significantly reverse it.After irradiation,CD45 staining deepened and PCNA staining became lighter,which could be effectively reversed by OMT.The results showed that the OMT into the oral cavity of mice irradiated by the head could effectively reverse the RIOM.16S rRNA sequencing analysis showed that local head irradiation could not significantly change the abundance of oral flora in mice,but significantly changed the composition of oral flora.The oral flora of mice with OMT were analyzed by 16S rRNA sequencing after local irradiation of the head.OMT could significantly change the abundance and composition distribution of oral flora in mice irradiated locally with the head.2.Effect of radical Intensity Modulated Radiation Therapy(IMRT)on oral flora in patients with HNC.The oral flora of 49 patients with HNC during radiotherapy were analyzed by 16S rRNA gene sequencing.The abundance of oral flora in patients with HNC treated with radical IMRT was slightly higher than that before radiotherapy(no statistical difference),but the composition and distribution of oral flora changed significantly during and after radiotherapy.The same genus of bacteria fluctuated greatly before,during and after radiotherapy.ConclusionOMT from normal mice into the oral cavity of mice irradiated locally by the head can effectively reverse the RIOM.Local head irradiation slightly increased the abundance of oral flora in mice over time(no statistical difference),but significantly changed the composition of oral flora.OMT from normal mice can significantly change the abundance and composition distribution of oral flora in mice after local head irradiation.The abundance of oral flora in patients with HNC treated with radical IMRT was slightly higher than that before radiotherapy(no statistical difference),but the composition and distribution of oral flora changed significantly during and after radiotherapy.The same genus of bacteria fluctuated greatly before,during and after radiotherapy.The relationship between oral flora and immune levelBackground and ObjectionImmunotherapy has become a promising method for the treatment of HNC.Radiotherapy plays an important role in tumor-immune circulation.Immunoglobulin and complement components are parts of humoral immunity.T lymphocyte subsets are the main form of cellular immunity.Through the changes of immune cells before and after radical IMRT in patients with HNC,we can understand the immune level of the body.There is a close relationship between flora and immunity.The relationship between the changes of oral flora and immune level and immunotherapy before and after radiotherapy needs to be further studied.The purpose of this study was to reveal the changes of immunoglobulin,complement and T lymphocyte subsets in peripheral blood of patients with HNC before and after radical IMRT,and to analyze the correlation between oral flora and the changes of immune cells,so as to provide basis for HNC immunotherapy.Methods54 cases from Nanfang Hospital,Southern Medical University,received IMRT.The concentrations of immunoglobulin G(IgG),A(IgA),M(IgM),complement 3(C3),complement 4(C4)and serum total complement activity(CH50)were measured by Elisa before and after radiotherapy in patients with HNC.The percentage of T lymphocyte subsets in peripheral blood of patients with HNC before and after radiotherapy was detected by flow cytometry.The changes of the above indexes before and after radiotherapy were compared.The 16s rRNA sequencing of oral flora before and after radiotherapy was selected from 54 patients,and the relationship between oral flora and immune cells before and after radiotherapy was analyzed.ResultsIn all 54 patients with different types of HNC,C3,C4 and CH50 increased after radiotherapy,and the difference of C4 was statistically significant.IgG,IgA and IgM decreased after radiotherapy.Treg(CD4+CD25+),CD3+CD8+and NK increased after radiotherapy,in which the difference of CD3+CD8+and NK was statistically significant.The percentage of CD3+,CD3+CD4+,CD4+CD45RO+,CD4+CD45RA+,CD8+CD28+T cells and CD3+CD4+/CD3+CD8+ decreased after radiotherapy,in which the differences of CD3+,CD3+CD4+,CD4+CD45RA+T cells and CD3+CD4+/CD3+CD8+were statistically significant.The abundance of specific flora in the oral cavity of 10 patients with HNC before and after radical IMRT was correlated with the concentration or proportion of immune cells.ConclusionAfter radical IMRT,the immunoglobulin of HNC patients decreased,the complement in serum increased,and there was no absolute activation or inhibition of cellular immunity.It is necessary to further study the effect of radiotherapy on immunity in HNC comprehensive therapy.There is a correlation between the abundance of specific oral flora and the concentration or proportion of immune cells in HNC patients before and after radical IMRT,suggesting that the regulation of specific oral flora before and after radiotherapy may affect the immune level of the body.