| Background:Periodontitis is a chronic immune inflammatory disease which is characterized by the destruction of periodontal connective tissue and alveolar bone absorption.It is one of the main reasons for the loss of teeth in Chinese adults and the prevalence rate is as high as 80% to 90%.Porphyromonas gingivalis(P.gingivalis,Pg)is considered a keystone pathogen in the initiation of periodontitis.It could induce a large number of phagocytic cells to accumulate after triggering periodontal tissue to cause inflammation,resulting in periodontal connective tissue destruction and alveolar bone loss.Cyclic diguanylate,which is released after P.gingivalis planting into periodontal tissue usually exists in gram negative bacteria,as a bacterial second messenger.The second messenger is a target molecule that transfers the receptor signal from the cell surface to the cytoplasm or nucleus of eukaryotic cells.It is a part of the cell signal transduction cascade,and could amplify the initial signal by several times to produce the biochemical reaction inside the cell.Immune inflammatory response,especially macrophages,plays an important role in the process of periodontal tissue destruction.In the pro-inflammatory or anti-inflammatory microenvironment,macrophages can be differentiated into two types called macrophage polarization,namely M1 type macrophages and M2 type macrophages.Macrophage polarization plays a critical role in the body’s resistance to pathogenic microorganism infection and tissue repair.In conclusion,c-di-GMP,secreted by P.gingivalis,regulates M1 macrophages polarization to produce the inflammatory and bone resorption cytokines which lead to the periodontal tissue destruction.Methods:In this study,RAW264.7 macrophages derived mice in the logarithmic growth period were stimulated with different concentrations(1μg / mL,5μg / mL,10μg / mL,50μg / mL)c-di-GMP for different time(3h,6h,12 h and 24h).The following various tests are carried out:1.The effect of c-di-GMP on the growth morphology of macrophages was observed by optical microscope.2.After extracing the total RNA of the cell samples,the mRNA expression of inflammatory cytokines IL-1β,IL-6,TNF-α and M1 and M2 macrophage polarization related cytokines IL-23,iNOS and IL-10 were detected by real-time quantitative PCR.3.Collecting the cell culture supernatant,the protein secretion of the above cytokines were measured by enzyme linked immunosorbent assay(ELISA).4.The expression of the surface marker molecules of M1 macrophages,F4/80,CD86 and CD206,was detected by flow cytometry(FCM).Results:1.1.After RAW264.7 cells line and c-di-GMP co-culturing for 24 h,optical microscope observed: in negative control group(c-di-GMP concentration: 0 μg/mL),the cells morphology were mainly round,others were irregular polygons,and a small number of cells had pseudo foot;the cells stimulated with c-di-GMP showed long spindle shape with extending a large number of pseudo foot,and the spreading area of the cells increased obviously.2.After different concentrations(1 μg/mL,5 μg/mL,10 μg/mL,50μg/mL)c-di-GMP stimulated RAW264.7 cells for 24 h,qPCR results showed the mRNA expression of inflammatory cytokines IL-1β、IL-6、TNF-α and M1 macrophage polarization related cytokines IL-23,iNOS increased gradually with the increasing concentration of c-di-GMP in a dose dependent manner;the mRNA expression of M2 macrophage polarization related cytokines IL-10 was not significantly increased expression level in each concentration group.According to the above results,we identified the best co-culture concentration of c-di-GMP was 50 μg/mL.Next,we used this concentration to stimulate RAW 264.7 cells for 3,6,12,24 h,respectively.The mRNA expression of IL-1β,IL-6,TNF-α and IL-23,iNOS were increased all the time,with the time dependent manner,and the peak time of the expression of different cytokine genes was slightly different.While there was no significant increase in the mRNA expression level of IL-10 in each time group,without statistical significance.3.Co-culturing in different concentrations of c-di-GMP and RAW 264.7 cells for different time,ELISA results showed that the protein secretion of inflammatory cytokines IL-1β、IL-6、TNF-α and M1 and M2 macrophage polarization related cytokines IL-23,NO and IL-10 were according with the correlative mRNA expression level.The protein secretion of inflammatory cytokines IL-1β、IL-6、TNF-α and M1 macrophage polarization related cytokines IL-23,NO were increased gradually with the increase of c-di-GMP concentration and time in each group,with a dose and time dependence.And the protein secretion of M2 macrophage polarization related cytokines IL-10 in each concentration group was basically the same at all time points,and the difference was not statistically significant.4.Flow cytometry results showed that positive expression rate of F4/80 on the surface of RAW264.7 cells cultured on H-MDEM medium was 96.67%.In addition,the positive expression rate of M1 macrophage marker surface factor CD86 and M2 macrophage marker surface factor CD206 were significantly different from that of the same type control group.Therefore,RAW264.7 cells can be certified to be undifferentiated macrophages,as M0 state of macrophages.After high concentration group(50 μg/mL c-di-GMP)stimulating RAW264.7 cells for 24 h,the single positive rate of the surface marker CD86 of RAW 264.7 cells increased from 78.42% to 93.66% compared with the blank control group,and the distinction was statistically significant.These results suggested that M0 state of macrophages had successfully differentiated into M1 type macrophages.There was no significant difference between the low concentration group(5 μg/mL c-di-GMP)and the negative control group,but there was an increasing trend.Conclusions:1.The c-di-GMP secreted by P.gingivalis could stimulate macrophages to produce representative periodontitis inflammatory factors such as IL-1β,IL-6 and TNF-α.2.M1 Macrophage phenotypes can be induced by c-di-GMP in vitro,secreting M1 macrophage polarization related cytokines IL-23,iNOS and surface marker CD86. |
PDF Full Text Request