| Context:Chronic periodontitis is one kind of inflammatory disease characterized by destruction of periodontal soft and hard tissues.It can not only result in loosening and loss of teeth,but also interact with the progress of a mass of systemic diseases,including diabetes,heart disease and Alzheimer’s disease,which as a consequence,reducing life quality of patients.However,the etiology and mechanism of chronic periodontitis have not been fully explained so far.Apart from being a traditional component of innate immune response,macrophages have been gradually found to have new immunomodulatory effects.At the same time,macrophages are often divided into M1 macrophages and M2 macrophages due to their strong plasticity.Studies have confirmed that different types of polarized macrophages play different roles in the process of periodontitis,the proportion of M1/M2 also varies in the progress of periodontitis.PGRN,one kind of protein widely expressed in various tissues,has been proved to play a vital role in various diseases,including osteoarthritis,sugar metabolism,neurodegenerative lesions and tumor progress.Additionally,PGRN has also been found to be involved in the infiltration and recruitment of macrophages in inflammation.However,few studies focus on the regulation of PGRN on the polarization of macrophages.In addition,our previous studies have confirmed that PGRN can inhibit the polarization of LPS-stimulated macrophages to M1 type.Therefore,this study aims to figure out the relationship between PGRN and macrophage polarization in the process of periodontitis,and further verify whether PGRN can affect macrophage polarization by binding to TNFRs,so as to provide new ideas for the research of periodontitis pathogenesis.Methods:(1)Detection of PGRN and macrophage polarization related markers in human gingival specimens:gingival tissues from both healthy candidates and patients with periodontitis(untreated)were collected,and PGRN and M1&M2 macrophage polarization related markers,including CD68,iNOS and CD206 in gingival tissues,were detected by immunohistochemical staining.Image J was used to analyze the distribution of macrophages and SPSS software was used to analyze the correlation between the polarization related markers and the expression level of PGRN.(2)Detection of the effect of PGRN on the proliferation of RAW264.7:CCK8 experiment was used to detect the effect of PGRN with different concentration gradients on the proliferation of RAW264.7,and to further verify the concentration of PGRN used in the later experiment.(3)Detection of the effect of PGRN on the polarization of RAW264.7:40 ng/ml and 80 ng/ml PGRN were used to stimulate RAW264.7 macrophages with addition of LPS/IL-4 24 hours before.After 24 hours,cell samples and supernatant samples were collected.The expression of TNF-α,iNOS,CD206,IL-10 and Arginase-1 were detected by qPCR.The expression of CD86 and CD206 was detected by flow cytometry.Enzyme linked immunosorbent assay was used to detect the secretion of IL-10 protein.(4)Detection of the effect of PGRN on the polarization of RAW264.7 with pre-incubation of TNFR2 neutralizing antibody:40 ng/ml PGRN was used to stimulate RAW264,7 with addition of LPS/IL-4 24 hours before,with or without TNFR2 neutralizing antibody(1 μg/ml)pretreatment for 2 hours,and samples were collected after 24 hours.The expression of CD206,IL-10 and Arg-1 genes was detected by qPCR,and the secretion of IL-10 protein was detected by ELISA.Results:(1)Expression of PGRN,CD68,iNOS,CD206 andCD206/iNOS ratio in gingival tissue of chronic periodontitis was significantly higher than those of healthy gingival tissue:compared with healthy gingival tissue,the expression of PGRN in gingival tissue of chronic periodontitis was significantly increased(P<0.01),and expression of CD68(P<0.01),iNOS(P<0.01)and CD206(P<0.05)was also significantly increased.Meanwhile,the ratio of CD206/iNOS in gingival tissue of chronic periodontitis was significantly higher than that of healthy gingival tissue(P<0.01).(2)The expression of PGRN was positively correlated with the expression of CD68,CD206,iNOS and the ratio of CD206/iNOS in gingival tissue of periodontitis:the datas obtained by Image J were tested for normality(K-S test),and the results didn’t coincided with normal distribution.As a result,Spearman test was selected for bivariate correlation analysis.The results showed that the expression of PGRN was positively correlated with the expression of CD68(P<0.05),iNOS(P<0.01)and CD206(P<0.05)in inflammatory gingival tissue,while in normal gingival tissue,the expression of PGRN was only correlated with the expression of iNOS(P<0.05).There was no significant correlation of PGRN with the expression of CD68(P>0.05)and CD206(P>0.05).In addition,PGRN was positively correlated with CD206/iNOS ratio(P<0.05).(3)CCK8 results showed that PGRN had no significant cytotoxicity to RAW264.7.PGRN of 20ng/ml,40ng/ml and 80ng/ml could promote proliferation of RAW264.7(P<0.05),while PGRN of 160ng/ml and 320ng/ml had no significant effect on the proliferation of RAW264.7(P>0.05).(4)PGRN inhibited the M1 phenotype polarization of RAW264.7 induced by LPS,and promoted the M2 phenotype polarization of RAW264.7 induced by IL-4:qPCR results showed that PGRN could inhibit the M1 phenotype polarization of RAW264.7 stimulated by LPS,and significantly reduce the expression of TNF-α and iNOS(P<0.01).Accordingly PGRN could promote RAW264.7 stimulated by LPS polarizing to M2 phenotype,and the expression of CD206(P<0.05)and IL-10(P<0.01)increased significantly.At the same time,PGRN synergistically promoted the M2 polarization of RAW264.7 stimulated by IL-4,and increased the expression of CD206(P<0.01),IL-10(P<0.05)and Arg-1(P<0.05),The results of flow cytometry showed similar results where PGRN could inhibit the polarization of RAW264.7 stimulated by LPS to M1 phenotype,decreasing the expression of CD86,and promote the polarization of RAW264.7 stimulated by LPS to M2 phenotype,increasing the expression of CD206.In addition,PGRN can synergistically promote the polarization of RAW264.7 stimulated by IL-4 to M2 phenotype,and increase the expression of CD206.The result of ELISA further showed that PGRN could promote the polarization of RAW264.7 stimulated by LPS to M2 phenotype and increase the secretion of IL-10 protein(P<0.01).PGRN could also synergistically promote the polarization of RAW264.7 stimulated by IL-4 to M2 and increase the secretion of IL-10(P<0.05).The effect of 40ng/ml PGRN worked the most obviously.(5)TNFR2 neutralizing antibody inhibited the polarization of RAW264.7 to M2 phenotype whether induced by LPS or IL-4:qPCR results showed that RAW264.7 pretreated with TNFR2 neutralizing antibody decreased the expression of CD206(P<0.01),IL-10(P<0.01)and Arg-1(P<0.05)after LPS and PGRN co-stimulation,and similarly decreased the expression of IL-10 after IL-4 and PGRN co-stimulation(P<0.01).In addition,ELISA results showed that the secretion of IL-10 in RAW264.7 pretreated with TNFR2 neutralizing antibody decreased both in LPS and IL-4 groups(P<0.01).Conclusions:(1)The expression of CD68,iNOS,CD206 and PGRN in periodontitis gingival tissues was significantly higher than that of normal gingiva.The expression of CD68,iNOS,CD206 and CD206/iNOS ratio was positively correlated with the expression of PGRN.It indicates that periodontitis characterized by macrophage infiltration accompanies with an increased expression of PGRN,and PGRN may play a protective role in the related process.(2)PGRN can inhibit the polarization of macrophages to M1 phenotype and promote the polarization to M2 phenotype under stimulation of LPS.Meanwhile,PGRN can enhance the effect of IL-4 stimulation on macrophage to M2 phenotype.Further,these effects beneath are related to the activation of TNFR2. |
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