The Effects Of The Regulation Of Local Serum Amyloid A On The Chemotaxis Of Immune Cells In Periodontitis

Objective: Periodontitis is a group of chronic infectious diseases including gingivitis and destruction of periodontal tissue.Currently,the prevalence of periodontits in the population is very high,which affects people's quality of life.In previous studies,it was found that serum amyloid A(SAA)is closely related to periodontitis,but the specific regulatory mechanism needs further research.The purpose of this study was to investigate the recruitment of immune cells(leukocytes)by SAA during the development of periodontitis using human gingival fibroblasts(HGFs)in vitro and mice in vivo,and to find out the important role of SAA in periodontitis,in order to provide a new reference for clarifying the pathogenesis of periodontitis and improving the control methods.Methods: 1.According to the inclusion and exclusion criteria of the study,patients who were treated in the Oral and Maxillofacial Surgery and Periodontal Department of Tianjin Medical University were selected,and gingival tissues were collected after signing the informed consent,and stored in RNA protection solution or 10% penicillin-streptomycin-containing phosphate buffer solution at low temperature.2.The Gene Chip microarray was used to detect the expression of chemokines with human recombinant Apolippprotein-SAA(Apo-SAA)stimulated for 24 h in HGFs.The results were verified by real-time polymerase chain reaction(PCR)and chemokine solid protein chip.3.Detection of chemokines expression in healthy gum tissue and inflammatory gum tissue by real-time PCR.4.Real-time PCR was used to detect different of chemokine expression in healthy HGFs with 1 ?g/m L lipopolysaccharide(LPS)stimulated,which pretreated with SAA-si RNA.5.Real-time PCR was used to detect the expression of chemokines in healthy HGFs stimulated with 10 ?g/m L Apo-SAA,with TLR2 antibodies or TLR2 inhibitors(C29)pre-treated.6.Western blot experiments were used to detect major proteins in mitogen-activated protein kinase(MAPK)and nucleic acid factor-?B(NF-?B)signal pathway after 10 ?g/m L Apo-SAA treatment in healthy HGFs.7.Detection the expression of chemokines in healthy HGFs stimulated with 10 ?g/m L Apo-SAA,pre-treated with or without c-Jun N-terminal kinase(JNK),extracellular regulated protein kinases(ERK),p38,NF-?B inhibitor ?(I?B?)or NF-?Bp65 inhibitors,respectively using real-time PCR.8.Transwell test to detect different conditioned medium of HGFs(LPS + si RNA-NC group,LPS + si RNA-SAA group,LPS + Ig G group,LPS + anti-SAA group,Ig G group,anti-SAA group,Apo-SAA + Ig G group,Apo-SAA + anti-SAA group)for chemotaxis of white blood cells,counted by CCK8,and analyzed the neutrophils,lymphocytes(T cells,B cells)by flow cytometry in recruited cells.9.Animal experiments: SAA antibodies were given for periodontitis mouse model.Detection the alveolar bone and gingival tissues using micro-computed tomography(micro-CT)and Hematoxylin-eosin(HE)staining,respectively.Results: 1.HGFs were observed in a long spindle-shaped and radial arrangement under a microscope,showing vigorous growth and good condition.2.Compared with untreated HGFs,chemokine(C-C motif)ligand 2(CCL)2,CCL5,CCL7,CCL8,CCL13,CCL20,chemokine(C-X-C motif)ligand 2(CXCL2),CXCL3,CXCL5,CXCL8,CXCL10,and CXCL11 mRNA expressions were significantly increased after Apo-SAA stimulation for 24 h.The results were verified by chemokine solid antibody chip experients.3.Compared with healthy gum tissue,CCL2,CCL5,CCL7,CCL8,CCL13,CCL20 and CXCL2,CXCL3,CXCL5,CXCL8,CXCL10,CXCL11 mRNA were significantly up-regulated in inflammatory gum tissue.4.Compared with the untreated HGFs,CCL2,CCL5,CCL7,CCL8,CCL13,CCL20 and CXCL2,CXCL3,CXCL5,CXCL8,CXCL10,CXCL11 mRNA were significantly up-regulated in 1 ?g/m L lipopolysaccharide-treated cells and this upregulation could be inhibited by si RNA-SAA.5.Apo-SAA can significantly promote the expression of TLR2 mRNA,and promote the phosphorylation of JNK,ERK,p38 and I?B?,NF-?Bp65.TLR2 inhibitors and JNK,ERK,p38 and I?B?,NF-?Bp65 inhibitors could significantly decrease the up-regulation of Apo-SAA-induced chemokines.6.Compared with LPS-stimulated HGFs culture medium,LPS-stimulated HGFs pre-treated with si RNA-SAA and anti-SAA antibodies could cause a significant decrease in the number of leukocytes recruited,and the Apo-SAA treated HGFs culture medium induced a significant increase leukocytes recruited,however anti-SAA antibodies could inhibit this effect.The proportion of neutrophils and lymphocytes(T cells,B cells)in recruited white blood cells did not different significantly.7.Compared with the untreated periodontitis mice,using SAA antibody locally could relieve alveolar bone resorption;HE staining shown that the infiltration of inflammatory cell reduced significantly after using SAA antibody(Ig G group: 1.674 % ± 0.1757%,SAA-antibody group: 0.8714% ± 0.1727%).Conclusions: 1.The increasing of chemokines in inflammatory gum tissue indicates that chemokines were involved in the occurrence and development of periodontitis.2.SAA could induce the expression of chemokines by regulating and activating HGFs surface receptors TLR2,MAPK and NF-?B signaling pathway,thereby promoting the chemotaxis of white blood cells and participating in the periodontal inflammation process.3.Inhibition of the SAA-TLR2-MAPK/NF-?B signaling pathway could reduce the expression of chemokines,and local inhibition of the affection of SAA could relieve periodontal inflammation and alveolar bone resorption,indicating that this pathway maybe a target for the prevention and treatment of periodontitis.