Preliminary Study On The Role And Mechanism Of Serum Amyloid A2 In Chronic Periodontitis

Objective: The aim of this study was to compare the differences in the expression of serum amyloid A2(SAA2)in human gingival tissues from healthy volunteers and patients with chronic periodontitis,to detect the expression of SAA2 and inflammatory cytokines in human gingival fibroblasts(HGFs)in vitro in presence of Toll-like receptor(TLR)2 ligand Pam3CSK4 or TLR4 ligand Escherichia coli(E.coli)lipopolysaccharide(LPS),to test the expression of SAA2 after TLR2 or TLR4 inhibition with antibodies,to measure the expression of inflammatory cytokines after using siRNA-SAA2 technology,in order to explore the role and function of SAA2 in chronic periodontitis and to explore the relationship between SAA2 and TLRs signaling pathway,and to provide experimental information for further elucidating the occurrence and development mechanism of periodontitis.Methods:1.Gingival tissues were collected from healthy volunteers and patients with chronic periodontitis during extracting the third molar or periodontal surgery needed after informed consent at Hospital of Stomatology,Tianjin Medical University.After obtaining the gingival tissues,they were placed in the centrifuge tube containing RNA protection solution or formalin solution.2.The expression of SAA2 mRNA in the gingival tissues from healthy or chronic periodontitis was detected by reverse transcription polymerase chain reaction(RT-PCR).Immunohistochemistry was used to detect the expression of SAA2 protein in two kinds of gingival tissues.3.After informed consent,healthy gingival tissue were collected during extracting the third molar,immediately stored in phosphate buffer solution containing 10% double antibiotics.HGFs were cultured in vitro.4.Real-time PCR was used to detect SAA2 and cytokines IL-1?,IL-6,IL-8,TNF-? m RNA expression in HGFs at 6 h,12 h,24 h after stimulation with Pam3CSK4(0,0.1,1,5,10 ?g / ml)or E.coli LPS(0,0.1,1,5,10 ?g / ml).5.The expression of SAA2 and cytokine IL-1? mRNA was detected by real-time PCR after TLR2 antibody/1 ?g/ml Pam3CSK4 exposure.And the expression of SAA2 and cytokine IL-1? m RNA in HGFs was detected by real-time PCR after TLR4 antibody/1 ?g/ml E.coli LPS exposure.6.After siRNA-SAA2 transfection in HGFs,SAA2 m RNA and protein level was detected by RT-PCR and Western blotting,in order to detect siRNA inhibitory efficiency.7.The expression of inflammatory cytokines and TLR4,TLR2,cluster of differentiation 14(CD14)in HGFs was detected by real-time PCR after siRNA-SAA2 transfection and 1 ?g/ml Pam3CSK4 or E.coli LPS treatment.Results:1.The expression of SAA2 m RNA was found in gingival tissues from healthy and chronic periodontitis,while the expression of SAA2 in gingival tissues of chronic periodontitis was significantly higher than that in healthy gingival tissues(p<0.05).The immunohistochemistry results showed that the expression of SAA2 protein could be find in epithelium and connective tissue of gingival tissue and in gingival tissue of chronic periodontitis,and it was significantly higher than that in healthy gingival tissue.2.The primary cells were migrated from the edge of tissue,tended to be consistent,long spindle and thrived.The growth activity is good.The results of real-time PCR showed that the levels of SAA2,interleukin(IL)-1?,IL-6,IL-8 and tumor necrosis factor(TNF)-? m RNA were increased in HGFs after E.coli LPS or Pam3CSK4 stimulation.3.When TLR2 antibody blocked Pam3CSK4(1 ?g/ml)stimulation,the expression of IL-1? decreased(p < 0.05),but SAA2 had no significant difference(p > 0.05).However,the expression of SAA2 and IL-1? was decreased(p<0.05),when TLR4 antibody blocked E.coli LPS(1 ?g/ml)stimulation,.4.siRNA-SAA2 was successfully transfected into HGFs.The inhibitory efficiency in SAA2 m RNA level is about 99%,and the protein level of SAA2 decreased significantly after transfection.5.The expression of SAA2,IL-1? and IL-6 was significantly down-regulated(p<0.05)at 6 h,12 h after siRNA-SAA2 transfection in HGFs with 1 ?g/ml Pam3 CSK4 stimulation,and IL-8 decreased at 6 h and 12 h,and were significantly up-regulated at 24 h(p<0.05).The expression of TNF-? m RNA increased at 6 h,12 h and 24 h(p<0.05).The expression of TLR4 did not change at 6 h,but was up-regulated at 12 h and 24 h after siRNA-SAA2 transfection.CD14 decreased at 6 h(p<0.05),and increased at 12 h and 24 h(p<0.05).6.The expression of SAA2,IL-1?,IL-6 and IL-8 was significantly down-regulated for 6 h,12 h and 24 h after siRNA-SAA2 transfection in HGFs with 1 ?g/ml E.coli LPS stimulation(p<0.05),while the expression of TNF-? increased at 6 h and 24 h(p<0.05)without significant change at 12 h(p>0.05).The expression of TLR2 m RNA decreased at 6 h and 12 h(p<0.05),and there was no significant difference between the two groups at 24 h(p>0.05).The expression of TLR4 decreased at 6 h(p<0.05)and increased at 12 h and 24 h(p<0.05).CD14 showed no difference at 6 h(p>0.05),up-regulated at 12 h and 24 h(p<0.05).Conclusion:1.SAA2 may be involved in the inflammatory process of chronic periodontitis.2.The induction of SAA2 may be closely related to TLR4.3.SAA2 can affect the expression of inflammatory factors in HGFs,possibly through regulating TLRs pathway.