Hypoxia Augments Lipopolysaccharide-Induced Cytokine Expression In Periodontal Ligament Cells

Background and Objective:Periodontal disease is a type of common oral diseases and is one of the main causes of tooth loss in adults,but it is often overlooked by people.Periodontal disease can be caused by various pathogenic factors,the most common cause is bacterial infection.It is just an inflammatory disease caused by plaque whose metabolites acting on periodontal tissues can activate the host’s inflammatory immune response leading to the destruction of periodontal tissues.When human periodontal ligament cells(hPDLCs)are stimulated by pathogenic bacteria and their metabolites,hPDLCs will secrete a variety of cytokines and chemical mediators by themselves to participate in local immune responses.Lipopolysaccharides(LPS)are recognized as inflammatory priming factors,which can increase the expression of inflammatory factors by stimulating immune cells and local adjacent tissue cells,thereby inducing a series of immune responses in the host.Interleukin-6(IL-6)is closely related to inflammation and autoimmune diseases.After the inflammatory immune response occurring,IL-6 is first produced and participates in the chronic inflammatory processof periodontal tissues.Interleukin-8(IL-8),also known as the chemokine CXCL-8,is a kind of cytokine of the chemokine family.IL-8-binding receptor a(CXCR-1)and receptor B(CXCR-2)release the lysosomal enzyme to promote the release of inflammatory mediators by chemotaxis,aggregation and activation of neutrophils,to regulate the inflammatory response and to achieve the purposes of sterilization and cell damage.TLR-2(Toll-like receptor-2)and TLR-4(Toll-like receptor-4)are I transmembrane proteins,which are pattern recognition receptors,play an important role in the inflammatory immune response induced by LPS,which also are key pathways for bacteria to destroy cells.When inflammation occurs,local inflammatory tissues and cells are in hypoxia micro-environment.Therefore,the study under hypoxia conditions is closer to the true micro-environment of periodontal tissues in periodontitis.At present,researches on the effects of hypoxia and LPS of hPDLCs have been extensive and in-depth,but cytokine expression in hPDLCs induced by hypoxia condition and LPS has rarely been reported.Based on these,we established a local hypoxia pathological model by culturing LPS-induced hPDLCs in hypoxic environment,simulating the environment of inflammatory periodontal tissues under real condition.And then we analyzed the effect of cytokine expression in LPS-induced under hypoxia condition by detecting the expression of IL-6,IL-8,TLR-2 and TLR-4,in order to provide new ideas for the treatment of periodontal disease.Materials and Methods:1.Primary culture of hPDLCs by tissue block method;2.Took the 4th generation of hPDLCs and identify the cell molecular phenotype by flow cytometry;3.Took the 3th generation of hPDLCs and detect cell proliferation by CCK-8 method;4.Model establishment and experimental grouping:The 4th generation of well-growth hPDLCs were randomly divided into 4 groups:(group A)normoxia + no LPS,(group B)normoxia + LPS,(group C)hypoxia + no LPS,(group D)hypoxia +LPS.After 6h,12h,24h and 48h,the cells of each group were collected.The expressions of IL-6,IL-8,TLR-2 and TLR-4 mRNA were detected by Realtime-PCR.5.Model establishment and experimental grouping:The 4th generation of well-growth hPDLCs were randomly divided into 4 groups:(group A)normoxia + no LPS,(group B)normoxia + LPS,(group C)hypoxia + no LPS,(group D)hypoxia +LPS.After 6h,12h,24h and 48h,the cells and supernatants of each group were collected.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of IL-6 and IL-8 in each group and the expression of TLR-2 and TLR-4 were detected by flow cytometry.Results:1.hPDLCs were successfully obtained by tissue block method in vitro culture;2.the results of flow cytometry showed:the cultured cells were mesenchymal cells,in line with the biological characteristics of fibroblasts;,there was a significant promotion on the expression of inflammatory factors IL-6,IL-8,TLR-2,TLR-4 mRNA of hPDLCs induced by hypoxia(1%O2)or LPS.Moreover,hypoxia(1%O2)could enhance the expression of IL-6,IL-8,TLR-2,TLR-4 mRNA induced by LPS.5.ELISA and flow cytometry results from the protein level showed:Compared with the control group,there was a significant promotion on the expression of inflammatory factors IL-6,IL-8,TLR-2,TLR-4 protein of hPDLCs induced by hypoxia(1%O2)or LPS.Moreover,hypoxia(1%O2)could enhance the expression of IL-6,IL-8,TLR-2,TLR-4 protein induced by LPS.Conclusion:1.Hypoxia and LPS can induce the expression of inflammatory cytokines in hPDLCs;2.Hypoxia can enhance the expression of inflammatory cytokines induced by LPS in hPDLCs.