Study On Signal Pathway Of IL-17 Regulating Bone Destruction In Periodontitis Under Hypoxia Microenvironment

Objective:The aim of this study was to investigate the role of interleukin-17（IL-17）in the regulation of human periodontal ligament cells（hPDLCs）via TRAF6-NF-κB and PI3K/Akt signaling pathways in a hypoxic microenvironment and the specific mechanism of osteoclast differentiation to promote alveolar bone resorption in periodontitis.Methods:Human periodontal ligament fibroblasts（hPDLCs）and mouse bone marrow-derived mononuclear cells（BMMNCs）were obtained.The hPDLCs were infected with TRAF6,NF-κB,Akt1 interfering lentiviral vectors and overexpressing lentiviral vectors.The infection efficiency was observed by inverted fluorescence microscope,and the best lentivirus was screened by RT-PCR and Western Blot method,and the signal molecular interference and over expression construction were verified.The cells infected with lentiviral vectors were divided into normal oxygen plus IL-17 group,hypoxia plus IL-17 group and hypoxia group.Each group was divided into signal molecule interference LV-NC group,interference group,and overexpression LV-NC group,overexpression group.Western Blot methods were used to observe the expression of RANKL/OPG and HIF-1α in hPDLCs.In addition,a direct co-culture system of BMMNCs and hPDLCs infected with lentivirus was constructed under the conditions of normoxia and hypoxia.At the same time,BMMNCs were cultured separately as control.The morphological observation of osteoclast like cells was observed by optical microscopy and TRAP staining on the induced third days,seventh days and fourteenth days.Results:（1）After the overexpression of TRAF6 and RelA signal molecules,compared with LV-NC group,the ratio of the relative expression of RANKL and OPG protein in the overexpressed group increased and the HIF-la increased in the condition of hypoxia plus IL-17.And the ratio of RANKL/OPG in the hypoxic plus IL-17 group was higher than that in the hypoxic group and normoxia plus IL-17 group.（2）After the overexpression of Aktl signal molecules,the ratio of the relative expression of RANKL and OPG protein in the overexpressed group increased and the HIF-la decreased in the condition of hypoxia plus IL-17.And the ratio of RANKL/OPG in the hypoxic plus IL-17 group was lower than that in the hypoxic group but higher that in the normoxia plus IL-17 group.（3）After the interference of TRAF6 signal,the ratio of the relative expression of RANKL and OPG protein in the interference group decreased in the condition of hypoxia plus IL-17.And the ratio of RANKL/OPG in the hypoxic plus IL-17 group was higher than that in the hypoxic group.（4）In morphological experiments,the number of TRAP positive cells in TRAF6,RelA and Aktl signal interference groups decreased compared with those in LV-NC group.Conclusions:The TRAF6-NF-κB and Akt signaling pathway not only participates in the normoxic state IL-17 to regulate the hPDLCs expression promoting bone absorption related molecules,also participates in the hypoxia microenvironment in which IL-17 regulates the hPDLCs expression of bone resorption related molecules.When the signal pathway is activated,hPDLCs promotes the differentiation of the osteoclast cells into mature osteoclasts,which leads to the alveolar bone absorption.