Hypoxia-inducible Factor-1α Inhibits Osteogenic Differentiation Of Periodontal Ligament Stem Cells By Noggin

Periodontitis is a chronic inflammatory disease involving damage to the supporting periodontal tissues,especially the loss of alveolar bone,resulting in the gradual loosening of the affected teeth.Without timely and effective treatment,the affected teeth will be inevitably lost.At present,a variety of treatment methods for the treatment of periodontitis,e.g.from basic treatments to periodontal surgeries,can block the progress of the disease and relieve the symptoms of periodontitis,but for the missing periodontal tissue,especially the alveolar bone,there is still no satisfactory treatment effect.The incidencd of periodontitis is rather high,and multiple epidemiological surveys indicate that periodontitis is widespread worldwide and affects most people from young to old around the world.This suggests that the study of the pathological mechanism of periodontitis is still very important.Periodontitis occurs in an inflammatory microenvironment,and this microenvironment not only has a variety of inflammatory components,but also is accompanied by the reduction of oxygen content.And plateau periodontitis is a common disease caused by hypoxia.More specifically,periodontal tissue is an inflammatory hypoxic microenvironment in patients with periodontitis.It is necessary to reveal the effects of hypoxia on the course of periodontitis and the related molecular mechanisms.Hypoxia-inducible factor-1α（HIF-1α）is the key transcription factor for regulating oxygen balance in vivo.It can sensitively react to the changes of oxygen content in local microenvironment,initiate or participate in a series of signaling pathways to deal with the decrease of oxygen level,and affect the proliferation,metabolism and apoptosis of cells.Bone immunology plays an important role in the course of periodontal disease,and several studies have confirmed that there is a close correlation between hypoxia and inflammation.For example,HIF-1αenhances the transcription of inflammatory factors such as nuclear factor-κB（NF-κB）,which also increases the expression of HIF-1α.In addition,periodontal ligament stem cells（PDLSCs）are present in the periodontal microenvironment,which have the potential to self-renew and differentiate into bone-like or periodontal ligament-like tissues.Therefore,PDLSCs were used as the subject to study the role of hypoxia in the course of periodontitis,especially the effect of hypoxia on bone balance in periodontal tissues and the corresponding molecular mechanism,in the study.Method:1.Using tissue block combined with enzymatic digestion method,the periodontal membrane cells were isolated and cultured from human periodontal ligament,and the mesenchymal stem cells were identified by colony forming assay,osteogenic,adipogenic,and chondrogenic differentiation,and flow cytometry detection of mesenchymal stem cell surface markers.2.Cell culture model of hypoxia（3%O₂）-vs.-normoxia（20%O₂）was established,and high-throughput sequencing method was adopted to detect hypoxia-induced differentially expressed genes in PDLSCs,from which GO and Pathway enrichment assays were adopted to screen genes that can be expressed by PDLSCs,regulated by HIF-1α,and was associated with bone balance,where the gene NOG was confirmed.3.The expression of Noggin in PDLSCs was measured through cell immunofluorescence,qPCR,and Western blot.After treating PDLSCs with low oxygen（3%O₂）,or cobalt chloride（CoCl₂）,an agonist of HIF-1α,the hypoxia-induced expression of Noggin in PDLSCs was measued.4.Osteogenic differentiation of PDLSCs was induced under normoxic or hypoxic conditions,and changes in osteogenic genes were detected.By regulating the expression of HIF-1αor Noggin,the changes of osteogenic genes during osteogenic differentiation of PDLSCs under normoxic or hypoxic conditions were examined.Then,by respectively regulating the expression of HIF-1αor Noggin,the signal transfer pattern between the two molecules was studied,and the mode of interaction between HIF-1αand NOG was confirmed by chromatin immunoprecipitation（ChIP）.Results:1.The isolated human periodontal ligament cells in the study have the ability of cloning,and to differentiate into bone,fat and cartilage.Through flow cytometry,these cells were confirmed to show the expression of mesenchymal stem cell surface markers CD29and CD44,and not the expression of hematopoietic stem cell surface markers CD34 or CD45.2.High-throughput sequencing found that PDLSCs expressed 156 differentially expressed genes in low oxygen conditions,where the bone balance-related gene NOG in was screened out.3.HIF-1αincreased the expression of Noggin gene and protein in PDLSCS.4.The osteogenic differentiation of PDLSCs was suppressed under hypoxia,and the inhibition of osteogenic-related genes was alleviated after the disturbance of Noggin expression.HIF-1αworks as an upstream factor of Noggin.ChIP confirmed that HIF-1αbinds directly to the promoter of NOG,and the binding position is located at-1505 to-1502bp.Conclusion:1.In this experiment,human PDLSCs were successfully isolated and identified,and there were 156 differentially expressed genes being confirmed in human PDLSCs under normoxia-vs-hypoxia by high-throughput sequencing.From the perspective of osteogenesis,bone balance-related gene NOG was screened out,and its expression in periodontal ligament cells was confirmed.2.The osteogenic differentiation of human PDLSCs was inhibited under hypoxic conditions,and the interference of Noggin expression could alleviate the decrease of osteogenic related genes,suggesting that HIF-1α/Noggin can affect the osteogenic differentiation of PDLSCs.3.HIF-1αcan work as an upstream regulator of Noggin expression in human PDLSCs.ChIP confirmed that HIF-1αcan directly bind to NOG promoter,and the binding site is-1505 to-1502 bp in NOG promoter region.