Effects Of Interleukin 1β On Differentiation Of Cementoblasts And Its Mechanism

Cementum,a layer of mineralized tissue attached to the surface of the tooth root,is an important part of periodontal tissue and the basis for orthodontic treatment of tooth movement.After the tooth root development is completed,the cementum usually has not physiologically altered and lacks the remodeling ability like bone tissue.Orthodontic treatment usually leads to the absorption of cementum,and cementoblasts become the key to the restoration of cementum.With the proliferation,differentiation and mineralization of cementoblasts restorative cementum is formed gradually.Therefore,it is very important to improve the function of cementoblasts for the restoration of cementum.And it also provides a theoretical basis for the prevention and treatment of orthodontic root resorption and periodontal tissue repair.IL1β is a common cytokine in the early stage of infection,which can regulate immune response-mediated inflammation and is highly expressed in periodontal tissues of patients with orthodontics and periodontitis.It may play a role in inflammatory damage,bone resorption and osteoblast differentiation.However,the role of IL1β in the metabolism of cementum,especially the mechanism of cementoblasts,is still unclear.Consequently,the following experiments were conducted to investigate the effects of IL1β on the differentiation of cementoblasts and its related mechanisms.1.Effects of IL1β on cementoblastsObjective: To clarify the effects of IL1β on proliferation,differentiation and mineralization of cementoblasts.Methods: The cementoblasts(OCCM-30)was induced by IL1β recombinant protein(10 ng/m L).The effect of IL1β on the proliferation of cementoblasts was detected by MTT.The flow cytometry was used to observe the apoptosis of cementoblasts.Real-time PCR and western blot were used to determine the expressions of genes and proteins of runx2 and osterix.The activity of alkaline phosphatase was also detected,and the alizarin red staining was used to analyze the formation of VI mineralized nodules.Results: IL1β had no effect on the proliferation of cementoblasts within 1 to 3 days.It did not induce the apoptosis of cementoblasts in 1 or 3 days.IL1β down-regulated the expression of runx2 and osterix and inhibited the formation of mineralized nodule formation and alkaline phosphatase.Conclusion: IL1β had no effects on the proliferation and apoptosis of cementoblasts.IL1β inhibited the differentiation and mineralization of cementoblasts.2.Mi R-325-3p mediated IL1β-induced cementoblast differentiationObjective: To verify the role of micro RNAs in IL1β-induced cementoblasts differentiation.Methods: Using bioinformatics technology,we screened and verified that mi R-325-3p participated in the differentiation of cementoblasts under the action of IL1β.The target gene of mi R-325-3p was verified by double luciferase assay.The mimics and inhibitors of mi R-325-3p were synthesized.The effects of micro RNA-325-3p on the differentiation of cementoblasts were detected by real-time PCR and western blot,and the effect of mi R-325-3p on the differentiation of cementoblasts was verified by heterotopic osteogenesis animal model.Results: IL1β up-regulated the expression of mi R-325-3p during the differentiation of cementoblasts.The mi R-325-3p down-regulated the levels of runx2 and osterix,and had a negative regulatory effect on the differentiation of cementoblasts.In vivo osteogenesis model,mi R-325-3p inhibitors could significantly increase the volume,density,calcium content and alkaline phosphatase activity of newly formed cementoid tissues.The mi R-325-3p inhibitor,could attenuate the inhibitory effects of IL1β on the differentiation of cementoblasts.Conclusion: IL1β could inhibit the differentiation of osteoblasts by up-regulating the expression of mi R-325-3p.The inhibitor of mi R-325-3p could attenuate the inhibitory effects of IL1β on the differentiation of cementoblasts.3.Transcriptome sequencing analysis of the mechanism of IL1β on the differentiation of cementoblastsObjective: To elucidate the mechanisms of IL1β on cementoblasts at transcriptional levels.Methods: The osteogenic induction of cementoblasts was performed by IL1β recombinant protein(10 ng/m L).Cell samples were collected,RNA was extracted,RNA-seq libraries were constructed,libraries were sequenced,sequencing data processing and bioinformatics analysis were performed.Results: The database of cementoblasts differentiation regulated by IL1β was successfully constructed.Based on RNA-seq,85 up-regulated genes and 27 downregulated genes were screened.GO enrichment analysis revealed that 1735 GO functional items were significantly enriched(p < 0.05),and were highly enriched in immune response and extracellular stimulation.KEGG enrichment analysis showed that 12 pathways were significantly enriched(p < 0.05).TNF signaling pathway,cytokine-receptor interaction pathway,chemokine signaling pathway,PI3K-Akt signaling pathway,Toll-like receptor signaling pathway and Jak-STAT signaling pathway are the most important enrichment pathways.Conclusion: IL1β could stimulate cementoblasts to happen many biological reactions like immune responses,which might play a role through TNF,PI3K-Akt and other signaling pathways.The results provided valuable new clues for exploring the mechanism of IL1β-inhibited cementoblasts differentiation.4.Effects of lcn2 on the differentiation of cementoblastsObjective: To reveal the effects of lcn2 on the differentiation of cementoblasts.Methods: A system for transfecting osteoblasts into si RNA and plasmid was established by transient transfection and the transfection efficiency was tested.Realtime PCR and western blot were used to observe the overexpression and knockdown efficiency of lcn2,and to detect the effects of interleukin on the expression of differentiation-related genes such as runx2 and osterix.Results: The transfection system of osteoblasts transfected with si RNA and plasmid was successfully established.Overexpression of lcn2 at the m RNA level was significantly higher than that of the control group,and after knockdown was about half of the control group.Overexpression of lcn2 at the protein level was approximately doubled,and approximately half of the control group after knockdown.Lcn2 negatively regulates the expressions of runx2,osterix,alp and ocn m RNAs in osteoblast differentiation,and positively regulates opn m RNA expression,and negatively regulates the expression of runx2 and osterix.Conclusion: IL1β might inhibit the differentiation of cementum cells by upregulating the expression of lcn2.In summary,IL1β inhibited cementoblasts differentiation,which might be mediated by activating mi R-325-3p and targeting transcription factor runx2.Perhaps,the expression of lcn2,a key protein screened by sequencing,was activated to inhibit the differentiation of cementoblasts.The results of transcriptome sequencing provide clues for future research and need to be further explored.