Influence Of Let-7c-5p On DPSCs Differentiation Ability And Its Mechanism In Pulpitis Condition

Background:Pulpitis is an irreversible damage to the pulp tissue caused by bacterial infection and external mechanical and chemical stimulation.It is a common disease in the oral cavity.At present,the clinical diagnosis and treatment is mainly symptomatic treatment such as endodontic therapy.More than 14 million cases of endodontic therapy are performed in the United States each year.Although endodontic therapy can effectively slow down the further progression of pain symptoms and inflammation of pulpitis,it is inevitable to completely destroy the biological activity of pulp tissue in the operation,so that the support of dental pulp tissue to the dental hard tissue is completely cut off.Therefore,even if an effective endodontic therapy is taken,the life of the teeth is greatly reduced.Dental pulp stem cells(DPSCs)are one type of mesenchymal stem cells(MSCs)and are vital cells in the pulp tissue that have the ability to differentiate.In the inflammatory reaction,some inflammatory factors may affect the functions of dental pulp stem cells in cell proliferation,cell differentiation,and deposition of calcium.The irreversible damage of pulpitis is mainly caused by inflammation,which destroys the differentiation ability of pulp tissue.Therefore,the study on the influence of pulp inflammation on the differentiation of dental pulp stem cells has important significance for the treatment of pulp inflammation.Micro RNA is a small molecular nucleotide sequence involved in the regulation of various gene expression in eukaryotic cells,and has raised a heated research in recent years.Let-7c-5p(mature,5’-UGAGGUAGUAGGUUGUAUGGUU-3’)was found to have a decreased expression in dental pulp inflammatory reaction and be involved in the differentiation of DPSCs,the release of inflammatory factors,calcium deposition and other physiological functions by directly acting on its target gene DMP1 and HMGA2.Therefore,with reference to the relevant research literature,taking Let-7c-5p as an entry point of pulp inflammatory reaction to study the anti-inflammatory mechanism has important guidance for the study of pulpitis significance.Methods:In this study,DPSCs are isolated from primary cells culture of SD rat dental pulp tissue,and identified by immunofluorescence assay.The dental inflammatory model is established by inducing DPSCs inflammatory response with LPS solution.The lentivirus is transfected with Let-7c-5p to increase the expression of Let-7c-5p in DPSCs.Experiments of Cell viability,immunohistochemistry,real-time quantitative PCR,Western-blot and alizarin red staining are performed on DPSCs in different experimental conditions to test the affects of let-7c-5p on inflammatory factors,osteogenic differentiation of DPSCs and calcium ion deposition through the interaction of its downstream DMP1-NF-κB/p65 pathway and HMGA2/PI3K/Akt pathway.The experimental data are used to explain the effect of Let-7c-5p on pulp inflammation and its mechanism.Results:1.The cell morphology of primary isolation culture of SD rat dental pulp tissue is polymorphic and most shapes of cells are fusiform.The immunofluorescence results are positive for CD90,CD105,CD29 and CD146,and negative for CD34 and CD45,which are consistent with DPSCs.2.The expression of micro RNA Let-7c-5p has significantly decreased after LPS induced inflammatory reaction in DPSCs of SD rats(p<0.05),and the expression of inflammatory factors TNF-α and IL-1β are significantly increased(p<0.05).The expression of DMP1,p-Ik Bα.p-IKKβ and NF-κB p65 significantly increases,but the expression of Ik Bα decreases,indicating that DMP1-NF-κB/p65 signaling pathway is activated in inflammatory conditions.3.After transfection with lentivirus,the expression of Let-7c-5p is overexpressed in DPSCs,and the expression of inflammatory factors TNF-α and IL-1β are significantly lower than those in LPS inflammation group(p<0.05).The expression of DMP1,p-Ik Bα,p-IKKβ NF-κB p65 is significantly lower than that of LPS inflammation group,but the expression of Ik Bα increases,indicating that the overexpression of Let-7c-5p has inhibited the activity of DMP1-NF-κB signaling pathway in inflammatory conditions.4.After LPS induced inflammatory response of DPSCs in SD rats.Alizarin red staining shows significant reduction in calcium ion deposition in DPSCs in inflammatory conditions.ALP activity also significantly decreases in alkaline phosphatase activity test.The expression of factors related to osteogenic gene detection such as Osteocalcin(OCN),Osteopontin(OPN),zinc finger structural transcription factor(Osterix,OSX),MSX2 gene,RUNX2 transcription factor is significantly decreased(p<0.05).The expression of HMGA,p-PI3 K and p-Akt in DPSCs which induced by LPS was significantly increases(p<0.05),indicating that after LPS induced inflammation in DPSC,the ability of osteogenic differentiation is attenuated by the activation of the HMGA2/PI3K/Akt pathway.5.By transfecting with lentivirus,Let-7c-5p in DPSCs is overexpressed.In relevant detections of DPSCs,calcium salt deposition,ALP activity increase,OCN,OPN,OSX,MSX2,RUNX2 related factors increase.The expression of HMGA2,pPI3 K and p-Akt decrease,indicating that the overexpression of Let-7c-5p enhances the osteogenic capacity of DPSCs in inflammatory conditions by inhibiting the activity of HMGA2/PI3K/Akt pathway.6.The results of experiments in SD rats further verify the data in vitro test,and immunohistochemistry suggested that overexpression of Let-7c-5p can significantly reduce neutrophil infiltration after LPS induced inflammation,suggesting that overexpression of Let-7c-5p has an anti-inflammatory effect.Conclusion:1.let-7c-5p in DPSCs can participate in anti-inflammatory effects by regulating its activity of downstream DMP1-NF-κB/p65 pathway.2.In the DPSCs,let-7c-5p can affect the ability of osteogenesis of cells in inflammatory conditions by regulating the activity of its downstream HMGA2/ PI3K/Akt pathway.