A Preliminary Study Of Human Dental Pulp Stem Cell Exosomes Regulating Pulpitis Factor Expression In Vitro

Chronic Pulpitis is one of the common diseases in oral clinical work,with a high prevalence and a wide range of people involved.Although root canal treatment can effectively solve pain and preserve affected teeth,the dental pulp and dentin complex also loses its ability to respond to external stimuli and to form restorative dentin.Pulp has its unique immune microenvironment,which contains complex cell composition,including dental pulp stem cells（DPSCs）,macrophages and vascular endothelial cells.After being stimulated by pulpitis microenvironment,a large number of inflammatory factors will be released to aggravate pulpitis.To explore a mediator or drug that can inhibit inflammatory stimulation and regulate host’s immune response to intervene in the early stage of pulpitis might be an alternative treatment method for chronic pulpitis and a supplement to the treatment concept of pulpitis.Studies have shown that DPSCs are a kind of mesenchymal stem cells（MSCs）derived from the neural ridge,and play a certain biological role in tissue damage repair and immune inflammation regulation.Exosomes derived from dental pulp stem cells（DPSC-exo）secreted by paracrine function have similar functions in terms of immune regulation,and have more advantages compared with the use of cells.It is rich in mi RNAs and can adjust the phenotype and activity of receptor cells to play a role in both innate and adaptive immune responses.Based on the possible immunomodulatory potential of DPSC-Exo,We speculated whether DPSC-Exo could regulate the expression of inflammatory cytokines in three major cells during LPS-induced pulpitis:DPSCs,macrophages and vascular endothelial cells.To study the effect of DPSC-Exo on cellular inflammation and its possible downstream molecular signals;In addition,mi RNAs that may be involved in the regulation of cellular inflammation were screened and verified by RNA-seq,so as to preliminarily evaluate the feasibility of DPSC-Exo in the improvement of chronic pulpitis,and provide a theoretical basis for the clinical application of DPSC-Exo.Objective To explore whether exosomes derived from dental pulp stem cell（DPSC-Exo）could affect the expression of inflammatory factors in DPSCs,macrophages and vascular endothelial cells induced by LPS and the possible downstream molecular signals of their effects,and to screen and verify whether mi RNA highly expressed in DPSC-Exo is involved in the regulation of the expression of inflammatory factors in cells.Methods 1.Isolate and culture DPSCs from the young permanent teeth,and use flow cytometry for identification.Culture U937 macrophage with RPMI 1640 medium and use 100ng/m L PMA to induce macrophage differentiation and maturation.DPSC-Exo were isolated from the supernatant of DPSCs using membrane affinity column method and identified by transmission electron microscopy,western blot and NTA particle size detection.2.DPSC-Exo stained with membrane dye was co-culture with cells to trace the entry of DPSC-Exo into cells by laser confocal detection.The effect of DPSC-Exo on migration of DPSCs and HUVEC was detected by scratch test.LPS was used to construct U937 macrophage inflammation model,and the secretion of inflammatory cytokines including IL-6,IL-1βand TNF-αwere detected by ELISA.The expression of inflammatory cytokines and CD206 were detected by q PCR.The inflammatory models of DPSCs and HUVEC were constructed,and the expressions of inflammatory cytokines were detected by q PCR.3.Using mRNA sequencing to detect DPSC-Exo regulated LPS-induced DPSCs,macrophages and HUVEC inflammation groups,macrophages were used as a representative to analyze partial genes that significantly differentially expressed and their enrichment pathways involved.Use mi RNA sequencing to detect DPSC-Exo mi RNA expression profile and analyze it.Transfect Let-7a-5p mimics and inhibitors into macrophages,q PCR detects the expression of inflammatory cytokines,and explore its relationship with inflammation.Results 1.The DPSCs were successfully isolated in this study and expressed CD73,CD90,CD146 without CD45.The differentiation and maturation of U937 macrophages were successfully induced.Transmission electron microscopy showed that DPSC-Exo presented a typical double-layer lipid membrane with a saucer-like structure.Western blot results showed positive expression of CD9 and CD63 and NTA results showed that the particle size of DPSC-Exo was uniform and concentrated between30nm and 150nm,conformed to the characteristics of exosomes.2.DPSC-Exo could be endocytotic in the cytoplasm and significantly promoted the migration of DPSCs and HUVEC（P<0.05）.For U937macrophages,DPSC-Exo intervention could significantly decreased the secretion of IL-6 and IL-1β.Also,DPSC-Exo intervention could significantly decrease the expression of IL-6,IL-1β,TNF-αand increased the expression of CD206 compared with LPS group（P<0.05）.For DPSCs,the expression of IL-6,IL-1βand TNF-αin DPSC-Exo intervention group were significantly decreased compared with LPS group（P<0.05）.And for HUVEC,the expression of IL-6 and TNF-αin DPSC-EXO intervention group were significantly decreased compared with LPS group（P<0.05）.3.The results of mRNA sequencing（U937 macrophage cells）showed that IL-1βand TNF,MMPs and CCLs,M1 macrophages marker protein CD86,CLEC5A expression were significantly lower and the expression of heat shock protein HSPA1A/B and GADD45βwere increased in the DPSC-Exo intervention group compared with LPS group（|㏒₂FC|≥1,FDR≤0.001）.They were significantly enriched in inflammation-related signaling pathways such as PI3K-Akt,IL-17,Toll-like,TNF,NF-KB and autophagy,and the PI3K-Akt signaling pathway was enriched in the process of DPSC-Exo regulating LPS-induced inflammation of the three kinds of cells.Let-7a-5p was significantly high expressed in DPSC-Exo enriched mi RNAs（|㏒₂FC|≥1,FDR≤0.001）and the target genes of the top16 mi RNAs were mainly enriched in the autophagy pathway.Overexpression Let-7a-5p could reduce the expression of inflammatory cytokines TNF-α,but IL-6 and IL-1βdid not reduce after overexpression（P<0.05）.Conclusions DPSC-Exo could inhibit the expression of inflammatory factors in DPSCs,U937 and HUVEC cells and promote the migration of DPSCs and HUVEC;The highly expressed Let-7a-5p in DPSC-Exo could down-regulate TNF-αin U937 macrophage cells;However,the mechanism of DPSC-Exo and Let-7a-5p in the regulation of pulpitis remains to be further studied.